kiss1-protein--human and Breast-Neoplasms

Breast cancer is the most frequently diagnosed cancer in women and the first cause of cancer death in France. Among the different subtypes of breast cancer, the predominant form is characterized by positive hormone receptors (more than 70% of breast cancers). Hormone therapy thus plays a key role in the strategy of management of these cancers both in adjuvant and metastatic situations. The two types of adjuvant hormone therapy used are selective estrogen receptor modulators and aromatase inhibitors. Fulvestrant, an anti-estrogen, is used alone or in combination with other molecules in metastatic situations. Hot flashes are one of the symptoms most frequently reported by patients under hormone therapy. Hormone replacement therapy, which is currently the most effective treatment for hot flashes, is contraindicated in patients with a personal history of breast cancer. Various therapeutic classes of drugs have been tested in this indication but without real efficacy in the various studies carried out to date, and moreover associated with non-negligible side effects. The recent discovery of the implication of the kisspeptin system located at the hypothalamic level in the mechanism of genesis of hot flashes opens the way to possible new symptomatic treatments for hot flashes. Neurokinin 3 receptor antagonists have shown encouraging preliminary results in postmenopausal cancer-free patients and could be considered in patients in hormonal therapy for breast cancer. Broader additional studies are needed to confirm these initial results.

Topics: Antineoplastic Agents, Hormonal; Aromatase Inhibitors; Breast Neoplasms; Contraindications, Drug; Estrogen Replacement Therapy; Female; Fulvestrant; Hot Flashes; Humans; Kisspeptins; Ovary; Receptors, Neurokinin-3; Selective Estrogen Receptor Modulators; Tamoxifen

Tumor suppressors are cellular proteins typically expressed in normal (non-cancer) cells that not only regulate such cellular functions as proliferation, migration and adhesion, but can also be secreted into extracellular space and serve as biomarkers for pathological conditions or tumor progression. KISS1, a precursor for several shorter peptides, known as metastin (Kisspeptin-54), Kisspeptin-14, Kisspeptin-13 and Kisspeptin-10, is one of those metastasis suppressor proteins, whose expression is commonly downregulated in the metastatic tumors of various origins. The commonly accepted role of KISS1 in metastatic tumor progression mechanism is the ability of this protein to suppress colonization of disseminated cancer cells in distant organs critical for the formation of the secondary tumor foci. Besides, recent evidence suggests involvement of KISS1 in the mechanisms of tumor angiogenesis, autophagy and apoptosis regulation, suggesting a possible role in both restricting and promoting cancer cell invasion. Here, we discuss the role of KISS1 in regulating metastases, the link between KISS1 expression and the autophagy-related biology of cancer cells and the perspectives of using KISS1 as a potential diagnostic marker for cancer progression as well as a new anti-cancer therapeutics.

Topics: Animals; Autophagy; Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Kisspeptins

Topics: Animals; Breast Neoplasms; Female; Humans; Kisspeptins; Neoplasm Metastasis; Neoplasm Recurrence, Local; Receptors, Kisspeptin-1; Signal Transduction

Topics: Breast Neoplasms; Cadherins; Female; Humans; Kangai-1 Protein; Kisspeptins; Mutation; Neoplasm Metastasis; Neoplasm Proteins; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Osteopontin; Receptors, Chemokine; Repressor Proteins; rho GTP-Binding Proteins; Tumor Suppressor Proteins

Metastatic disease is the most critical impediment to cancer patient survival. However, comparatively little is known concerning the intricate pathways which govern the complex phenotypes associated with metastasis. The KISS1 metastasis suppressor gene inhibits metastasis in both in vivo melanoma and breast carcinoma models. Despite its clear physiological activity, the mechanism of KISS1 remains unclear. Recent identification of a 54 amino acid peptide of KISS1, termed metastin or kisspeptin-54, and its cognate G-protein coupled receptor (hOT7T175, AXOR12, GPR54) have provided additional clues and avenues of research. While studies have attributed KISS1 with modulation of NFkappaB regulation, experiments with metastin and its receptor implicate MAP kinase pathways and also suggest the potential of autocrine, paracrine and endocrine roles. Impacts on motility, chemotaxis, adhesion and invasion have each been documented in disparate cell lines and conflicting observations require resolution. Nevertheless, mounting clinical evidence, particularly the loss of KISS1 in metastases, correlates KISS1 and metastin receptor expression with human tumor progression. Together, the data substantiate roles for these molecules in metastasis regulation.

Topics: Breast Neoplasms; Chromosomes, Human, Pair 6; Genes, Tumor Suppressor; Humans; JNK Mitogen-Activated Protein Kinases; Kisspeptins; MAP Kinase Kinase 4; Melanoma; Mitogen-Activated Protein Kinase Kinases; Neoplasm Metastasis; Proteins; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; Receptors, Neuropeptide; Signal Transduction; Tumor Suppressor Proteins

The present is an overview of recent data that describes the genetic underpinnings of the suppression of cancer metastasis. Despite the explosion of new information about the genetics of cancer, only six human genes have thus far been shown to suppress metastasis functionally. Not all have been shown to be functional in breast carcinoma. Several additional genes inhibit various steps of the metastatic cascade, but do not necessarily block metastasis when tested using in vivo assays. The implications of this are discussed. Two recently discovered metastasis suppressor genes block proliferation of tumor cells at a secondary site, offering a new target for therapeutic intervention.

Topics: Antigens, CD; Breast Neoplasms; Cadherins; Carcinoma; Cell Adhesion; Disease Progression; Female; Forecasting; Genes, Tumor Suppressor; Humans; Kangai-1 Protein; Kisspeptins; MAP Kinase Kinase 4; Membrane Glycoproteins; Mitogen-Activated Protein Kinase Kinases; Monomeric GTP-Binding Proteins; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Proteins; Proto-Oncogene Proteins; Repressor Proteins; Transcription Factors; Tumor Suppressor Proteins

GDF15 has been recently recognized as a tumor-suppressive gene. However, the underlying mechanism by which GDF15 affects breast carcinogenesis is not well understood. Here, we showed that the inhibitory effect of GDF15 on cell proliferation was dependent on the nuclear localization of the protein. Dynamic translocation of GDF15 into the nucleus altered expression of a number of genes, including KISS-1, and resulted in inhibition of cell growth and invasive behavior. Using KISS-1 promoter-driven luciferase reporter and chromatin immunoprecipitation assays, we demonstrated that, in highly malignant breast cancer cells, GDF15 directly interacts with specific protein-1 (Sp1) at the Sp1-binding sites of the KISS-1 promoter, leading to upregulated KISS-1 expression. Our study indicates that nuclear GDF15 could serve as a transcriptional coactivator to mediate the expression of particular genes to reduce cell proliferation.

