u-0126 and Breast-Neoplasms

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a class B G protein-coupled receptor with the neuropeptide VIP as a ligand. Increased VIPR2 mRNA expression and/or VIPR2 gene copy number has been documented in several cancers including breast carcinoma. However, the pathophysiological role of increased VIPR2 in the proliferation of breast cancer cells remains largely unknown. In this study, we found that VIPR2 overexpression in MCF-7 and MDA-MB-231 cells, human breast cancer cell lines, promoted cell proliferation. Increased VIPR2 also exacerbated intraperitoneal proliferation of breast cancer MDA-MB-231 cells in a tumor nude mouse model in vivo. Treatment with KS-133, a VIPR2-selective antagonist peptide, significantly inhibited VIP-induced cell proliferation in VIPR2-overexpressing MCF-7 and MDA-MB-231 cells. Overexpressed VIPR2 caused increases in the levels of cAMP and phosphorylated extracellular signal-regulated kinase (ERK), which involves a VIPR2 signaling pathway through Gs protein. Additionally, phosphorylation of vasodilator-stimulated phosphoprotein (Ser157) and cAMP response element binding protein (Ser133) in VIPR2-overexpressing MCF-7 cells was greater than that in control cells, suggesting the increased PKA activity. Moreover, an inhibitor of mitogen-activated protein kinase kinase, U0126, attenuated tumor proliferation in exogenous VIPR2-expressing MCF-7 and MDA-MB-231 cells at the same level as observed in EGFP-expressing cells treated with U0126. Together, these findings suggest that VIPR2 controls breast tumor growth by regulating the cAMP/PKA/ERK signaling pathway, and the excessive expression of VIPR2 may lead to an exacerbation of breast carcinoma.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Female; Humans; MAP Kinase Signaling System; Mice; Receptors, Vasoactive Intestinal Peptide, Type II; Signal Transduction; Vasoactive Intestinal Peptide

In humans, breast cancer affects a large number of females and causes a high rate of mortality worldwide. Chemokine (C-C motif) ligand 5 (CCL5) is one of the cytokines that is highly correlated to the invasive and metastatic stages of breast cancer. Our previous study has suggested the prognostic value of CCL5 expression in luminal B (HER2 - ) breast cancer. In this study, CCL5 expression was upregulated or knockdown in a luminal B breast cancer cell line, ZR7530. Further, we elucidated the effects of CCL5 on the differentiation of THP-1 monocytes into M2 macrophages. Overexpression of CCL5 affected THP-1-M2 differentiation and phosphorylation of MEK1/2, ERK1/2, and STAT2 in the cocultivated cell lines. We report that the knockdown of CCR5, a receptor of CCL5 in THP-1, inhibited the effect of ZR7530 in promoting THP-1-M2 differentiation. Furthermore, our data revealed that the inhibition of MEK1/2 and STAT3 in THP-1 cells produced equivalent results similar to those of CCL5 knockdown. In summary, we revealed the role of CCL5 in the polarization of M2 macrophages. Furthermore, we studied its interaction with CCR5 and MEK/STAT3 signaling members. These targets could be used as key regulatory members in human breast cancer therapy.

Topics: Breast Neoplasms; Butadienes; Cell Differentiation; Cell Line, Tumor; Cell Polarity; Chemokine CCL5; Coculture Techniques; Cyclic S-Oxides; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Macrophages; MAP Kinase Signaling System; Nitriles; STAT3 Transcription Factor; THP-1 Cells

Heparan sulfate proteoglycans (HSPGs) act as signaling co-receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2-O-sulfotransferase (HS2ST1), the enzyme mediating 2-O-sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF-7 and MDA-MB-231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF-2) to HS2ST1-expressing cells compared with control cells. HS2ST1-overexpressing cells showed reduced MAPK signaling responses to FGF-2, and altered expression of epidermal growth factor receptor (EGFR), E-cadherin, Wnt-7a, and Tcf4. The increased viability of HS2ST1-depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1-dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E-cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways.

Topics: Antigens, CD; Breast Neoplasms; Butadienes; Cadherins; Cell Movement; Cell Survival; ErbB Receptors; Female; Fibroblast Growth Factor 2; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; MCF-7 Cells; Neoplasm Invasiveness; Nitriles; RNA, Small Interfering; Sulfotransferases

TGFβ-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased TGFβ-SMAD3 and a reduced BMP-SMAD1/5 targeted gene expression signature correlated with shortened distant metastasis free survival and overall survival of patients. At molecular levels, we discovered that TGFβ abolished BMP-induced SMAD1/5 activation in the highly-invasive breast cancer MDA-MB-231 cells, but to a less extent in the non-invasive cancer and normal breast cells. This suggests an inverse correlation between BMP signaling and invasiveness of tumor cells and TGFβ signaling acts in a double whammy fashion in driving cancer invasion and metastasis. Sustained ERK activation by TGFβ was specifically observed in MDA-MB-231 cells, and MEK inhibitor (MEKi) treatment restored BMP-SMAD1/5 signaling while not affecting SMAD2/3 activation. FK506 potently activated BMP, but not TGFβ signaling in breast cancer cells. MEKi or FK506 alone inhibited MDA-MB-231 extravasation in a zebrafish xenograft cancer model. Importantly, when administrated at suboptimal concentrations MEKi and FK506 strongly synergized in promoting BMP-SMAD1/5 signaling and inhibiting cancer cell extravasation. Furthermore, this combination of suboptimal concentrations treatment in a mouse tumor model resulted in real-time reduction of BMP-SMAD1/5 signaling in live tumors, and consequently potently inhibited tumor self-seeding, liver and bone metastasis, but not lung and brain metastasis. Mechanistically, it is the first time to identify BMP-SMAD1/5 signaling as an underlying molecular driver for organ-specific metastasis. Combining of MEKi and FK506, or their analogues, may be explored for clinical development of breast cancer.

Topics: Animals; Bone Morphogenetic Proteins; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice; Neoplasm Metastasis; NIH 3T3 Cells; Nitriles; Organ Specificity; Protein Kinase Inhibitors; Signal Transduction; Tacrolimus; Xenograft Model Antitumor Assays; Zebrafish

B7-H3 is a tumor-promoting glycoprotein that is expressed at low levels in most normal tissues, but is overexpressed in various human cancers which is associated with disease progression and poor patient outcome. Although numerous publications have reported the correlation between B7-H3 and cancer progression in many types of cancers, mechanistic studies on how B7-H3 regulates cancer malignancy are rare, and the mechanisms underlying the role of B7-H3 in drug resistance are almost unknown. Here we report a novel finding that upregulation of B7-H3 increases the breast cancer stem cell population and promotes cancer development. Depletion of B7-H3 in breast cancer significantly inhibits the cancer stem cells. By immunoprecipitation and mass spectrometry, we found that B7-H3 is associated with the major vault protein (MVP) and activates MEK through MVP-enhancing B-RAF and MEK interaction. B7-H3 expression increases stem cell population by binding to MVP which regulates the activation of the MAPK kinase pathway. Depletion of MVP blocks the activation of MEK induced by B7-H3 and dramatically inhibits B7-H3 induced stem cells. This study reports novel functions of B7-H3 in regulating breast cancer stem cell enrichment. The novel mechanism for B7-H3-induced stem cell propagation by regulating MVP/MEK signaling axis independent of the classic Ras pathway may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

Topics: Animals; B7 Antigens; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Polarity; CRISPR-Cas Systems; Drug Resistance, Neoplasm; Enzyme Activation; Female; Humans; MAP Kinase Kinase Kinases; Mice; Mice, Nude; Nanog Homeobox Protein; Neoplasm Invasiveness; Neoplasm Proteins; Neoplastic Stem Cells; Nitriles; Protein Domains; Protein Interaction Mapping; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Recombinant Proteins; RNA Interference; RNA, Guide, Kinetoplastida; RNA, Small Interfering; Sequence Deletion; SOXB1 Transcription Factors; Spheroids, Cellular; Transfection; Up-Regulation; Vault Ribonucleoprotein Particles

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Enhancer of Zeste Homolog 2 Protein; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Resveratrol; Signal Transduction

Topics: Antigens, CD; Antioxidants; Breast Neoplasms; Butadienes; Cadherins; Cell Line; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase 1; Histones; Humans; Hydrogen Peroxide; Isoenzymes; Kaplan-Meier Estimate; MCF-7 Cells; Nitriles; Pentacyclic Triterpenes; Signal Transduction; Sitosterols; Snail Family Transcription Factors

The extracellular matrix protein fibronectin (FN) facilitates tumorigenesis and the development of breast cancer. Inhibition of the FN-induced cellular response is a potential strategy for breast cancer treatment. In the present study, we investigated the effects of the flavonoid baicalein on FN-induced epithelial-mesenchymal transition (EMT) in MCF-10A breast epithelial cells and in a transgenic mouse MMTV-polyoma middle T antigen breast cancer model (MMTV-PyMT). Baicalein inhibited FN-induced migration, invasion, and F-actin remodeling. Baicalein also suppressed FN-induced downregulation of the epithelial markers E-cadherin and ZO-1 and upregulation of the mesenchymal markers N-cadherin, vimentin, and Snail. Further investigation revealed that calpain-2 was involved in baicalein suppression of FN-induced EMT. Baicalein significantly decreased FN-enhanced calpain-2 expression and activation by suppressing its plasma membrane localization, substrate cleavage, and degradation of its endogenous inhibitor calpastatin. Overexpression of calpain-2 in MCF-10A cells by gene transfection partially blocked the inhibitory effect of baicalein on FN-induced EMT changes. In addition, baicalein inhibited calpain-2 by decreasing FN-increased intracellular calcium ion levels and extracellular signal-regulated protein kinases activation. Baicalein significantly decreased tumor onset, growth, and pulmonary metastasis in a spontaneous breast cancer MMTV-PyMT mouse model. Baicalein also reduced the expression of FN, calpain-2, and vimentin, but increased E-cadherin expression in MMTV-PyMT mouse tumors. Overall, these results revealed that baicalein markedly inhibited FN-induced EMT by inhibiting calpain-2, thus providing novel insights into the pharmacological action and mechanism of baicalein. Baicalein may therefore possess therapeutic potential for the treatment of breast cancer though interfering with extracellular matrix-cancer cell interactions.

Topics: Animals; Breast Neoplasms; Butadienes; Cadherins; Calcium; Calpain; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Female; Fibronectins; Flavanones; Humans; Kaplan-Meier Estimate; Mice; Mice, Transgenic; Nitriles; Up-Regulation; Zonula Occludens-1 Protein

High insulin levels in obese people are considered as a risk factor to induce breast carcinogenesis. And consumption of fish oils which mainly contain omega-3 fatty acids is associated with a reduced risk of breast cancer. However, whether omega-3 free fatty acids (FFAs) modulate insulin signaling pathway to prevent breast cancer is poorly understood. The current study tested the hypothesis that omega-3 FFAs attenuate insulin-induced breast cancer cell proliferation and regulate insulin signaling pathway. We show here that omega-3 FFAs attenuate MCF-7 cell proliferation and Akt and Erk1/2 phosphorylation levels stimulated by insulin. Knockdown Shp2 by siRNA resulted in significantly elevated omega-3 FFAs-activated Akt phosphorylation but failed to change insulin-stimulated Akt and Erk1/2 phosphorylation. And viable cell number was not affected by either downregulation of Shp2 expression or Erk1/2 inhibitor U0126 treatment. These observations indicated that omega-3 FFAs attenuate insulin-promoted breast cancer cell proliferation and insulin-activated Akt phosphorylation.

Topics: Breast Neoplasms; Butadienes; Cell Proliferation; Fatty Acids, Omega-3; Female; Gene Expression Regulation; Humans; Insulin; MCF-7 Cells; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Proto-Oncogene Proteins c-akt; Signal Transduction

Aberrant fibronectin (FN) expression is associated with poor prognosis, cell adhesion, and cell motility in a variety of cancer cells. In this study, we investigated the relationship between p53 and FN expression in breast cancer cells. Basal FN expression was significantly decreased by treatment with the p53 activator III, RITA, in MCF7 breast cancer cells with wild-type p53. In addition, overexpression of wild-type p53 markedly decreased the level of FN expression in p53-mutant breast cancer cells. To examine the mechanism underlying the relationship between p53 and FN expression, we treated MCF7 breast cancer cells with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). Our results showed that basal FN expression was increased by TPA treatment in a time-dependent manner. In contrast, the level of p53 expression was decreased by TPA treatment. However, the expression of FN and p53 was not altered by TPA in p53-mutant breast cancer cells. Furthermore, the alterations in FN and p53 expression in response to TPA were prevented by a specific MEK inhibitor, UO126. Finally, we demonstrated that TPA triggers degradation of p53 through the proteasomal pathway in MCF7 cells. TPA-induced FN expression was decreased by the proteasome inhibitor MG132. Under the same condition, p53 protein expression, but not mRNA expression, was reversed by MG132. Taken together, our data demonstrate that the level of FN expression is associated with the status and expression of p53 in breast cancer cells.

Topics: Breast Neoplasms; Butadienes; Drug Resistance, Neoplasm; Female; Fibronectins; Furans; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Nitriles; Protein Kinase Inhibitors; Tetradecanoylphorbol Acetate; Tumor Suppressor Protein p53

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the two most popular surfactants among perfluorinated compounds (PFCs), with a wide range of uses. Growing evidence suggests that PFCs have the potential to interfere with estrogen homeostasis, posing a risk of endocrine-disrupting effects. This in vitro study aimed to investigate the estrogenic effect of these compounds on T47D hormone-dependent breast cancer cells. PFOS and PFOA (10(-12) to 10(-4)  M) were not able to induce estrogen response element (ERE) activation in the ERE luciferase reporter assay. The ERE activation was induced when the cells were co-incubated with PFOS (10(-10) to 10(-7)  M) or PFOA (10(-9) to 10(-7)  M) and 1 nM of 17β-estradiol (E2). PFOS and PFOA did not modulate the expression of estrogen-responsive genes, including progesterone (PR) and trefoil factor (pS2), but these compounds enhanced the effect of E2-induced pS2 gene expression. Neither PFOS nor PFOA affected T47D cell viability at any of the tested concentrations. In contrast, co-exposure with PFOS or PFOA and E2 resulted in an increase of E2-induced cell viability, but no effect was found with 10 ng ml(-1) EGF co-exposure. Both compounds also intensified E2-dependent growth in the proliferation assay. ERK1/2 phosphorylation was increased by co-exposure with PFOS or PFOA and E2, but not with EGF. Collectively, this study shows that PFOS and PFOA did not possess estrogenic activity, but they enhanced the effects of E2 on estrogen-responsive gene expression, ERK1/2 activation and the growth of the hormone-deprived T47D cells. Copyright © 2015 John Wiley & Sons, Ltd.

Topics: Alkanesulfonic Acids; Breast Neoplasms; Butadienes; Caprylates; Carcinogens, Environmental; Cell Line, Tumor; Cell Proliferation; Cell Survival; Endocrine Disruptors; Estradiol; Estrogens; Female; Fluorocarbons; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; MAP Kinase Signaling System; Neoplasm Proteins; Nitriles; Osmolar Concentration; Protein Kinase Inhibitors; Response Elements; Surface-Active Agents; Trefoil Factor-1

Kisspeptins (KPs), peptide products of the KISS1 gene are endogenous ligands for the kisspeptin receptor (KISS1R), a G protein-coupled receptor. In numerous cancers, KISS1R signaling plays anti-metastatic roles. However, we have previously shown that in breast cancer cells lacking the estrogen receptor (ERα), kisspeptin-10 stimulates cell migration and invasion by cross-talking with the epidermal growth factor receptor (EGFR), via a β-arrestin-2-dependent mechanism. To further define the mechanisms by which KISS1R stimulates invasion, we determined the effect of down-regulating KISS1R expression in triple negative breast cancer cells. We found that depletion of KISS1R reduced their mesenchymal phenotype and invasiveness. We show for the first time that KISS1R signaling induces invadopodia formation and activation of key invadopodia proteins, cortactin, cofilin and membrane type I matrix metalloproteases (MT1-MMP). Moreover, KISS1R stimulated invadopodia formation occurs via a new pathway involving a β-arrestin2 and ERK1/2-dependent mechanism, independent of Src. Taken together, our findings suggest that targeting the KISS1R signaling axis might be a promising strategy to inhibit invasiveness and metastasis.

