| Research BackgroundRenal cell carcinoma (RCC) is one of the most lethal types of urological cancer. Recent studies have markedly increased the understanding of the cell molecular biology of RCC, dominated by the inactivation of VHL in ubiquitin-mediated proteolysis pathway (UMPP) and alteration of PBRM1 involved in chromatin regulation. The increased understanding of RCC biological pathways has led to the development of molecularly targeted therapeutic agents that have improved patient outcomes. However, the advanced and metastatic RCC (mRCC) remains incurable, therefore further studies are highly needed to understand the mechanisms of the molecular basis of resistance and response, thus leading to the discovery of novel targets for the treatment of mRCC.In addition to UMPP3, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway has also been identified as an important pathway in RCC. The PI3K/AKT pathway begins with the involvement of growth factors binding to the receptor tyrosine kinases. PI3K is activated through attachment to receptors anchored on plasma membrane and generates phosphatidylinositol-3-phosphate (PIP3) by phosphorylating phosphatidylinositol 4,5-bisphosphate. Through a pleckstrin homology domain, AKT binds to PIP3 and is phosphorylated to pAKT. Class IA PI3Ks are heterodimers that consist of a catalytic subunit (p110α, p110β and p110δ) and a regulatory subunit (p85α, p55α, p50α, p85β, and p55γ). The catalytic subunit p110α is encoded by PIK3CA, while the regulatory subunit p85a is encoded by PIK3R1. The PI3K/AKT pathway regulates many aspects of cellular functions, including cell growth, proliferation, translation and survival, and is also involved in pathological conditions. Deregulation of the pathway can lead to disturbance of cell growth, proliferation and survival, thus resulting in growth advantage and metastatic competence of cancer cells.The alteration of PI3K/AKT pathway has been found in a broad spectrum of cancers. Members of the pathway such as PIK3CA and PTEN are frequently altered in RCC. Since the pathway plays an important role in RCC pathogenesis, it has been showing a great promise for molecularly targeted treatment of RCC. However, only a small number of patients benefit from single-agent PI3K targeted therapy. The related mechanism of unsatisfied effect of PI3K targeted therapy remains to be clarified. Can, in addition to PIK3CA and PTEN, other member of the pathway play a role and be a target of therapy in RCC?Accumulating studies have showed an important role of PIK3R1 in human carcinogenesis. PIK3R1 has been reported as an oncogene in ovarian and colon tumors, whereas it has been shown as a tumor suppressor in hepatocellular carcinomas. The underexpression of PIK3R1 has been reported to be associated with poor prognosis of breast cancers. A missense mutation of PIK3R1 which resulted in decrease of PIK3R1 expression has also been strongly linked with colon cancers. We have reported a nonsense mutation in PIK3R1 in an mRCC, while the mutation was absent in the corresponding primary renal cell carcinoma (pRCC). Therefore, we hypothesize that the downregulation of PIK3R1 may confer renal cancer cells a selective advantage to translocate, colonize and develop as mRCC.We speculate that ectopic expression of PIK3R1 may be associated with progression and metastasis of RCC. To examine our hypothesis, we firstly analyzed the expression of PIK3R1 in RCC including both pRCC and mRCC by immunohistochemistry (IHC) and real-time polymerase chain reaction (RT-PCR). We discovered that the expression of PIK3R1 in RCC negatively correlated with tumor progression and metastasis. In addition, we induced deletion mutations of PIK3R1 in renal cancer cell lines (786-0 and A-704 cell lines) using a CRISPR/Cas9 system to achieve haploid knockout of PIK3R1 which significantly decreased the expression of P85a. The mutated renal cancer