Inhibitory Effects Of Metformin As An Antipyretic-detoxicate Drug On Prostate Cancer Cell Migration And The Underlying Mechanisms

I.BackgroundAccording to the report in JAMA Oncology,prostate cancer(PCa)has been the main reason for cancer incidence worldwide for men.In China,there were 26.6thousand deaths and 60.3 thousand incident cancer cases in 2015.Meanwhile,both incidence rate and death rate are rising.The 5-years relative survival rate of prostate cancer is almost 100% for local and region stages,but sharply drops to 28% for distant stage prostate cancer.A medicine ancient book,“Su Wen” record Shen(a term like kidney in traditional Chinese medicine)governs reproduction and “Shen supports bone”.So bone will also suffer from the harm transferred from Shen when Shen feeds bone.The most common site of PCa metastasis is the bone with skeletal metastases identified at autopsy in up to 91.1% of patients dying from PCa.Currently,when prostate cancer has spread to bone or other organs,it is incurable and causes significant mortality.It remains critical to find effective approach on inhibiting prostate cancer cell migration and invasion to prevent its spreading to other organs.Galegine is the main active component of both Biebersteinia heterostemon Maxim.and Galega officinalis L..However,it could not be used as a clinical drug due to its significant toxic effects for animals,including human.According to the molecular structure of galegine,the biguanide metformin was synthesized.Metformin(1,1-dimethylbiguanide)as the first-line oral medication for the treatment of type-2diabetes is cheap and wildly used,which shows the heat-clearing effect of metformin as an antipyretic-detoxicate drug.Metformin can have a heat-clearing and detoxifying effect like Biebersteinia heterostemon Maxim.Indeed,multiple epidemiological studies and accumulated evidences have shown that metformin may reduce cancer risk and improve cancer prognosis and survival,including prostate cancer.Our previous work indicated that metformin inhibits salivary adenocarcinoma grow in vitro through cell cycle arrest and apoptosis,and targets c-MYC oncogene to prevent prostate cancer growth in vitro and in vivo.In addition,metformin significantly inhibited breast and melanoma cancer cell migration that is a critical step to cancer metastasis,which supports that metformin may inhibit prostate cancer metastasis and progression in addition to its regulation on cancer cell proliferation and apoptosis.It has been reported that metformin may inhibit prostate cancer growth via repressing mTOR and androgen receptor signaling pathways.However,much remains to be understood about metformin's anticancer properties and the direct molecular mechanism by which metformin inhibits PCa cell migration remains unclear.II.ObjectiveThe aim of this study was to investigate the effect of metformin to prostate cancer cells and uncover its direct molecular mechanism,so that found the potential target for inhibiting prostate cancer metastasis.Further support the application of metformin alone or in combination in prostate cancer therapy as an antipyretic-detoxicate drug.III.Methods1.Wound healing assay was used to check the effects of metformin to cell migration of two metastatic prostate cancer cellsTwo metastatic prostate cancer cell lines,an osteolytic(PC-3)and an osteoblastic(C4-2B)metastatic cell line,were treated with metformin for 24 h and 48 h,separately.Wound healing assays were performed to measure the cell migration.2.SUV39H1 gene expression in prostate cancer cells under regulation of metformin was assessed2.1 Real-time PCR was used to measure SUV39H1 mRNA levels in PC-3 and C4-2B cells treated with metformin.2.2 With actinomycin D,we checked whether SUV39H1 mRNA reduction by metformin was caused through inhibiting transcription of SUV39H1 or m RNA stability.2.3 With cycloheximide(CHX),we checked whether metformin regulated SUV39H1 transcription directly or indirectly.2.4 Western blotting assay was used to measure SUV39H1 protein levels in prostate cancer cells treated with metformin for 24,48 and 72 h.3.Made stable cell lines over-expressing SUV39H1 or SUV39H1?SET to investigate the effects of SUV39H1 over-expression to cell migration of prostate cancer cell line C4-2B3.1 Generated stable cell lines over-expressing SUV39H1 or SUV39H1?SET.3.2 Measured SUV39H1 protein levels in over-expression cell lines treated with metformin,with Western blotting assays and immunofluorescence assays.3.3 Wound healing assay was performed to measure the cell migration of SUV39H1 or SUV39H1?SET over-expressing cell lines.3.4 Measure the cell proliferation of SUV39H1 or SUV39H1?SET over-expressing cell lines with crystal violet staining.4.The determination SUV39H1 and H3K9me2/3 levels in tissue microarrayImmunohistochemistry(IHC)was used to measure SUV39H1 and H3K9me2/3levels in normal prostate tissues and prostate cancer tissues.5.Using both loss-of-function and rescue systems,we investigated the role of SUV39H1 in prostate cancer cell migration5.1 SUV39H1 knockout(KO)in PC-3 