Topics: Arrestins; beta-Arrestin 2; beta-Arrestins; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; Cofilin 1; Cortactin; ErbB Receptors; Female; Humans; Matrix Metalloproteinase 14; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Podosomes; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; RNA Interference; RNA, Small Interfering; Signal Transduction

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) induces ER stress which is observed in many human diseases, including breast cancer. Cellular adaptation to ER stress is mediated by the unfolded protein response (UPR), which aims at restoring ER homeostasis. Higher levels of GRP78 expression indicates constitutive activation of the UPR in breast cancer leading to breast cancer cells that are relatively resistant to ER stress-induced apoptosis. Tunicamycin (TM), an ER stress inducer, constitutively activates the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK), and (MEK)/ERK pathway which plays a role in upregulation of GRP78 by ER stress in that inhibition of MEK by U0126 reduces the levels of GRP78 and blocks its upregulation by TM. Inhibition of the MEK/ERK pathway by U0126 sensitizes breast cancer cells to TM-induced apoptosis. Inhibition of GRP78 by siRNA knockdown enhances TM- and U0126-induced apoptosis in breast cancer cells. This sensitization of breast cancer cells to TM-induced apoptosis by inhibition of MEK/ERK and GRP78 is caspase-dependent, at least in part, by activation of caspase-4. These results seem to indicate that GRP78 has potential as a chemotherapeutical target and have important implications for new treatment strategies in breast cancer by combination with agents that induce ER stress with inhibitors of the MEK/ERK pathway.

Topics: Apoptosis; Breast Neoplasms; Butadienes; Caspases; Cell Line, Tumor; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Female; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; MAP Kinase Signaling System; MCF-7 Cells; Nitriles; Tunicamycin; Unfolded Protein Response

The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. However, the exact contributions of MEK1 and MEK2 to the development of cancer remain to be established. We studied the effects of MEK small-molecule inhibitors (PD98059 and U0126) and MEK1 and MEK2 knock-down on cell proliferation, apoptosis and MAPK activation. We showed a diminution of cell viability that was associated with a downregulation of cyclin D1 expression and an increase of apoptosis marker in MEK2 silenced cells; by contrast, a slight increase of cell survival was observed in the absence of MEK1 that correlated with an augment of cyclin D1 expression. These data indicate that MEK2 but not MEK1 is essential for MDA-MB-231 cell survival. Importantly, the role of MEK2 in cell survival appeared independent on ERK1/2 phosphorylation since its absence did not alter the level of activated ERK1/2. Indeed, we have reported an unrevealed link between MEK2 and MKK3/MKK6-p38 MAPK axis where MEK2 was essential for the phosphorylation of MKK3/MKK6 and p38 MAPK that directly impacted on cyclin D1 expression. Importantly, the MEK1 inhibitor PD98059, like MEK1 silencing, induced an augment of cyclin D1 expression that correlated with an increase of MDA-MB-231 cell proliferation suggesting that MEK1 may play a regulatory role in these cells. In sum, the crucial role of MEK2 in MDA-MB-231 cell viability and the unknown relationship between MEK2 and MKK3/MKK6-p38 axis here revealed may open new therapeutic strategies for aggressive breast cancer.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Enzyme Activation; Female; Flavonoids; Gene Knockdown Techniques; Gene Silencing; Humans; MAP Kinase Kinase 2; MAP Kinase Kinase 3; MAP Kinase Kinase 6; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction

Septins are a novel class of GTP-binding cytoskeletal proteins evolutionarily conserved from yeast to mammals and have now been found to play a contributing role in a broad range of tumor types. However, their functional importance in breast cancer remains largely unclear. Here, we demonstrated that pharmaceutical inhibition of global septin dynamics would greatly suppress proliferation, migration and invasiveness in breast cancer cell lines. We then examined the expression and subcellular distribution of the selected septins SEPT2 and SEPT7 in breast cancer cells, revealing a rather variable localization of the two proteins with cell cycle progression. To determine the role of both septins in mediating malignant behavior of cancer cells, we used RNA silencing to specifically deplete endogenous SEPT2 or SEPT7 in highly invasive breast cancer cell line MDA-MB-231. Our findings showed that SEPT2/7 depletion had virtually identical inhibitory effects on cellular proliferation, apoptosis, migration and invasion. Moreover, the opposite performance in migration and invasion was observed after enforced expression of SEPT2/7 in the same cell line. We further demonstrated MEK/ERK activation, but not other MAPKs and AKT, was positively correlated with the protein levels of SEPT2 and SEPT7. Additionally, in SEPT2/7-overexpressing cells, the MEK specific inhibitor U0126 was able to correct the high active status of MEK/ERK while normalizing the increased invasive behaviors of these cells. Taken together, these results strongly suggest that SEPT2 and SEPT7 are involved in breast carcinogenesis and may serve as valuable therapeutic targets for breast cancer.

Topics: Apoptosis; Breast Neoplasms; Butadienes; Carcinogenesis; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme Inhibitors; Female; Gene Knockdown Techniques; Human Umbilical Vein Endothelial Cells; Humans; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Microscopy, Fluorescence; Neoplasm Invasiveness; Nitriles; Phenylurea Compounds; Proto-Oncogene Proteins c-akt; Pyridines; RNA Interference; RNA, Small Interfering; Septins

The mitogen-activated protein kinase (MAPK) pathway is aberrantly activated in many human cancers, including breast cancer. Activation of MAPK signaling is associated with the increased expression of a wide range of genes that promote cell survival, proliferation, and migration. This report investigated the influence of MAPK signaling on the regulation and expression of JUNB in human breast cancer cell lines. JUNB has been associated with tumor suppressor and oncogenic functions, with most reports describing JUNB as an oncogene in breast cancer. Our results indicated that JUNB expression is elevated in MCF10A(met), SKBR3, and MDA-MB-231 human breast cancer cell lines compared to nontransformed MCF10A mammary epithelial cells. Increased RAS/MAPK signaling in MCF10A(met) cells correlates with the increased association of RNA polymerase II (Pol II) phosphorylated on serine 5 (Pol IIser5p) with the JUNB proximal promoter. Pol IIser5p is the "transcription initiating" form of Pol II. Treatment with U0126, a MAPK pathway inhibitor, reduces Pol IIser5p association with the JUNB proximal promoter and reduces JUNB expression. Oncostatin M (OSM) enhances MAPK and STAT3 signaling and significantly induces JUNB expression. U0126 treatment reduces OSM-induced Pol IIser5p binding to the JUNB proximal promoter and JUNB expression, but does not reduce pSTAT3 levels or the association of pSTAT3 with the JUNB proximal promoter. These results demonstrate that the MAPK pathway plays a primary role in the control of JUNB gene expression by promoting the association of Pol IIser5p with the JUNB proximal promoter.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Extracellular Space; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Nitriles; Oncostatin M; Phosphoserine; Promoter Regions, Genetic; RNA Polymerase II; STAT3 Transcription Factor; Transcription Factors

Diabetes is a risk factor for breast cancer development and is associated with poor prognosis for breast cancer patients. However, the molecular and biochemical mechanisms underlying the association between diabetes and breast cancer have not been fully elucidated. Here, we investigated estradiol response in MCF-7 breast cancer cells with or without chronic exposure to insulin. We found that insulin priming is necessary and specific for estradiol-induced cancer cell growth, and induces anaplerotic shunting of glucose into macromolecule biosynthesis in the estradiol treated cells. Treatment with ERK or Akt specific inhibitors, U0126 or LY294002, respectively, suppressed estradiol-induced growth. Interestingly, molecular analysis revealed that estradiol treatment markedly increases expression of cyclin A and B, and decreases p21 and p27 in the insulin-primed cells. In addition, estradiol treatment activated metabolic genes in pentose phosphate (PPP) and serine biosynthesis pathways in the insulin-primed cells while insulin priming decreased metabolic gene expression associated with glucose catabolism in the breast cancer cells. Finally, we found that anti-diabetic drug metformin and AMPK ligand AICAR, but not thiazolidinediones (TZDs), specifically suppress the estradiol-induced cellular growth in the insulin-primed cells. These findings suggest that estrogen receptor (ER) activation under chronic hyperinsulinemic condition increases breast cancer growth through the modulation of cell cycle and apoptotic factors and nutrient metabolism, and further provide a mechanistic evidence for the clinical benefit of metformin use for ER-positive breast cancer patients with diabetes.

Topics: Breast Neoplasms; Butadienes; Cell Proliferation; Chromones; Diabetes Complications; Diabetes Mellitus; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Glucose; Humans; Insulin; MCF-7 Cells; Mitogen-Activated Protein Kinase 3; Morpholines; Nitriles; Oncogene Protein v-akt; Risk Factors

Metabolic reprogramming is increasingly being viewed as a hallmark of cancer. Accordingly, metabolic readouts can serve as biomarkers of response to therapy. The goal of this study was to investigate some of the MRS-detectable metabolic consequences of mitogen-activated protein kinase kinase (MEK) inhibition. We investigated PC3 prostate cancer, MCF-7 breast cancer and A375 melanoma cells, and determined that, consistent with previous studies, MRS-detectable levels of phosphocholine decreased significantly in all cell lines (to 63%, 50% and 18% of the control, respectively) following MEK inhibition with U0126. This effect was mediated by a decrease in the expression of choline kinase α, the enzyme that catalyzes the phosphorylation of choline. In contrast, the impact of MEK inhibition on glycolysis was cell line dependent. A375 cells, which express mutant BRAF, demonstrated significant decreases in glucose uptake (to 36% of control) and lactate production (to 42% of control) in line with positron emission tomography data. In contrast, in PC3 and MCF-7 cells, increases in glucose uptake (to 198% and 192% of control, respectively) and lactate production (to 177% and 212% of control, respectively) were observed, in line with a previous hyperpolarized (13) C MRS study. This effect is probably mediated by the activation of the phosphoinositide 3-kinase pathway and AMP-activated protein kinase. Our findings demonstrate the value of translatable non-invasive MRS methods for the provision of information on cellular metabolism as an indication of the activation of potential feedback loops following MEK inhibition.

Topics: AMP-Activated Protein Kinases; Breast Neoplasms; Butadienes; Cell Line, Tumor; Glycolysis; Humans; Magnetic Resonance Spectroscopy; Male; Melanoma; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Prostatic Neoplasms; Protein Kinase Inhibitors

The pro-apoptotic protein Bnip3 is induced by hypoxia and is present in the core regions of most solid tumors. Bnip3 induces programmed necrosis by an intrinsic caspase independent mitochondrial pathway. Many tumor cells have evolved pathways to evade Bnip3-mediated death attesting to the physiological relevance of the survival threat imposed by Bnip3. We have reported that acidosis can trigger the Bnip3 death pathway in hypoxic cells therefore we hypothesized that manipulation of intracellular pH by pharmacological inhibition of the vacuolar (v)ATPase proton pump, a significant pH control pathway, may activate Bnip3 and promote death of hypoxic cells within the tumor. Here we confirm that bafilomycin A1 (BafA1), a selective vATPase inhibitor, significantly increased death of breast cancer cells in a hypoxia and Bnip3-dependent manner and significantly reduced tumor growth in MCF7 and MDA-MB-231 mouse xenografts. Combined treatment of cells with BafA1 and the ERK1/2 inhibitor U0126 further augmented cell death. Combined treatment of mice containing MDA-MB-231 xenografts with BafA1 and the ERK1/2 inhibitor sorafenib was superior to either treatment alone and supported tumor regression. BafA1 and sorafenib treatments alone reduced MDA-MB-231 cell metastasis and again the combination was significantly more effective than either treatment alone and was without apparent side effects. These results present a novel mechanism to destroy hypoxic tumor cells that may help reverse the resistance of hypoxic tumors to radiation and chemotherapy and perhaps target tumor stem cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Butadienes; Cell Hypoxia; Enzyme Inhibitors; Gene Knockdown Techniques; Heterografts; Humans; Hydrogen-Ion Concentration; Macrolides; MAP Kinase Signaling System; MCF-7 Cells; Membrane Proteins; Mice; Neoplasm Metastasis; Niacinamide; Nitriles; Phenylurea Compounds; Proto-Oncogene Proteins; Sorafenib; Tumor Burden; Vacuolar Proton-Translocating ATPases

Overproduction of prostaglandin E2 (PGE2) has been reported to be implicated in carcinogenesis. The intracellular level of PGE2 is maintained not only by its biosynthesis, but also by inactivation/degradation. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the conversion of oncogenic PGE2 to a biologically inactive keto metabolite. In the present study, we demonstrate that 15-deoxy-Δ(12,14)-prostaglandin J2 (15 d-PGJ2), one of the terminal products of cyclooxygenase-2, updregulates the expression and the activity of 15-PGDH in human breast cancer MDA-MB-231 cells. By using deletion constructs of the 15-PGDH promoter, we have found that E-twenty six (Ets) is the most essential determinant for 15-PGDH induction. 15 d-PGJ2 induced phosphorylation of Elk-1, one of Ets transcription factor family members, in the nucleus. Knockdown of Elk-1 abolished the ability of 15 d-PGJ2 to upregulate 15-PGDH expression. Furthermore, 15 d-PGJ2-mediated activation of Elk-1 was found to be dependent on activation of extracellular-signal related kinase (ERK) 1/2. Treatment of U0126, a pharmacological inhibitor of MEK1/2-ERK, abolished phosphorylation and DNA binding of Elk-1 as well as 15-PGDH induction in 15 d-PGJ2-treated MDA-MB-231 cells. Moreover, 15 d-PGJ2 generated reactive oxygen species (ROS), which contribute to the expression of 15-PGDH as well as phosphorylation of ERK1/2 and Elk-1. 15 d-PGJ2 inhibited the migration of MDA-MB-231 cells, which was attenuated by transient transfection with 15-PGDH siRNA. Taken together, these findings suggest that 15 d-PGJ2 induces the expression of 15-PGDH through ROS-mediated activation of ERK1/2 and subsequently Elk-1 in the MDA-MB-231 cells, which may contribute to tumor suppressive activity of this cyclopentenone prostaglandin.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; ets-Domain Protein Elk-1; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hydroxyprostaglandin Dehydrogenases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Prostaglandin D2; Reactive Oxygen Species

The Na(+)/I(-) symporter (NIS (SLC5A5)) is a transmembrane glycoprotein that mediates active iodide uptake into thyroid follicular cells. NIS-mediated iodide uptake in thyroid cells is the basis for targeted radionuclide imaging and treatment of differentiated thyroid carcinomas and their metastases. Furthermore, NIS is expressed in many human breast tumors but not in normal non-lactating breast tissue, suggesting that NIS-mediated radionuclide uptake may also allow the imaging and targeted therapy of breast cancer. However, functional cell surface NIS expression is often low in breast cancer, making it important to uncover signaling pathways that modulate NIS expression at multiple levels, from gene transcription to posttranslational processing and cell surface trafficking. In this study, we investigated NIS regulation in breast cancer by MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) signaling, an important cell signaling pathway involved in oncogenic transformation. We found that MEK inhibition decreased NIS protein levels in all-trans retinoic acid/hydrocortisone-treated MCF-7 cells as well as human breast cancer cells expressing exogenous NIS. The decrease in NIS protein levels by MEK inhibition was not accompanied by a decrease in NIS mRNA or a decrease in NIS mRNA export from the nucleus to the cytoplasm. NIS protein degradation upon MEK inhibition was prevented by lysosome inhibitors but not by proteasome inhibitors. Interestingly, NIS protein level was correlated with MEK/ERK activation in human breast tumors from a tissue microarray. Taken together, MEK activation appears to play an important role in maintaining NIS protein stability in human breast cancers.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Humans; Hydrocortisone; Iodides; Lysosomes; Mitogen-Activated Protein Kinase Kinases; Nitriles; Protein Kinase Inhibitors; Proteolysis; RNA, Messenger; Symporters; Tretinoin

Since its isolation from Tripterygium wilfordii in 1972, triptolide has been shown to possess potent anticancer activity against a variety of cancers, and has entered phase I clinical trial. It is a diterpenoid triepoxide that acts through multiple molecular targets and signaling pathways. The mitogen-activated protein kinases are well known for their modulation of cell survival and proliferation. In particular, the ERK pathway has a dual role in cell proliferation and cell death. Thus far, data on the effect of triptolide on ERK signaling remain limited. In our current study, we have shown for the first time that ERK activation rather than inhibition occurred in a dose- and time-dependent manner following triptolide treatment in MDA-MB-231 breast cancer cells. ERK activation was crucial in mediating triptolide-induced caspase-dependent apoptosis. Tritpolide-induced ERK activation modulated the expression of the Bcl-2 protein family member Bax but was not involved in the downregulation of Bcl-xL expression. Signals acted upstream of ERK activation included generation of reactive oxygen species (ROS) and endoplasmic reticulum stress predominantly via the PERK‑eIF2α pathway, as the MEK inhibitor U0126 did not inhibit the phosphorylation of PERK and eIF2α or the generation of ROS.