cells displayed increased abilities of colony formation, tumor formation, migration, epithelial-mesenchymal transition and oncosphere formation.Chapter one The expression of PIK3R1 in renal cell carcinoma and normal renal tissuesObjective In order to investigate the expression of PIK3R1 in RCC and to examine relation bettween the downregulation of PIK3R1 in RCCs and their progression and metastasis.Methods The protein expression of PIK3R1 in normal kidney (n=13), pRCC (n=13) and mRCC (n=21) was determined by IHC. The mRNA expression of PIK3R1 was then determined by real-time polymerase chain reaction (RT-PCR).Results Normal kidney tissues displayed high level of PIK3R1 expression, whereas the expression of PIK3R1 was decreased in pRCC and was further reduced to a lower level in mRCC. Compared with normal kidney tissue group, the mRNA expression of PIK3R1 was significantly decreased in RCC group (n=18). The epithelial-mesenchymal transition (EMT) is considered to be crucial to tumor progression and metastasis, in which NCAD is the hallmark of EMT. To determine whether the downregulation of PIK3R1 could affect the expression of NCAD, the mRNA expression of NCAD was examined, and data showed that the expression of NCAD had a negative correlation with the mRNA expression of PIK3R1 (Correlation =0.6929, P=0.0014). Additionally, the mRNA expression of PIK3R1 negatively correlated with the T category of tumor.Conclusions Our data showed that PIK3R1 expression was reduced in RCC, especially in advanced and metastatic RCC, and the downregulation of PIK3R1 correlated with advanced or metastatic RCC, indicating that the reduction of PIK3R1 expression may acquired tumorigenicity in RCC, and thus supporting the view that PIK3R1 may function as a potential cancer suppressor, and that the downregulation of PIK3R1 may promote progression and metastasis of RCC.Chapter two Haploid knockout of PIK3R1 by CRISPR/Cas9 promotes tumor proliferationObjective To achieve the PIK3R1 depletion in renal cancer cells and determine the effects of PIK3R1 depletion on the growth and tumor formation capability of the cells.Methods Firstly, we analyzed the expression levels of PIK3R1 in normal renal cell line (HK2) and RCC cell lines (786-0, A-498, A-704, and ACHN) by using RT-PCR. The CRISPR/Cas9 strategy was designed and applied to induce a haploid deletion mutation in order to achieve haploid knockout of PIK3R1. We also confirmed the sequence character of chr5:67576819 in PIK3R1 in 786-0 and A-704 cell lines with Sanger sequencing. We then applied the CRISPR/Cas9 technology to screen 786-0 cells and A-704 cells with haploid deletion mutation of PIK3R1. After Sanger sequencing validation, we obtained muted 786-0 cells (786-mutl and 786-mut2) and A-704 cells (A-704-mutl and A-704-mut2) with haploid deletion mutation. To determine the knockout efficiency in the 786 and A-704 mutated cells, RT-PCR and western blot (WB) were carried out to examine PIK3R1 expression in 786-0 WT cells (786-0), mutated 786-0 cells, A-704 WT cells (A-704), and mutated A-704 cells. To determine the effects of haploid knockout of PIK3R1 on the growth of renal cancer cells, we carried out colony formation assays for the wild type and mutant cells. In order to evaluate the tumor formation capability of the WT and muted cells, they were respectively inoculated subcutaneously into NOD/SCID mice to carry out tumor formation assays.Results 786-0 and A-704 cell lines showed higher expression of PIK3R1 compared with that of HK2 cell line. Therefore, we performed depletion of PIK3R1 in 786-0 and A-704 cell lines. The 786-0 and A-704 cell lines harbored the wild type (WT) sequence of chr5:67576819C in PIK3R1. we obtained muted 786-0 cells (786-mutl and 786-mut2) and A-704 cells (A-704-mutl and A-704-mut2) with haploid deletion mutation. The mRNA expression of PIK3R1 was significantly decreased in 786 and A-704 mutated cells