Topics: Breast; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 15; Humans; Kisspeptins; Mastectomy; Promoter Regions, Genetic; RNA-Seq; Sp1 Transcription Factor; Transcriptional Activation; Up-Regulation

Brain metastases manifest the advanced stage of breast cancer disease with poor prognosis for patient survival. Recent reports demonstrate that some therapeutic agents can activate the expression of several breast cancer-associated genes, whose products are involved in the onset and development of brain metastases. In this study, we discovered a functional link between KISS1 and E-cadherin that could be observed in both primary brain metastatic lesions and paired cell lines, such as parental CN34TGL and MDA-MB-231 and their respective brain metastatic subclones CN34Brm2Ctgl and MDA-MB-231Br. Remarkably, expression of KISS1 and E-cadherin genes consistently showed an inverse correlation in all of the above cell/tissue types. While E-cadherin expression was strongly upregulated in metastatic clones isolated from blood and brain, the levels of this protein in parental MDA-MB-231 cell line was low. Furthermore, E-cadherin upregulation can be artificially induced in MDA-MB-231Br and CN34Brm2Ctgl cell populations by knocking down KISS1 expression directly or through overexpressing the miR345 mimic. In the aggregate, our data suggest that the tumor microenvironment, which controls breast cancer spreading via miR345-regulated KISS1 expression, might modulate metastatic spreading by a mechanism(s) involving upregulation of E-cadherin production.

Topics: Adult; Antigens, CD; Brain Neoplasms; Breast Neoplasms; Cadherins; Carcinoma, Ductal, Breast; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Kisspeptins; MicroRNAs; Middle Aged; Transcriptional Activation; Tumor Microenvironment; Up-Regulation

NME1 and KISS1 genes are two tumor metastasis suppressor genes, mapped to chromosomes 17q21.3 and 1q32 respectively. Here, we analyzed the association of EcoR1 (rs34214448-G/T) polymorphism in NME1 gene and 9 del T (rs5780218-A/-) polymorphism in KISS1 gene with breast cancer development and metastasis.. The study included 75 women newly diagnosed with breast cancer recruited from Oncology Center at Mansoura University Hospitals and 37 age-matched healthy female volunteers as a control group. DNA was extracted from peripheral blood samples and genotyping of rs34214448 and rs5780218 SNPs was carried out by PCR-RFLP technique. NME1 EcoR1 (rs34214448) polymorphism has a statistically significant association with breast cancer risk (P < 0.001). Most of breast cancer group (55%) had heterozygous (G/T) genotype while most of control group (95%) had homozygous wild (G/G) genotype (P < 0.0005). Also, KISS1 rs5780218 polymorphism has a statistically significant association with breast cancer risk. The wild (A/A) genotype was associated with lower risk of breast cancer (A/- + -/- vs. A/A: OR = 3.1, 95% CI = 1.15-8.36, P = 0.025). EcoR1 (rs34214448) polymorphism revealed a significant association with tumor stage and distant metastasis as patients. Carriers of the wild (G/G) genotype were more likely to present with advanced disease stages and distant metastasis.. Both EcoR1 (rs34214448) polymorphism of NME1 gene and rs5780218 polymorphism of KISS1 gene revealed significant association with increased risk of breast cancer development. The (G/G) genotype of EcoR1 polymorphism was associated with higher risk of breast cancer metastasis.

Topics: Adult; Breast Neoplasms; Case-Control Studies; Female; Humans; Kisspeptins; Middle Aged; NM23 Nucleoside Diphosphate Kinases; Polymorphism, Single Nucleotide

KISS1 tumor suppressor protein regulates cancer cell invasion via MMP9 metalloproteinase. Downregulation of KISS1 gene expression promotes progression of breast cancer and melanoma, resulting in the development of distant metastases. In the current study, we investigated whether restoration of KISS1 expression in KISS1-deficient human metastatic breast cancer cells holds potential as an advanced anticancer strategy. To this end we engineered an infectivity-enhanced conditionally-replicative human adenovirus type 5 encoding KISS1 as an "arming" transgene in the Ad5 E3 region for an ectopic KISS1 expression in transduced cancer cells. The oncolytic potential of the vector was examined using brain-invading metastatic clones of CN34 and MDA-MB-231 breast cancer cells, which supported high levels of AdKISS1 replication, correlating with a robust CRAd-mediated cytotoxicity. Secretion of cellular factors responsible for tumor angiogenesis, cell-to-cell communication and anti-tumoral immune responses upon KISS1 expression in breast cancer cells was analyzed by a RayBiotech Kiloplex Quantibody array. Overall, our results indicate that KISS1 transgene expression provides an important benefit for CRAd-mediated cytotoxicity in breast cancer cells and holds potential as an anticancer treatment in conjunction with oncolytic virotherapy of breast and other metastatic cancers.

Topics: A549 Cells; Adenoviridae; Brain Neoplasms; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Survival; Cells, Cultured; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Genetic Vectors; Humans; Kisspeptins; Neovascularization, Pathologic; Oncolytic Virotherapy

Topics: Adolescent; Adult; Aging; Benzhydryl Compounds; Breast Neoplasms; Child; Disease Susceptibility; Female; Flame Retardants; Humans; Kisspeptins; Leptin; Male; Menarche; Obesity; Phenols; Puberty; Time Factors; Uterine Neoplasms

Formation of metastases, also known as cancer dissemination, is an important stage of breast cancer (BrCa) development. KISS1 expression is associated with inhibition of metastases development. Recently we have demonstrated that BrCa metastases to the brain exhibit low levels of KISS1 expression at both mRNA and protein levels. By using multicolor immunofluorescence and coculture techniques here we show that normal adult astrocytes in the brain are capable of promoting metastatic transformation of circulating breast cancer cells localized to the brain through secretion of chemokine CXCL12. The latter was found in this study to downregulate KISS1 expression at the post-transcriptional level via induction of microRNA-345 (MIR345). Furthermore, we demonstrated that ectopic expression of KISS1 downregulates ATG5 and ATG7, 2 key modulators of autophagy, and works concurrently with autophagy inhibitors, thereby implicating autophagy in the mechanism of KISS1-mediated BrCa metastatic transformation. We also found that expression of KISS1 in human breast tumor specimens inversely correlates with that of MMP9 and IL8, implicated in the mechanism of metastatic invasion, thereby supporting the role of KISS1 as a potential regulator of BrCa metastatic invasion in the brain. This conclusion is further supported by the ability of KISS1, ectopically overexpressed from an adenoviral vector in MDA-MB-231Br cells with silenced expression of the endogenous gene, to revert invasive phenotype of those cells. Taken together, our results strongly suggest that human adult astrocytes can promote brain invasion of the brain-localized circulating breast cancer cells by upregulating autophagy signaling pathways via the CXCL12-MIR345- KISS1 axis.

Topics: Adult; Aged; Animals; Astrocytes; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Brain Neoplasms; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Chemokine CXCL12; Female; Humans; Interleukin-8; Kisspeptins; Matrix Metalloproteinase 9; Mice; Microglia; MicroRNAs; Middle Aged; Xenograft Model Antitumor Assays

To investigate the protein expressions of metastasis-associated in colon cancer 1 (MACC1), KISS-1 (a cancer ruppressor gene) and Snail (the marker of epithelial-mesenchymal transition) in infiltrating breast carcinoma (IBC) and explore the role of them in invasion, metastasis and prognosis in IBC.. Expressions of MACC1, Snail and KISS-1 were examined by immunohistochemistry in 250 specimens of IBC and 80 specimens of normal breast tissues. Their clinicopathological features were analyzed, and their influence on patients' survival was identified.. The positive rate of MACC1, Snail and KISS-1 in normal breast tissues and IBC tissues was 7.5%, 5.0%, 87.5% and 63.6%, 58.8%, 38.0%, respectively. And there was a significant difference between the IBC group and control group (. Abnormal expression of MACC1, Snail and KISS-1 should be involved in the invasion and metastasis of IBC. The combined detection in the expressions of MACC1, Snail and KISS-1 at the early stage may play an important role in predicting the progression and prognosis of IBC.

