afimoxifene and Neoplasm-Metastasis

Most breast cancers (BCs) express estrogen receptor α (ERα) and are treated with the endocrine therapy (ET) drugs 4OH-tamoxifen (Tam) and fulvestrant (ICI 182,780; ICI). Unfortunately, a high fraction of ET treated women relapses and becomes resistant to ET. Therefore, additional anti-BC drugs are needed. Recently, we proposed that the identification of novel anti-BC drugs can be achieved using modulation of the intracellular ERα content in BC cells as a pharmacological target. Here, we searched for Food and Drug Administration (FDA)-approved drugs that potentially modify the ERα content in BC cells.. We screened in silico more than 60,000 compounds to identify FDA-approved drugs with a gene signature similar to that of ICI. We identified mitoxantrone and thioridazine and tested them in primary, Tam-resistant and genome-edited Y537S ERα-expressing BC cells.. We found that mitoxantrone and thioridazine induced ERα downmodulation and prevented MCF-7 BC cell proliferation. Interestingly, while mitoxantrone was found to be toxic for normal breast epithelial cells, thioridazine showed a preferential activity towards BC cells. Thioridazine also reduced the ERα content and prevented cell proliferation in primary, Tam-resistant and genome-edited Y537S ERα expressing BC cells.. We suggest that modulation of the intracellular ERα concentration in BC cells can be exploited in in silico screens to identify anti-BC drugs and uncover a re-purposing opportunity for thioridazine in the treatment of primary and metastatic ET resistant BCs.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Proliferation; Computer Simulation; Down-Regulation; Drug Resistance, Neoplasm; Estrogen Receptor alpha; Female; Fulvestrant; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mitoxantrone; Neoplasm Metastasis; Reproducibility of Results; Tamoxifen; Thioridazine

Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in metastasis. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment of breast cancer. Tamoxifen and its metabolites have been shown to directly impact platelet function, suggesting that this drug has additional mechanisms of action. The purpose of this study was to determine whether tamoxifen exerts antitumor effects through direct platelet inhibition.. This study found that pretreatment with tamoxifen leads to a significant inhibition of platelet activation. Platelets exposed to tamoxifen released significantly lower amounts of proangiogenic regulator vascular endothelial growth factor. In vitro angiogenesis assays confirmed that tamoxifen pretreatment led to diminished capillary tube formation and decreased endothelial migration. Tamoxifen and its metabolite, 4-hydroxytamoxifen, also significantly inhibited the ability of platelets to promote metastasis in vitro. Using a membrane-based array, we identified several proteins associated with angiogenesis metastasis that were lower in activated releasate from tamoxifen-treated platelets, including angiogenin, chemokine (C-X-C motif) ligand 1, chemokine (C-C motif) ligand 5, epidermal growth factor, chemokine (C-X-C motif) ligand 5, platelet-derived growth factor dimeric isoform BB, whereas antiangiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished vascular endothelial growth factor release, and lower angiogenic and metastatic potential.. We demonstrate that tamoxifen and its metabolite 4-hydroxytamoxifen directly alter platelet function leading to decreased angiogenic and metastatic potential. Furthermore, this study supports the idea of utilizing targeted platelet therapies to inhibit the platelet's role in angiogenesis and malignancy.

Topics: Angiogenesis Inhibitors; Blood Platelets; Breast Neoplasms; Cell Movement; Cell Proliferation; Coculture Techniques; Female; Human Umbilical Vein Endothelial Cells; Humans; MCF-7 Cells; Neoplasm Metastasis; Neovascularization, Physiologic; Platelet Activation; Platelet Aggregation Inhibitors; Signal Transduction; Tamoxifen; Vascular Endothelial Growth Factor A

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cell Proliferation; Cohort Studies; Demography; Disease-Free Survival; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Incidence; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Phenotype; Phosphorylation; Polymorphism, Single Nucleotide; Receptors, Metabotropic Glutamate; Tamoxifen; Tissue Array Analysis; Young Adult

The Wnt–β-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt–β-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of β-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear β-catenin content. Nuclear β-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt–β-catenin signaling. In the presence of high nuclear β-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the β-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.

Topics: Animals; Apoptosis; beta Catenin; Cell Line, Tumor; Cell Nucleus; Chlorpropamide; Colonic Neoplasms; Doxycycline; Drug Resistance, Neoplasm; Female; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Mice; Mice, SCID; Neoplasm Metastasis; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Tamoxifen; Wnt Signaling Pathway

