The transcription factor KLF6 has an essential role in the development and metastasis of multiple human cancers. Paradoxically, KLF6 expression was found to be attenuated in primary metastatic clear cell renal cell carcinoma (ccRCC), such that it is unclear how KLF6 affects malignant progression in this setting. In this study, we demonstrate that KLF6 attenuation in renal cells is sufficient to promote E2F1-mediated epithelialmesenchymal transition and metastatic prowess. In a mouse xenograft model of human ccRCC, silencing KLF6 increased tumor cell proliferation and malignant character, whereas E2F1 silencing reversed these properties. These effects were corroborated in a metastatic model system, where we observed a greater number of pulmonary metastatic lesions formed by ccRCC cells where KLF6 was silenced and E2F1 enforced. Analysis of clinical specimens of ccRCC revealed that low levels of KLF6 and high levels of E2F1 correlated closely with ccRCC development. Overall, our results established the significance of activating the KLF6-E2F1 axis in aggressive ccRCC, defining a novel critical signaling mechanism that drives human ccRCC invasion and metastasis. Cancer Res; 77(2); 330-42. Ó2016 AACR.

BackgroundTranscription factor E2F1 exerts effects on many types of cancers. As an upstream regulator of a host of genes, E2F1 can trigger diverse aberrant transcription processes that may dominate malignancy. Clear cell renal cell carcinoma (ccRCC) is the most common subtype in renal cell carcinoma which displays high malignancy and has a shortage of biomarkers in clinics. Our study aimed to explore the function of E2F1 in ccRCC and its correlation with clinicopathological parameters.Methodology/Principle FindingsTranscription factor E2F1 was mainly distributed in cancer cell nucleus and mRNA expression significantly increased in 72 cases of clear cell renal cell carcinoma (ccRCC) tissues compared with adjacent non-cancerous kidney tissues (p<0.001). The protein expression was consistent with mRNA expression. Further analysis in 92 cases indicated that E2F1 mRNA level expression was associated with the tumor pathologic parameters embracing diameter, Fuhrman tumor grade, pT stage, TNM stage grouping and macrovascular infiltration (MAVI). These surgical specimens had high grade tumors accompanied with an elevated E2F1 expression. Moreover, E2F1 transfection was found to contribute significantly to cancer cell proliferation, migration and invasion in vitro.Conclusions/SignificanceOverexpression of E2F1 may be a key event in the local and vascular infiltration of ccRCC indicated by the activation of matrix metalloproteinase (MMP) 2 and MMP9. These findings highlighted the implication of E2F1’s function in the metastatic process. Furthermore, the clinical relevance of E2F1 in ccRCC pointed to a potential new therapeutic target.

Clear cell renal cell carcinoma (ccRCC) is a common genitourinary malignancy with high mortality. However, the molecular pathogenesis of ccRCC remains unclear and effective biomarkers for daily practice are still limited. Thus, we aimed to identify the potential crucial genes and pathways associated with carcinogenesis of ccRCC and further analyze the molecular mechanisms implicated in tumorigenesis. In the present study, expression profiles GSE 66270, GSE 53757, GSE 36895, and GSE 76351 were downloaded from GEO database, including 244 matched primary and adjacent normal tissues, furthermore, the level 3 RNAseq dataset (RNAseqV2 RSEM) of KIRC was also downloaded from The Cancer Genome Atlas (TCGA), which consist of 529 ccRCC tumors and 72 normal tissues. Then, differentially expressed genes (DEGs) and pathway enrichment were analyzed by using R software. A total of 129 up- and 123 down-regulated genes were identified, which were aberrantly expressed both in GEO and TCGA data. Second, Gene ontology (GO) analyses revealed that most of the DEGs were significantly enriched in integral component of membrane, extracellular exosome, plasma membrane, cell adhesion, and receptor binding. Signaling pathway analyses indicated that DEGs had common pathways in signal transduction, metabolism, and immune system. Third, hub genes were identified with protein-protein interaction (PPI) network, including PTPRC, TGFB1, EGF, MYC, ITGB2, CTSS, FN1, CCL5, KNG1, and CD86. Additionally, sub-networks analyse was also performed by using MCODE plugin. In conclusion, the novel DEGs and pathways in ccRCC identified in this study may provide new insight into the underlying molecular mechanisms that facilitates RCC carcinogenesis.

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Microtubules (Mts), which consist of a/b-tubulin heterodimers, are involved in cancer development and metastasis. Tubulin cofactor A (TBCA) plays crucial roles in modulating tubulin folding and a/b-tubulin heterodimer polymerization. Here, we identified the aberrant expression of TBCA in clear cell renal cell carcinoma (ccRCC) specimens as well as cell lines and revealed the function of TBCA as a novel positive regulator in ccRCC progression, invasion and metastasis. qRT-PCR, Western blot and immunohistochemistry assays confirmed that TBCA was significantly highly expressed in ccRCC specimens and cell lines compared to their corresponding normal kidney tissues and HKC. Accordingly, the influence of TBCA on cell proliferation, apoptosis and invasion/migration was detected through overexpression and knockdown of endogenous TBCA protein level in ccRCC cells via plasmids. Silencing of TBCA expression inhibited the proliferation of 786-O cells and Caki-1 cells and promoted the apoptosis of 786-O cells. Down-regulation of TBCA expression also reduced the invasion and migration ability of 786-O cells. Interestingly, overexpression of TBCA did not induce biocharacteristics that directly contrasted to those of TBCA knockdown. Importantly, exploration of the mechanism showed that TBCA could function via modulating cytoskeleton integration and influencing cell cycle progress. Furthermore, down-regulation of TBCA expression in 786-O and Caki-1 cells affected cytoskeleton integration and cell size, induced S/G2 cell cycle arrest and led to cyclineA/E and CDK2 aberrant expression. By investigating novel roles of TBCA in regulation of ccRCC cell progression, invasion and metastasis, our study identified that TBCA may be a potential molecular target for ccRCC therapy.Clear cell renal cell carcinoma (ccRCC) accounts for about 3% of all malignancies. It is the second leading cause of death among all types of urologic tumors. 1 At present, radical or partial nephrectomy is still the most effective treatment for local ccRCC. However, prognosis remains poor for advanced and metastatic ccRCCs because of low sensitivity to chemotherapy and radiotherapy. Recently, molecular targeted therapy has evolved rapidly with the clinical application of tumor-targeted agents such as sorafenib and sunitinib. Unfortunately, the risk of adverse events and disparate treatment outcomes limits the use of these agents. 2,3 Therefore, it is of great importance to find new molecular targets or markers to establish effective strategies for the treatment and early diagnosis of ccRCC.Recently, researchers established that cytoskeletal components including actin, microtubules and intermediate filaments play key roles in cancer development and metastasis. 4,5 Microtubules are a crucial structure for living cells and are involved in many biological functions, such as cell shape, polarity, cell motility, cell division, intracellular transport and cellular architecture. 6 It is composed of a heterodimer, one a-tubulin polypeptide and one b-tubulin polypeptide. A ...