Topics: Breast Neoplasms; Humans; Kisspeptins; Lymphatic Metastasis; Prognosis; Snail Family Transcription Factors; Trans-Activators; Transcription Factors

Kisspeptins (KPs) are major regulators of trophoblast and cancer invasion. Thus far, limited and conflicting data are available on KP-mediated modulation of breast cancer (BC) metastasis; mostly based on synthetic KP-10, the most active fragment of KP. Here, we report for the first time comprehensive functional effects of term placental KPs on proliferation, adhesion, Matrigel invasion, motility, MMP activity and pro-inflammatory cytokine production in MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). KPs were expressed at high level by term placental syncytiotrophoblasts and released in soluble form. Placental explant conditioned medium containing KPs (CM) significantly reduced proliferation of both cell types compared to CM without (w/o) KP (CM-w/o KP) in a dose- and time-dependent manner. In MDA-MB-231 cells, placental KPs significantly reduced adhesive properties, while increased MMP9 and MMP2 activity and stimulated invasion. Increased invasiveness of MDA-MB-231 cells after CM treatment was inhibited by KP receptor antagonist, P-234. CM significantly reduced motility of MCF-7 cells at all time points (2-30 hr), while it stimulated motility of MDA-MB-231 cells. These effects were reversed by P-234. Co-treatment with selective ER modulators, Tamoxifen and Raloxifene, inhibited the effect of CM on motility of MCF-7 cells. The level of IL-6 in supernatant of MCF-7 cells treated with CM was higher compared to those treated with CM-w/o KP. Both cell types produced more IL-8 after treatment with CM compared to those treated with CM-w/o KP. Taken together, our observations suggest that placental KPs differentially modulate vital parameters of estrogen receptor-positive and -negative BC cells possibly through modulation of pro-inflammatory cytokine production.

Topics: Adult; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Movement; Cell Proliferation; Female; Humans; Immunoenzyme Techniques; Immunoprecipitation; Kisspeptins; Placenta; Pregnancy; Real-Time Polymerase Chain Reaction; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Wound Healing; Young Adult

Breast cancer is the commonest female cancer worldwide and its propensity to metastasize negatively impacts on therapeutic outcome. Several clinicopathological parameters with prognostic/predictive significance have been associated with metastatic suppressor expression levels. The role of metastatic suppressor gene (MSG) KiSS1 in breast cancer remains unclear. Our goal was to investigate the possible clinical significance of KiSS1 breast cancer.. The study was conducted on 87 histologically proven cases of breast cancer and background normal tiisue. Quantitative reverse transcriptase polymerase chain reaction (qRT PCR) and immunohistochemistry (IHC) were used to investigate KiSS1 at gene and protein levels, respectively, for correlation with several patient characteristics including age, family history, hormonal receptor status, stage, tumor size, nodal involvement and metastatic manifestation and finally with median overall survival (OS).. Our study revealed (i) KiSS1 levels were generally elevated in breast cancer vs normal tissue (< 0.05). (ii) however, a statistically significant lower expression of KiSS1 was observed in metastatic vs non metastatic cases (P = 0.04). (iii) KiSS1 levels strongly correlated with T,N,M category, histological grade and advanced stage (<0.001) but not other studied parameters. (iv) Lastly, a significant correlation between expression of KiSS1 and median OS was found (P = 0.04).. Conclusively, less elevated KiSS1 expression is a negative prognostic factor for OS, advancing tumor stage, axillary lymph node status, metastatic propensity and advancing grade of the breast cancer patient. Patients with negative KiSS1 expression may require a more intensive therapeutic strategy.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Breast; Breast Neoplasms; Case-Control Studies; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Kisspeptins; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Survival Rate; Young Adult

Breast cancer is the leading cause of cancer-related death in women. Kisspeptin-10 (KP-10) is a shorter fragment of KISS1. In the present study, we demonstrated the antitumor effects of KP-10 on human breast cancer cell lines, MDA-MB-231 and MDA-MB-157, both in vitro and in vivo. KP-10 was observed to induce apoptosis and inhibit the mobility of MDA-MB-231 and MDA-MB-157 cells. Correspondingly, KP-10 suppressed tumor growth in established xenograft tumor models and improved the survival rate of tumor-bearing mice. The formation of intratumoral microvessels was inhibited following treatment with KP-10. Finally, we confirmed that KP-10 inhibited cell mobility via epithelial-mesenchymal transition (EMT). Overall, the present study demonstrated that KP-10 suppressed breast cancer and human umbilical vein endothelial cell (HUVEC) growth both in vivo and in vitro. KP-10 is a novel regulator of EMT in breast cancer cells. However, additional studies are needed to confirm these results in other cell types.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chick Embryo; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Kisspeptins; Mice; Neovascularization, Pathologic; Xenograft Model Antitumor Assays

Recent studies have raised doubts about the protective role of KiSS1/KiSS1R in breast malignancy progression. However, the role of the KiSS1/KiSS1R system in primary breast cancer remains largely unknown. The aim of the present study was to characterize the biology and invasiveness potential of primary breast cancer through evaluation of KiSS1/KiSS1R protein expression and cellular localization with regard to lymph node metastasis status, receptor status (ERs, PR and HER-2/neu), and expression of aromatase, MMP-9, Ki-67 and Cyclin D1 in primary invasive breast cancer tissues. We showed increased protein expression of both KiSS1/KiSS1R and MMP-9 in the cancerous tissues compared with noncancerous tissue adjacent to the breast tumour. In the studied group of breast cancer samples, we observed a positive correlation between KiSS1 and MMP-9. We also showed a positive correlation between KiSS1R and aromatase expression in all studied breast cancers. We did not notice any associations between system and cell cycle regulators. KiSS1/KiSS1R did not correlate either with Cyclin D1 and Ki-67 or with receptor status. However, we showed higher levels of KiSS1R expression in ERα-negative cases than in ERα-positive cases in patients with lymph node metastasis. Present data do not confirm the protective role of KiSS1/KiSS1R in breast cancer progression, but our results do support the hypothesis that the KiSS1/KiSS1R system is activated even in primary breast cancer and sustained during invasion to local lymph nodes.

