The Role Of BKCa In Prostate Cancer Growth And Metastasis And Its Molecular Mechanism

Prostate cancer is the second most frequently diagnosed cancer in men worldwide, with 1.1 million new casesestimated to have occurred in 2012. It is the most frequently diagnosed cancer among men in highly developed countries. New cases of prostate cancer ranked the first in more developed countries, and the fourth in developing countries, among all diagnosed cancer cases in man. The occurrence of prostate cancer is increasing because of the aging of the population and shifting of lifestyle associated with China’s economic development. Although prostate cancer mortality is currently declining along with the development of comprehensive diagnosis and therapy, including screening, early diagnosis, surgery, radiotherapy, chemotherapy, endocrine and immune therapy, there is still a considerable number of patients who suffer from relapse and metastasis. Therefore, it is constantly needed to explore the molecular mechanisms of prostate cancer recurrence and metastasis, which may help to identify potential targets for intervention. Recent studies suggest that ion channels play an important role in oncogenesis and progression.About 13% of currently known drugs whose primary therapeutic targets are ion channels are being used for the treatment of a variety of human conditions, including cardiovascular and neurological disorders. Therefore, it is promising to explore the therapeutic and prognostic values of targeting ion channels in certain cancer.BKCa is a large conductance calcium activated potassium channel ubiquitously expressed in a variety of tissues and organs, such as cardiac and vascular smooth muscle and nervous system. BKCa can modulate vasomotor and nerve excitability and other physiological functions mainly by regulating membrane potential and calcium signaling in excitable cells. Recent studies demonstrate that BKCa is overexpressed in various tumors and is involved in many malignant behaviors of cancer cell, including proliferation, apoptosis, migration and invasion. The BKCa coding genes is amplifed in nearly 16% of advanced prostate cancer, while no amplification is detected in the early stage samples. BKCa also promotes the proliferation of prostate cancer cell, but the molecular mechanisms is not fully elucidated. In addition, it is unknown whether BKCa participate in the metastasis of prostate cancer. Moreover, whether BKCa can promote prostate cancer progression in vivo remains to be explored. In view of this, we detected the expression of BKCa in prostate cancer cell lines and clinical samples. We also investigated the role and molecular mechanism of BKCa in regulating prostate cancer cell proliferation, migration, invasion, tumorigenicity and metastasis through in vitro experiments and tumor-bearing nude mice.Part 1. Expression of BKCa in prostate cancer cells and tissuesObjective: To compare the difference of BKCa expression in prostate cancer cells and tissues with that in normal cells and adjacent benign tissues and to investigate the correlation between BKCa expression and the clinical stage and Ki67 expression of prostate cancer.Methods: Real-time PCR, western blot and immunofluorescence were performed to detect the expression of BKCa in prostate cancer PC3 and C4-2 cells and normal prostate epithelial RWPE-1 cells. Real-time PCR, western blot and immunohistochemistry were performed to detect the expression of BKCa in prostate cancerous and adjacent benign tissues. Immunohistochemistry was performed to analyze the correlation between BKCa expression and clinical stage and Ki67 expression in prostate cancer.Results: The expression of BKCa in prostate cancer cells and tissues was significantly higher compared with their non-cancerous counterparts both in m RNA and protein levels. BKCa expression was positively correlated with Ki67 expfession in prostate cancer tissues(r = 0.461, p = 0.0032). The expression of BKCa was significantly higher in stage IV prostate cancers than that in stage I-III samples.Conclusion: The expression of BKCa was significantly higher in cancerous prostate cells and tissues than its expression in non-cancerous prostate epithelial cells and adjacent benign tissues. BKCa expression was positively associated with Ki67 expression and the clinical stage of prostate cancer.Part 2. BKCa regulates the growth and metastasis of prostate cancer cellsObjective: To investigate the effects of BKCa regulation in prostate cancer growth and metastasis.Methods: The plasmid-mediated BKCa upregulating system and the lentivirus-mediated BKCa silencing system were used to modulate BKCa expression in prostate cancer PC3 and C4-2cells. MTT, Ed U, colony formation, scratches wound healing and transwell assays were performed to detect the effects of BKCa modulation on cell proliferation, migration and invasion. Prostate cancer PC3 cells were injected subcutaniously and intravenously into nude mice to explore the effects of BKCa downregulation on prostate cancer growth and lung metastasis. The expression of Ki67 in PC3 cell xenografts was also examined by immunohistochemistry.Results: BKCa upregulation resulted in enhanced cell viability, DNA synthesis and colony formation efficiency in prostate cells. The scratch wound healing rate and the numbers of migrated and invaded cells were also increased. When BKCa was downregulated, the above mentioned biological behaviors of prostate cancer cells were significantly inhibited. In vivo studies suggested that BKCa surppression significantly reduced the tumor volume,quality and Ki67 expression of PC3 cells xenografts. Morever, the number of lung metastasis form intravenously injected PC3 cells was also significantly reduced by BKCa downregulation.Conclusion: BKCa upregulation promotes prostate cancer cell proliferation, migration and invasion. BKCa downregulation inhibits prostate cancer cell growth and metastasis both in-vitro and in-vivo.Part 3.The molecular mechanismof BKCa in regulating prostate cancer cell growth and metastasisObjective: To investigate the molecular mechanism of BKCa in regulating prostate cancer lell growth and metastasis.Methods:BKCa channel activity were detect in PC3 cells in the presence or absense of its specific agonist NS1619 and inhibitor IBTX through patch-clamp assaay. The effects of NS1619 and IBTX on PC3 cell proliferation and migration were also examined.Laser confocal immunofluorescence and co-immunoprecipitation were performed to explore the detect co-localization and interaction between BKCa, FAK and integrin αvβ3.The impact of BKCa modulation on the expression of integrin αvβ3, FAK and p-FAK(Tyr397) was also detected by westernblotting. Cell proliferation and migration were examined in the presence or absense of LM609(an integrin αvβ3 blocking antibody) and Y15(a specific FAK inhibitor) in BKCa upregulated of PC3 cells to determine the role of integrin αvβ3 / FAK signaling pathway in BKCa-promoted prostate cancer cell growth and metastasis. Immunofluorescence was performed to determinethe correlation between BKCa and p-FAK(Tyr397) expression in prostate cancer clinical specimens.The results:Thepotassium-conducting function of BKCa channelscontributed slightly to proste cancer PC3 cell proliferation and migration, but it is not the main mechanism. BKCa co-localize with both integrin αvβ3 and FAK in the membrane of PC3 cells. They can be reciprocally immunoprecipitated by each other. Upregulation of BKCa enhanced the interaction between integrin αvβ3 and FAK in PC3 cells, and resulted in increased expression of p-FAK(Tyr397) which could be abrogated by LM609. Downregulation of BKCa showed the opposite effects. However, the expression of integrin αvβ3 and FAK was not changed in both cases. When PC3 cells were treated with LM609 or Y15, BKCa-promoted cell proliferation and migration were significantly abrogated. Furthermore, the expression of BKCa was positively correlated with p-FAK(Tyr397)in clinical prostate cancer specimens(r=0.517, p=0.0008).sConclusion: BKCa promotes prostate cancer cell growth and metastasis by forming a functional complex with FAK and integrin αvβ3 and facillitating thesignal coupling between these two molecules.