Androgen/androgen Receptor Axis Effect On Cancer Cells Stemness Through Regulating Pluripotency Factor Nanog

Androgen/androgen receptor axis is a widely scattered pathway in human cells. The conventional role of the axis is promoting male genital development and maintaining sexuality, as well as it has been identified to take part in cell metabolism and immuneregulation. Also, more and more researches demonstrated it could regulate many cell physiological activities, like proliferation and apoptosis. But, because of the widely regulation effect, which has been considered as a double-edged sword, the axis also had been reported to involved in cancer development.In the early stage of cancer research, because the androgen signal axis itself is closely related to the physiological regulation of the male, a large number of studies had focused on the male reproductive system tumors, such as prostate cancer. But with the deepening researches, more and more hormone related tumors were found to be closely related to the androgen signaling axis. Among these, as a female reproductive system malignant tumor, ovarian cancer was seldom considered associated with androgen. But as an important sex hormone synthesis and secretion organ, ovarian tissue indeed concludes a large number of estrogen, progesterone, as well as high levels of androgen. In recent years, the relationship between ovarian cancer and androgen/androgen receptor axis has been constantly explored. On the other hand, there is another special tumor, hepatocellular carcinoma(HCC), which the occurrence rate gets obvious gender bias around the world. The epidemiological investigation showed, after excluding extrinsic factors like environment and habits, the incidence of male also significantly higher than female. Although liver is an important metabolism organ of the human body that is not directly involved in sex hormones synthesis, it is an important organ for sex hormone metabolism, and the androgen level in it is higher than other non-gonad organs.At present, the 5 year survival rate of ovarian cancer patients is only 40%, which mortality rate is highest in gynecological tumors. This poor prognosis closely related to drug resistance and recurrence of ovarian cancer cells, but the exactly reasons and mechanisms are still not fully understood. At the same time, HCC is also one of the most fatal malignant tumors, although recently years many therapeutic measures had been taken, its 5 year survival rate is still low. So, it gets important theory and application value to study the influencing factors and mechanisms in the ovarian cancer and liver cancer occurrence and development. And in this study, according to the relationship between these two cancers and androgen, it makes us interested in the common role of androgen/ androgen receptor axis in the ovarian and liver cancer processes.In recent years, with the understanding of tumor biological behavior, a large number of studies have confirmed that cancer stem cells(CSCs) played an important role in the tumor development. CSCs get the capacities of continuous self-renewal and effectively resist external damage, which make them to become the key initiating factors in tumorigenesis and drug resistance. Therefore, it is one of the key parts in anti-tumor therapy to study the mechanism of cancer cells stemness regulation.Considering the association of androgen signaling axis with ovarian and liver cancer, as well as the CSCs role in tumor development, we wondered if the androgen/androgen receptor axis is involved in the regulation of ovarian and liver cancer cells stemness. Our previous study had confirmed the pleuripotency factor Nanog can be a reliable cancer stem cell marker, and played an important role as a "switch" in the regulation of CSCs. On the other hand, it has reported that androgen receptor can promote the Nanog expression. So, with the relevant theoretical basis, in this study, we went to identify the effect of androgen/androgen receptor axis on cancer cells stemness, as well as the behind mechanisms.The main methods and results of this paper are as follows:1. Androgen receptor(AR) was highly expressed in ovarian and HCC tissue, and its expression was associated with the Nanog.(1) The AR expression was significantly higher in ovarian cancer and HCC tissue than the normal tissues. Through analysising the ovarian cancer and HCC clinical tissue specimens, the immunohistochemical staining and Western blot detection confirmed: AR expression in ovarian cancer and HCC tissues were significantly higher than the corresponding normal tissues.(2) The expression of AR and Nanog got correlation in both tumor tissues. Then through Western blot, we detected the expression of Nanog, which expression was also higher in tumor tissues than normal tissue. And the correlation between AR and Nanog expression in tumor tissue was confirmed by Pearson correlation analysis.2. Constructed endogenous Nanog gene labeled cells model by CRISPR/Cas9 system, and confirmed that AR and Nanog expression in the cell are consistent and co-localization.(1) Constructed the endogenous Naong gene labeled cell model by CRISPR/Cas9 system. First of all, through a series of molecular cloning processes, we constructed the Nanog Gene CRISPR/Cas9+gRNA targeting vector; at the same time, by PCR, restriction enzyme digestion and Gibson clone method, we constructed the Nanog-2A-GFP recombined homologous arm plasmid. Through transfecting ovarian cancer and HCC cells with the two plasmid simultaneously, and utilized the homologous recombination repair mechanism in cell itself, we recombined the green fluorescent protein(GFP) encoding sequence into the coding region of Nanog. Then, by flow cytometry sorting GFP positive single cell and clone cultured, we got some Nanog labeled cells trains. And, by enzyme digesting and sequencing, we identified the corrected labeled cells to followed studies.