Much research effort has been directed toward understanding how estrogen [17beta-estradiol (E2)] regulates cell proliferation and motility through the rapid, direct activation of cytoplasmic signaling cascades (i.e. nongenomic signaling). Cell migration is critical to cancer cell invasion and metastasis and involves dynamic filamentous actin cytoskeletal remodeling and disassembly of focal adhesion sites. Although estrogen is recognized to induce cell migration in some model systems, very little information is available regarding the underlying pathways and potential influence of selective estrogen receptor modulators such as 4-hydroxytamoxifen on these processes. Using the human endometrial cancer cell lines Hec 1A and Hec 1B as model systems, we have investigated the effects of E2 and Tam on endometrial nongenomic signaling, cytoskeletal remodeling, and cell motility. Results indicate that both E2 and Tam triggered rapid activation of ERK1/2, c-Src, and focal adhesion kinase signaling pathways and filamentous actin cytoskeletal changes. These changes included dissolution of stress fibers, dynamic actin accumulation at the cell periphery, and formation of lamellipodia, filopodia, and membrane spikes. Longer treatments with either agent induced cell migration in wound healing and Boyden chamber assays. Agent-induced cytoskeletal remodeling and cell migration were blocked by a Src inhibitor. These findings define cytoskeletal remodeling and cell migration as processes regulated by E2 and 4-hydroxytamoxifen nongenomic signaling in endometrial cancer. This new information may serve as the foundation for the development of new clinical therapeutic strategies.

Topics: Actins; Antineoplastic Agents, Hormonal; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; CSK Tyrosine-Protein Kinase; Cytoplasm; Cytoskeleton; Dose-Response Relationship, Drug; Endometrial Neoplasms; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Ligands; Microscopy, Confocal; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Protein-Tyrosine Kinases; Signal Transduction; src-Family Kinases; Tamoxifen; Time Factors; Wound Healing

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreER(T2)). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apoptosis; Blotting, Western; DNA Primers; Flow Cytometry; Fluorescent Antibody Technique; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Deletion; Genotype; Hydroxytestosterones; Immunohistochemistry; Integrases; Keratin-14; Keratinocytes; Keratins; Mice; Mice, Transgenic; Neoplasm Metastasis; Protein-Tyrosine Kinases; Receptors, Estrogen; Skin Neoplasms; Tamoxifen

Thirty tumors from metastatic breast cancer patients were screened for mutations in the estrogen receptor (ER) gene using single-strand conformation polymorphism and sequence analysis. Three missense mutations, Ser47Thr, Lys531Glu, and Tyr537Asn, were identified in these lesions. To investigate these mutated ERs or altered transcriptional activation function, expression vectors containing wild-type (wt) and mutant ERs were constructed and cotransfected with different estrogen response element reporter gene constructs into HeLa cells and MDA-MB-231 human breast cancer cells. The first two ER mutants were similar to wt ER. However, the Tyr537Asn ER mutant possessed a potent, estradiol-independent transcriptional activity, as compared to wt ER. Moreover, the constitutive activity of the Tyr537Asn ER mutant was virtually unaffected by estradiol, tamoxifen, or the pure antiestrogen ICI 164,384. Tyr537 is located at the beginning of exon 8 in the COOH-terminal portion of the hormone-binding domain of the ER, to which dimerization and transcription activation functions have also been ascribed. It has been identified as a phosphorylation site implicated in hormone binding, dimerization, and hormone-dependent transcriptional activity. Our results suggest that the Tyr537Asn substitution induces conformational changes in the ER that might mimic hormone binding, not affecting the ability of the receptor to dimerize, but conferring a constitutive transactivation function to the receptor. If present in other metastatic breast tumors, this naturally occurring ER mutant may contribute to breast cancer progression and/or hormone resistance.

Topics: Breast Neoplasms; Estradiol; Female; HeLa Cells; Humans; Models, Genetic; Mutation; Neoplasm Metastasis; Polymorphism, Single-Stranded Conformational; Receptors, Estrogen; Tamoxifen; Transcription, Genetic; Transfection

As previously reported, ovarian epithelial carcinomas may respond to endocrine therapy. We examined the direct effect of progesterone, medroxyprogesteroneacetate, gestoneron, 17-beta-estradiol, tamoxifen, 4-OH-tamoxifen, or N-desmethyltamoxifen on the proliferative capacity of ovarian carcinoma cells by means of the colony assay described by Hamburger and Salmon. The growth rate of 25 tested tumors (ascitic fluid, primary tumor, metastases) was 68%. The plating efficiency was 0.078%. Beside the drug testing estrogen and progesterone receptor levels were determined. The inhibition of colony survival was slightest with 17-beta-estradiol, more pronounced with medroxyprogesteroneacetate, gestoneron, N-desmethyltamoxifen, and progesterone, and greatest with 4-OH-tamoxifen and tamoxifen. Significant and dose-dependent inhibition of greater than 70% was observed with tamoxifen and 4-OH-tamoxifen in 80% of the tested tumors. There was no significant correlation between the in vitro responsiveness and the level of hormonal act not only via an estrogen receptor but also via an antiestrogen-binding site.

Topics: Adenocarcinoma; Aged; Cell Division; Cells, Cultured; Estradiol; Estrogen Antagonists; Ethanol; Female; Gestonorone Caproate; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; Middle Aged; Neoplasm Metastasis; Neoplastic Stem Cells; Ovarian Neoplasms; Progesterone; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen