293T cells were transfected with the AHNAK2-pGL4.17-firefly reporter construct and a control Renilla expression vector. AHNAK2 was greatly upregulated, at least in part, by hypoxia in cancer cells. By using chromatin immune-precipitation (CHIP) and promoter-luciferase reporter assays, we identified that upregulation of AHNAK2 induced by hypoxia was hypoxia-inducible factor-1 (HIF1)-dependent. Knockdown of AHNAK2 impaired hypoxia-induced epithelial-mesenchymal transition (EMT) and stem cell-like properties. Considered together, we reveal that AHNAK2 is upregulated in cancer cells and hypoxic upregulation of AHNAK2 can drive tumorigenesis and progression by supporting EMT and cancer cell stemness. Thus, AHNAK2 is a novel prognostic marker and an oncogenic protein for ccRCC. < 0.05, ** < 0.01, *** < 0.001. Results Identification of AHNAK2 as an upregulated gene in ccRCC To identify novel oncogenic genes in ccRCC, we performed data-mining analyses from 4 microarray datasets in the Oncomine database, all of which were analyzed using the Human Genome U133A Array 22-25. We examined the top 200 genes upregulated in each data set, and found that AHNAK2 is one of the 45 overlapping genes among all of the 4 datasets (Figure ?(Figure1A).1A). While some of them, such as CXCR4, TGFB1 and HEY1 are already implicated in tumorigenesis 26-28, the role of AHNAK2 in cancer Rabbit polyclonal to KCTD1 is unknown. Thus, we decided to focus on AHNAK2. Notably, the mRNA level of AHNAK2 was enhanced significantly in human ccRCC samples as compared to their adjacent normal renal tissues (Beroukhim Renal, n = 32, fold change = 9.856, p p p < 0.01, *** < 0.001. (B) Immunofluorescence staining analysis for AHNAK2 protein level in ccRCC and adjacent normal tissues. Scale bar = 20 m. (C) Representative immunohistochemistry micrographs of AHNAK2 expression from the large cohort of ccRCC patients paraffin samples (N = adjacent normal tissue, T = ccRCC tissue). Scale bar: left = 1 mm, right = 25 m. (D) Quantitative analysis of sample numbers in different AHNAK2 expression levels in 355 ccRCC samples collected from our own Institution. (E) Analysis of AHNAK2 staining scores in association with histopathological grades (n=355). (F) Probability of patients with metastasis in different levels of AHNAK2 expression group (n = 266 in AWZ1066S the low-expression group; n = 89 in the high-expression group). (G) Kaplan-Meier curve of comparing overall survival (OS) in the high expression of AHNAK2 group with the low expression group (n= 89 in the AHNAK2 high-expression group, n = 264 in the AHNAK2 low-expression group). The log-rank (Mantel-Cox) test was used. In addition, we further assessed the expression of AHNAK2 in the immortalized renal tubular epithelial cell line HK-2, the human embryonic kidney cell line 293T and 4 renal cancer cell lines, including CAKI-1, 769-P, 786-O, and ACHN. Both mRNA and protein levels of AHNAK2 were higher in the renal cancer cell lines than HK-2 and 293T cells (Figure S1A, S1B). We made attempts to perform western blots, but failed due to the large size of the AHNAK2 protein (~ 600 kDa). Nevertheless, our quantitative RT-PCR (Figure ?(Figure2A,2A, Figure S1A), immunofluorescence (Figure ?(Figure2B,2B, Figure S1B) and immunohistochemistry data (Figure ?(Figure2C)2C) support the notion that AHNAK2 is upregulated in ccRCC cells and tissues. High expression of AHNAK2 correlates with the disease progression and shortened patient survival in ccRCC To determine the clinical relevance of AHNAK2 expression in human ccRCC, we carried out immunohistochemical analysis of AHNAK2 on primary human tumors from a large cohort of 355 ccRCC patient samples collected from our own Institution (Table ?(Table1).1). Expression of AHNAK2 was observed within the cytoplasm of cancer cells (Figure ?(Figure2C).2C). A significantly increased intensity of AHNAK2 staining was seen in ccRCC tumor tissues compared to the adjacent normal tissues (Figure ?(Figure2C,2C, 2D). Although AHNAK2 AWZ1066S expression did not correlate with age, gender, tumor size, or TNM stage (> 0.05), The AHNAK2 staining scores of G3-G4 tumors were higher than that of G1 tumors (valueand < 0.001; Figure ?Figure3E).3E). Moreover, we performed time-lapse tracking to observe directional migration patterns by single cells (Figure ?(Figure3F).3F). Cell migration velocity and distance were significantly decreased in AHNAK2-knockdown CAKI-1 cells compared to the control (Figure ?(Figure3G).3G). These in vitro data were consistent with our finding that increased AHNAK2 is associated with a higher occurrence of human ccRCC metastasis in patients. Open in a separate window Figure 3 AHNAK2 knockdown inhibits the growth of ccRCC in vitro and in vivo. (A) CAKI-1 cells stably transfected with shRNAs were treated with doxycycline (1 g/ml) for 7 days and performed by quantitative RT-PCR analysis. AWZ1066S *** p < 0.001. (B) Representative immunofluorescence staining of AHNAK2 expression in each of the indicated cell.