Topics: Apoptosis; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Clinical Trials as Topic; Diterpenes; eIF-2 Kinase; Endoplasmic Reticulum Stress; Epoxy Compounds; Eukaryotic Initiation Factor-2; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinases; Nitriles; Oxidative Stress; Phenanthrenes; Reactive Oxygen Species; Signal Transduction

Granulocyte colony-stimulating factor (G-CSF) affects granulopoiesis and is important for mobilizing neutrophils into blood circulation. Due to the hematopoietic properties of G-CSF, it has been widely used to clinically treat chemotherapy-induced neutropenia. However, G-CSF can promote tumors by inhibiting innate and adaptive immunity and enhancing angiogenesis and neoplastic growth. Most G-CSF-producing tumors are associated with a poor prognosis. This indicates that G-CSF promotes cancer progression. Thus, identifying regulatory molecules involved in tumor-derived G-CSF expression may provide therapeutic targets for cancer treatment. This study identified considerable G-CSF expression in malignant breast, lung and oral cancer cells. However, G-CSF expression was barely detectable in non-invasive cell lines. Expression of G-CSF mRNA and protein increased during exposure to tumor necrosis factor-α (TNF-α). Treatment with U0126 (a mitogen-activated protein kinase inhibitor) drastically reduced basal levels of G-CSF and TNF-α-induced G-CSF in aggressive cancer cells. This study also showed that knockdown of extracellular signal-regulated kinase (ERK) 2 by shRNA was necessary and sufficient to eliminate the expression of tumor-derived G-CSF. This did not apply to ERK1. Therefore, ERK2 (but not ERK1) is responsible for the transcriptional regulation of tumor-derived G-CSF. The results indicate the pharmaceutical value of specific ERK2 inhibitors in treating patients with G-CSF-producing tumors.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Breast Neoplasms; Butadienes; Carcinoma, Squamous Cell; Cell Line, Tumor; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasms; Nitriles; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tumor Necrosis Factor-alpha

We have recently identified tumor necrosis factor (TNF)-α-induced phosphorylation of epidermal growth factor receptor (EGFR) at Thr-669 and Ser-1046/1047 via ERK and p38 pathways, respectively. In the present study, we investigated the roles of ligand-induced phosphorylation of serine and threonine residues in EGFR-overexpressing MDA-MB-468 breast cancer cells. Epidermal growth factor and heregulin, an ErbB3 ligand, induced the phosphorylation of Thr-669 and Ser-1046/1047. Inversely, constitutive tyrosine phosphorylation of the C-terminal domain, including Tyr-1068, was significantly downregulated on ligand stimulation. Inhibition of the ERK pathway by U0126 blocked ligand-induced Thr-669 phosphorylation as well as Tyr-1068 dephosphorylation. Downregulation of constitutive tyrosine phosphorylation of EGFR in HEK293 cells stably expressing the wild type was abolished by substitution of Thr-669 for Ala. In an asymmetric EGFR homodimer structure, one Thr-669 in the receiver kinase of the dimer was involved in downregulation. Similarly, Thr-669 in an EGFR-ErbB3 heterodimer also participated in tyrosine dephosphorylation. These results indicate that ERK-mediated Thr-669 phosphorylation suppresses constitutive tyrosine phosphosphorylation in the homo- and heterodimer asymmetric conformations of the EGFR.

Topics: Adenocarcinoma; Breast Neoplasms; Butadienes; Cell Line, Tumor; Dimerization; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Female; Humans; Ligands; MAP Kinase Signaling System; Neoplasm Proteins; Neuregulin-1; Nitriles; Phosphorylation; Phosphothreonine; Protein Conformation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Recombinant Proteins; Tumor Necrosis Factor-alpha

Nuclear hormone receptor family member PPARγ plays an important role in mammary gland tumorigenesis. Previous studies have shown PPARγ has cytoplasmic activities upon tetradecanoyl phorbol acetate (TPA) stimulation. However, the clinical pathological significance of cytoplasmic PPARγ is not completely understood in human breast cancer. Skp2 is oncogenic, and its frequent amplification and overexpression correlated with the grade of malignancy. In this study, the role of cytoplasmic PPARγ and Skp2 expression was investigated in human breast cancer progression. Therefore, immunohistochemical analysis was performed on formalin-fixed paraffin sections of 70 specimens. Furthermore, Western blot and immunofluorescence microscopy analysis were used to study the relationship between expression of cytoplasmic PPARγ and Skp2 expression in human breast cancer cells in vitro. Results showed that the expression of cytoplasmic PPARγ was positively correlated with Skp2 expression (p < 0.05), and correlated significantly with estrogen receptor (p = 0.026) and pathological grade (p = 0.029), respectively. In addition, Skp2 overexpression can provoke cytoplasmic localization of PPARγ upon MEK1-dependent mechanisms in human breast cancer cells by nuclear-cytosolic fractionation technology and immunofluorescence microscopy analysis. Using RNA interference technology, we also found that down-regulated Skp2 reduced the phosphorylation level of MEK1 and significantly reversed TPA-induced nuclear export of PPARγ in MDA-MB-231 cells. The changes in the subcellular localization of PPARγ may represent a novel target for selective interference in patients with breast cancer.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Enzyme Inhibitors; Female; Flavonoids; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; MCF-7 Cells; Nitriles; Phosphorylation; PPAR gamma; Protein Kinase Inhibitors; Receptors, Estrogen; RNA Interference; RNA, Small Interfering; S-Phase Kinase-Associated Proteins; Tetradecanoylphorbol Acetate

Alterations in cell metabolism are increasingly being recognized as a hallmark of cancer and are being exploited for the development of diagnostic tools and targeted therapeutics. Recently, ¹³C MRS-detectable hyperpolarized pyruvate to lactate conversion has been validated in models as a noninvasive imaging method for the detection of tumors and treatment response, and has successfully passed phase I clinical trials. To date, response to treatment has been associated with a decrease in hyperpolarized lactate production. In this study, we monitored the effect of treatment with the mitogen-activated protein kinase (MEK) inhibitor U0126 in prostate and breast cancer cells. Following treatment, we observed a 31% decrease in the flux of hyperpolarized ¹³C label in treated MCF-7 breast cancer cells relative to controls. In contrast, and unexpectedly, the flux increased to 167% in treated PC3 prostate cancer cells. To mechanistically explain these observations, we investigated treatment-induced changes in the different factors known to affect the pyruvate to lactate conversion. NADH (nicotinamide adenine dinucleotide, reduced form) levels remained unchanged, whereas lactate dehydrogenase expression and activity, as well as intracellular lactate, increased in both cell lines, providing an explanation for the elevated hyperpolarized lactate observed in PC3 cells. The expression of MCT1, which mediates pyruvate transport, decreased in treated MCF-7, but not PC3, cells. This identifies pyruvate transport as rate limiting in U0126-treated MCF-7 cells and explains the decrease in hyperpolarized lactate observed in these cells following treatment. Our findings highlight the complexity of interactions between MEK and metabolism, and the need for mechanistic validation before hyperpolarized ¹³C MRS can be used to monitor treatment-induced molecular responses.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Female; Humans; Lactic Acid; Male; MCF-7 Cells; Mitogen-Activated Protein Kinase Kinases; Nitriles; Prostatic Neoplasms; Pyruvic Acid

Compared to other cancers affecting women, breast cancer is significantly associated with alcohol consumption. However, the principles underlying the carcinogenesis of alcohol-induced breast cancer and the related metastatic mechanisms have yet to be established. To observe the effect of alcohol on the growth regulation in breast cancer cells, we identified differentially expressed proteins in alcohol-exposed human breast cancer T47D cells using gel-based proteomics analysis. The expression of c-ros receptor tyrosine kinase (ROS1) was increased and activated by autophosphorylation, thereby activating mitogen- and stress-activated protein kinase 1 (MSK1) through the mitogen‑activated protein kinase (MAPK) pathway; activated MSK1, in turn, phosphorylated histone 3 serine 10 (H3S10p) residues in the nucleus. The increase in H3S10 phosphorylation consequently increased the level of expression of immediate-early gene such as c-fos. This study demonstrated that when breast cancer cells are exposed to alcohol, phosphorylated ROS1 activates MSK1 via Erk1/2 in the MAPK pathway, which then induces modifications to histone residues that regulate gene expression by 14-3-3 protein recruitment, leading to a lack of control of breast cancer cell proliferation.

Topics: Alcohol Drinking; Breast Neoplasms; Butadienes; Cell Line, Tumor; Ethanol; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Up-Regulation

Matrix metalloproteinases (MMPs) play an important role in cancer metastasis, cell migration and invasion. Herein, we investigated the effects of silibinin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration and MMP-9 expression in thyroid and breast cancer cells. Our results revealed that the levels of MMP-9 mRNA and protein expression were significantly increased by TPA but not MMP-2 in TPC-1 and MCF7 cells. To verify the regulatory mechanism of TPA-induced MMP-9 expression, we treated TPC-1 and MCF7 cells with the MEK1/2 inhibitor, UO126, and TPA-induced MMP-9 expression was significantly decreased. We also found that TPA-induced cell migration and MMP-9 expression was significantly decreased by silibinin. In addition, TPA-induced phosphorylation of MEK and ERK was also inhibited by silibinin. Taken together, we suggest that silibinin suppresses TPA-induced cell migration and MMP-9 expression through the MEK/ERK-dependent pathway in thyroid and breast cancer cells.

Topics: Breast Neoplasms; Butadienes; Cell Movement; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; MCF-7 Cells; Neoplasm Invasiveness; Nitriles; Phosphorylation; Silybin; Silymarin; Tetradecanoylphorbol Acetate; Thyroid Neoplasms

Recent studies have suggested that progestins play a role in the etiology of breast cancer; however, the mechanisms by which progestins promote tumor formation/progression have not been defined. Progestin action, in target tissues such as the breast, is mediated by the progesterone receptor (PR). PR signaling is complex and PR regulates transcription of target genes through a variety of mechanisms. Many cell signaling pathways are activated inappropriately in breast cancer cells and these pathways can regulate PR activity. For example, the p42/p44 MAPK pathway can regulate PR function by altering phosphorylation of PR, as well as its coregulators. We found that inhibition of the p42/p44 MAPK signaling pathway with a MEK inhibitor (U0126) impairs PR-mediated gene induction, but not gene repression. In addition, the effects of U0126 on PR-mediated gene transcription are much greater with long-term versus short-term inhibition and are gene-specific. Finally, treatment with U0126 delays phosphorylation of Ser294, but does not block phosphorylation completely, suggesting that p42/p44 MAPK kinase is not the dominant kinase responsible for phosphorylating this site. Collectively, these studies suggest that in addition to the p42/p44 MAPK pathway, other signaling pathways are also important for PR transcriptional activity in breast cancer cells. The integration of PR transcriptional effects and cell signaling pathways has implications for the initiation or progression of breast cancer. Understanding how these pathways interact may aid in the development of prevention and/or treatment strategies for the disease.

Topics: Breast Neoplasms; Butadienes; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Real-Time Polymerase Chain Reaction; Receptors, Progesterone; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured

The potential benefits of drugs directly targeting the ErbB receptors for cancer therapy have led to an extensive development within this field. However, the clinical effects of ErbB receptor-targeting drugs in cancer treatment are limited due to a high frequency of resistance. It has been reported that, when inhibiting the epidermal growth factor receptor (EGFR) with the tyrosine kinase inhibitor gefitinib, increased activation of ErbB3 via MET, or by re-localization of ErbB3 mediates cell survival. Here we show further evidence that members of the ErbB receptor family facilitate resistance to EGFR inhibitor treatment in ErbB2 overexpressing breast cancer cells. We found that gefitinib treatment increased ErbB3 expression, both at protein and mRNA levels. ErbB3 expression was upregulated not only by gefitinib but also by a panel of different EGFR inhibitors, suggesting that inhibition of EGFR in general affects ErbB3 expression. In addition, we found that gefitinib treatment increased ErbB2 expression levels while EGFR inhibitors decreased the activity of ErbB2. Concentrations of gefitinib that decreased phospho-ErbB2 reversely increased ErbB3 levels. We further examined changes induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Chromones; ErbB Receptors; Erlotinib Hydrochloride; Female; Gefitinib; Gene Expression; Gene Expression Profiling; Humans; Imidazoles; MAP Kinase Kinase 1; Morpholines; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyridines; Quinazolines; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; RNA, Messenger; Signal Transduction; Tyrphostins; Up-Regulation

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a differential role on the regulation of the cell cycle in a variety of tumor cells. The mechanism between TPA and the cell cycle in breast cancer is not fully understood. Therefore, we investigated the regulatory mechanism of TPA on control of the cell cycle of breast cancer cells. Our results showed that TPA increased the level of p21 expression in MCF-7 cells with wild-type p53 and MDA-MB-231 cells with mutant p53 in a dose-dependent manner. In contrast, TPA decreased the expression of p53 in MCF-7 cells, but did not affect MDA-MB-231 cells. We next examined the regulatory mechanism of TPA on p21 and p53 expression. Our results showed that the TPA-induced up-regulation of p21 and down-regulation of p53 was reversed by UO126 (a MEK1/2 inhibitor), but not by SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor), although TPA increased the phosphorylation of ERK and JNK in MCF-7 cells. In addition, the TPA-induced arrest of the G2/M phase was also recovered by UO126 treatment. To confirm the expression of p21 through the MEK/ERK pathway, cells were transfected with constitutively active (CA)-MEK adenovirus. Our results showed that the expression of p21 was significantly increased by CA-MEK overexpression. Taken together, we suggest that TPA reciprocally regulates the level of p21 and p53 expression via a MEK/ERK-dependent pathway. The up-regulation of p21 in response to TPA is mediated through a p53-independent mechanism in breast cancer cells.

Topics: Breast Neoplasms; Butadienes; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Inhibitors; Female; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; M Phase Cell Cycle Checkpoints; MAP Kinase Signaling System; Nitriles; Phosphorylation; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Suppressor Protein p53

Although it has been suggested that smad7 blocks downstream signaling of TGF-β, the role of smad7 in the EGF signaling pathway has not been fully elucidated. We determined the effect of smad7 on EGF-induced MMP-9 expression in SKBR3 breast cancer cells. The expression of smad7 and MMP-9 was increased by EGF or TGF-β1, respectively, and further increased by EGF and TGF-β1 co-treatment. EGF induced the phosphorylation of EGFR, smad3, ERK, and JNK, and MMP-9 expression was decreased by the EGFR inhibitor, AG1478. In addition, EGF-induced MMP-9 expression was inhibited by UO126 (a MEK1/2 inhibitor) or SIS3 (a smad3 inhibitor), but not by SP600125 (a JNK inhibitor). Interestingly, EGF-induced smad3 phosphorylation was completely blocked by smad7 over-expression, but not the phosphorylation of ERK and JNK. EGF- or TGF-β1-induced MMP-9 expression was completely decreased by adenoviral-smad7 (Ad-smad7) over-expression. We also investigated the role of smad3 on EGF-induced MMP-9 expression and showed that EGF-induced MMP-9 expression was decreased by smad3 siRNA transfection, whereas EGF-induced MMP-9 expression was further increased by smad3 over-expression, as expected. This study showed that EGF-induced smad3 phosphorylation mediates the induction of MMP-9, whereas smad7 inhibits TGF-β1 as well as the EGF signaling pathway in SKBR3 cells.