Topics: Aged; Aged, 80 and over; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Cell Proliferation; Disease Progression; Estrogen Receptor alpha; Female; Humans; Immunohistochemistry; Kisspeptins; Lymphatic Metastasis; Matrix Metalloproteinase 9; Middle Aged; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; Receptors, Progesterone

KiSS1 is a metastasis suppressor gene associated with inhibition of cellular chemotaxis and invasion attenuating the metastasis in melanoma and breast cancer cell lines. Along the KiSS-1 gene at least 294 SNPs have been described; however the association of these polymorphisms as genetic markers for metastasis in breast cancer studies has not been investigated. Here we describe two simple PCR-RFLPs protocols to identify the rs5780218 (9DelT) and the rs12998 (E20K) KiSS1 polymorphisms and the allelic, genotypic, and haplotypic frequencies in Mexican general population (GP) and patients with benign breast disease (BBD) or breast cancer (BC).. The rs5780218 polymorphism was individually associated with breast cancer (P = 0.0332) and the rs12998 polymorphism shows statistically significant differences when GP versus case (BC and BBD) groups were compared (P < 0.0001). The H1 Haplotype (G/-) occurred more frequently in BC group (0.4256) whereas H2 haplotype (G/T) was the most prevalent in BBD group (0.4674).. Our data indicated that the rs5780218 polymorphism individually confers susceptibility for development of breast cancer in Mexican population and a possible role as a genetic marker in breast cancer metastasis for H1 haplotype (Wt/variant) in KiSS1 gene must be analyzed in other populations.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma; Case-Control Studies; Female; Humans; Kisspeptins; Mexico; Middle Aged; Polymorphism, Single Nucleotide

Recently we have shown that breast cancer cell invasion was dramatically increased when co-cultured with MG63 cells. In addition we have generated mesenchymal transformed MCF-7 breast cancer cells (MCF-7-EMT), showing significantly increased invasion in contrast to wild type MCF-7 cells (MCF-7 WT). In this study we have analyzed whether stromal derived factor-1 (SDF-1) is responsible for MCF-7 and T-47-D breast cancer cell invasion and epithelial-mesenchymal-transition (EMT). In addition we have analyzed whether kisspeptin-10 (KP-10) treatment affects SDF-1-induced invasion and EMT. Invasion was quantified by assessment of MCF-7 and T-47-D breast cancer cell migration rate through an artificial basement membrane in a modified Boyden chamber during co-culture with MG63 cells or after treatment with SDF-1α, SDF-1β or the combination of both isoforms. Induction of EMT was verified by analysis of protein expression of epithelial marker E-cadherin (CDH1) and mesenchymal markers N-cadherin (CDH2) and Vimentin (VIM). The role of SDF-1 for invasion and induction of EMT in breast cancer cells was analyzed by blocking SDF-1 secretion during co-culture with MG63 cells. In addition effects of KP-10 treatment on SDF-1-induced invasion and EMT were analyzed. Breast cancer cell invasion was significantly increased when co-cultured with MG63 cells. During co-culture SDF-1 protein expression of MG63 cells was significantly induced. The increased breast cancer cell invasion could be blocked by anti-SDF-1 antibodies. Treatment of breast cancer cells in monoculture (without MG63) with SDF-1α, SDF-1β or the combination of both isoforms resulted in a significant escalation of breast cancer cell invasion and induction of EMT. Protein expression of mesenchymal markers CDH2 and VIM was clearly elevated, whereas protein expression of epithelial marker CDH1 was clearly decreased. The SDF-1-induced increase of cell invasion was significantly reduced after treatment with KP-10. In addition, induction of EMT was inhibited. Furthermore, protein expression of the binding site of SDF-1, CXC-motive-chemokine receptor 4 (CXCR-4), was reduced by KP-10. Treatment of MCF-7-EMT cells with KP-10 resulted in a significant drop of cell invasion and CXCR-4 protein expression. Our findings suggest that SDF-1 plays a major role in breast cancer invasion and EMT. SDF-1-induced invasion and EMT can be inhibited by KP-10 treatment by down-regulating CXCR-4 expression.

Topics: Breast Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Chemokine CXCL12; Coculture Techniques; Epithelial-Mesenchymal Transition; Female; Humans; Kisspeptins; MCF-7 Cells; Receptors, CXCR4

Numerous studies have shown that the Kiss-1 gene countervails the metastatic aptitude of several cancer cell lines and solid-tumor neoplasias. However, there still remains ambiguity regarding its role in breast cancer and literature has arisen asserting that Kiss-1 expression may be linked to an aggressive phenotype and malignant progression. Herein, we investigated the protein expression of Kiss-1 and its receptor GPR54 in breast cancer tissues compared to non-cancerous mammary tissues.. Paraffin-fixed cancer tissues from 43 women with resected breast adenocarcinomas and 11 specimens derived from women suffering from fibrocystic disease, serving as controls, were immunostained with Kiss-1 and GPR54 antibodies.. Kiss-1 and GPR54 protein expression levels were significantly higher in breast cancer compared to fibrocystic tissues (p<0.05). No significant correlation was established between Kiss-1 or GRP54 expression and tumor grade, tumor size, lymph node positivity, histological type or ER status. Kiss-1 expression significantly and positively correlated with GPR54 expression in both breast cancer and fibrocystic disease specimens.. Kiss-1/GPR54 expression was found to be significantly higher in breast cancer compared to non-malignant mammary tissues.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Case-Control Studies; Female; Follow-Up Studies; Humans; Kisspeptins; Lymphatic Metastasis; Middle Aged; Neoplasm Grading; Neoplasm Staging; Prognosis; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1

The metastasis suppressor protein Kisspeptin regulates cancer cell proliferation and motility through its receptor, GRP54. However, the critical downstream effectors remain unclear. In this study, we investigated GPR54 signaling in breast cancer cells. Kisspeptin stimulation caused a decrease in migration of multiple breast cancer cell lines. Also, Kisspeptin inhibited MDA-MB-231 cell colony formation in 3D matrigel culture and in soft agar. Kisspeptin treatment elevated phosphorylated PKD1 in a PKC-dependent manner. However, knockdown of either GPR54 or PKD1 increased breast cancer cell migration and invasion. Furthermore, GPR54 knockdown blocked Kisspeptin-induced phosphorylation of PKD1. Finally, Kisspeptin stimulation induced a PKD1 phosphorylation-dependent decrease in expression of Slug, a transcription factor that drives epithelial-mesenchymal transition (EMT), and a concomitant increase in E-cadherin expression. Therefore, KiSS1/GPR54 signaling through PKD1 acts to maintain the epithelial state and to inhibit breast cancer cell invasiveness, and exerts functions associated with its role as a metastasis suppressor.

Topics: Breast Neoplasms; Cadherins; Cell Line; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Kisspeptins; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; TRPP Cation Channels

The WASF3 gene promotes invasion and metastasis in breast cancer cells, which have undergone epithelial-to-mesenchyme transition (EMT). Overexpression of WASF3 in cells that do not show EMT increases their invasion potential as a result of increased ZEB1/2 levels, which specifically suppress the anti-invasion chromosome 1 miR-200a/200b/429 cluster. ZEB1/2 upregulation by WASF3 results from downregulation of KISS1, leading to the release of inhibition of nuclear factor (NF)κB by IκBα. We further show that ZEB1 expression is regulated by the NFκB transcription factor. Knockdown of WASF3 in breast cancer cells leads to reduced ZEB1 levels and increased miR-200 and E-cadherin levels, resulting in loss of invasion potential. The central regulation of this interactive pathway by WASF3 accounts for the increased invasion associated with increased WASF3 expression seen in aggressive breast cancer cells. WASF3, therefore, is a potential target to suppress invasion and metastasis in breast cancer cells.

