Progesterone Inhibits Cell Migration And Invasion Of Human Breast Cancer And Lung Adenocarcinoma Cells Through MPRα Mediated Pathway And Its Underlying Mechanisms

ObjectivesThe role of sex hormone and its receptors in tumor genesis and development has attached substantial attention of cancer research-ers, especially in breast cancer. The cancer-promoting role of estrogen has been confirmed by a number of studies, while the understanding of progesterone and its receptors in breast cancer is mostly based on researches associated with estrogen. Exploring roles of progesterone and its receptors in breast cancer from its own perspective may expand new directions for targeted therapy in breast cancer. Studies on progesterone receptor-mediated progestin effects in breast cancer have made some progress in recent years, but still remained controver-sial. Dif-ferent progesterone receptors may exert different influences on biological characteristics of breast cancer. The newly disco-vered membrane progesterone receptor alpha (mPRa) can mediate rapid non-genomic actions of progesterone, whose mode of action is quite different from progesterone nuclear receptor; this has raised great interests of cancer researchers. Till now, studies of mPRa have mainly focused on basic researches in fish and amphibians; limited studies have taken place in malignant tumors, especially in breast cancer; and no studies reported in the non-sex hormone target organs such as in lung cancer. Tumor metastasis is the leading cause of death of cancer patients; epithelial-mesenchymal transition (EMT) is a key mechanism involving in cancer invasion and metastasis. Our previous study proposed that progesterone can reverse EMT process via an mPRa mediated pathway in basal phenotype breast cancer MB468cells. This gave rise to a question whether progesterone can impact cancer cell migration, invasion and metastasis in breast cancer cells via a similar mPRα mediated pathway as well as in lung adenocarcinoma cells. There are no reported studies regarding this. In this study, we aim to observe the expression and cellular location of mPRα in breast cancer MB231Br (MB231Brain-seeking) cells and MB231cells, as well as in lung adenocarcinoma A549cells and H1299cells, to explore role of mPRα in breast and lung adenocarcinoma cell migration and invasion and its underlying molecular pathway mechanisms, to analyze the expression of mPRα in breast cancer tissue and its relationship with clini-cal pathological features.Chapter1:expression and cellular location of mPRα in breast cancer and lung adenocarcinoma cell linesMethods:1) Expression of mPRα mRNA and protein in breast and lung cancer cells were detected by RT-PCR and western blot. Cellular location and P4binding of mPRα on breast cancer MB231Br cells and lung adenocarcinoma A549cells were investigated by confocal microscope using a P4-BSA-FITC which can't penetrate cell membrane.Results:1) Set MB231cell as a negative control, MB468cells and MB231w/mPRα cells as positive controls, our results demonstrated that breast cancer MB231Br cells were mPRα mRNA and protein moderately positive, lung adenocarcinoma A549cells were positive while H1299cells were negative.2) Clear green fluorescent signals were detected in the membrane of breast cancer MB231Br cells and MB231w/mPRα cells, not in the plasma or nuclear, no signals in the MB231cells. Similar fluorescent signals were also seen in the membrane of lung adenocarcinoma A549cells, but not in H1299cells. After co-incubating MB231Br cells and A549cells with P4-BSA-FITC conjugate and excessive un-conjugated free P4, no fluorescent signals were shown in these cells any more.Conclusion:We firstly found that the expression of mPRa was turned-on and up-regulated from negative to moderately positive both at transcriptional (mRNA) and translational levels (protein) in breast cancer MB231Br cells. Expression of mPRa was positive in lung adenocarcinoma A549cells, but not H1299cells. We firstly confirmed the specific membrane binding of P4and mPRa on breast cancer MB231Br cells and lung adenocarcinoma A549cells. Our results may imply an essential mediating role of mPRa in P4function.Chapter2:mPRa mediates progesterone's influence on cell migration and invasion of human breast cancer and lung adenocarcinoma cells and its underlying mechanismsMethods:1) Cell proliferation were analyzed by MTT assay cells in presence of P4(30ng/ml or60ng/ml) for24hrs or48hrs. Cell morphology was observed by microscope with P4at a series of concentrations (0,15,30, and60ng/ml) for48hrs.2) Different effects of P4and/or PP1(Src inhibitor) on cell migration of breast cancer cell lines and lung adenocarcinoma cell lines were investigated by cell migration