Study On Epigenetic Mechanism Of Androgen Receptor In Prostate Cancer Stem Cells

Prostate cancer is the most commonly diagnosed malignancy accounting for 9%of male cancers of western developed country and is projected to be the second frequent cause of mal cancer-related death.Conventional chemotherapy therapies and/or androgen deprivation therapy remains the standard therapeutic option for advanced PCa.Although initially effective at blocking tumor growth,ADT eventually fails,leading to a state of hormone-refractory PCa and wide-spread metastasis.Progressive prostate cancer is always a difficult issue in the urological clinical treatment.Also the molecular mechanism of the initiation and progression of androgen independent prostate cancer is the hot topic in this area.The androgen receptor plays an important role in the development and progression of PCa.Prostate cancer stem cell theory postulates that a very small population of cells exist prostate cancer tissue,which has been recognized as having self-renewal and differentiation capabilities and,hence,is referred to as cancer stem cell.According to this hypothesis,prostate cancer stem cell is responsible for the occurrence of distant metastases and for tumor recurrence after ADT treatment.In our previous studies,cancer stem cell population was observed increased expressions after castration.In recent years,cancer epigenetics regulation becomes another hot area of research.Regulation of DNA methylation in transcription region CG can affect the tumor-associated gene expression.Some papers report that AR promotor methylated affect AR gene expression.We found that AR no/low expression in prostate cancer stem cell due to many CpG island methylation status in AR promotor.So we believe that epigenetic regulated prostate cancer stem cells to silencing AR expression effect PCa progression.Therefore,we try to isolate the populations of prostate cancer stem cells and identify their biological characteristics.Explore the AR role in prostate cancer stem cells and the process of their epigenetic regulation;clarify the AR in the maintenance of epigenetic regulation of prostate cancer stem cell properties,and the molecular mechanism concerning the progressive prostate cancer.Our goal is to provide experimental evidence to support the targeted therapy for prostate cancer stem cells.Methods:This study is divided to 3 parts:1.The aim of this study is to isolate the populations of cells with different cell surface markers in androgen dependent prostate cancer LNCap cell line:?Using CD133 as special marker sorted stem cell populations from LNCaP by FACs and MACs.? Using PCR and Immunohistological to investigate which population of cells have the stem cell property,to identify the tumor stem cells and their different gene expression profile from LNCaP S/P and non-S/P population.?Test prostate cancer stem cells renewal ability by Matrigel sphere formation assay.To study prostate cancer stem cell tumorrigenic in vitro by softagar assay.Construct mouse prostate orthotopic models by cancer stem cells and non S/P population,compare two different tumor formation ability in these two populations.To confirm the sorted cells have self-renewal?multilineage differentiation and infinite proliferation ability.2.Investigation of AR role in LNCaP stem cell line:? To test AR mRNA and protein expression level in LNCaP sorted stem cells by Q-PCR and Western-blot.?Overexpression AR in cancer stem cells though Lentiviral system.Analysis overexprssion AR effect cancer stem cell proliferation?differentiation and tumorgenetic ability by MTT?Soft-agar and invivo experiments.3.Study how DNA methylatedion inhibitor(5-Aza)regulates AR expression in LNCaP stem cell populations:?To analysis AR promotor methylated status in LNCaP stem cells by specific methylation PCR.?To further study on the epigenetic machanism of AR in LNCaP stem cells.Analysis stem or differentiation marks such as CK5?CK8 and AR exprsssion in LNCaP S/P cell line upon difference dose 5-Aza treatment by immunohistological.? To test AR affect self-renewal and tumorgenetic ability of stem cells by Matrigel sphere-formation assay and soft-agar assay.Result:1.Using magnetic activated cell sorting and flow cytometry system isolated cancer stem cell from LNCaP.? We found that integrina2?1+ and CD133+V expression rates were 0.7%?1.2%;89.2%?93.7%;CD44 negative expression.So we choose CD 133 as a stem cell sorting marker.?The clone formation ability in CD133+ cells in LNCaP cell line is higher than that of CD133-population cells.(P<0.01);?Stem marker such as Integrina2?1?CD133?CK5 expression higher than CD 133-cells,meanwhile,differentiation marker CK8.AR?PSA expression higher than CD133+ cells.?Nude mice experiments showed that:tumor formation ability of LNCaP S/P in nude mice(100%rate)was significantly higher than CD133-group(60%).There is significant difference between the two groups(P<0.05)2.Investigation of AR role in LNCaP stem cell line:?Q-PCR and Western-blot results showed that in LNCaP stem/progenitor cells,AR expression was reduced;?the overexpressing AR of LNCaP S/P cells show that the stem cell marker CD133,integrin and basal cell characteristic markers CK5 expression was significantly decreased;cell differentiation specific markers CK8 and AR expression was increased.?The results showed that overexpression AR significantly inhibited LNCaP S/P cell proliferation and colony formation.?Our data confirmed overexpression AR can inhibit LNCaP S/P cells tumor formation capacity,and promote their differentiation in vivo.3.?Results show that ARmRNA and protein expression significantly increase upon treatment 5?M-5Aza.The IC50 of 5-Aza treating LNCaP Stem cells is 8.5?M.?Use immunofluorescence staining LNCaP S/P cells to test different dose 5-Aza effect on LNCaP S/P cell.After treatment 5-Aza 5 days,we can observe that differentiate marker AR and CK8 expression increase,at the same time stem cell marker CK5 expression decrease.5-Aza induce AR expression increase,AR is key gene push stem cell differentiate.? 5-Aza inhibit LNCaP S/P self-renewal and tumorgenetic ability and have dose-dependent.Conclusion:1.We have successfully isolated cancer stem cell populations in human hormone-dependent LNCaP prostate cancer cell lines by magnetic activated cell sorting system and flow cytometry sorting system;identified CD133 as a cancer stem cell surface marker;CD133+populations has a strong self-renewal capacity and in vivo tumor formation ability of cancer stem cell characteristics.VI2.Androgen receptor mRNA level and protein level expression both very low in LNCaP stem cell population;AR inhibited cell proliferation/self-renewal ability and tumor formation in vivo of LNCaP stem cells.3.AR expression is very low due to the epigenetic modification.After the DNA methylation inhibitor(5-Aza)treatment,make AR promotor is demethylated,lead to AR expression increase and push LNCaP stem cells go to differentiate.This study provides importatnt evidences for AR epigenetic modification of prostate cancer stem cells maintaining the biological characteristics,and prostate cancer stem cells can serve as targets for the initiation of prostate tumorigenesis and development.