cell with CRISPR-Cas9.5.2 Check the effects of SUV39H1 KO to cell migration of PC-3 with wound healing assay and transwell assay.5.3 Cell proliferation of SUV39H1 KO cells was assessed with crystal violet staining.5.4 Check the inhibitory effects of metformin to cell migration of PC-3 WT and KO cells with wound healing assays.5.5 Re-expressed SUV39H1 or SUV39H1?SET in KO cells via stable transfection.5.6 Wound healing assay was used to measure the cell migration of SUV39H1 or SUV39H1?SET re-expressing cells.6.RNA sequencing was used to analyze gene differential expression and pathway impact in SUV39H1 KO cells6.1 Total RNA extracted from PC-3 WT and KO cells,was used for RNA sequencing.6.2 Gene differential expression and pathway impact analysis were performed with RNA sequencing data.6.3 Confirm the change of integrin ?V and ?1 levels between PC-3 WT and KO cells with Western blotting assay.7.The determination of FAK and p-FAK(Y397)levelsWestern blotting assays were performed to measure the levels of FAK and p-FAK(Y397)in KO,re-expressing and metformin-treated cells.8.The mining of inhibitory transcription factorsUse RNA sequencing data and Transcription Factor ChIP-seq data from ENCODE to mine inhibitory transcription factors with R,as well as function analysis.IV.Results1.The impacts of metformin on prostate cancer cell migration and SUV39H1expression1.1 Metformin inhibited the cell migration of prostate cancer cell.With wound healing assay,we found the cell migration of both PC-3 and C4-2B were inhibited by metformin,by 26% in PC-3 cells and 59% in C4-2B cells,separately.1.2 Metformin inhibited SUV39H1 expression in prostate cancer cells.Real-time PCR data showed that SUV39H1 mRNA and protein levels were both decreased in PC-3 and C4-2B cells treated with metformin for 48 h.With actinomycin D and CHX,we found that metformin caused SUV39H1 mRNA decrease through inhibiting its transcription indirectly.2.The effects of SUV39H1 over-expression on prostate cancer cell migration and relevance in prostate cancer development and metastasis2.1 SUV39H1 over-expression promoted C4-2B cell migration.Using C4-2B cells,we made SUV39H1 or SUV39H1?SET over-expressing cell lines through stable transfection.And we found SUV39H1 over-expression promoted C4-2B cell migration,but SUV39H1?SET over-expression did not.However,SUV39H1 and SUV39H1?SET over-expression did not affect the proliferation of C4-2B cell.2.2 SUV39H1 expression showed a clinical relevance in prostate cancer development and metastasis.IHC staining in prostate cancer tissue microarrays showed that levels of SUV39H1 and H3K9me2/3 were positively correlated with the progression of prostate cancer stages in patients.Especially in metastatic prostate cancer samples,the levels of SUV39H1 and H3K9me2/3 were both elevatedsignificantly and dramatically.3.The effects of SUV39H1 KO and re-expression on prostate cancer cell migration3.1 SUV39H1 KO decreased PC-3 cell migration.Using CRISPR-Cas9 system,we knocked out SUV39H1 from PC-3 cell and obtained two individual SUV39H1-KO cell lines.With wound healing assays,we found that SUV39H1 KO decreased the cell migration of PC-3 cells,but did not affect their cell proliferation.In addition,the wound healing assays measuring the cell migration of PC-3 WT and KO cells treated with metformin showed that the inhibitory effect of metformin to KO cell migration was not significant.3.2 SUV39H1 re-expression rescued the cell migration of KO cells.Via stable transfection,SUV39H1 and SUV39H1?SET were re-expressed in KO cells,separately.According to the results of transwell assays,we found SUV39H1re-expression rescued KO cell migration,but SUV39H1?SET re-expression could not.4.The molecular mechanism of SUV39H1 regulated prostate cancer cell migration4.1 SUV39H1 regulated integrin-FAK signaling.According to gene differential expression and pathway impact analysis,we found that cell adhesion molecules and ECM-receptor interaction related to cell mobility,were both in the top 10 impacted pathways.Among the genes in these two pathways,ITGAV and ITGB1 were down regulated significantly in KO cells.It was confirmed that their protein levels were also decreased in KO cells.And integrin signaling activated FAK auto-phosphorylation(p-FAK)at tyrosine 397(Y397)was decreased in KO and metformin treated cells.Notably,p-FAK levels were also closely associated with SUV39H1 levels in SUV39H1 or SUV39H1?SET re-expressing cells.4.2 Inhibitory transcription factors mediated SUV39H1's regulating ITGAV and ITGB1 expression.We obtained 6 inhibitory transcription factors that may mediate SUV39H1's regulating ITGAV and ITGB1 expression,through bioinformatic analysis and function analysis.V.ConclusionOur data revealed a molecular mechanism by which SUV39H1 modulates prostate cancer cell migration and mobility and demonstrated that the antipyretic-detoxicate drug metformin reduced SUV39H1 to inhibit migration of PCa cells via disturbing the integrin-FAK signaling.Our study suggests SUV39H1 as a novel target to inhibit PCa cell migration and invasion.