Topics: Active Transport, Cell Nucleus; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Female; Homeodomain Proteins; Humans; Kisspeptins; MicroRNAs; Neoplasm Invasiveness; NF-kappa B; Promoter Regions, Genetic; Transcription Factors; Wiskott-Aldrich Syndrome Protein Family; Zinc Finger E-box-Binding Homeobox 1

Kisspeptins (KPs), peptide products of the KISS1 metastasis-suppressor gene, are endogenous ligands for a G protein-coupled receptor (KISS1R). KISS1 acts as a metastasis suppressor in numerous human cancers. However, recent studies have demonstrated that an increase in KISS1 and KISS1R expression in patient breast tumors correlates with higher tumor grade and metastatic potential. We have shown that KP-10 stimulates invasion of estrogen receptor α (ERα)-negative MDA-MB-231 breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR). Here, we report that either KP-10 treatment of ERα-negative nonmalignant mammary epithelial MCF10A cells or expression of KISS1R in MCF10A cells induced a mesenchymal phenotype and stimulated invasiveness. Similarly, exogenous expression of KISS1R in ERα-negative SKBR3 breast cancer cells was sufficient to trigger invasion and induced extravasation in vivo. In contrast, KP-10 failed to transactivate EGFR or stimulate invasiveness in the ERα-positive MCF7 and T47D breast cancer cells. This suggested that ERα negatively regulates KISS1R-dependent breast cancer cell migration, invasion, and EGFR transactivation. In support of this, we found that these KP-10-induced effects were ablated upon exogenous expression of ERα in the MDA-MB-231 cells, by down-regulating KISS1R expression. Lastly, we have identified IQGAP1, an actin cytoskeletal binding protein as a novel binding partner of KISS1R, and have shown that KISS1R regulates EGFR transactivation in breast cancer cells in an IQGAP1-dependent manner. Overall, our data strongly suggest that the ERα status of mammary cells dictates whether KISS1R may be a novel clinical target for treating breast cancer metastasis.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Movement; Chick Embryo; Epithelial Cells; Epithelial-Mesenchymal Transition; ErbB Receptors; Estrogen Receptor alpha; Female; Humans; Inositol Phosphates; Kisspeptins; Mammary Glands, Human; MCF-7 Cells; Microscopy, Fluorescence; Neoplasm Invasiveness; ras GTPase-Activating Proteins; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; RNA Interference

Breast cancer is the most frequently diagnosed cancer in women. In these studies, a metastasis suppressor gene, KISS1 and its truncated fragment, were overexpressed in the breast cancer cell line MDA-MB-231. In addition, KISS1 expression was downregulated in MDA-MB-157 cell line using a KISS1-specific siRNA. The effects of KISS1 on breast cancer cells both in vivo and in vitro were then identified. Our results indicate that KISS1 can induce apoptosis and inhibit mobility of breast cancer cells. Moreover, the expression of KISS1 in established xenografted tumors was associated with a decrease in tumor size and weight. Accordingly, the survival rate of these mice was significantly higher compared to that of mice bearing tumors that did not express KISS1. We also confirm that KISS1 could decrease the number of circulating tumor cells (CTCs). The plasma levels of metastin and the number of CTCs were significantly positively correlated. Furthermore, we found that KISS1 can inactivate p-MEK and p-ERK. Overall, these studies demonstrate the antitumor activity of KISS1 in the breast cancer cell lines and provide insight into relevant mechanisms that may lead to novel treatments for breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Kisspeptins; Mice; Molecular Targeted Therapy; Neoplastic Cells, Circulating; Xenograft Model Antitumor Assays

Kisspeptins are peptides derived from the metastasis suppressor gene KISS1 interacting with GPR54 as their corresponding receptor. The KISS1/GPR54 system is one regulator of cellular motility mechanisms leading to decreased migration and invasion. Its role in cell proliferation processes is not clearly understood. In this study, breast cancer cell lines, T47D, ZR75-1, MDA‑MB‑231, MDA‑MB‑435s, MDA‑MB‑453, HCC 70, HCC 1806, HCC 1937 and MCF‑7, were investigated for their endogenous GPR54 expression by immunocytochemistry, RT‑PCR and western blot analysis. The effect of kisspeptin‑10 on proliferation was measured in MDA‑MB‑231, MDA‑MB‑435s, HCC 1806 and MCF‑7 cells. Further experiments on proliferation were carried out with cells transfected with GPR54. All of the tested breast cancer cell lines expressed GPR54 in different amounts. No effects on proliferation were detected in the breast cancer cells expressing the receptor endogenously. In transfected neuronal cells overexpressing GPR54, proliferation was significantly inhibited by kisspeptin‑10. The results indicate that the antiproliferative action of kisspeptin depends on the nature of GPR54 expression. The effect was detected in an artificial system of cells transfected with GPR54 and not in cells expressing the receptor endogenously. Thus, the antiproliferative action of kisspeptin seems not to be important for pathophysiological processes.

Topics: Breast Neoplasms; Cell Proliferation; Female; Humans; Kisspeptins; MCF-7 Cells; Neoplasm Proteins; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; RNA, Messenger; Transfection

Metastases to the brain represent a feared complication and contribute to the morbidity and mortality of breast cancer. Despite improvements in therapy, prognostic factors for development of metastases are lacking. KISS1 is a metastasis suppressor that demonstrates inhibition of metastases formation in several types of cancer. The purpose of this study was to determine the importance of KISS1 expression in breast cancer progression and the development of intracerebral lesions.. In this study, we performed a comparative analysis of 47 brain metastases and 165 primary breast cancer specimens by using the antihuman KISS1 antibody. To compare KISS1 expression between different groups, we used a 3-tier score and the automated score computer software (ACIS) evaluation. To reveal association between mRNA and protein expression, we used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Significance of immunohistochemistry stainings was correlated with clinicopathological data.. We identified that KISS1 expression is significantly higher in primary breast cancer compared with brain metastases (P < .05). The mRNA analysis performed on 33 selected ductal carcinoma brain metastatic lesions and 36 primary ductal carcinomas revealed a statistically significant down-regulation of KISS1 protein in metastatic cases (P = .04). Finally, we observed a significant correlation between expression of KISS1 and metastasis-free survival (P = .04) along with progression of breast cancer and expression of KISS1 in primary breast cancer specimens (P = .044).. In conclusion, our study shows that breast cancer expresses KISS1. Cytoplasmic expression of KISS1 may be used as a prognostic marker for increased risk of breast cancer progression.

Topics: Adult; Brain Neoplasms; Breast Neoplasms; Carcinoma, Ductal, Breast; Disease Progression; Disease-Free Survival; Female; Humans; Kisspeptins; Middle Aged; Prognosis

Nodal staging in breast cancer is a key predictor of prognosis. This paper presents the results of potential clinicopathological predictors of axillary lymph node involvement and develops an efficient prediction model to assist in predicting axillary lymph node metastases. Seventy patients with primary early breast cancer who underwent axillary dissection were evaluated. Univariate and multivariate logistic regression were performed to evaluate the association between clinicopathological factors and lymph node metastatic status. A logistic regression predictive model was built from 50 randomly selected patients; the model was also applied to the remaining 20 patients to assess its validity. Univariate analysis showed a significant relationship between lymph node involvement and absence of nm-23 (p = 0.010) and Kiss-1 (p = 0.001) expression. Absence of Kiss-1 remained significantly associated with positive axillary node status in the multivariate analysis (p = 0.018). Seven clinicopathological factors were involved in the multivariate logistic regression model: menopausal status, tumor size, ER, PR, HER2, nm-23 and Kiss-1. The model was accurate and discriminating, with an area under the receiver operating characteristic curve of 0.702 when applied to the validation group. Moreover, there is a need discover more specific candidate proteins and molecular biology tools to select more variables which should improve predictive accuracy.