Topics: Anthracenes; Breast Neoplasms; Butadienes; Cell Line, Tumor; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Matrix Metalloproteinase 9; Nitriles; Phosphorylation; RNA, Messenger; Signal Transduction; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta1

Molecularly targeted therapies have emerged as the leading theme in cancer therapeutics. Multi-cytotoxic drug regimens have been highly successful, yet many studies in targeted therapeutics have centered on a single agent. We investigated whether the Src/Abl kinase inhibitor dasatinib displays synergy with other agents in molecularly heterogeneous breast cancer cell lines. MCF-7, SKBR-3, and MDA-MB-231 display different signaling and gene signatures profiles due to expression of the estrogen receptor, ErbB2, or neither. Cell proliferation was measured following treatment with dasatinib±cytotoxic (paclitaxel, ixabepilone) or molecularly targeted agents (tamoxifen, rapamycin, sorafenib, pan PI3K inhibitor LY294002, and MEK/ERK inhibitor U0126). Dose-responses for single or combination drugs were calculated and analyzed by the Chou-Talalay method. The drugs with the greatest level of synergy with dasatinib were rapamycin, ixabepilone, and sorafenib, for the MDA-MB-231, MCF-7, and SK-BR-3 cell lines respectively. However, dasatinib synergized with both cytotoxic and molecularly targeted agents in all three molecularly heterogeneous breast cancer cell lines. These results suggest that effectiveness of rationally designed therapies may not entirely rest on precise identification of gene signatures or molecular profiling. Since a systems analysis that reveals emergent properties cannot be easily performed for each cancer case, multi-drug regimens in the near future will still involve empirical design.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzenesulfonates; Breast Neoplasms; Butadienes; Cell Line, Tumor; Chromones; Dasatinib; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Epothilones; Female; Humans; Morpholines; Niacinamide; Nitriles; Paclitaxel; Phenylurea Compounds; Pyridines; Pyrimidines; Signal Transduction; Sirolimus; Sorafenib; Tamoxifen; Thiazoles

In the current study, we investigated whether 17beta-estradiol (E2) induces cyclin E expression and triggers cyclin E processing via calpain in MCF-7 breast cancer cells. We found that E2 induced increased expression of cyclin E in a slow and persistent manner, and a rapid yet sustained processing of cyclin E. In addition, estrogenic ethanol was able to stimulate cyclin E truncation. Calpeptin or ALLN greatly suppressed the E2-triggered cyclin E processing and its expression, suggesting a calpain-mediated action for E2. Finally, the E2-induced effects could also be significantly suppressed by BAPTA or U0126, indicating involvement of calcium/ERK signaling. Taken together, these results show that estrogen may contribute to both up-regulation and proteolysis of cyclin E through calpain in MCF-7 cells.

Topics: Breast Neoplasms; Butadienes; Calcium; Calpain; Cell Line, Tumor; Chelating Agents; Cyclin E; Egtazic Acid; Estradiol; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Nitriles; Signal Transduction; Up-Regulation

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin- β1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral- MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HRG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Lapatinib; MAP Kinase Signaling System; Matrix Metalloproteinase 1; Matrix Metalloproteinase 9; MCF-7 Cells; Neuregulin-1; Nitriles; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Protein Multimerization; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Receptor, ErbB-3

The presence of mesothelin (encoded by the mesothelin [MSLN] gene) in breast cancer is associated with tumour infiltration of the lymph node. This study evaluated whether MSLN overexpression promotes breast cancer cell invasiveness and metastasis.. This study evaluated the effects of overexpression of MSLN on extracellular signal-regulated kinase (ERK1/2) and matrix metalloproteinase (MMP)-9 levels, and the invasiveness and angiogenesis of the breast cancer cell line MCF-7 in vitro, and on MCF-7-derived tumour development in vivo.. MSLN overexpression significantly increased ERK1/2 and MMP9 protein levels and activity, and the invasive and angiogenic capability of MCF-7 cells, in vitro. Inhibition of ERK1/2 suppressed MMP-9 and the invasive and angiogenic capability of MSLN overexpressing MCF-7 cells. MSLN overexpression also increased MCF-7-derived tumour metastasis in vivo.. MSLN overexpression promoted the invasive potential of MCF-7 cells through ERK1/2-dependent upregulation of MMP-9; this association may have contributed to metastasis of MCF-7 cells in vivo. Mesothelin may be a useful biomarker for cancer progression and a novel therapeutic or chemopreventive target in human breast cancer.

Topics: Animals; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Lymphatic Metastasis; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; MCF-7 Cells; Mesothelin; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Neovascularization, Pathologic; Nitriles; Phenylalanine; Protease Inhibitors; Thiophenes; Transplantation, Heterologous

α-Tocopherol ether-linked acetic acid (α-TEA) is a promising agent for cancer prevention/therapy based on its antitumour actions in a variety of cancers.. Human breast cancer cells, MCF-7 and HCC-1954, were used to study the effect of α-TEA using Annexin V/PI staining, western blot analyses, and siRNA knockdown techniques.. α-Tocopherol ether-linked acetic acid suppressed constitutively active basal levels of pAKT, pERK, pmTOR, and their downstream targets, as well as induced both cell types to undergo apoptosis. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin suppressed pAKT, pERK, pmTOR, and their downstream targets, indicating PI3K to be a common upstream mediator. In addition, α-TEA induced increased levels of pIRS-1 (Ser-307), a phosphorylation site correlated with insulin receptor substrate-1 (IRS-1) inactivation, and decreased levels of total IRS-1. Small interfering RNA (siRNA) knockdown of JNK blocked the impact of α-TEA on pIRS-1 and total IRS-1 and impeded its ability to downregulate the phosphorylated status of AKT, ERK, and mTOR. Combinations of α-TEA+MEK or mTOR inhibitor acted cooperatively to induce apoptosis and reduce basal levels of pERK and pmTOR. Importantly, inhibition of MEK and mTOR resulted in increased levels of pAKT and IRS-1, and α-TEA blocked them.. Downregulation of IRS-1/PI3K pathways via JNK are critical for α-TEA and α-TEA+MEK or mTOR inhibitor-induced apoptosis in human MCF-7 and HCC-1954 breast cancer cells.

Topics: 1-Phosphatidylinositol 4-Kinase; alpha-Tocopherol; Androstadienes; Anthracenes; Antioxidants; Apoptosis; Blotting, Western; Breast Neoplasms; Butadienes; Drug Synergism; Drug Therapy, Combination; Enzyme Inhibitors; Female; Humans; Immunosuppressive Agents; Insulin Receptor Substrate Proteins; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Wortmannin

Activation of epidermal growth factor receptor (EGFR)-induced signaling pathways has been correlated with tumor progression, invasion and metastasis in a variety of cancers including breast carcinoma, but the underlying mechanism is not well understood. Matrix metalloproteinases (MMPs) have been implicated in cancer invasion and metastasis for their extracellular matrix (ECM)-proteolytic activity. However, the correlation of EGFR pathway with MMP expression in breast cancer has not been established. The aim of this study was to elucidate the interaction between EGFR ligands and their signaling pathway and MMP expression which might be closely related with breast cancer pathogenesis. We investigated the effect of EGF ligands on the MMP1 expression in SK-BR3 cell lines using RT-PCR, Western blot, ELISA and EMSA. Treatments with EGFR ligands, EGF and TGF-α enhanced MMP1 expression at the level of both transcription and translation in SK-BR3 breast cancer cells. EGF and TGF-α treatment resulted in phosphorylation of EGFR, and consequent activation of ERK1/2 pathway. Tyrosine kinase inhibitors of HER family, erlotinib, lapatinib and canertinib suppressed EGF-ligands mediated MMP1 overexpression. The specific MEK inhibitor, U0126, significantly blocks EGF and TGF-α-mediated ERK1/2 activation and subsequent MMP1 induction in SK-BR3 cells. Inhibition of the Akt pathway with LY294002 paradoxically augmented MMP1 expression by reciprocal activation of ERK1/2 pathway. These data suggest that invasive potential of SK-BR3 cell would be affected by these drugs by suppression of EGFR ligands-induced MMP1 expression.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Chromones; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ligands; Matrix Metalloproteinase 1; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Neuregulin-1; Nitriles; Proto-Oncogene Proteins c-akt; Transforming Growth Factor alpha

Naturally-occurring chemopreventive agent phenethyl isothiocyanate (PEITC), derived primarily from watercress, has been shown to inhibit cell growth and induce apoptosis in cancer cells. In this study, we examined the potential of PEITC in enhancing cisplatin-induced apoptosis in cervical cancer cells and its mechanisms.. HeLa cells were exposed to PEITC, cisplatin or both. Pretreatment of cells with PEITC strongly enhanced cisplatin-induced cytotoxicity. PEITC activated the mitogen-activated protein kinases, including c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38. Caspase-3 activity assay demonstrated that the synergistic induction of apoptosis was significantly attenuated by MEK1/2 inhibitor U0126, but not by JNK or p38 inhibitor, suggesting that ERK activation is responsible for the synergistic effect. We found that NF-κB signaling pathway is not involved in the synergistic effect. Sulforaphane and benzyl isothiocyanate, two other members of the isothiocyanate family, also sensitize HeLa cells to apoptosis induced by cisplatin. Furthermore, we found that the synergistic effect was also observed in cervical cancer C33A and breast cancer MCF-7 cells but not in normal mammary epithelial MCF-10A cells. Finally, we demonstrated that Noxa induction was associated with apoptosis induced by PEITC plus cisplatin.. Taken together, this study shows that PEITC can sensitize cancer cells to apoptosis induced by cisplatin and this effect is mediated through ERK activation, suggesting the potential of PEITC to be used as an adjuvant with cisplatin in combination therapeutic treatments.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Butadienes; Caspase 3; Cell Line, Tumor; Cisplatin; Drug Synergism; Female; HeLa Cells; Humans; Isothiocyanates; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 1; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Uterine Cervical Neoplasms

Carcinoma progression is associated with the loss of epithelial features, and the acquisition of a mesenchymal phenotype by tumour cells. Herein we show that exposure of MCF-7 cells to epidermal growth factor (EGF) resulted in morphological alterations characteristic of epithelial-to-mesenchymal transition (EMT). EGF treatment resulted in increased motility along with an up-regulation of transcription factors Slug, Zeb1, Zeb2, and mesenchymal markers Vimentin and N-cadherin. Treatment of MCF-7 cells with a combined stimulation of EGF and resveratrol, a naturally occurring stilbene with antitumor properties, failed to alter cell morphology, motility and overexpression of EMT markers induced by EGF. Using specific chemical inhibitors, we demonstrated that EGF-induced EMT is mediated by extracellular signal-regulated kinase 1/2 (ERK 1/2) signalling pathway and that resveratrol is able to repress EGF-induced ERK activation. In summary, these data provide new evidence of the inhibitory effect of resveratrol on EGF-induced EMT cell transformation.

Topics: Actin Depolymerizing Factors; Angiogenesis Inhibitors; Breast Neoplasms; Butadienes; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Shape; Cell Survival; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Immunoblotting; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Repressor Proteins; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; Snail Family Transcription Factors; Stilbenes; Transcription Factors; Vimentin; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

Hypoxia-inducible factor 1 (HIF-1α) expression induced by hypoxia plays a critical role in promoting tumor angiogenesis and metastasis. However, the molecular mechanisms underlying the induction of HIF-1α in tumor cells remain unknown.. In this study, we reported that hypoxia could induce HIF-1α and VEGF expression accompanied by Rac1 activation in MCF-7 breast cancer cells. Blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1 (T17N) or Rac1 siRNA downregulated hypoxia-induced HIF-1α and VEGF expression. Furthermore, Hypoxia increased PI3K and ERK signaling activity. Both PI3K inhibitor LY294002 and ERK inhibitor U0126 suppressed hypoxia-induced Rac1 activation as well as HIF-1α expression. Moreover, hypoxia treatment resulted in a remarkable production of reactive oxygen species (ROS). N-acetyl-L-cysteine, a scavenger of ROS, inhibited hypoxia-induced ROS generation, PI3K, ERK and Rac1 activation as well as HIF-1α expression.. Taken together, our study demonstrated that hypoxia-induced HIF-1α expression involves a cascade of signaling events including ROS generation, activation of PI3K and ERK signaling, and subsequent activation of Rac1.

Topics: Breast Neoplasms; Butadienes; Cell Hypoxia; Cell Line, Tumor; Chromones; Extracellular Signal-Regulated MAP Kinases; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; rac1 GTP-Binding Protein; Reactive Oxygen Species

Although the multi-functional, prosurvival protein, Bcl-2-associated anthanogene 1 (BAG-1) is frequently overexpressed in breast cancers, its role in the development or maintenance of the malignant state remains unclear. Here, we have used the established MCF-10A 3-dimensional (3D) model of mammary morphogenesis as a biologically relevant system to determine how BAG-1 expression may influence the development of breast cancer. When cultured in 3D, MCF-10A cells undergo a highly regulated morphogenic program leading to the development of polarized acinar structures containing a central, hollow lumen formed, in part, through the induction of BIM-dependent apoptosis. BAG-1 overexpression resulted in an attenuation of this normal apoptotic program characterized by a significantly increased number of acini with filled lumens-a phenotype commonly observed in ductal carcinoma in situ. BAG-1's effects were associated with an activation of RAF-1-a known binding partner of BAG-1, enhanced signaling through the MAP kinase pathway and a decrease in BIM expression. Reversal of the BAG-1-associated survival phenotype by the mitogen-activated kinase/ERK kinase inhibitor, U0126, implicates the RAF-1-extracellular signal-regulated kinase signaling pathway as a major mediator of BAG-1's effects in this model. As BAG-1 expression is often elevated in preinvasive breast cancers, these findings support a possible role for BAG-1 as an early contributor to the malignant process in the breast.

Topics: Apoptosis; Breast Neoplasms; Butadienes; Cell Culture Techniques; Cell Line; Cell Line, Tumor; DNA-Binding Proteins; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Human; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Neoplasm Invasiveness; Nitriles; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-raf; Transcription Factors

Tumor cells that are grown in three-dimensional (3D) cell culture exhibit relative resistance to cytotoxic drugs compared with their response in conventional two-dimensional (2D) culture. We studied the effects of targeted agents and doxorubicin on 2D and 3D cultures of human breast cell lines that represent the progression from normal epithelia (modeled by MCF10A cells) through hyperplastic variants to a dysplastic/carcinoma phenotype (MCF10.DCIS cells), variants transformed by expression of activated Ras, and also a basal-subtype breast carcinoma cell line (MDA-MB-231). The results showed the expected relative resistance to the cytotoxic agent doxorubicin in 3D cultures, with greater resistance in normal and hyperplastic cells than in carcinoma models. However, the response to the targeted inhibitors was more complex. Inhibition of mitogen-activated protein kinase kinase (MEK) by either 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) or 2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide (CI-1040, PD184352) produced a similar inhibition of the growth of all the MCF10 cell lines in 2D. In 3D culture, the normal and hyperplastic models exhibited some resistance, whereas the carcinoma models became far more sensitive to MEK inhibition. Increased sensitivity to MEK inhibition was also seen in MDA-MB-231 cells grown in 3D compared with 2D. In contrast, inhibition of phosphatidylinositol 3'-kinase activity by wortmannin had no significant effect on the growth of any of the cells in either 2D or 3D. Our conclusion is that 3D culture models may not only model the relative resistance of tumor cells to cytotoxic therapy but also that the 3D approach may better identify the driving oncogenic pathways and critical targeted inhibitors that may be effective treatment approaches.

Topics: Androstadienes; Antineoplastic Agents; Benzamides; Breast Neoplasms; Butadienes; Cell Culture Techniques; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Drug Resistance, Neoplasm; Female; Genes, ras; Humans; Hyperplasia; Mammary Glands, Human; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Wortmannin

Lapatinib, a dual HER2 and EGFR tyrosine kinase inhibitor is highly active in HER2+ breast cancer. However, its efficacy is limited by either primary or acquired resistance. Although mutations in ras genes are rarely found in breast cancer, H-ras overexpression is frequently observed. Moreover, genetic alterations that do not directly involve ras such as Brk amplification, ultimately result in increased ras signaling. Using SKBR3 cells, a HER2+ breast cancer cell line that is naturally devoid of mutations in PI3KCA, PTEN, BRAF, and ras we show that both H-ras overexpression and expression of an oncogenic ras allele (ras V12) reduce susceptibility to lapatinib in analogy to what observed with activating PI3KCA mutations and with a constitutively active form of Akt. Importantly, we found that resistance to lapatinib due to ras overexpression or to ras V12 is overcome by MEK inhibition with U0126, suggesting a key role for the MEK-Erk pathway in ras-induced resistance. Similar results were obtained in BT474 cells, another HER+ breast cancer cell line. Therefore, our data indicate that overexpressed/mutated ras may act as a biological modifier of the response to lapatinib. Combining MEK inhibitors with lapatinib may help overcome this form of resistance and increase the efficacy of lapatinib in these tumors.

Topics: Antineoplastic Agents; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Genes, ras; Humans; Immunoblotting; Lapatinib; Mitogen-Activated Protein Kinase Kinases; Mutation; Nitriles; Quinazolines; Receptor, ErbB-2; Signal Transduction

Expression of fibroblast growth factor 8 (FGF-8) is increased in several forms of hormonal cancer. It was previously shown to regulate expression of thrombospondin 1 (TSP-1), an inhibitor of angiogenesis, in S115 breast cancer cells. Here, we studied the FGF-8-activated signalling pathways mediating TSP-1 repression in S115 cells and in non-tumorigenic MCF10A cells. Inhibition of FGF receptors or of MEK1/2 and PI3K with specific inhibitors (PD173074, U0126 or LY294002, respectively) restored TSP-1 mRNA expression in the presence of FGF-8 in S115 cells. Furthermore, U0126 and LY294002 increased TSP-1 mRNA expression in S115 cells over-expressing FGF-8. In MCF10A cells, FGF-8 treatment also decreased TSP-1 expression and the effect was dependent on active MEK1/2. In conclusion, FGF-8 suppresses TSP-1 expression through two independent pathways, MEK1/2 and PI3K. Repression of TSP-1 may be an important mechanism involved in induction of an angiogenic phenotype and growth of FGF-8-expressing breast cancer.