compared with 786-0 and A-704 WT cells. The protein levels of PIK3R1 were also significantly decreased in 786 and A-704 mutated cells respectively (Fig.2b).786 and A-704 mutated cells displayed enhanced colony formation capability under the condition of separated single cells than that of 786-0 and A-704 WT cells. The mutated cells formed bigger tumors than those of the wild type cells in NOD/SCID mice.Conclusions We generated cell lines with the insufficiency of PIK3R1 by CRISPR/Cas9.786 and A-704 mutated cells displayed enhanced colony formation capability under the condition of separated single cells than that of 786-0 and A-704 WT cells. The mutated cells formed bigger tumors than those of the wild type cells in NOD/SCID mice. Haploid knockout of PIK3R1 by CRISPR/Cas9 promotes tumor proliferation.Chapter three Haploid knockout of PIK3R1 promotes renal cancer cells migration and EMT in vitroObjective To determine the effects of haploid knockout of PIK3R1 on the migration and epithelial-mesenchymal transition (EMT) of renal cancer cells.Methods Wound-healing and transwell assays for 786-0,786-mutl,786-mut2, A-704, A-704-mutl, and A-704-mut2 cells were carried out. RT-PCR and WB were used to determine the expression of the EMT related gene such as ECAD, NCAD, VIM, ZEB1, CD44 and POU5F1.Results Eighteen hours after artificial wounding,786-0 and A-704 WT cells demonstrated slight migration, whereas 786 and A-704 mutated cells showed enhanced migration ability and healed more than half of the wound. The 786 and A-704 mutated cells showed higher ability of migration in the transwell assay than that of 786-0 and A-704 WT cells.Additionally,786 and A-704 mutated cells displayed a mesenchymal morphology that was different from the epithelial morphology of 786-0 and A-704 WT cells. Cmpared with 786-0 and A-704 WT cells, in 786 and A-704 mutated cells with the downregulation of PIK3R1, the mRNA expression of ECAD was decreased, whereas the mRNA expression of NCAD, VIM and ZEB1 was increased. These observed changes were also found in the protein levels of ECAD, NCAD, VIM and ZEB1 in 786 and A-704 mutated cells respectively. However, no differences of CD44 and POU5F1 were observed between the wild type and mutant cells.Conclusions Through the activation of the PI3K/AKT pathway which phosphorylates several cytoskeleton-regulating and EMT-activating proteins, the downregulation of PIK3R1 promotes migration and EMT in renal cancer.Chapter four Haploid knockout of PIK3R1 promotes a cancer stem cell phenotypeObjective To evaluate whether the reduction of PIK3R1 influences the cancer stem cell phenotype of renal cancer cells and uncover the stem cell signaling pathway activated by the downregulation of PIK3R1.Methods Cell sphere formation assays of 786-0,786-mutl,786-mut2, A-704, A-704-mutl, and A-704-mut2 cells were performed. CSCs have been identified based on expression of various markers such as CD44, CD133 and CXCR4. By using anti-CD44 antibodies, anti-CD 133 antibodies and anti-CXCR4 antibodies, we analysize the CD44+, CD133+ and CXCR4+ subset of 786 and A-704 mutated and WT cells. The mRNA level of CTNNB1, HES1, GLI1, and NANOG were analyzed in 786-0,786-mutl,786-mut2, A-704, A-704-mutl, and A-704-mut2 cells by RT-PCR.Results After two weeks of cultivation,786 and A-704 mutated cells developed more and bigger spheres than that of 786-0 and A-704 WT cells under the medium supplemented with 20 ng/mL EGF, FGF, N2, and B27. CSCs have been identified based on expression of various markers such as CD44, CD133 and CXCR4. By using anti-CD44 antibodies, anti-CD133 antibodies and anti-CXCR4 antibodies we observed that 786 and A-704 mutated cells comprised more CD44+, CD133+ and CXCR4+ subset, respectively, compared with 786-0 and A-704 WT cells. With the downregulation of PIK3R1 in 786 and A-704 mutated cells, the mRNA expression of CTNNB1 was higher than that in 786-0 