(2). Confirmed different biological function between Nanog labeled GFP(+) and(-) cells. First, by flow cytometry sorting the cell strain into GFP(+) and(-) cells, RT-PCR and Western blot assays confirmed that in GFP(+) cells, Nanog gene expression were significantly higher than that of GFP(-) cells at both m RNA and protein level; secondly, through the cell sphere and clone formation assay, it confirmed the GFP(+) cells got higher sphere and clone formation abilities than GFP(-) ones; finally, the subcutaneously transplanted tumor model of mice further confirmed that GFP(+) cells got higher tumorigenesis ability than the GFP(-) cells.(3) The expressions of AR and Nanog were consistent in the monoclonal cell strains, and they were co-localization in the cells. RT-PCR and Western blot analysis confirmed the AR and Nanog expression at the transcription and protein level has consistency in cells, as in GFP(+) cells them were higher than that of GFP(-) cells; then, immunofluorescence identified the AR and Nanog got co-localizationin GFP(+) cells, which were not detected in GFP(-) cells.3. Revealed Androgen/AR axis could promote cancer cells stemness through increasing the expression of Nanog by binding to and activing its promoter.(1). Confirmed androgen/AR axis could regulate the expression of Nanog gene. Using androgen(DHT) stimulation or androgen receptor degradation enhancer agent(ASC-J9) inhibits androgen signaling activity, we detected the AR and Nanog expression by RT-PCR and Western blot. The result confirmed that in DHT treated groups, both AR and Nanog expression of GFP(+) and(-) cells were increased significantly at m RNA and protein levels, and this effect can be inhibited by ASC-J9.(2). Confirmed that the androgen/AR axis can regulate the tumor cell stemness. By DHT treated with or without ASC-J9 on both GFP(+) and(-) cells, it confirmed the activation of DHT treatment can obviously enhance cell colony and sphere formation, and inhibition of the signal by ASC-J9 reversed the DHT effect.(3). Confirmed the Androgen/ AR axis effect on cells stemness is mediated by Nanog. By using lentivirus vector, we over-expressed or knocked down Nanog expression in target monoclonal cell lines. Then, treated Nanog knock-down cell lines by DHT or Nanog over expressing cells by ASC-J9, respectively, and detected their sphere and clone format abilities. The results showed that DHT could no longer increase the cells stemness, nor the ASC-J9 could down-regulated the Nanog over-expression cells stemness obviously.(4). Proved that AR could directly bind to the Nanog promoter region, activated its promoter and increased its expression. Firstly, through sequence analysis, we found the Nanog promoter region exactly existed AR specific binding motifs. Then by Chromatin Immunoprecipitation(Ch IP), we confirmed the specific bandings between AR and Nanog promoter regions. Followed, By constructing Nanog promoter different regions luciferase reporter lentiviral vector and infecting corresponding cell strains, we got Nanog promoter activity report cell line system. Then treated the cells by DHT with or without ASC-J9 and detected luciferase activity, we proved that the DHT could enhance Nanog promoter activity and ASC-J9 could reverse this effect.4. Confirmed the androgen/AR axis could promote the tumor growth in vivo.(1) Constructed the castrated nude mouse model. By removing the testes of nude mice to construct castrated nude mice model, then the castrated mice were divided into castrated group and added androgen treatment group.(2) Established the subcutaneous transplantation tumor model in each group. Tumor volumes in castration group were significantly smaller than the other two groups.(3) Confirmed that androgen can promote Nanog expression, and AR and Nanog were co-localization in transplantation tumor tissues. The transplanted tumor serial sections immunohistochemistry staining confirmed that the Nanog and AR expression were significantly higher in untreated or castrated added androgen treatment groups than the only castrated ones, and Nanog was co-localization with AR in these tissues.In summary, the main conclusions of this study can be summarized as follows:1. AR was highly expressed in ovarian cancer and HCC tissues and cells, and its expression got coincidence with pleuripotency gene Nanog, also their expressions got association.2. Androgen signaling axis could activate Nanog promoter through AR binding to it directly, up-regulated Nanog expression and promoted the cancer cells stemness. And the axis effect on cancer cells stemness is Nanog dependent.3. Androgen signaling axis could promote tumorigenesis and cancer cell growth in vivo.Above all, this study investigated the relationship between androgen/AR axis and ovarian cancer or HCC, two hormone high associated tumors, and confirmed the androgen/AR axis could promote the cancer cells stemness. By investigating the mechanism under it, we revealed this effect is Nanog dependent, and the axis could directly regulate Nanog expression. On the one hand, this study enriched our knowledge of the androgen/AR axis effect on cancer process, and revealed relevant mechanisms at the cancer stemness level; on the other hand, it may provide some new strategies for the ovarian cancer and HCC therapies.