Topics: Angiogenesis Modulating Agents; Animals; Blotting, Western; Breast Neoplasms; Butadienes; Cell Line; Cell Line, Tumor; Chromones; Down-Regulation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblast Growth Factor 8; Gene Expression; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Polymerase Chain Reaction; Pyrimidines; Recombinant Proteins; Signal Transduction; Thrombospondin 1

The pathways downstream of ErbB-family proteins are very important in BC, especially when considering treatment with onco-protein inhibitors. We studied and implemented dynamic simulations of four downstream pathways and described the fragment of the signaling network we evaluated as a Molecular Interaction Map. Our simulations, enacted using Ordinary Differential Equations, involved 242 modified species and complexes, 279 reversible reactions and 111 catalytic reactions. Mutations within a single pathway tended to be mutually exclusive; only inhibitors acting at, or downstream (not upstream), of a given mutation were active. A double alteration along two distinct pathways required the inhibition of both pathways. We started an analysis of sensitivity/robustness of our network, and we systematically introduced several individual fluctuations of total concentrations of independent molecular species. Only very few cases showed significant sensitivity. We transduced the ErbB2 over-expressing BC line, BT474, with the HRAS (V12) mutant, then treated it with ErbB-family and phosphorylated MEK (MEKPP) inhibitors, Lapatinib and U0126, respectively. Experimental and simulation results were highly concordant, showing statistical significance for both pathways and for two respective endpoints, i.e. phosphorylated active forms of ERK and Akt, p one tailed = .0072 and = .0022, respectively. Working with a complex 39 basic species signaling network region, this technology facilitates both comprehension and effective, efficient and accurate modeling and data interpretation. Dynamic network simulations we performed proved to be both practical and valuable for a posteriori comprehension of biological networks and signaling, thereby greatly facilitating handling, and thus complete exploitation, of biological data.

Topics: Antineoplastic Agents; Breast Neoplasms; Butadienes; Cell Line, Tumor; Computational Biology; Computer Simulation; Female; G1 Phase; Humans; Lapatinib; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Mutant Proteins; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); Quinazolines; Receptor, ErbB-2; Receptors, Growth Factor; Resting Phase, Cell Cycle; Signal Transduction

Phosphatase and tensin homolog (PTEN) is a tumor suppressor that inhibits PI3K/Akt signaling. To examine the effect of PTEN on breast cancer cell proliferation, we expressed PTEN in MDA-MB-468 cells (MDA-MB-468 PTEN) by retroviral infection and tested the cell proliferation rate. We found that the growth rate of MDA-MB-468 PTEN cells was significantly lower than that of MDA-MB-468 control vector cells (MDA-MB-468 vec). When the PI3K/Akt signaling inhibitor LY294002 and the MEK/Erk signaling inhibitor U0126 were applied, LY294002 reduced cell proliferation in MDA-MB-468 PTEN and MDA-MB-468 vec by 20%, while U0126 led to a >60% reduction in MDA-MB-468 PTEN and a 20% reduction in MDA-MB-468 vec cells. FACS analysis demonstrated that the subG0/G1 apoptotic fraction was significantly increased in MDA-MB-468 PTEN cells after U0126 treatment, while LY294002 treatment in both cell lines and U0126 treatment in MDA-MB-468 vec cells led to a modest increase in the apoptotic fraction. This phenomenon was accompanied by the down-regulation of p-Erk. p-Erk levels were significantly lower after U0126 treatment in MDA-MB-468 PTEN cells. Similar results were observed in MDA-MB-231 cells, which express endogenous PTEN. The growth of MDA-MB-231 cells was significantly inhibited after U0126 treatment, compared to LY294002, while PTEN-null ZR-75-1 cells did not show increased sensitivity to U0126 over LY294002. Taken together, these findings suggest that blockade of PI3K/Akt signaling by PTEN may render breast cancer cells more dependent on the MEK/Erk pathway for their proliferation and survival.

Topics: Antineoplastic Agents; Breast Neoplasms; Butadienes; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Extracellular Signal-Regulated MAP Kinases; Female; Humans; MAP Kinase Kinase Kinases; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Time Factors; Transfection

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca2+. Here, we show that extracellular Ca2+ (Ca2+(o)) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca2+(o) compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca2+(o) in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cbeta (PLCbeta) led to an abolition of the Ca2+(o)-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca2+(o)-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca2+(o) concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca2+-rich environment.

Topics: Bone Neoplasms; Breast Neoplasms; Butadienes; Calcium; Calcium Signaling; Cell Line, Tumor; Chemotaxis; Estrenes; Extracellular Signal-Regulated MAP Kinases; Extracellular Space; Female; Humans; Nitriles; Phospholipase C beta; Pyrrolidinones; Receptors, Calcium-Sensing

Phytoestrogens are the natural compounds isolated from plants, which are structurally similar to animal estrogen, 17beta-estradiol. Tectoridin, a major isoflavone isolated from the rhizome of Belamcanda chinensis. Tectoridin is known as a phytoestrogen, however, the molecular mechanisms underlying its estrogenic effect are remained unclear. In this study we investigated the estrogenic signaling triggered by tectoridin as compared to a famous phytoestrogen, genistein in MCF-7 human breast cancer cells. Tectoridin scarcely binds to ER alpha as compared to 17beta-estradiol and genistein. Despite poor binding to ER alpha, tectoridin induced potent estrogenic effects, namely recovery of the population of cells in the S-phase after serum starvation, transactivation of the estrogen response element, and induction of MCF-7 cell proliferation. The tectoridin-induced estrogenic effect was severely abrogated by treatment with U0126, a specific MEK1/2 inhibitor. Tectoridin promoted phosphorylation of ERK1/2, but did not affect phosphorylation of ER alpha at Ser(118). It also increased cellular accumulation of cAMP, a hallmark of GPR30-mediated estrogen signaling. These data imply that tectoridin exerts its estrogenic effect mainly via the GPR30 and ERK-mediated rapid nongenomic estrogen signaling pathway. This property of tectoridin sets it aside from genistein where it exerts the estrogenic effects via both an ER-dependent genomic pathway and a GPR30-dependent nongenomic pathway.

Topics: Adenocarcinoma; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cyclic AMP; Drug Interactions; Estradiol; Estrogen Receptor alpha; Female; Genistein; Humans; Isoflavones; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Phytoestrogens; Signal Transduction

Breast cancer resistance protein (BCRP)/ABCG2 is a drug efflux pump responsible for multidrug resistance in cancer cells. We report that dephosphorylation of extracellular signal-regulated kinase (ERK) by treatment with mitogen-activated protein kinase/ERK kinase (MEK) inhibitors causes two opposing effects, transcriptional upregulation and prompted protein degradation of endogenous BCRP in breast cancer MCF-7 cells. Endogenous BCRP was eventually found to be upregulated. Conversely, treatment with epidermal growth factor was associated with its downregulation in the cells. MEK inhibitors also caused prompted degradation of exogenous BCRP in MCF-7 and gastric cancer NCI-N87 cells that express exogenous BCRP without affecting its transcriptional levels, and potentiated anticancer agents in the cells. A lysosomal inhibitor abolished this prompted degradation of exogenous BCRP, but a proteasome inhibitor did not. Inhibition of p90 ribosomal protein S6 kinase (RSK), one of the downstream effectors of ERK, resulted in transcriptional upregulation of endogenous BCRP but did not affect the protein degradation of exogenous BCRP. The data suggest that BCRP expression is differentially regulated downstream of the MEK-ERK pathway, transcriptionally upregulated through the inhibition of the MEK-ERK-RSK pathway, and posttranscriptionally downregulated through the inhibition of the MEK-ERK-non-RSK pathway. Although the immediate downstream effector of ERK remains to be elucidated, the data provide new insights into regulatory mechanisms of BCRP activity and may assist the development of BCRP-specific expression modulators.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Butadienes; Cell Line, Tumor; DNA Primers; Enzyme Inhibitors; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Neoplasm Proteins; Nitriles; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stomach Neoplasms; Up-Regulation

Superoxide dismutase (SOD) occurs in two intracellular forms in mammals, copper-zinc SOD (CuZnSOD), found in the cytoplasm, mitochondria and nucleus, and manganese superoxide dismutase (MnSOD), in mitochondria. Changes in MnSOD expression (as compared to normal cells) have been reported in several forms of cancer, and these changes have been associated with regulation of cell proliferation, cell death, and metastasis. We have found that progestins stimulate MnSOD in T47D human breast cancer cells in a time and physiological concentration-dependent manner, exhibiting specificity for progestins and inhibition by the antiprogestin RU486. Progestin stimulation occurs at the level of mRNA, protein, and enzyme activity. Cycloheximide inhibits stimulation at the mRNA level, suggesting that progestin induction of MnSOD mRNA depends on synthesis of protein. Experiments with the MEK inhibitor UO126 suggest involvement of the MAP kinase signal transduction pathway. Finally, MnSOD-directed siRNA lowers progestin-stimulated MnSOD and inhibits progestin stimulation of migration and invasion, suggesting that up-regulation of MnSOD may be involved in the mechanism of progestin stimulation of invasive properties. To our knowledge, this is the first characterization of progestin stimulation of MnSOD in human breast cancer cells.

Topics: Base Sequence; Breast Neoplasms; Butadienes; Cell Line, Tumor; DNA Primers; Female; Hormone Antagonists; Humans; MAP Kinase Signaling System; Mifepristone; Neoplasm Invasiveness; Nitriles; Progesterone Congeners; Promegestone; Receptors, Progesterone; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Superoxide Dismutase

The expression of matrix metalloproteinase-9 (MMP-9) and cyclooxygenase-2 (COX-2) are pivotal steps in breast cancer pathogenesis. In a previous study, we reported that silibinin suppresses TPA-induced MMP-9 expression through the Raf/MEK/ERK pathway.. Herein we determined the co-relationship between MMP-9 and COX-2, as well as the effect of silibinin on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 and COX-2 expression in the human breast cancer cells, MCF-7 and MDA-MB231.. The toxicity of silibinin was evaluated by Quick Cell Proliferation Assay Kit II. MMP-9 and COX-2 expression were analyzed by Zymography and Western blotting, respectively. Adenoviral constitutively active (CA)-MEK was used to activate MEK/ERK pathway.. The expression of MMP-9 and COX-2 in response to TPA was increased, whereas TPA-induced MMP-9 and COX-2 expression was decreased by silibinin. Our results showed that TPA-induced MMP-9 expression was inhibited by celecoxib in a dose-dependent fashion, but not MMP-1-expression. Both MMP-9 and COX-2 expression were significantly increased by CA-MEK overexpression. In contrast, TPA-induced MMP-9 and COX-2 expression was decreased by UO126 (MEK 1/2 inhibitor).. Silibinin down-regulates TPA-induced MMP-9 expression through inhibition of COX-2 expression in breast cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Antioxidants; Breast Neoplasms; Butadienes; Celecoxib; Cell Line, Tumor; Cyclooxygenase 2; Down-Regulation; Humans; MAP Kinase Kinase Kinases; Matrix Metalloproteinase 1; Matrix Metalloproteinase 9; Nitriles; Phytotherapy; Protein Kinase Inhibitors; Pyrazoles; Seeds; Silybin; Silybum marianum; Silymarin; Sulfonamides; Tetradecanoylphorbol Acetate

Growth factor (GF) deprivation and/or blocking of cognate signaling can induce apoptosis and is the basis of several cancer treatment paradigms. We observed that RXR agonists (rexinoids) induce apoptosis of tumor cells when GF support is abrogated. This "rexinoid apoptosis" involves activation of both iNOS and eNOS by RXR-PPARgamma and results in production of apoptogenic NO. IGF/EGF-induced IGF receptor 1-mediated MAP kinase blocks rexinoid apoptosis by RXR phosphorylation. Combining rexinoids with the MAPK inhibitor U0126 induced apoptosis in human cancer cells in vitro and ex vivo and blocked xenograft growth in vivo. Our results suggest a regulatory mechanism in which GF signaling antagonizes RXR-PPARgamma-mediated default apoptosis to sustain cell life.

Topics: Animals; Apoptosis; Breast Neoplasms; Butadienes; Cell Line, Tumor; Dimerization; Enzyme Inhibitors; Female; Genetic Vectors; HCT116 Cells; HT29 Cells; Humans; Intercellular Signaling Peptides and Proteins; Lentivirus; Mice; Mice, Nude; Mutagenesis, Site-Directed; Nitric Oxide; Nitriles; Plasmids; PPAR gamma; Random Allocation; Retinoid X Receptors; RNA, Small Interfering; Signal Transduction; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Cell migration is a critical step in cancer cell invasion. Recent studies have implicated the importance of the extracellular signal-regulated kinase (ERK) signaling pathway in cancer cell migration. However, the mechanism associated with ERK-regulated cell migration is poorly understood. Using a panel of breast cancer cell lines, we detected an excellent correlation between ERK activity and cell migration. Interestingly, we noticed that a 48-hour treatment with U0126 [specific mitogen-activated protein/ERK kinase (MEK)-1/2 inhibitor] was needed to significantly inhibit breast cancer cell migration, whereas this inhibitor blocked ERK activity within 1 hour. This observation suggests that ERK-dependent gene expression, rather than direct ERK signaling, is essential for cell migration. With further study, we found that ERK activity promoted the expression of the activator protein-1 (AP1) components Fra-1 and c-Jun, both of which were necessary for cell migration. Combination of U0126 treatment and Fra-1/c-Jun knockdown did not yield further reduction in cell migration than either alone, indicating that ERKs and Fra-1/c-Jun act by the same mechanism to facilitate cell migration. In an attempt to investigate the role of Fra-1/c-Jun in cell migration, we found that the ERK-Fra-1/c-Jun axis regulated slug expression in an AP1-dependent manner. Moreover, the occurrence of U0126-induced migratory inhibition coincided with slug reduction, and silencing slug expression abrogated breast cancer cell migration. These results suggest an association between ERK-regulated cell migration and slug expression. Indeed, cell migration was not significantly inhibited by U0126 treatment or Fra-1/c-Jun silencing in cells expressing slug transgene. Our study suggests that the ERK pathway regulates breast cancer cell migration by maintaining slug expression.

Topics: Animals; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Neoplasm Invasiveness; Nitriles; Oligonucleotide Array Sequence Analysis; Phosphorylation; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors

Several studies have indicated the potential value of targeting HER-2 signaling to enhance the anti-tumor activity of ionizing radiation. However, therapeutic resistance resulting from several factors, including activation of the downstream pathway, represents a major obstacle to treatment. Here, we investigated whether inhibitors targeting downstream of HER-2 signaling would radiosensitize SKBR3 breast cancer cells that exhibit overamplification of HER2. Selective inhibition of MEK-ERK signaling using pharmacologic inhibitors (PD98059, UO126) did not increase the radiosensitivity of SKBR3 cells. Selective inhibition of the PI3K-AKT-mTOR pathway using pharmacologic inhibitors (LY294002, AKT inhibitor VIII, Rapamycin) significantly attenuated expression of p-AKT and p-70S6K, respectively and radiosensitized SKBR3 cells. MCF-7 cells those did not overexpress HER-2, showed less radiosensitization compared to SKBR3 cells by inhibition of this pathway. Pre-treatment with these inhibitors also caused significant abrogation of typical G(2) arrest following ionizing radiation and induced marked prolongation of gammaH2AX foci indicating impairment of DNA damage repair. A dual inhibitor of Class I PI3K and mTOR, PI103 effectively radiosensitized SKBR3 cells and showed significant prolongation of gammaH2AX foci. Inhibition of PI3K-AKT signaling was associated with downregulation of DNA-PKs, respectively. While apoptosis was the major mode of cell death when the cells were pretreated with LY294002 or AKT inhibitor VIII, the cells were pretreated by rapamycin or PI103 showed mixed mode of cell death including autophagy. Our results suggest possible mechanisms to counteract the HER-2 prosurvival signaling implicated in radioresistance, and offer an alternative strategy to overcome resistance to HER-2 inhibitors combined with radiation.