Topics: Breast Neoplasms; Female; Humans; Kisspeptins; Logistic Models; Lymphatic Metastasis; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Receptor, ErbB-2; Receptors, Estrogen; ROC Curve

Loss of WASF3 function in breast cancer cells results in loss of invasion phenotypes and reduced metastatic potential. By using oligonucleotide arrays, we now demonstrate that knockdown of WASF3 leads to the upregulation of the KISS1 metastasis suppressor gene with concomitant reduced invasion and loss of matrix metalloproteinases (MMP)-9 activity. Using a luciferase reporter, KISS1 transcription is significantly increased in the absence of WASF3. Knockdown of KISS1 in WASF3-silenced cells resulted in the recovery of the invasion phenotype. WASF3 knockdown also resulted in elevated IκBα levels in the cytoplasm and reduced levels of nuclear factor-kappa-B (NF-κB) p65/50 subunits in the nucleus. Tumor necrosis factor-alpha (TNF-α) has been associated with cell invasion through induction of MMP-9 production via KISS1 regulation of the NF-κB pathway. When WASF3 knockdown cells are treated with TNF-α, no effect is seen on invasion or nuclear translocation of NF-κB. Thus, coordinated expression patterns of the WASF3 metastasis promoter gene and the KISS1 metastasis suppressor gene appear to exert their influence through inhibition of NF-κB signaling, which in turn regulates MMP-9 production facilitating invasion.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Gene Knockdown Techniques; Humans; Kisspeptins; Male; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; NF-kappa B; Prostatic Neoplasms; Signal Transduction; Up-Regulation; Wiskott-Aldrich Syndrome Protein Family

Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Enzyme Activation; ErbB Receptors; Female; Humans; Kisspeptins; Matrix Metalloproteinase 9; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1

Metastasis suppressor genes are involved in the inhibition of a cancer cell's ability to metastasize. Down expression of such genes may contribute to pathogenesis of breast cancer. The aim of current study was firstly to evaluate expression of two examples, KAI1 and KISS1, and then to determine relationships with stages of breast cancer in a Pakistani population.. Fresh biopsy tissues were collected from different hospitals and oncology research institutes. The semi quantitative reverse transcriptase polymerase chain reaction was used to investigate KAI1 and KISS1 expression in 25 breast tumor tissues and 25 normal tissues. Statistical analysis was performed to explore its association with breast cancer risk.. The present study revealed that KAI1 and KISS1 mRNA expression was markedly reduced in tissues of breast cancer compared to adjacent normal tissue. In present study a splice variant of KAI1 during a screen for its expression analysis was also observed. This splice variant has not been reported previously.. Metastasis suppressor genes demonstrate reduced expression in breast cancers in Pakistan.

Topics: Base Sequence; Biopsy; Breast Neoplasms; Down-Regulation; Female; Genes, Tumor Suppressor; Humans; Kangai-1 Protein; Kisspeptins; Molecular Sequence Data; Pakistan; Protein Isoforms; RNA, Messenger; Sequence Analysis, DNA

KiSS-1 is ametastasis suppressor gene, its inactivation linked to advanced tumor stage and dismal prognosis. We studied its mutational status ,transcription and protein expression in human cancer cell lines and patients with early breast cancer.
Tumor tissue DNA and messenger RNA (mRNA) of KiSS1 exons III and IV from the human cancer cell lines Hela, Jurkat, A549, W138t, MCF-7 and from formalin-fixed resected breast adenocarcinomas from 50 women were analysed by means of PCR-SSCP, RT-PCR and sequencing. Tumor tissue was stained for KiSS1 protein expression by means of the streptavidin-biotin complex immunoperoxidase assay. Presence of KiSS1 mutation, mRNA levels and protein staining were examined for correlations with patient/tumor characteristics.
A transversion in exon IVa replacing cytosine with guanine was identified 242 base pairs from the translation start site (242C>G) in the cell lines MCF-7, A549 and in 5/50 tumors (10%), resulting in substitution of proline by arginine (P81R) and alteration of the protein tertiary structure. As the substitution was present in germ-line DNA in 3/5 breast cancer patients harbouring the polymorphism in their tumor, the incidence of tumour-specific somatic mutation was 4% among the 50 patiens with early breast cancer. Although the P81R substitution was associated with reduced KiSS1 protein immunoreactivity (56% in wild-type tumors versus 20% in KiSS1-variant tumours) and with axillary nodal involvement (55% in wild-type versus 80% in KiSS1-variant tumors), the correlations did not reach statistical significance. KiSS1 mRNA was detected in only 15/48 tumours (31%) and showed no correlation with mutation or protein expression. Twenty-six tumors stained for KiSS1 protein, in contrast to the universal strong staining seen in normal breast parenchyma and placental tissues. At amedian follow-up of 38 months, relapses occurred in 20% of women with non wild-type tumors versus 13% of women with wild-type KiSS1 tumors (p=0.7). Presence of KiSS1 mutation, mRNA levels and protein expression did not have prognostic significance for relapse-free survival.
In conclusion, altered nucleotide sequence and repression of transcription are two potential mechanisms of suppression of the anti-metastatic effects of KiSS1 in early breast cancer: Confirmation in larger cohorts and study of functional effects of the 242C>G exon IVa mutation are warranted.. KiSS1, metastasis-suppressor gene, breast cancer, mutation, transcription.

Topics: Amino Acid Sequence; Base Sequence; Breast Neoplasms; Female; Genetic Variation; Humans; Immunohistochemistry; Kisspeptins; Middle Aged; Molecular Sequence Data; Mutation; RNA, Messenger; Transcription, Genetic; Tumor Suppressor Proteins

Kisspeptins are hypothalamic neuropeptides encoded by KISS1 and recently described as major regulators of GnRH release from hypothalamic neurons. Although 17beta-estradiol (E2)-induced up-regulation of KISS1 expression has been documented in anteroventral periventricular nucleus neurons, E2 down-regulates KISS1 expression in arcuate nucleus neurons via the estrogen receptor alpha by unknown molecular mechanisms. Because KISS1 was initially described as a metastasis inhibitor, notably in breast tumors, we used the MDA-MB-231 breast cancer cell line, which expresses high levels of KISS1, to characterize the molecular mechanism underlying KISS1 regulation by E2. E2 rapidly down-regulated endogenous KISS1 in a stable ERalpha-expressing MDA-MB-231 cell line. Promoter analysis revealed that E2 down-regulation was determined by a short 93-bp sequence devoid of estrogen response element and Sp1 sites. E2 down-regulation persisted with an ERalpha that was unable to bind DNA and in the presence of histone deacetylase inhibitor. In the absence of E2, unliganded ERalpha and RNA polymerase II (RNAPII) were present on the proximal promoter. E2 stimulation induced recruitment of ERalpha and loss of RNAPII at the proximal promoter. Along the gene body, total RNAPII amounts were similar in E2-treated and untreated cells, whereas the active form was significantly less abundant in E2-treated cells. Thus, E2-induced down-regulation of KISS1 is mediated by a pathway combining RNAPII loss at the proximal promoter and modulation of active RNAPII along the gene body, which is a novel mechanism in the complex process of E2-induced repression of gene expression.

