The Study On Apoptosis-inducing Factor Downregulationand Mechanism In Renal Cell Carcinoma And Related Function

Renal cell carcinoma(RCC) is one of adult malignancies which threatening human health. The annual incidence of RCC in Chinahas been increasing especially in developed areas. Since it is relatively resistant to radiation therapy and chemotherapy, nephrectomy is commonly used in removing early tumors. However, when metastasis occurs, RCC is very difficult to be treated and has poor prognosis. Currently, the early detection and diagnosis of RCC remains a challenge to oncologists. Roughly 30% of RCC cases present with metastatic disease at the time of initial diagnosis. Patients with metastatic RCC have a much poorer prognosis compared with patients with early-stage diseases, with a 5-year survival rate of 23% for stage IV disease as compared to a 5-year survival rate of 96% for stage I presentation. Nowadays, it is well accepted that Von Hippel-Lindau(VHL) gene mutation is the hallmark of clear cell renal cell carcinoma. It is also clear that p VHL loss is not sufficient step in RCC, which suggests other molecular events included in renal carcinogenesis.Apoptosis-inducing factor(AIF) was initially identified as a death effector which locates on chromosome X. Currently, AIF is considered as a dual roles protein: in normal cell, AIF plays a vital function in maintaining mitochondrial structure and respiratory chain as a NADH oxidoreductase. In response to apoptotic stimuli, AIF is cleaved by calpains or cathepsins to yield a truncated form that translocates to the nucleus to induce DNA condensation and large-scale fragmentation in the presence of its interacting proteins.In this study, we report significant AIF downregulation in the majority of renal cell carcinomas(RCC) which is caused by genomic and epigenetic mechanism. We also explored its interacting proteins and roles in RCC. In Cancer profiling array II analysis, AIF c DNA probe was used to detected RNA level of AIF in 154 pairs of cancer/normal cases of 19 kinds of tissue types. The result showed AIF is significant downregulation in RCC than that in normal kidney. 17(85%) cases of clear cell RCC clinical tissues had AIF downregulation when detected by Western Blot in 20 pairs of matched cancer and adjacent normal tissues. In a group of RCC specimens,84%(43 out of 51) had AIF downregulation by immunohistochemistry stain. All these findings demonstrate that AIF is common and significant downregulation in RCC. It is well known that RCC arises from the cells of the proximal renal tubular epithelium in which AIF expression significant downregulated than that in normal kidney. These facts may suggest that AIF downregulation is a common event in kidney tumor development.The mechanisms of the AIF downregulation include both genetics and epigenetics variations. We performed fluorescence in situ hybridization(FISH) analysis using a BAC clone containing the entire AIF genome sequence, and identified AIF allelic reduction in 2 out of 11 tumors. Southern blot results showed 3 out of 11 RCC tissues exhibited genomic deletion in AIF 3′UTR. Furthermore, to circumvent the interference of hypermethylated loci on the inactive X-chromosome in females, 4 pairs of male CCRCC samples with downregulated AIF(no apparent AIF genomic loss) were selected for AIF promoter methylation studies. Bisulfite sequencing showed a complicated DNA methylation profiling. Multiple DNA methylation sites, including non-Cp G methylation, were detected in all 4 tumor samples. In addition, we detected a unique constant Cp T methylation at +561 in Caki-1 cell. To study whether AIF promoter methylation affects AIF expression, Caki-1 cells were treated with a DNA demethylating agent 5-aza-2’-deoxycytidine(5-aza-d C), which led to almost three-fold increase of AIF. Taken together, these results demonstrate that aberrant promoter methylation and genomic deletion in RCC contribute to AIF downregulation.To study the interacting factor of AIF in RCC, a series of GST pull-down and Mass-spectrum analysis were performed. Some proteins were found in the complex including Serine/threonine protein kinase 3(STK3, also known as MST2), and the interaction between AIF and STK3 was confirmed by Co IP. STK3 is an important protein in control proliferation and apoptosis by activating multiple cell signaling pathways such as Hippo pathway, FOXO pathway and RASSF1 A pathway.To understand the role of AIF in carcinogenesis and development of RCC, AIF was overexpressed in RCC cells. As AIF increasing, a significant increment of STK3 phosphorylation at Thr180 was detected with the total STK3 level constant. And overexpressed AIF induced massive apoptosis in both Caki-1 and 786-O cells. Indeed, most transfected cells were dead and detached from culture flasks within 24 h after transfection. As determined by immunofluorescence stain, Caki-1 cells overexpressing pc DNA3.1-AIF had AIF signals in nuclei, consistent with its role in apoptosis. These findings suggest AIF has complex function in RCC cell by interacting with multiple cell signaling pathways.In summary, our finding that AIF is frequently downregulated in RCC indicate that loss of AIF is involved in RCC development. The interaction between AIF and STK3 also reveals a novel role of AIF in regulating programmed cell death. Further research on AIF function in RCC may allow us a better understanding of RCC carcinogenesis and development. Targeting the AIF signaling pathway therefore is a viable option for RCC therapy.