Topics: Antineoplastic Agents; Breast Neoplasms; Butadienes; Cell Cycle; Cell Line, Tumor; Chromones; Dose-Response Relationship, Radiation; Drug Resistance, Neoplasm; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Morpholines; Nitriles; Radiation-Sensitizing Agents; Receptor, ErbB-2; RNA Interference; Signal Transduction

Parathyroid hormone-related protein (PTHrP) regulates growth and migration of adherent breast cancer cells. Here, we show that PTHrP also interferes with the ability of breast cancer cells to aggregate in suspension cultures. Cell colonies were significantly smaller when the expression of PTHrP or its target genes, integrin alpha6 or KISS-1, was suppressed by RNA interference. TGFbeta1, a stimulator of PTHrP transcription, abolished the effect of PTHrP and KISS-1 specific siRNAs and increased ERK1/2 phosphorylation, whereas inhibition of ERK1/2 phosphorylation by U0126 reduced colony size. PTHrP and KISS-1 may regulate colony formation in 3D by influencing ERK1/2 phosphorylation.

Topics: Breast Neoplasms; Butadienes; Cell Aggregation; Cell Line, Tumor; Cell Proliferation; Female; Humans; Integrin alpha6; Kisspeptins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Parathyroid Hormone-Related Protein; Phosphorylation; Protein Kinase Inhibitors; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Spheroids, Cellular; Transfection; Transforming Growth Factor beta1; Tumor Suppressor Proteins

Mesothelin (MSLN) is a glycoprotein that is overexpressed in various tumors. MSLN is present on the cell surface and is also released into body fluids or culture supernatants from MSLN-positive tumor cells. Despite intensive study of MSLN as a diagnostic marker or target for immunotherapy, its biological function is largely unknown. In the present study, we examined the effects of ectopic expression of MSLN in human breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). We found that overexpression of MSLN promoted anchorage-independent growth in soft agar. In addition, MDA-MB-231 cells expressing high levels of MSLN exhibited resistance to anoikis (a type of apoptosis induced by detachment from substratum), as indicated by decreased DNA fragmentation and down-regulation of the proapoptotic protein Bim. Incubating MSLN-expressing MDA-MB-231 cells in the presence of U0126, an inhibitor of mitogen-activated protein/extracellular-signal-regulated kinase kinase, induced accumulation of Bim and restored susceptibility to anoikis. Western blot analysis also revealed that overexpression of MSLN resulted in sustained activation of extracellular signal-regulated kinase 1/2 and suppression of Bim. The present results constitute novel evidence that MSLN enables cells to survive under anchorage-independent conditions by suppressing Bim induction via the extracellular signal-regulated kinase signaling pathway.

Topics: Anoikis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Breast Neoplasms; Butadienes; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; MAP Kinase Signaling System; Membrane Glycoproteins; Membrane Proteins; Mesothelin; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Proto-Oncogene Proteins

Phosphorylation of estrogen receptor-alpha (ERalpha) at specific residues in transcription activation function 1 (AF-1) can stimulate ERalpha activity in a ligand-independent manner. This has led to the proposal that AF-1 phosphorylation and the consequent increase in ERalpha activity could contribute to resistance to endocrine therapies in breast cancer patients. Previous studies have shown that serine 118 (S118) in AF-1 is phosphorylated by extracellular signal-regulated kinases 1 and 2 (Erk1/2) mitogen-activated protein kinase (MAPK) in a ligand-independent manner. Here, we show that serines 104 (S104) and 106 (S106) are also phosphorylated by MAPK in vitro and upon stimulation of MAPK activity in vivo. Phosphorylation of S104 and S106 can be inhibited by the MAP-erk kinase (MEK)1/2 inhibitor U0126 and by expression of kinase-dead Raf1. Further, we show that, although S118 is important for the stimulation of ERalpha activity by the selective ER modulator 4-hydroxytamoxifen (OHT), S104 and S106 are also required for the agonist activity of OHT. Acidic amino acid substitution of S104 or S106 stimulates ERalpha activity to a greater extent than the equivalent substitution at S118, suggesting that phosphorylation at S104 and S106 is important for ERalpha activity. Collectively, these data indicate that the MAPK stimulation of ERalpha activity involves the phosphorylation not only of S118 but also of S104 and S106, and that MAPK-mediated hyperphosphorylation of ERalpha at these sites may contribute to resistance to tamoxifen in breast cancer.

Topics: Animals; Breast Neoplasms; Butadienes; Chlorocebus aethiops; COS Cells; Estradiol; Estrogen Receptor alpha; HeLa Cells; Humans; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NIH 3T3 Cells; Nitriles; Phosphorylation; Proto-Oncogene Proteins c-raf; Serine; Transfection; Tumor Cells, Cultured

In breast cancer, the presence of estrogen receptor alpha (ER) denotes a better prognosis and response to antiestrogen therapy. Lack of ERalpha correlates with overexpression of epidermal growth factor receptor or c-erbB-2. We have shown that hyperactivation of mitogen-activated protein kinase (MAPK) directly represses ERalpha expression in a reversible manner. In this study, we determine if inhibition of MAPK in established ERalpha(-) breast cancer cell lines and tumors results in reexpression of ERalpha, and further, if reexpression of ERalpha in these ERalpha(-) tumors and cell lines could restore antiestrogen responses.. Established ERalpha(-) breast cancer cell lines, ERalpha(-) breast tumors, and tumor cell cultures obtained from ERalpha(-) tumors were used in this study. Inhibition of hyperactive MAPK was accomplished via the MAPK/ERK kinase 1/2 inhibitor U0126 or via upstream inhibition with Iressa or Herceptin. Western blotting or reverse transcription-PCR for ERalpha was used to assess the reexpression of ERalpha in cells treated with U0126. Growth assays with WST-1 were done to assess restoration of antiestrogen sensitivity in these cells.. Inhibition of MAPK activity in ERalpha(-) breast cancer cell lines results in reexpression of ERalpha; upstream inhibition via targeting epidermal growth factor receptor or c-erbB-2 is equally effective. Importantly, this reexpressed ERalpha can now mediate an antiestrogen response in a subset of these ERalpha(-) breast cancer cell lines. Treatment of ERalpha(-) tumor specimens with MAPK inhibitors results in restoration of ERalpha mRNA, and similarly in epithelial cultures from ERalpha(-) tumors, MAPK inhibition restores both ERalpha protein and antiestrogen response.. These data show both the possibility of restoring ERalpha expression and antiestrogen responses in ERalpha(-) breast cancer and suggest that there exist ERalpha(-) breast cancer patients who would benefit from a combined MAPK inhibition/hormonal therapy.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; ErbB Receptors; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Fulvestrant; Humans; Mitogen-Activated Protein Kinases; Nitriles; Phenotype; Protein Kinase Inhibitors; Receptor, ErbB-2; RNA, Messenger; Tamoxifen

Obesity is considered a risk factor for many cancers, including breast cancer. Our laboratory has previously shown that leptin is mitogenic in many cancer cell lines, including breast. Information regarding the effects of high leptin levels on leptin receptor expression and signaling is lacking. The purpose of this study was to characterize leptin receptor expression in response to leptin in breast cancer cells. In addition, SOCS-3 expression (a leptin inducible inhibitor of leptin signaling), plus MAPK and PI3K signaling, were examined to determine their role in leptin-induced cell proliferation. Breast cancer cell lines, ZR75-1 and HTB-26, were treated with 0, 4, 40 or 80 ng/ml of leptin. Multiplex RT-PCR was performed to determine relative mRNA expression levels of the human short (huOB-Ra) or long (huOB-Rb) leptin receptor isoforms, or SOCS-3. MAPK and PI3K signaling was analyzed by phosphorylation of ERK and Akt, respectively, via Western blotting. Cell proliferation and inhibitor studies were analyzed by MTT assay. HTB-26 and ZR75-1 both expressed huOB-Ra, huOB-Rb and SOCS-3 mRNA; however, mRNA expression levels generally remained unchanged over time with leptin treatment. MAPK and PI3K pathways were activated in the presence of leptin over time. MAPK and PI3K inhibitors significantly blocked leptin-induced proliferation. Higher levels of circulating leptin contribute to breast cancer proliferation by activation of the MAPK and PI3K signaling pathways involved in cell growth and survival. The mitogenic effects of leptin are not a consequence of altered leptin receptor or SOCS-3 mRNA expression.

Topics: Blotting, Western; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Leptin; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Receptors, Cell Surface; Receptors, Leptin; Signal Transduction; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Time Factors

Synuclein-gamma is aberrantly expressed in more than 70% of stage III/IV breast and ovarian carcinomas. Ectopic overexpression of synuclein-gamma enhanced MDA-MB-435 cell migration in vitro and metastasis in a nude mouse model. However, the mechanism of how synuclein-gamma promotes cell motility is not clear. In our previous studies, we showed that synuclein-gamma overexpression activates ERK. In the present study, we overexpressed synuclein-gamma in several breast and ovarian cancer cell lines and evaluated the effect of synuclein-gamma on the activity of small G-protein RHO family members. We found that at least one of the RHO/RAC/CDC42 GTPases showed a higher level of the GTP-bound active form. Consistent with their role in regulating the intracellular motile machinery, inhibition of the RHO/RAC/CDC42 by C. difficile Toxin B blocked cell migration in both parental cells and synuclein-gamma overexpressing cells. The ERK inhibitor U0126 also blocked the cell migration in both parental cells and synuclein-gamma overexpressing cells. Collectively, our data indicate that synuclein-gamma might be involved in late stage breast and ovarian cancer metastasis by enhancing cell motility through activation of the RHO family small-GTPases and ERK.

Topics: Bacterial Proteins; Bacterial Toxins; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Movement; Extracellular Signal-Regulated MAP Kinases; Female; gamma-Synuclein; Humans; Nitriles; Ovarian Neoplasms; Protein Kinase Inhibitors; rho GTP-Binding Proteins; Transcriptional Activation; Transfection

One of the intriguing questions regarding cell motility concerns the mechanism that makes stationary cells move. Here, we provide the first physical evidence that the onset of breast cancer cell motility in response to insulin-like growth factor I (IGF-I) correlates with lowering of adhesion strength from 2.52 +/- 0.20 to 1.52 +/- 0.13 microdynes/microm2 in cells attached to fibronectin via alpha5beta1 integrin. The adhesion strength depends on the dose of IGF-I and time of IGF-I treatment. Weakening of cell-matrix adhesion is blocked significantly (p < 0.01) by the catalytically inactive IGF-I receptor (IGF-IR) and the phosphoinositide 3-kinase (PI-3 kinase) inhibitor LY-294002, but it is unaffected by mitogen-activated protein kinase kinase inhibitor UO-126 and Src kinase inhibitor PP2. Sustained blockade of Rho-associated kinase (ROCK) with Y-27632 down-regulates adhesion strength in stationary, but not in IGF-I-treated, cells. Jasplakinolide, a drug that prevents actin filament disassembly, counteracts the effect of IGF-I on integrin-mediated cell adhesion. In the absence of growth factor signaling, ROCK supports a strong adhesion via alpha5beta1 integrin, whereas activation of the IGF-IR kinase reduces cell-matrix adhesion through a PI-3K-dependent, but ROCK-independent, mechanism. We propose that disassembly of the actin filaments via PI-3 kinase pathway contributes to weakening of adhesion strength and induction of cell movement. Understanding how cell adhesion and migration are coordinated has an important application in cancer research, developmental biology, and tissue bioengineering.

Topics: Actins; Amides; Blotting, Western; Breast Neoplasms; Butadienes; Carcinoma; Catalysis; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chromones; CSK Tyrosine-Protein Kinase; Culture Media, Serum-Free; Depsipeptides; Down-Regulation; Fibronectins; Flow Cytometry; Humans; Immunoprecipitation; Insulin-Like Growth Factor I; Integrin alpha5beta1; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Microscopy, Phase-Contrast; Models, Biological; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Pyridines; Pyrimidines; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; src-Family Kinases; Time Factors

Epidermal growth factor (EGF) is a mitogen for estrogen receptor (ER)-positive breast tumor cells, and it has been proven that EGF occasionally mimicked estrogen action and cross-talks with ER-alpha to exert its activity. Therefore, the present study was undertaken to explore whether EGF is able to modulate the expression of Wnt-1-induced signaling protein-2/connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed 5 (WISP-2/CCN5), an estrogen-responsive gene, in normal and transformed cell lines of the human breast and, if so, whether this induction is critical for EGF mitogenesis and what downstream signaling pathways are associated with this event. Here, we show that EGF-induced WISP-2 expression in ER- and EGF receptor-positive noninvasive MCF-7 breast tumor cells was dose and time dependent and that expression was modulated at transcription level. A synergism was seen in combination with estrogen. Moreover, small interfering RNA-mediated inhibition of WISP-2/CCN5 activity in MCF-7 cells resulted in abrogation of proliferation by EGF. The multiple molecular cross-talks, including the interactions between phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways and two diverse receptors (i.e., ER-alpha and EGFR), were essential in the event of EGF-induced WISP-2/CCN5 up-regulation in MCF-7 cells. Moreover, EGF action on WISP-2/CCN5 is restricted to ER- and EGFR-positive noninvasive breast tumor cells, and this effect of EGF cannot be instigated in ER-alpha-negative and EGFR-positive normal or invasive breast tumor cells by introducing ER-alpha. Finally, regulation of phosphorylation of ER-alpha and EGFR may play critical roles in EGF-induced transcriptional activation of WISP-2 gene in breast tumor cells.

Topics: Blotting, Northern; Blotting, Western; Breast Neoplasms; Butadienes; CCN Intercellular Signaling Proteins; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Cloning, Molecular; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Microscopy, Confocal; Microscopy, Fluorescence; Neoplasm Invasiveness; Neoplasm Proteins; Nitriles; Phosphatidylinositol 3-Kinases; Phosphorylation; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Subcellular Fractions; Time Factors; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Up-Regulation

Several mitogen-activated protein kinase (MAPK) signaling inhibitors are currently undergoing clinical trial as part of novel mechanism-based anticancer treatment strategies. This study was aimed at detecting biomarkers of MAPK signaling inhibition in human breast and colon carcinoma cells using magnetic resonance spectroscopy. We investigated the effect of the prototype MAPK kinase inhibitor U0126 on the (31)P-MR spectra of MDA-MB-231, MCF-7 and Hs578T breast, and HCT116 colon carcinoma cells. Treatment of MDA-MB-231 cells with 50 micromol/L U0126 for 2, 4, 8, 16, 24, 32, and 40 hours caused inhibition of extracellular signal-regulated kinases (ERK1/2) phosphorylation from 2 hours onwards. (31)P-MR spectra of extracted cells indicated that this was associated with a significant drop in phosphocholine levels to 78 +/- 8% at 8 hours, 74 +/- 8% at 16 hours, 66 +/- 7% at 24 hours, 71 +/- 10% at 32 hours, and 65 +/- 10% at 40 hours post-treatment. In contrast, the lower concentration of 10 micromol/L U0126 for 40 hours had no significant effect on either P-ERK1/ 2 or phosphocholine levels in MDA-MB-231 cells. Depletion of P-ERK1/2 in MCF-7 and Hs578T cells with 50 micromol/L U0126 also produced a drop in phosphocholine levels to 51 +/- 17% at 40 hours and 23 +/- 12% at 48 hours, respectively. Similarly, in HCT116 cells, inhibition with 30 micromol/L U0126 caused depletion of P-ERK1/2 and a decrease in phosphocholine levels to 80 +/- 9% at 16 hours and 61 +/- 4% at 24 hours post-treatment. The reduction in phosphocholine in MDA-MB-231 and HCT116 cells correlated positively with the drop in P-ERK1/2 levels. Our results show that MAPK signaling inhibition with U0126 is associated with a time-dependent decrease in cellular phosphocholine levels. Thus, phosphocholine has potential as a noninvasive pharmacodynamic marker for monitoring MAPK signaling blockade.

Topics: Biomarkers, Tumor; Breast Neoplasms; Butadienes; Cell Line, Tumor; Colorectal Neoplasms; Enzyme Inhibitors; HCT116 Cells; Humans; Magnetic Resonance Spectroscopy; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylcholine

Tumor hypoxia is associated with cancer invasiveness, metastasis and treatment failure. Recent data suggest that the major target for endocrine treatment in breast cancer, ERalpha, is downregulated during hypoxia, but the mechanism behind this remains unknown. MAPK signaling as well as ERalpha regulation has earlier been independently linked to hypoxia and we now demonstrate HIF-1alpha and ERK1/2-activation in vivo towards the necrotic zone in DCIS of the breast, parallel with ERalpha downregulation. Hypoxia further caused transcriptional downregulation of ERalpha via activation of ERK1/2 in cell lines and, importantly, MEK1/2 inhibitors (U0126 or PD184352) or ERK1/2 suppression by siRNA partially restored the ERalpha expression. U0126 combined with tamoxifen accordingly produced an increased efficacy of the anti-estrogens during hypoxia. Based on these findings, we suggest a promising novel therapy for ERalpha-positive breast cancer where a combination of endocrine treatment and ERK1/2 inhibitors may increase treatment response by improved targeting of dormant hypoxic tumor cells.