Topics: Binding Sites; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Estradiol; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Kisspeptins; Promoter Regions, Genetic; Protein Binding; RNA Polymerase II; Signal Transduction; Tumor Suppressor Proteins

The KiSS-1 gene product is absent or expressed at low level in metastatic breast cancer compared with their nonmetastatic counterparts. A deca-peptide derived from the KiSS-1 gene product, designated kisspeptin-10 (Kp-10), activates a receptor coupled to Gαq subunits (GPR54 or KiSS-1R). In this study we have analyzed whether Kp-10 treatment affects bone-directed migration of GPR54-positive breast cancer cells.. GPR54 expression was analyzed using immune cytochemistry. Bone-directed breast cancer cell invasion was measured by assessment of the breast cancer cell migration rate through an artificial basement membrane. Chemokine receptor CXCR4 and stromal cell-derived factor-1 (SDF-1) mRNA expression was quantified using semi-quantitative RT-PCR. CXCR4 protein expression and SDF-1 protein secretion were measured using the western blot technique.. Breast cancer cell invasion was increased when cocultured with MG63 osteoblast-like cells. Treatment with KP-10 reduced the ability to invade a reconstituted basement membrane and to migrate in response to the cellular stimulus. This effect was significant in a dose-window of 10⁻⁹ M to 10⁻¹¹ M. Searching for the molecular mechanisms we found that KP-10 treatment significantly reduces expression of the chemokine receptor CXCR4 by the breast cancer cells. In addition, expression and secretion of its ligand SDF-1 by the MG63 cells were significantly reduced. Furthermore, SDF-1-induced CXCR4 signaling was down-regulated.. These data represent the first report that KP-10 inhibits bone-directed migration of GPR54-positive breast cancer cells. In addition, we found evidence for a KP-10 dose-window effect. Furthermore, the SDF-1/CXCR4 system seems to be involved in the anti-migratory action of KP-10.

Topics: Bone Neoplasms; Breast Neoplasms; Cell Communication; Cell Line, Tumor; Cell Movement; Chemokine CXCL12; Coculture Techniques; Female; Humans; Immunohistochemistry; Kisspeptins; Oncogene Protein v-akt; Osteoblastoma; Osteoblasts; Phosphorylation; Receptors, CXCR4; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; RNA, Messenger; Signal Transduction; Tumor Suppressor Proteins

Tumor necrosis factor-alpha (TNFalpha) induces cancer development and metastasis, which is prominently achieved by nuclear factor-kappa B (NF-kappaB) activation. TNFalpha-induced NF-kappaB activation enhances cellular mechanisms including proliferation, migration, and invasion. KiSS1, a key regulator of puberty, was initially discovered as a tumor metastasis suppressor. The expression of KiSS1 was lost or down-regulated in different metastatic tumors. However, it is unclear whether KiSS1 regulates TNFalpha-induced NF-kappaB activation and further tumor cell migration. In this study, we demonstrate that KiSS1 suppresses the migration of breast cancer cells by inhibiting TNFalpha-induced NF-kappaB pathway and RhoA activation. Both KiSS1 overexpression and KP10 (kisspeptin-10) stimulation inhibited TNFalpha-induced NF-kappaB activity, suppressed TNFalpha-induced cell migration and cell attachment to fibronectin in breast cancer cells while KP10 has little effect on cancer cell proliferation. Furthermore, KP10 inhibited TNFalpha-induced cell migration and RhoA GTPase activation. Therefore, our data demonstrate that KiSS1 inhibits TNFalpha-induced NF-kappaB activation via downregulation of RhoA activation and suppression of breast cancer cell migration and invasion.

Topics: Breast Neoplasms; Cell Adhesion; Cell Movement; Cell Proliferation; Down-Regulation; Female; Humans; Kisspeptins; Neoplasm Invasiveness; NF-kappa B; Oligopeptides; rhoA GTP-Binding Protein; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins

To study the expression of KiSS-1, nuclear factor kappa B (NF-kappaB) p50 and matrix metalloproteinase 9 (MMP-9) in breast cancer tissue and the relationship with clinicpathological factors.. Immunohistochemical staining for KiSS-1, NF-KappaBp50, and MMP-9 protein was performed in 152 cases of human breast tissue [92 cases of BC, 30 cases of epithelial hyperplasia, and 30 cases of peritumoral breast tissue (PMT)] and 54 cases of axillary lymph node metastases. In-situ hybridization for KiSS-1 mRNA was done in 50 cases of breast cancer, and 20 cases of PMT.. (1) The expression of KiSS-1 gene was significantly higher in well-differentiated breast cancer than in PMT, and this expression progressively decreased with decreasing degree of tumor differentiation, increasing pathological grade, TNM stage and the presence of lymph node metastases. The expression of KiSS-1 gene in lymph node metastasis was markedly lower than the corresponding primary tumor. There was correlation between the expression of KiSS-1 mRNA and KiSS-1 protein in breast cancer group. (2) The expression of NF-kappaKBp50 and MMP-9 increased progressively with decreasing degree of tumor differentiation, increasing TNM stage, large tumor size ( >2 cm) and the presence of lymph node metastases.. The expression of KiSS-1 protein showed negative correlation with that of NF-kappaBp50 and MMP-9 respectively. MMP-9 protein expression was positively correlated with NF-kappap50 protein expression. These suggest that the genes of KiSS-1, NF-kappaBp50 and MMP-9 could be involved in the progression and metastasis of breast cancer.

Topics: Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Kisspeptins; Lymphatic Metastasis; Matrix Metalloproteinase 9; NF-kappa B; RNA, Messenger; Statistics as Topic; Tumor Suppressor Proteins

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

Mammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntington's disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below.. Expression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L).. We found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding alpha2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes.. Collectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.

Topics: Animals; Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Movement; Disease Progression; DNA, Complementary; Female; Gene Expression Regulation, Neoplastic; HSP40 Heat-Shock Proteins; Humans; Immunoblotting; Kisspeptins; Mice; Mice, Nude; Microarray Analysis; Microscopy, Confocal; Molecular Chaperones; Neoplasm Invasiveness; Nerve Tissue Proteins; Nucleophosmin; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Tumor Suppressor Proteins; Up-Regulation; Wound Healing