assay in presence of different combination of mPRa antibody, mPRa antibody specific binding peptide, a nuclear progesterone receptor antagonist mifepriston and an anti-PGRMC1antibody, depend on their status of mPRa. 3) Different effects of P4and/or PP1on cell invasion of breast cancer cell lines and lung adenocarcinoma cell lines were investigated by Transwell Chamber invasion assay.4) Expression of p-FAK, MMP-9and KCNMA1were analyzed in presence of P4and/or PP1by western blot.Results:1) MB231Br cell and A549cell proliferation were inhibited but not significantly by30ng/ml for24hrs, but inhibited by30ng/ml for48hrs and60ng/ml for24or48hrs. Cell morphological study showed that MB231Br cells without P4incubation appeared apparent mesenchymal phenotypes, characterized by diverse sizes and spindle or elongated shapes; while with P4treatment, most of the cells appeared epithelial-like phenotypes, featured by large and polygonal shapes or small oval shapes. No changes in cell proliferation or morphology in MB231cells.2) In presence of both P4and PPl, breast cancer MB231Br and MB468cells, but not MB231cells, displayed inhibited horizontal migration. This inhibition was blocked by an anti-mPRα antibody, and resumed by adding an anti-mPRa antibody specific peptide, but not hampered by mifepriston or an anti-PGRMC1antibody. Introduction of the exogenous mPRa cDNA into MB231cells enabled its responsive to P4/PP1treatment. Similar results were obtained in lung adenocarcinoma A549cells, but not in H1299cells.3) In presence of both P4and PP1, breast cancer MB231Br and lung adenocarcinoma A549cells, but not MB231nor H1299cells, displayed inhibited invasion of three-dimensional matrices.4) In presence of both P4and PP1, breast cancer MB231Br and lung adenocarcinoma A549cells, but not MB231nor H1299cells, displayed dephosphorylation of focal adhesion kinase (FAK) in Src/FAK pathway, reduced expressions of MMP-9and KCNMA1. Conclusion:1) We firstly demonstrated that mPRa functioned as an essential mediator for P4and/or PP1induced inhibitory effects on cell migration and invasion of breast cancer MB231Br cells.2) mPRa may also mediate P4and/or PP1's inhibitory effects on cell migration and invasion of lung adenocarcinoma A549cells.3) The induced inhibitory effect of P4and/or PP1was realized dephosphorylation of FAK, down-regulation of MMP-9and KCNMA1expressions.Chapter3:mPRa and its relationship with clinical pathological features in breast cancer tissue microarraysMethods:Immunohistochemistry was performed to detect the expression levels of mPRa in breast cancer tissue microarrays and its relationship with ER, PR and HER-2expression as well as pathological features such as age, grade, TNM, lymph node metastasis.Results:1) The average intensity of mPRa expression, but not percentage of breast cancer with high level of mPRa expression (mPRa-HiEx), was significantly lower in the TNM stage4patients compared to those with TNM1-3patients.2) Both average intensities of mPRa expression and mPRa-HiEx rates were significantly higher in cancers negative for ER, as compared with those cancers with ER+. However, after adjusting for age at diagnosis and/or TNM stage, only average intensities of mPRa expression were associated with ER status.3) Both mPRa-HiEx rate and average intensity of mPRa expression were significantly higher in HER2+subtype cancers (i.e. HER2+ER-PR-) as compared to ER+subtype cancers.Conclusion:mPRα expression was associated with low TNM stage and ER expression, and higher in HER-2+subtype breast cancer. mPRα may serve as a potential biomarker for breast cancer following ER, PR and HER-2.Overall conclusions1) We firstly demonstrated that mPRα was expressed at a moderate level in a brain metastatic breast cancer MB231Br cells derived from mPRα negative MB231cells. We firstly detected the expression of mPRα in lung adenocarcinoma cell lines, in which A549were positive while H1299negative. We firstly confirmed the specific membrane binding of P4and mPRα on breast cancer MB231Br cells and lung adenocarcinoma A549cells; mPRα may mediate effects of P4and exert influence on biological characters breast cancer and lung adenocarcinoma cells.2) We firstly proposed that mPRα functioned as an essential mediator for P4and/or PP1induced inhibitory effects on cell migration and invasion of breast cancer MB231Br and lung adenocarcinoma A549cells. This inhibitory effect is independent of nPR and may be realized by induced dephosphorylation of FAK, down-regulation of MMP-9and KCNMA1expressions.3) mPRα expression was associated with low TNM stage and ER expression, and higher in HER-2+subtype breast cancer. mPRα may serve as a potential biomarker for breast cancer following ER, PR and HER-2.