Topics: Benzamides; Breast Neoplasms; Butadienes; Cell Hypoxia; Down-Regulation; Drug Therapy, Combination; Enzyme Inhibitors; Estrogen Receptor alpha; Estrogen Receptor Modulators; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Tamoxifen

To study the expression of proline-rich Akt-substrate PRAS40 in the cell survival pathway and tumor progression.. The effects of three key kinase inhibitors on PRAS40 activity in the cell survival pathway, serum withdrawal, H(2)O(2) and overexpression of Akt were tested. The expression of PRAS40, Akt, Raf and 14-3-3 in normal cells and cancer cell lines was determined by Western blot.. The PI3K inhibitors wortmannin and Ly294002, but not rapamycin, completely inhibited the phosphorylation of Akt and PRAS40. The phosphorylation level of Akt decreased after serum withdrawal and treatment with the MEK inhibitor Uo126, but increased after treatment with H(2)O(2) at low concentration, whereas none of these treatments changed PRAS40 activity. 14-3-3 is a PRAS40 binding protein, and the expression of 14-3-3, like that of PRAS40, was higher in HeLa cells than in HEK293 cells; PRAS40 had a stronger phosphorylation activity in A549 and HeLa cancer cells than in HEK293 normal cells. In the breast cancer model (MCF10A/MCF7) and lung cancer model (BEAS/H1198/H1170) we also found the same result: PRAS40 was constitutively active in H1198/H1170 and MCF7 pre-malignant and malignant cancer cells, but weakly expressed in MCF10A and BEAS normal cell. We also discussed PRAS40 activity in other NSCLC cell lines.. The PI3K-Akt survival pathway is the main pathway that PRAS40 is involved in; PRAS40 is a substrate for Akt, but can also be activated by an Akt-independent mechanisms. PRAS40 activation is an early event during breast and lung carcinogenesis.

Topics: 14-3-3 Proteins; Adaptor Proteins, Signal Transducing; Breast Neoplasms; Butadienes; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Chromones; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hydrogen Peroxide; Lung Neoplasms; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Transfection

The phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are important integrators of growth and survival signals originating from extracellular stimuli. We assessed the importance of these signaling pathways in the growth and survival of 8 breast cell lines (MCF10A, an immortalized line; and 7 cancer cell lines). The cell lines expressed variable levels of both phosphorylated ERK and phosphorylated Akt, but these were unchanged by incubation in serum-free medium. Despite continued activity of these pathways, the cells arrested growth in the absence of serum demonstrating that additional pathways are required for growth. Incubation with the PI3K inhibitor LY294002 suppressed growth of all cell lines, but most remained viable for at least 7-14 days. This long-term survival may be attributable to recovery of phospho-Akt by 24-48 h despite the continued presence of active LY294002, suggesting that alternate pathways may be activating Akt. In contrast, incubation with the MEK inhibitor U0126 not only arrested growth, but also killed all the cell lines within 2-4 days in the absence of serum; the presence of serum only slighted extended viability, except in MCF10A and MDA-MB-468 cells, in which serum provided significantly greater protection. It is likely that these signaling pathways control the level of pro-and anti-apoptotic proteins, yet assessment of Bcl-2 and Bcl-X showed dramatic reduction in level only when large numbers of cells were dead suggesting this may be a consequence rather than cause of death. Overall, the results demonstrate that the MEK/ERK pathway represents the more critical pathway for cell survival of these breast cancer cell lines, and suggest this pathways represents the better target for cancer therapy.

Topics: Blotting, Western; Breast Neoplasms; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction

Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) has been shown to inhibit proliferation of breast cancer cells although its mechanism of action has not been fully elucidated. To address this issue, we tested the effects of DFMO on cell cycle variables of MDA-MB-435 human breast cancer cells in culture. We also focused on the possible mediatory role of the mitogen-activated protein kinase (MAPK) pathway on the cell cycle effects of DFMO because this compound has been shown to activate MAPK signaling. We found that DFMO caused a p53-independent increase in p21 and its association with cyclin-dependent kinase (cdk)-2 and decreased cdk-2 protein as well as its phosphorylation on Thr160. In addition, DFMO markedly suppressed the expression of the full-length and low molecular weight forms of cyclin E. These effects of DFMO were reversible with exogenous putrescine, thus indicating that they are specifically mediated through polyamine depletion. Cdk-2 activity was drastically reduced in DFMO-treated breast cancer cells which exhibited a reduction in retinoblastoma (Rb) phosphorylation and protein. As a predictable consequence of these effects, DFMO caused a G1-S block. In addition, DFMO inhibited G2-M transition, most likely as a result of its induction of p21 expression. Inhibition of the MAPK pathway with PD98059 or U0126 blocked the DFMO-induced induction of p21 and the reduction of cdk-2 protein. PD98059 reversed the G2-M block induced by DFMO (probably as a result of suppression of p21) but not the G1-S arrest. MDA-MB-435 cells treated with PD98059 or U0126 in the presence and absence of DFMO exhibited a marked increase in the expression of p27 and its association with cdk-2, a decrease in phosphorylation of cdk-2 on Thr160, and a decrease in cyclin E expression. As predicted, PD98059 treatment reduced cdk-2 activity and Rb phosphorylation while reversing the decrease in Rb protein induced by DFMO. Neither DFMO nor PD98059, either alone or in combination, reduced cdk-4 activity despite a marked induction in p15 expression caused by DFMO. Our results indicate that activation of the MAPK pathway accounts for some of the effects of DFMO on cell cycle events of breast cancer cells. Inhibition of the MAPK pathway, however, does not reverse the cell cycle arrest induced by DFMO because of activation of alternative mechanisms leading to suppression of cdk-2 activity.

Topics: Breast Neoplasms; Butadienes; Calcium-Binding Proteins; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Drug Interactions; Eflornithine; Flavonoids; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Neoplasms, Hormone-Dependent; Nitriles; Phosphorylation; Putrescine; Retinoblastoma Protein

We have examined overexpression of the human epidermal growth factor receptor 2 (HER2) to determine if it modifies the anti-proliferative effect of transforming growth factor (TGF)-beta against MCF-10A human mammary epithelial cells. Exogenous TGF-beta inhibited cell proliferation and induced Smad-dependent transcriptional reporter activity in both MCF-10A/HER2 and MCF-10A/vector control cells. Ligand-induced reporter activity was 7-fold higher in HER2-overexpressing cells. In wound closure and transwell assays, TGF-beta induced motility of HER2-transduced, but not control cells. The HER2-blocking antibody trastuzumab (Herceptin) prevented TGF-beta-induced cell motility. Expression of a constitutively active TGF-beta type I receptor (ALK5(T204D)) induced motility of MCF-10A/HER2 but not MCF-10A/vector cells. TGF-beta-induced motility was blocked by coincubation with either the phosphatidylinositol 3-kinase inhibitor LY294002, the mitogen-activated protein kinase (MAPK) inhibitor U0126, the p38 MAPK inhibitor SB202190, and an integrin beta(1) blocking antibody. Rac1 activity was higher in HER2-overexpressing cells, where both Rac1 and Pak1 proteins were constitutively associated with HER2. Both exogenous TGF-beta and transduction with constitutively active ALK5 enhanced this association. TGF-beta induced actin stress fibers as well as lamellipodia within the leading edge of wounds. Herceptin blocked basal and TGF-beta-stimulated Rac1 activity but did not repress TGF-beta-stimulated transcriptional reporter activity. These data suggest that 1) overexpression of HER2 in nontumorigenic mammary epithelial is permissive for the ability of TGF-beta to induce cell motility and Rac1 activity, and 2) HER2 and TGF-beta signaling cooperate in the induction of cellular events associated with tumor progression.

Topics: Actins; Activin Receptors, Type I; Adenoviridae; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Blotting, Northern; Breast Neoplasms; Bromodeoxyuridine; Butadienes; Cell Cycle; Cell Division; Cell Line; Cell Line, Tumor; Cell Movement; Chromones; Disease Progression; DNA, Complementary; Enzyme Inhibitors; Epithelial Cells; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; Humans; Imidazoles; Immunoblotting; Integrin beta1; Ligands; Luminescent Proteins; Microscopy, Fluorescence; Models, Genetic; Morpholines; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Precipitin Tests; Protein Serine-Threonine Kinases; Pseudopodia; Pyridines; rac1 GTP-Binding Protein; Receptor, ErbB-2; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Retroviridae; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Trastuzumab; Wound Healing

Increased growth factor receptor signaling is implicated in antiestrogen-resistant breast tumors suggesting that abrogation of such signaling could restore or prolong sensitivity to antihormonal agents. Activation of the mitogen-activated protein/extracellular regulated kinase kinase (MEK)-extracellular regulated kinase (ERK)1/2 cascade is a common component of such pathways. We investigated the ability of the MEK activation inhibitor U0126 to block the increased growth of estrogen receptor-positive MCF-7 breast cancer cells caused by fibroblast growth factor 1 (FGF-1), heregulin beta1 (HRGbeta1), and epidermal growth factor (EGF) in the presence of the pure antiestrogen ICI 182780 (Faslodex; fulvestrant). We found that either FGF-1 or HRGbeta1 but not EGF substantially reduced the inhibitory effects of U0126 on growth and ERK1/2 activation, including the combined inhibitory effects of U0126 and ICI 182780. FGF-1 and HRGbeta1 also reduced the inhibition of ERK1/2 phosphorylation by the MEK inhibitors PD98059 and PD184161. Interestingly, a transiently transfected dominant-negative MEK1 completely abrogated activation of a coexpressed green fluorescent protein-ERK2 reporter by all three of the factors. Despite a short-lived activation of Ras and Raf-1 by all three of the growth factors, both FGF-1 and HRGbeta1, unlike EGF, induced a prolonged activation of MEK and ERK1/2 in these cells. Thus, activation of FGF-1- and HRGbeta1-specific signaling causes MEK-dependent prolonged activation of ERK1/2, which is incompletely susceptible to known MEK inhibitors. We also demonstrate that the cytosolic phospholipase A2 inhibitor arachidonyl trifluoro methyl ketone and the pan PKC inhibitor bisindolymaleimide abrogated U0126-resistant phosphorylation of ERK1/2 induced by HRGbeta1 but not by FGF-1. Phosphorylation of ERK5 by all three of the factors was also resistant to U0126 suggesting that its activation is not sufficient to overturn growth inhibition due to diminished ERK1/2 activation. Therefore, therapy combining antiestrogens and MEK inhibitors may be ineffective in some antiestrogen-resistant estrogen receptor-positive breast cancers.

Topics: Breast Neoplasms; Butadienes; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Fibroblast Growth Factor 1; Fulvestrant; Humans; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 7; Mitogen-Activated Protein Kinases; Neuregulin-1; Nitriles; Phosphorylation; Proto-Oncogene Proteins c-raf; ras Proteins; Ribosomal Protein S6 Kinases, 90-kDa

We have addressed the question of rapid, nongenomic mechanisms that may be involved in the mitogenic action of estrogens in hormone-dependent breast cancer cells. In quiescent, estrogen-deprived MCF-7 cells, estradiol did not induce a rapid activation of either the MAPK/ERK or phosphatidylinositol-3 kinase (PI-3K)/Akt pathway, whereas the entry into the cell cycle was documented by the successive inductions of cyclin D1 expression, hyperphosphorylation of the retinoblastoma protein (Rb), activity of the promoter of the cyclin A gene, and DNA synthesis. However, pharmacological inhibitors of the src family kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP1) or of the PI-3K (LY294002) did prevent the entry of the cells into the cell cycle and inhibited the late G1 phase progression, whereas the inhibitor of MAPK/ERK activation (U0126) had only a partial inhibitory effect in the early G1 phase. In agreement with these results, small interfering RNA targeting Akt strongly inhibited the estradiolinduced cell cycle progression monitored by the activation of the promoter of the cyclin A gene. The expression of small interfering RNA targeting MAPK 1 and 2 also had a clear inhibitory effect on the estradiol-induced activation of the cyclin A promoter and also antagonized the estradiol-induced transcription directed by the estrogen response element. Finally, transfection of the estrogen receptor into NIH3T3 fibroblasts did not confer to the cells sensitivity to a mitogenic action of estradiol. We conclude that the induction of the cell cycle by estradiol does not require a direct activation of MAPK/ERK or PI-3K signaling protein kinase cascades, but that these kinases appear to have a permissive role in the cell cycle progression.

Topics: Animals; Breast Neoplasms; Butadienes; Cell Line, Tumor; Chromones; Cyclin A; Estradiol; Estrogens; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; G1 Phase; Humans; Mice; Mitogens; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; RNA Interference; RNA, Small Interfering

To evaluate the effect of HER2/neu overexpression on wild p53 protein expression and to delineate the related signal pathways.. Lipofectin method was used to transfer HER2/neu into human breast tumor cell line MCF7. Overexpression of HER2/neu was then determined by Western blot. Western blot was also used to detect the quantity of p53, Akt, p-Akt, p-Raf, p-MEK, p-ERK protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect p53 mRNA expression. PI3K pathway inhibitor LY294002 and MEK inhibitor U0126 were used to block the two pathways. The subsequent effect on p53 protein expression was then determined.. HER2/neu-overexpressed MCF7 clone (MCF7-neu3) was successfully established, in which the amount of HER2/neu protein was 13 times more than that in parental MCF7 cells. The amount of p53 protein in MCF7-neu3 was 40% of that in parental MCF7 cells (P < 0.01), while there was no difference on p53 mRNA level. There were 2.5, 2.0, 1.6 and 1.6 fold increase in the amount of p-Akt, p-Raf, p-MEK, p-ERK protein respectively in MCF7-neu3 to that in parental MCF7 cells (P < 0.01). When treated with LY294002 or U0126 for 24 hours, the amount of wild p53 protein in MCF7-neu3 cells was 1.7 or 1.5 times higher than those in DMSO treated cells. There were 4.7 or 5.3 times increase in the p53 protein when MCF7-neu3 cells were treated with LY294002 or U0126 for 48 hours (P < 0.01). Similar results were not seen in MDA-MB-453 cells which contained mutant p53.. HER2/neu overexpression can activate PI3K and Ras/Raf/MEK/ERK pathways, resulting in reduction of wild p53 protein expression. This may be the molecular mechanism responsible for the poor prognosis and therapeutic non-responsiveness in HER2/neu-overexpressed breast cancer patients.

Topics: Breast Neoplasms; Butadienes; Cell Line, Tumor; Chromones; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Receptor, ErbB-2; RNA, Messenger; Signal Transduction; Tumor Suppressor Protein p53

Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.

Topics: Blotting, Western; Breast Neoplasms; Butadienes; COUP Transcription Factor I; COUP Transcription Factor II; COUP Transcription Factors; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Oncostatin M; Peptides; Receptors, Steroid; Reverse Transcriptase Polymerase Chain Reaction; Tetradecanoylphorbol Acetate; Transcription Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

Breast cancers often have increased MAPK activity; this pathway may drive breast cancer cell growth by targeting steroid hormone receptors. MAPK phosphorylates human progesterone receptors (PRs) on Ser294, thus regulating several aspects of PR activity. To study the role of PR Ser294 phosphorylation on subcellular distribution, we stably expressed wild-type (wt) or S294A (Ser294 to Ala) PR-B in several cell types. PRs phosphorylated on Ser294 were nuclear. Activation of MAPK induced Ser294 phosphorylation and rapid nuclear translocation of wt, but not S294A, PR-B; both receptors concentrated in the nucleus after progestin treatment. The MAPK kinase inhibitor, U0126, blocked epidermal growth factor but not progestin-induced Ser294 phosphorylation and translocation of wt PR, indicating a novel mechanism for nuclear localization. After progestin treatment, wt PR-B underwent ligand-dependent down-regulation, while S294A PR-B persisted in nuclei. Prolonged treatment with U0126 or the nuclear export inhibitor, leptomycin B, promoted nuclear accumulation of wt PR-B and blocked ligand-dependent PR down-regulation, suggesting that PR degradation occurs in the cytoplasm and requires MAPK-dependent nuclear export. Stabilization of PRs by leptomycin B also blocked PR transcriptional activity, indicating a link between nucleocytoplasmic shuttling, receptor stability, and function. These results support a regulatory role for MAPK in nuclear steroid hormone receptor subcellular localization and coupling to multiple PR functions.