KiSS1 is a putative metastasis suppressor gene in melanoma and breast cancer-encoding kisspeptins, which are also described as neuroendocrine regulators of the gonadotropic axis. Negative as well as positive regulation of KiSS1 gene expression by estradiol (E(2)) has been reported in the hypothalamus. Estrogen receptor alpha (ERalpha level is recognized as a marker of breast cancer, raising the question of whether expression of KiSS1 and its G-protein-coupled receptor (GPR54) is down- or upregulated by estrogens in breast cancer cells. KiSS1 was found to be expressed in MDA-MB-231, MCF7, and T47D cell lines, but not in ZR75-1, L56Br, and MDA-MB-435 cells. KiSS1 mRNA levels decreased significantly in ERalpha-negative MDA-MB-231 cells expressing recombinant ERalpha. In contrast, tamoxifen (TAM) treatment of ERalpha-positive MCF7 and T47D cells increased KiSS1 and GPR54 levels. The clinical relevance of this negative regulation of KiSS1 and GPR54 by E(2) was then studied in postmenopausal breast cancers. KiSS1 mRNA increased with the grade of the breast tumors. ERalpha-positive invasive primary tumors expressed sevenfold lower KiSS1 levels than ERalpha-negative tumors. Among ERalpha-positive breast tumors from postmenopausal women treated with TAM, high KiSS1 combined with high GPR54 mRNA tumoral levels was unexpectedly associated with shorter relapse-free survival (RFS) relative to tumors expressing low tumoral mRNA levels of both genes. The contradictory observation of putative metastasis inhibitor role of kisspeptins and RFS to TAM treatment suggests that evaluation of KiSS1 and its receptor tumoral mRNA levels could be new interesting markers of the tumoral resistance to anti-estrogen treatment.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma, Ductal, Breast; Disease-Free Survival; Down-Regulation; Estrogens; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Kisspeptins; Middle Aged; Neoplasm Recurrence, Local; Neoplasms, Hormone-Dependent; Prognosis; Receptors, Estrogen; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; Tamoxifen; Tumor Cells, Cultured; Tumor Suppressor Proteins

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2alpha (AP-2alpha) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2alpha binding elements, AP-2alpha-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2alpha into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2alpha wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2alpha regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2alpha directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2alpha and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2alpha in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.

Topics: Breast Neoplasms; Cell Line, Tumor; Consensus Sequence; Gene Expression Regulation, Neoplastic; Humans; Kisspeptins; Promoter Regions, Genetic; Proteins; Sp1 Transcription Factor; Transcription Factor AP-2; Transcriptional Activation; Tumor Suppressor Proteins

The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1-34 and 67-86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin alpha6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.

Topics: Biopsy; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colforsin; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha6; Kisspeptins; Parathyroid Hormone-Related Protein; Plasminogen Activator Inhibitor 1; RNA Interference; RNA, Small Interfering; Tumor Suppressor Proteins

Brain metastases are an increasingly common complication in breast cancer patients. The Metastasis Suppressor Genes (MSG) Nm23, KISS1, KAI1, BRMS1, and Mkk4 have been associated with the metastatic potential of breast cancer in vitro and in vivo.. The mRNA expression of Nm23, KISS1, KAI1, BRMS1, and Mkk4 in fresh frozen tissue samples of brain metastases from ductal invasive breast cancer specimens was examined in relation to primary tumors. In a first step, mRNA expression screening was carried out using a semi-quantitative RT-PCR approach, in a second step quantitative real-time RT-PCR was performed on selected specimens. By immunohistochemical staining, gene products were visualized on the protein level.. Semi-quantitative RT-PCR revealed reduced mRNA expression of Nm23, KISS1, KAI1, BRMS, and Mkk4 in brain metastases. Results for KISS1, KAI1, BRMS, and Mkk4 were confirmed by real-time RT-PCR. In detail, mRNA expression reduction in breast cancer brain metastases was tenfold. Expression of MSG could be confirmed by immunohistochemical staining on protein level.. Our investigations revealed significantly reduced mRNA expression of metastases suppressor genes KISS1, KAI1, BRMS1, and Mkk4 in breast cancer brain metastasis. Particularly, in the case of KISS1 and Mkk4, an important role for future treatment of patients with breast cancer brain metastatic lesions can be assumed.

Topics: Antigens, CD; Biomarkers, Tumor; Brain Neoplasms; Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Kangai-1 Protein; Kisspeptins; MAP Kinase Kinase 4; Membrane Glycoproteins; Neoplasm Proteins; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Proteins; Proto-Oncogene Proteins; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Suppressor Proteins

The KiSS-1 gene encodes a 145 amino acid residue peptide that is further processed to a final peptide, metastin, a ligand to a G-coupled orphan receptor (OT7T175/AXOR12). KiSS-1 has been identified as a putative human metastasis suppressor gene in melanomas and in breast cancer cell lines. This study aimed to determine the expression and distribution of KiSS-1 and its receptor in human breast cancer tissues and to identify a possible link between expression levels and patient prognosis. Frozen sections from breast cancer primary tumours (matched tumour 124 and background 33) were immuno-stained with KiSS-1 antibody. RNA was reverse transcribed and analyzed by Q-PCR (standardized using beta-actin, and normalized with cytokeratin-19 levels). Levels of expression of KiSS-1 were higher in tumour compared to background tissues (3,124+/-1,262 vs 2,397+/-1,181) and significantly increased in node positive tumours compared to node negative (3,637+/-1,719 vs 2,653+/-1,994, P = 0.02). KiSS-1 expression was also increased with increasing grade and TNM status. There were no such trends with the KiSS-1 receptor. Expression of KiSS-1 was higher in patients who had died from breast cancer than those who had remained healthy (4,631+/-3,024 vs 2,280+/-1,403) whereas expression of the receptor was reduced (480+/-162 vs 195+/-134). Immunohistochemical staining showed increased expression of KiSS-1 in tumour sections. Insertion of the KiSS-1 gene into the human breast cancer cell line MDA-MB-231, resulted in cells that were significantly more motile and invasive in behaviour, with reduced adhesion to matrix, using respective assays. In conclusion, KiSS-1 expression is increased in human breast cancer, particularly in patients with aggressive tumours and with mortality. Over-expression of KiSS-1 in breast cancer cells result in more aggressive phenotype. Together, it suggests that KiSS-1 plays a role beyond the initial metastasis repressor in this cancer type.

Topics: Breast Neoplasms; Cell Line, Tumor; Humans; Immunohistochemistry; Kisspeptins; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Proteins; Receptors, G-Protein-Coupled; Receptors, Galanin; Receptors, Kisspeptin-1; RNA, Messenger; Transfection; Tumor Suppressor Proteins

Based on the observation that chromosome 1q deletions are not infrequent in late-stage human breast carcinomas, we tested whether the recently discovered human melanoma metastasis suppressor gene, KiSS-1, which maps to chromosome 1q32-q41, could suppress metastasis of the human breast carcinoma cell line MDA-MB-435. Parental, vector-only transfectants and KiSS-1 transfectant clones were injected into the mammary fat pads of athymic nude mice and assessed for tumor growth and spontaneous metastasis to regional lymph nodes and lungs. Expression of KiSS-1 reduced metastatic potential by 95% compared to control cells but did not suppress tumorigenicity. Metastasis suppression correlated with a decreased clonogenicity in soft (0.3%) and hard (0.9%) agar. Although the overall rate of cell adhesion to extracellular matrix components was unaffected, KiSS-1 transfectants spread on immobilized type-IV collagen more rapidly than did control populations. Invasion and motility were unaffected by KiSS-1. Based on the predicted structure of the KiSS-1 protein, our results imply a mechanism whereby KiSS-1 regulates events downstream of cell-matrix adhesion, perhaps involving cytoskeletal reorganization. In addition to its already described role in melanoma, our results show that KiSS-1 also functions as a metastasis suppressor gene in at least some human breast cancers.

Topics: Animals; Blotting, Northern; Breast Neoplasms; Cell Adhesion; Cell Movement; Female; Genes, Tumor Suppressor; Humans; Kisspeptins; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Proteins; RNA, Messenger; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins