The antiapoptotic protein, FLIP, is regulated by heterogeneous nuclear ribonucleoprotein K and correlates with poor overall survival of nasopharyngeal carcinoma patients

Abstract

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) mediates antiapoptotic activity in part by inducing downstream antiapoptotic genes. To systematically identify hnRNP K targets in nasopharyngeal carcinoma (NPC), affymetrix chips were used to identify genes that were both overexpressed in primary NPC and downregulated by hnRNP K knockdown in NPC-TW02 cells. The resulting gene set included the antiapoptotic gene, FLIP, which was selected for further study. In cells treated with hnRNP K siRNA, TRAIL-induced apoptosis was enhanced and the FLIP protein level was reduced. Promoter, DNA pull-down and chromatin-immunoprecipitation assays revealed that hnRNP K directly interacts with the poly(C) element on the FLIP promoter, resulting in transcriptional activation. Through iTRAQ-mass spectrometric identification of proteins differentially associated with the poly(C) element or its mutant, nucleolin was determined to be a cofactor of hnRNP K for FLIP activation. Furthermore, FLIP was highly expressed in tumor cells, and this high-level expression was significantly correlated with high-level hnRNP K expression (P ¼ 0.002) and poor overall survival (P ¼ 0.015) as examined in 67 NPC tissues. A multivariate analysis confirmed that FLIP was an independent prognostic factor for NPC. Taken together, these findings indicate that FLIP expression is transcriptionally regulated by hnRNP K and nucleolin, and may be a potential prognostic and therapeutic marker for NPC. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) belongs to the hnRNP family of proteins. The members of this family interact directly with DNA and RNA through their K-homology domains and regulate gene expression at multiple levels, including transcription, RNA splicing, RNA stability and translation.1-2 The expression of hnRNP K has been shown to be aberrantly increased in numerous cancers, [3][4][5][6] and we recently reported that high-level hnRNP K expression is correlated with poorer overall survival (OS) and decreased metastasis-free survival among nasopharyngeal carcinoma (NPC) patients.5 Our findings were consistent with those from clinical correlation studies in oral squamous cell carcinoma 4 and prostate cancer. HnRNP K is a nucleocytoplasmic shuttling protein that is primarily located in the nucleus for transcriptional regulation. However, cytoplasmic accumulation of hnRNP K through ERK-mediated phosphorylation of hnRNP K serines-284 and -353 has been reported in cervical carcinoma HeLa, 7 chronic myelogenous leukemia 8 and NPC 9 cells. The tumorigenic activity of hnRNP K appears to be conferred through its ability to increase proliferation, 10 antiapoptotic effects, 9 clonogenic potential 8 and metastasis. 11 These functions may be due, at least in part, to the ability of hnRNP K to upregulate the c-myc, 8,12 thymidine phosphorylase (TP) 9 and eIF4E 10 genes through transcriptional or post-transcriptional regulation. However, the full spectrum of targets regulated by hnRNP K has not as yet been systematically examined.The acquisition of resistance to ...

Aberrant hnRNP K expression: All roads lead to cancer

“…These studies revealed that increased hnRNP K expression associated with poor clinical status in melanoma, prostate, breast, lung, colorectal, hepatocellular, and esophageal cancers. [14][15][16][17][18] Given the myriad of tumor types that overexpress hnRNP K in these studies and its correlation with disease prognosis, these data suggest that hnRNP K may have oncogenic functions when overexpressed.…”

“…[14][15][16][17][18] However, to directly test this notion, there is an absolute need for the development of transgenic animal models that overexpress wild-type hnRNP K. We have currently undertaken this task by generating tissuespecific Hnrnpk transgenic mice. These model systems will directly test whether hnRNP K is a bona fide oncogene and will be useful in identifying the genes and cellular programs critical for tumorigenesis when hnRNP K is overexpressed.…”

“…[8][9][10][11][12][13] In support of its potential oncogenic functions, clinical association studies suggest that hnRNP K overexpression correlates with poor clinical outcomes and advanced disease status in a variety of malignancies, including melanoma, prostate, breast, lung, colorectal, hepatocellular, and esophageal cancers. [14][15][16][17][18] In contrast, other clinical studies suggest reduced hnRNP K expression, due to deletion of or mutations in the HNRNPK gene, may underpin the pathogenesis of acute myeloid leukemia (AML). [19][20][21][22][23] In the case of mutation, there are further questions concerning whether specific mutations inactivate the protein or potentially stabilize or confer gain-of-function phenotypes, reminiscent of mutations observed in the c-Myc protein and mutant p53 proteins, respectively.…”

Embelin prevents LMP1-induced TRAIL resistance via inhibition of XIAP in nasopharyngeal carcinoma cells

“…This finding supports the hypothesis that the presence of death receptors in certain head and neck cancers may endow them with a greater susceptibility to TRAIL-mediated apoptosis. One study demonstrated the robust induction of apoptosis in the TW02 NPC cell line by TRAIL (11); however, another study indicated that inducing apoptosis in certain NPC cells lines by TRAIL may be challenging (12). The explanation for this discrepancy remains unknown.…”

Heterogeneous Nuclear Ribonucleoprotein K (hnRNP-K) Promotes Tumor Metastasis by Induction of Genes Involved in Extracellular Matrix, Cell Movement, and Angiogenesis

“…Consistently, a variety of cancers, including breast carcinoma (21), esophageal squamous cell carcinoma (22), head-and-neck/oral squamous cell carcinomas (23,24), lung adenocarcinoma (25,26), pancreatic carcinoma (27), melanoma (28), colorectal carcinoma (29), and chronic myeloid leukemia (30) were found to be enriched in hnRNP-K expression. It was shown to regulate the anti-apoptotic protein FLICE-like inhibitory protein (FLIP) and VEGF in cancer cells (31,32). The functional significance and molecular mechanism of the role of hnRNP-K in cancer cells yet remain unclear.…”