Topics: Acetylcysteine; Active Transport, Cell Nucleus; Breast Neoplasms; Butadienes; Cell Nucleus; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoplasm; Epidermal Growth Factor; Fatty Acids, Unsaturated; Humans; Ligands; Mitogen-Activated Protein Kinase Kinases; Multienzyme Complexes; Mutation; Nitriles; Phosphorylation; Promegestone; Proteasome Endopeptidase Complex; Receptors, Progesterone; Serine; Transcription, Genetic; Tumor Cells, Cultured

Long-term estrogen deprivation causes hypersensitivity of MCF-7 cells to the mitogenic effect of estradiol (E2) which is associated with activation of mitogen-activated protein kinase (MAPK). However, several lines of evidence indicate that MAPK activation is not the exclusive mechanism for E2 hypersensitivity and multiple signal pathways might be involved. The current study explores the possible role of the PI3 kinase (PI3K) pathway in development of E2 hypersensitivity. Basal PI3K activity in long-term estrogen deprived MCF-7 cells (LTED) was elevated as evidenced by increased phosphorylation of three downstream effectors, Akt, p70 S6 kinase, and eukaryotic initiation factor-4E binding protein (4E-BP1), which was blocked by the specific inhibitor of PI3K, LY294002. Dual blockade of both MAPK and PI3K completely reversed E2 hypersensitivity of LTED cells. Enhancement in aromatase activity is another phenomenon accompanied with E2 hypersensitivity. In aromatase over-expressing MCF-7 cells, aromatase activity was reduced by inhibitors of MAPK and PI3K suggesting the involvement of protein phosphorylation in the regulation of aromatase activity. Our data suggest that in addition to the MAP kinase pathway, activation of the PI3 kinase pathway is involved in E2 hypersensitivity, which develops during adaptation of MCF-7 cells to the low estrogen environment.

Topics: Adaptation, Physiological; Adaptor Proteins, Signal Transducing; Aromatase; Breast Neoplasms; Butadienes; Carrier Proteins; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Chromones; Drug Hypersensitivity; Enzyme Activation; Enzyme Inhibitors; Estradiol; Estrogens; Humans; Mitogen-Activated Protein Kinases; Mitogens; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction

We have previously shown that the G protein-coupled receptor (GPR)30 is critical for progestin-induced growth inhibition. In this study, we addressed signal transduction pathways involved in progestin-mediated signaling. Progestin could not provide any additional growth inhibitory effect to MCF-7 cells treated with specific MAPK kinase inhibitors, PD98059 and U0126. Medroxyprogesteroneacetate (MPA) induced a late (22-23 h) decrease in ERK-1 and -2 activities verified by immunoblotting and kinase assay. The inactivation was abrogated by antiprogestin. Transient expression of GPR30 decreased ERK-1 and -2 activity; and in the cells in which GPR30 expression was decreased by the antisense, ERK activities were increased. The antisense-expressing cells were able to significantly resist the growth-inhibitory effect of the MAPK kinase inhibitors PD98059 and U0126 but not that of other factors tested. Interestingly, the decrease of ERK activity induced by MPA was abrogated by GPR30 antisense. Collectively, these results show that MAPK activity is inhibited by progestin and GPR30 and suggest that progestin-induced ERK inactivation is mediated through GPR30. Coupled with our previous findings, the data imply that up-regulation of GPR30 by progestin leads to ERK-1 and -2 inactivation associated with MPA-induced growth inhibition.

Topics: Breast Neoplasms; Butadienes; Cell Division; DNA, Antisense; Enzyme Inhibitors; Flavonoids; Gene Expression; GTP-Binding Proteins; Humans; Medroxyprogesterone Acetate; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Progestins; Receptors, Cell Surface; Receptors, Estrogen; Receptors, G-Protein-Coupled; Signal Transduction; Tumor Cells, Cultured

Anchorage-independent growth is a hallmark of oncogenic transformation. We reported that the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 inhibited anchorage-independent growth of Ki-ras-transformed rat fibroblasts by simultaneously blocking both extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR)-p70(S6K) pathways. Here, we examined the effects of U0126 on the growth of eight human breast cancer cell lines. U0126 selectively repressed anchorage-independent growth of MDA-MB231 and HBC4 cells, two lines with constitutively activated ERK. Loss of contact with substratum triggers apoptosis in many normal cell types, a phenomenon termed anoikis. U0126 sensitized MDA-MB231 and HBC4 to anoikis, i.e., upon treatment with U0126, cells deprived of anchorage entered apoptosis, whereas adherent cells remained viable. Another MEK inhibitor PD98059 also induced anoikis sensitivity in MDA-MB231 cells but not in HBC4 cells. However, HBC4 cells were sensitized to anoikis when PD98059 was combined with the mTOR inhibitor rapamycin. To study the biochemical basis for induction of anoikis sensitivity, we examined the effects of the MEK inhibitors on ERK and p70(S6K) pathways in anchored versus nonanchored cells. As in Ki-ras-transformed rat fibroblasts, U0126 reduced activation of both ERK and p70(S6K) in MDA-MB231 and HBC4 cells, irrespective of anchorage. PD98059, in anchored cells, was more selective for the ERK pathway and did not significantly block the p70(S6K) pathway. Removal of anchorage substantially sensitized p70(S6K) to PD98059 in MDA-MB231 cells, whereas p70(S6K) in suspended HBC4 cells remained fairly refractory. U0126 was either without effect or less inhibitory on p70(S6K) in MDA-MB453 and SKBR3, two cell lines in which anoikis sensitivity was not induced. Thus, susceptibility of the p70(S6K) pathway to MEK inhibitors appeared to be an important determinant of anoikis sensitivity. The results indicate that concurrent inhibition of MEK-ERK and mTOR-p70(S6K) pathways induces apoptosis in MDA-MB231 and HBC4 cells when cells are deprived of anchorage but not when anchored. Inhibitors of MEK-ERK and mTOR-p70(S6K) pathways may provide a therapeutic strategy to selectively target neoplasms proliferating at ectopic locations, with acceptable effects on normal cells in their proper tissue context.

Topics: Animals; Anoikis; Antineoplastic Agents; Breast Neoplasms; Butadienes; Cell Division; Cell Line, Transformed; DNA Fragmentation; Dose-Response Relationship, Drug; Doxorubicin; Enzyme Inhibitors; Fibroblasts; Flavonoids; Immunoblotting; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Nucleosomes; Phosphorylation; Protein Kinases; Rats; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

The four receptor tyrosine kinases of the ErbB family play essential roles in several physiological processes and have also been implicated in tumor generation and/or progression. Activation of ErbB1/EGFR is mainly triggered by epidermal growth factor (EGF) and other related ligands, while activation of ErbB2, ErbB3, and ErbB4 receptors occurs by binding to another set of EGF-like ligands termed neuregulins (NRGs). Here we show that the Erk5 mitogen-activated protein kinase (MAPK) pathway participates in NRG signal transduction. In MCF7 cells, NRG activated Erk5 in a time- and dose-dependent fashion. The action of NRG on Erk5 was dependent on the kinase activity of ErbB receptors but was independent of Ras. Expression in MCF7 cells of a dominant negative form of Erk5 resulted in a significant decrease in NRG-induced proliferation of MCF7 cells. Analysis of Erk5 in several human tumor cell lines indicated that a constitutively active form of this kinase was present in the BT474 and SKBR3 cell lines, which also expressed activated forms of ErbB2, ErbB3, and ErbB4. Treatments aimed at decreasing the activity of these receptors caused Erk5 inactivation, indicating that the active form of Erk5 present in BT474 and SKBR3 cells was due to a persistent positive stimulus originating at the ErbB receptors. In BT474 cells expression of the dominant negative form of Erk5 resulted in reduced proliferation, indicating that in these cells Erk5 was also involved in the control of proliferation. Taken together, these results suggest that Erk5 may play a role in the regulation of cell proliferation by NRG receptors and indicate that constitutively active NRG receptors may induce proliferative responses in cancer cells through this MAPK pathway.

Topics: Active Transport, Cell Nucleus; Antineoplastic Agents; Breast Neoplasms; Butadienes; Cell Division; Enzyme Inhibitors; ErbB Receptors; Female; Humans; MAP Kinase Signaling System; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 7; Mitogen-Activated Protein Kinases; Neuregulins; Nitriles; Phosphorylation; Phthalimides; Receptor, ErbB-2; Recombinant Fusion Proteins; Tumor Cells, Cultured

The proteasome is emerging as a target for cancer therapy because small molecule inhibitors of its catalytic activity induce apoptosis in both in vitro and in vivo models of human malignancies and are proving to have efficacy in early clinical trials. To further elucidate the mechanism of action of these inhibitors, their impact on signaling through the p44/42 mitogen-activated protein kinase (MAPK) pathway was studied. Proteasome inhibition with either carbobenzoxy-leucyl-leucyl-phenylalaninal or lactacystin led to a loss of dually phosphorylated, activated p44/42 MAPK in A1N4-myc human mammary and MDA-MB-231 breast carcinoma cells in a dose- and time-dependent fashion. This correlated with an induction of the dual specificity MAPK phosphatases (MKP)-1 and -2, and blockade of MKP induction using either actinomycin D or Ro-31-8220 significantly decreased loss of activated p44/42 MAPK. Inhibition of p44/42 MAPK signaling by use of the MAPK kinase inhibitors PD 98059 or U0126, or by use of a dominant negative MAPK construct, enhanced proteasome inhibitor-mediated apoptosis. Conversely, activation of MAPK by epidermal growth factor, or use of a mutant MAPK resistant to MKP-mediated dephosphorylation, inhibited apoptosis. These studies support a role for inactivation of signaling through the p44/42 MAPK pathway in proteasome inhibitor-mediated apoptosis.

Topics: Apoptosis; Breast Neoplasms; Butadienes; Chymotrypsin; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dactinomycin; Enzyme Inhibitors; Female; Flavonoids; Humans; Kinetics; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Nitriles; Oligopeptides; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

We examined the role of Mcl-1 and Bcl-2 expression in the induction of apoptosis. through blocking protein tyrosine kinase (PTK), protein kinase C (PKC), phosphatidylinositol 3-kinase (P13-K) and mitogen-activated protein kinase (MAPK)/Erk kinase (MEK) signaling pathways by various kinase inhibitors in MCF-7 breast cancer cells. The PTK inhibitor genistein (GEN) and PKC inhibitor staurosporine (STP) down-regulated Mcl-1 and Bcl-2 expression, and induced growth inhibition by blocking at the G2/M phase of cell cycle, followed by apoptosis, leading to chromatin condensation and DNA fragmentation. LY294002 (LY)-mediated inhibition of P13-K activity down-regulated Bcl-2 but not Mcl-1 expression. triggered growth arrest at the G1/G0 phase of cell cycle and also led to apoptosis marked with chromatin condensation and DNA fragmentation. The MEK inhibitor U0126 (U0) decreased Bcl-2 expression but not Mcl-1 expression, inhibited cells growth and induced G1/G0 arrest. but in this case cell death occurred without significant apoptotic features. The kinase inhibitor concentration dependence of cytotoxicity correlated well with down-regulation of Bcl-2 but not with changes in Mcl-1 levels. This suggests that Bcl-2 plays a predominant role in the regulation of cell death induced by cell signaling alterations whereas Mcl-1 does not appear to control cell survival under these conditions in MCF-7 cells. Further studies showed that the combination of GEN, STP and LY with U0 can produce synergetic cytotoxic effects on MCF-7 cells. Our results suggest that PTK, PKC, P13-K and MEK signaling pathways can regulate Bcl-2 expression and form an integrated network that plays a critical role in cell survival.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Butadienes; Chromones; Down-Regulation; Enzyme Inhibitors; Female; Flow Cytometry; Genistein; Humans; Mitogen-Activated Protein Kinases; Morpholines; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Staurosporine; Tumor Cells, Cultured

Estrogen acts to promote DNA synthesis in the MCF-7 human breast cancer cell line via its interaction with high levels of estrogen receptor. The primary mode of estrogen action has been considered to be through transcriptional activation of genes containing estrogen response elements, including the immediate early genes c-myc and fos. Recent reports have indicated that estrogen, acting through the estrogen receptor, is capable of inducing the mitogen-activated protein kinase (MAPK) cytoplasmic signaling cascade. In this study, specific small molecule inhibitors of MAPK and phosphatidylinositol 3-kinase activity were used to determine the influence of these cascades on estrogen-mediated mitogenesis. Phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, as well as inhibitors of MAPK kinase-1, PD098059 and U0126, decreased the fraction of cells entering DNA synthesis after treatment with 17beta-estradiol. These compounds did not inhibit expression of myc or fos. However, the drugs did prevent the accumulation of cyclin D1 and hyperphosphorylated retinoblastoma protein, indicating that the block occurred at, or prior to, this point in the cell cycle. Although these compounds were effective in preventing estrogen-mediated mitogenesis, the downstream kinases extracellular signal-regulated kinase 1, extracellular signal-regulated kinase 2, and protein kinase B were not activated over basal levels by estrogen treatment. These studies suggest that estrogen initiates mitogenesis by inducing the transcription of immediate early genes, but cytoplasmic signaling pathways play an important role in the control of subsequent events in the cell cycle.

Topics: Adenocarcinoma; Androstadienes; Breast Neoplasms; Butadienes; Chromones; Culture Media, Serum-Free; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Depression, Chemical; DNA Replication; Enzyme Activation; Enzyme Inhibitors; Estradiol; Estrogen Receptor Modulators; Estrogens; Female; Flavonoids; Fulvestrant; Humans; Isoquinolines; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mitosis; Morpholines; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; Sulfonamides; Wortmannin

Growth factor modulation of estrogen receptor (ER) activity plays an important role in both normal estrogen physiology and the pathogenesis of breast cancer. Growth factors are known to stimulate the ligand-independent activity of ER through the activation of mitogen-activated protein kinase (MAPK) and the direct phosphorylation of ER. We found that the transcriptional activity of AIB1, a ligand-dependent ER coactivator and a gene amplified preferentially in ER-positive breast cancers, is enhanced by MAPK phosphorylation. We demonstrate that AIB1 is a phosphoprotein in vivo and can be phosphorylated in vitro by MAPK. Finally, we observed that MAPK activation of AIB1 stimulates the recruitment of p300 and associated histone acetyltransferase activity. These results suggest that the ability of growth factors to modulate estrogen action may be mediated through MAPK activation of the nuclear receptor coactivator AIB1.

Topics: Animals; Breast Neoplasms; Butadienes; Enzyme Inhibitors; Growth Substances; Histone Acetyltransferases; Humans; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Nitriles; Nuclear Proteins; Nuclear Receptor Coactivator 1; Phosphorylation; Protein Serine-Threonine Kinases; Receptors, Estrogen; Receptors, Steroid; Signal Transduction; Trans-Activators; Transcription Factors; Tumor Cells, Cultured

HER2/neu (erbB-2) overexpression has been causally associated with tamoxifen resistance in human breast cancer cells. Forced expression of HER2 in MCF-7 breast cancer cells resulted in mitogen-activated protein kinase (MAPK) hyperactivity and tamoxifen resistance. Inhibition of HER2 and MAPKs with AG1478 and U0126, respectively, as well as dominant-negative MEK-1/2 constructs restored the inhibitory effect of tamoxifen on estrogen receptor (ER)-mediated transcription and cell proliferation. Both AG1478 and U0126 also restored the tamoxifen-mediated association of ER with nuclear receptor corepressor (N-CoR) in the antiestrogen-resistant MCF-7 cells. Treatment with a combination of tamoxifen and a HER2 kinase inhibitor reduced tumor MAPK activity and markedly prevented growth of HER2-overexpressing MCF-7 xenografts in athymic mice. Thus, blockade of HER2 and MAPK signaling may enhance tamoxifen action and abrogate antiestrogen resistance in human breast cancer.

Topics: Animals; Breast Neoplasms; Butadienes; Drug Resistance, Neoplasm; Enzyme Inhibitors; Estrogen Receptor Modulators; Female; Gene Expression; Humans; MAP Kinase Signaling System; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Nitriles; Quinazolines; Receptor, ErbB-2; Receptors, Estrogen; Tamoxifen; Transcription, Genetic; Tyrphostins; Xenograft Model Antitumor Assays

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth an

Topics: Antibodies; Breast Neoplasms; Butadienes; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Carcinoma, Squamous Cell; Cell Death; Cell Division; DNA, Antisense; Dose-Response Relationship, Radiation; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Protein Kinases; Receptors, Tumor Necrosis Factor; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine