| Background and ObjectionRenal cell carcinoma is a common malignant neoplasm in the urinary system and the sixth leading cause of cancer deaths in Western countries. It is estimated 63,920 new cases of kidney cancer in 2014 in the United States, with 13860 new deaths due to this disease. Clear cell renal cell carcinoma is the most common type and accounts for 70% of RCC, which originating from the renal proximal tubule. Nearly 20-35% of patients with RCC have evidence of metastases at initial presentation. Renal cell carcinoma limited early warning signs render most patients diagnosed at an advanced state, and its resistance to chemotherapy or radiotherapy makes these patients’ therapies rather thorny. Although clinical management of clear cell renal cell carcinoma has changed in recent years with increased incidental diagnosis and by initiating therapy in localized stages, radical nephrectomy is effective to cure early and local RCCs,30% of patients develop metastatic disease after surgery and 40% of clear cell renal cell carcinoma patients die of cancer progression. Therefore, it is important to find novel approaches that can effectively inhibit clear cell renal cell carcinoma cell growth and metastasis. Targeting therapies have shown promise in achieving these goals.Human chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is a novel family consisting of nine genes, CKLF and CMTM1 to CMTM8. CMTMs, linking classical chemokines and the transmembrane-4 superfamily (TM4SF), play important roles in immune system, male reproductive system and tumor genesis. Among the CMTM family, CKLF and CMTM1-2 share various identical sequences with chemokines, while CMTM3-8 are share various identical sequences with more with TM4SF. Human chemokinelike factor (CKLF)-like MAL and related proteins for vesicle trafficking transmembrane, domain-containing member 5 (CMTM5), a member of the CMTM family, was firstly reported by Human Genomics Center of Peking University in 2003. As a member of CMTM family, the CMTM5 protein has a predicted MAL-related proteins for vesicle trafficking and membrane link (MARVEL) domain. MARVEL, a common feature of occludin, physins, gyrins, MAL and CMTM families, is a membrane-associating domain and may be a part of the machinery of membrane apposition events. The members of CMTM link to chemokines and the transmembrane 4 superfamily (TM4SF), having important roles in immune and male reproductive systems as well as disease pathogenesis including tumorigenesis. CMTM3, another member of the CMTM family, may inhibit tumor growth and invasion through promoting the transcription of P21, down-regulation the expression of MMP2 and phosphorylation of ERK1/2. In addition, CMTM7 also can represses oncogenic EGFR signaling through promoting EGFR internalization. Moreover, CMTM8 is also a regulator of the ERK1/2 signaling pathway. Thus, we suppose that the mechanism of CMTM5 acting on clear cell renal cell carcinoma cells might be also involved in membrane protein sorting and trafficking, such as EGFR and vascular endothelial growth factor receptor (VEGFR) signaling, both acknowledged targets of molecular targeted therapy of advanced clear cell renal cell carcinoma. Further evaluation of this hypothesis will provide additional information on molecular-targeted therapy. CMTM5 located at 14q11.2 is structurally similar to the TM4SF which can by collecting a variety of other molecules (such as AKT, EGFR) form four cross membrane protein network, thus mediated the migration, adhesion, cell proliferation and differentiation, apoptosis, and closely related to tumor formation and worse. It has been reported that CMTM5 acted as a tumor-suppressor gene (TSG) and was specifically down-regulated in many human cancers, such as cervical carcinoma, ovarian cancer, pancreatic cancer, myeloid leukemia and prostate cancer. But the role of CMTM5 in human RCC remains unclear. In this study, we aimed to detect the expression patterns of CMTM5 and their correlations with clinicopathological characteristics in CCRCC patients. Then, we also explored the effect of CMTM5 on cell proliferation and apoptosis based on RCC cell line in vitro.The difference expression between RCC cancer cells tissue and normal renal tissue were detected by immunohistochemistry. Establish pLentii6.3-CMTM5 recombinant lentivirus vectors and establish CMTM5 overexpression RCC cell line. Observe its effect on proliferation, cycle, apoptosis and invasion. Regulatory mechanism related signaling pathway were explored by western blot analysis.Methods and materialsTissue samples and cancer cells Tissue microarray (CCRCC and adjacent normal kidney tissues) were collected from 75 patients who underwent radical nephrectomy used for CMTM5 immunohistochemistry detection. The renal cell tumors consisted of stage Ⅰ 34, stage Ⅱ23, stage Ⅲ14, stage Ⅳ4, in accordance with 2009 World Health Organization(WHO) criteria. RCC cell lines ACHN, CAKI1 and OS-CA-2, and human kidney cell line HK2 Cells were maintained in DMEM media supplemented with 10% FBS at 37℃ with 5% CO2 humidified atmosphere.Lentivirus vector pLenti6.3-CMTM5-IRES-EGFP and The mock were transfected into ACHN cell line. Western blot and qPCR detection were used to detect difference between the CMTM5 over expression group, the negative control group (transfected pLentii6.3 empty plasmid) and the untreated control group.The function of CMTM5 in ACHN cell were detected by Cell proliferation analysis(cck8), Cell apoptosis and cell cycle analysis (flow cytometer) and migration and invasion analysis (Transwell)PI3K/AKT signaling pathway related proteins Bcl2, Bax, Bad, P21, cyclinD, cyclinE, caspase3 and MMPs were detected for regulatory mechanism of CMTM5 in RCC.ResultsWe examined the expression patterns of CMTM5 protein in CCRCC and adjacent non-cancerous kidney tissues using a tissue microarray by IHC. The positive immunostaining of CMTM5 protein was mainly localized in the membrane and cytoplasm of renal epithelial cells, and was found in 97%(73/75) of normal kidney tissues. However, CMTM5 protein was either absent or weakly expressed in 64% (48/75) of CCRCC tissues. There was a significant difference in the ratio of positive immunostaining expression of CMTM5 protein between CCRCC and adjacent normal kidney tissues (P<0.001). Then, we detected the expression levels of CMTM5 mRNA in three RCC cell lines. Compared with normal renal tubular epithelial cell line HK2, the expression level of CMTM5 mRNA was significantly down-regulated in ACHN, CAKI1 and OS-CA-2 RCC cell lines. Moreover, no statistically significant associations were found between various clinicopathological features, such as gender, age, clinical staging and histologic grade, and the expression of CMTM5 protein.After ACHN cell were infected by lentivirus vector pLenti6.3-CMTM5, the restoration of CMTM5 mRNA and protein in ACHN cells was confirmed by qRT-PCR and Western blotting.In CCK8 assay, the proliferation of ACHN-CMTM cells was decreased significantly compared to the untreated control and ad-null transfected control at the time point 48h and 72h (all P<0.05).The cell cycle analysis results generated by flow cytometer revealed that the CMTM5-infected group contained more cells in G1 phase and the cell proliferation index(cell rate of G2 and S phase) is lower (21.77±0.33%), compared with the ACHN group and control vector transfected cells(42.37±1.04%,42.98±0.43%, P<0.05) at the time point 48 h after transfection. The apoptotic effect of CMTM5 in RCC cells was tested by Annexin V/PI staining assay. Compared with the ACHN group (5.42±0.54%) and the control vector transfected cells (5.41±0.20%), the average apoptosis rate of CMTM5-ACHN cells (12.53±0.20%) was significantly higher at the time point 72 h after transfection. Using Transwell assay, we found that compared with the ACHN group and control vector transfected cells, the restoration of CMTM5 reduced penetration rate through the polycarbonate membrane in both migration(0.155±0.002,0.148±0.005,0.073±0.005) and invasion(0.137±0.001, 0.129±0.007,0.063±0.004)assays (both P<0.05).To find the molecular mechanisms underlying CMTM5 inhibit the cancer cell growth, apoptosis and migration inhibition, several proteins in PI3K-AKT signaling pathway were detected by Western blot. There were significant decreasing of p-AKT and its downstream Bcl2, cyclinD, cyclinE, MMP2 and MMP9 in CMTM5-transfected cells, and the increasing of Bax, Bad, cleaved caspase3, P21 compared with the control group suggesting enhancing of apoptosis and inhibiting of cell growth after CMTM5 transfecting. These results suggest that CMTM5 may inhibit RCC cells through regulating PI3K-AKT signaling.ConclusionOur data suggest that CMTM5 might function as a tumor suppressor in human RCC by suppressing the proliferation of RCC cells, implying its potentials as a therapeutic target for this malignancy. |
PDF Full Text Request