and A-704 WT cells. There were no significant differences in the mRNA expression of HES1 and GLI1 between the wild type and mutated cells. Though there were no significant differences in the NANOG mRNA expression between 786-0 WT and 786 mutated cells, significant difference was observed between A-704 WT and A-704 mutated cells.Conclusions In our current study, we observed that the down-expression of PIK3R1 promoted the colony formation, cell sphere and tumor formation of renal cancer cells.Chapter five Haploid knockout of PIK3R1 activates WNT/β-catenin pathway dependent on the phosphorylation of AKTObjective To determine the molecular mechanism underlying p-catenin (CTNNB1) activation mediated by PIK3R1 depletion, and to determine the function of AKT in the mutated cells.Methods The immunoprecipitation with PIK3R1 antibody was carried out by using lysates of 786-0,786-mutl,786-mut2, A-704, A-704-mutl, and A-704-mut2 cells. The depletion of AKT were performed in 786-mutl and A-704-mut2 cells.Results The protein levels of CTNNB1 were also increased in 786 and A-704 mutated cells respectively. The quantity of p110α immunoprecipitated by PIK3R1 was decreased due to knockout of PIK3R1 in 786 and A-704 mutated cells respectively, compared with 786-0 and A-704 WT cells. Additionally, more AKT was phosphorylated in 786 and A-704 mutated cells, compared with 786-0 and A-704 WT cells that had the amount of unchanged AKT. However, there was no significant change in the quantity of ERK and ERK with phosphorylation in 786 and A-704 mutated cells respectively.More GSK3β was phosphorylated in 786 and A-704 mutated cells compared with 786-0 and A-704 WT cells. To determine the function of AKT in the mutated cells, depletion of AKT were performed in 786-mut1 and A-704-mut2 cells. After the depletion of AKT, the expression of pAKT, p-GSK3β and CTNNBlwas decreased in 786-mutl shAKT and A-704-mut2 shAKT cells respectively, compared with 786-mutl shCtrl and A-704-mut2 sh Ctrl cells.Conclusions Our present study demonstrates that, the downregulation of PIK3R1 in RCC activates the WNT/β-catenin pathway dependent on the phosphorylation of AKT.Chapter six Haploid knockout of PIK3R1 promotes a cancer stem cell phenotype through WNT/β-catenin pathwayObjective To determine the function of CTNNB1 in the mutated cells.Methods Firstly, the depletion of CTNNB1 was performed in 786-mut1 and A-704-mut2 cells respectively. Furthermore, by using anti-CD44 antibodies, anti-CD133 antibodies and anti-CXCR4 antibodies, we determined the CD44+, CD133+ and CXCR4+ subset in 786-mut1 shCTNNB1, A-704-mut2 shCTNNB1, 786-mut1 shCtrl, and A-704-mut2 shCtr1 cells. Finally,786-mut1 shCTNNBl, A-704-mut2 shCTNNB1,786-mut1 shCtr1, and A-704-mut2 shCtrl cells were respectively inoculated subcutaneously into NOD/SCID mice to carry out tumor formation assays.Results The mRNA expression of CTNNB1 was decreased in 786-mut1 shCTNNB1 and A-704-mut2 shCTNNB1 cells, compared to 786-mutl and A-704-mut2 cells. The decrease of CTNNB1 protein was also observed in 786-mut1 shCTNNB1 and A-704-mut2 shCTNNB1 cells. After two weeks of cultivation, 786-mut1 shCTNNB1 and A-704-mut2 shCTNNB1 cells developed fewer and smaller spheres than that of 786-mut1 shCtr1 and A-704-mut2 shCtr1 cells. By using anti-CD44 antibodies, anti-CD133 antibodies and anti-CXCR4 antibodies, we observed that 786-mut1 shCTNNB1 and A-704-mut2 shCTNNB1 cells comprised fewer CD44+, CD133+ and CXCR4+ subset, respectively, compared with 786-mut1 shCtrl and A-704-mut2 shCtrl cells.786-mutl shCTNNBl and A-704-mut2 shCTNNBl cells formed smaller tumors than those of 786-mut1 shCtrl and A-704-mut2 shCtrl cells in NOD/SCID mice.Conclusions These results indicated that the haploid knockout of PIK3R1 enhanced the abilities of self-renewal and tumor formation dependent on the activation of the PI3K/AKT/CTNNB1 pathway in renal cancer cells. |
PDF Full Text Request