Aberrant Expression Of RNA Binding Protein Quaking In Colon Cancer And Its Role In Colon Cancer Development

Colon cancer is one of the most common cancers worldwide, causing half a million deaths each year. The model of colorectal tumorigenesis includes several genetic changes that are required for cancer initiation and progression, such as the APC pathway,β-catenin/Tcf pathway, RAS/RAF pathway and p53 pathway. Even though the above genetic changes have been characterized in most of the colon cancer patients, the initial factors corresponding to these changes besides the genetic reasons are not elucidated and it is yet to be established whether other important pathways are involved.Nowadays, posttranscriptional regulation is believed to be involved in the regulation of a wide variety of fundamental cellular processes, including cell growth and cell cycle progression, differentiation, and apoptosis. The RNA binding protein QKI belongs to the evolutionarily conserved STAR family and the QKI gene produces a diverse set of proteins by alternative splicing. The three well studied isoforms (QKI-5, -6, and -7) appear to have different roles in development. They are constructed with the same 311-amino acid body but have different carboxyl tails consisting of 30, 8, and 14 amino acids, respectively. QKI-5 is prominently expressed in early embryogenesis, whose mutation is believed to be responsible for the lethality at around 9.5 days of gestation. In contrast, QKI-6 and -7 are mainly expressed in CNS in late development when myelination starts. QKI-6 and -7 are believed to play a fundamental role in myelination through coordinately regulating mRNA targets such as MAG, p27, MBP. Besides these validated targets, Galarneau A et al has defined the QRE as a bipartite consensus sequence NACUAAY-N(1-20)-UAAY and predicted nearly 1,430 new putative mRNA targets, in which both p27 andβ-catenin are included. QKI are involved in the regulation of mRNA stability, nuclear retention, RNA transportation, and translational modulation through interaction with QREs locating in the UTR of target mRNAs in the heterodimer or homodimer form. Furthermore, evidence of alterations of QKI expression in neoplastic tissues, together with the identification of QKI as an important player in embryogenesis and a critical regulator of p27, suggests that aberration of this molecule may contribute to uncontrolled cell growth and transformation. It is thus interesting to test whether this RNA binding protein QKI is present in normal GI tract and its relevance with the colorectal carcinogenesis.【Aims】①To analyze the expression pattern of QKI in the colon along the crypt-villi axes, shedding light on the function of QKI in colon epithelium development;②To analyze the expression change of QKI between colon and colon cancer, and then to explore the underlying mechanism for the aberrant expression of QKI in colon cancers;③To examine the role of QKI in colon cancer cell proliferation and differentiation; ④To reveal the mechanism how QKI regulates cell proliferation and differentiation by screening and identifying the potential targets of QKI in this process.【Methods and Results】①we first analyzed the expression of QKI in normal colon along the crypt-villi axes. QKI, mainly the QKI-5 isoform was abundantly expressed in the crypt and with the cells migrating up to the villi and differentiated, QKI-6 became expressed and QKI-5 declines a little bit. In vitro data of HT29 differentiation revealed similar results.②Expression levels of QKI were compared among normal gastrointenstinal epithelial cell lines (GES-1 and IEC-6) and cancerous cell lines (HCT116, HT29, SW480, SW620 and Colo205 cells). High expression of QKI in normal GES-1 and IEC-6, median level in HT29 cells, and low level in HCT116, SW480 and SW620 were observed both at RNA and protein levels, while null expression was found in the poorly differentiated Colo205 cells. Furthermore, immunohistochemistry analysis in tissue samples revealed that QKI expression was detectable in about 60% of the adjacent normal colon samples (6/10), while only in 30% of colon cancer samples (3/10) and with a relatively lower level. Similar results were observed through western blot analysis. Besides, RT-PCR with specific primers revealed that both QKI5 and QKI6 are expressed in colon cells. Strikingly, in the QKI positive cancer tissue samples, QKI6 were greatly suppressed or nearly absent. All of these implicate that expression of QKI, especially QKI6 are aberrantly reduced in the colon cancer.③To deliberate the underlying mechanisms responsible for deregulation of QKI in cancer cells, we analyzed the promoter region of QKI. QKI promoter region is rich in CpG islands, especially in the 500bp upstream the putative transcription start site, leading us to ask whether CpG methylation accounts for the low and even null expression of QKI in colon cancers. Consistent with the varied expression of QKI in cell lines, methylation status of QKI promoter varied in these cell lines, which is reversely related with the expression level. Although methylation was found in the adjacent normal epithelium in about half of the patients, low or no methylation was found in normal colon epithelium while abundant methylation was found in the corresponding cancer samples in the remaining. Strikingly, 5-aza-dC treatment of the hypermethylated colo205 cells for 7 days rescued the expression of QKI, highly suggesting that hypermethylation of QKI lead to the reduced expression of QKI.④With the rescue of QKI expression under 5-aza-dC treatment, intestinal differentiation markers, such as lactase and IAP were also significantly upregulated, suggesting a differentiating role of QKI in colon epithelium. Overexpression of either QKI5 or QKI6 in HT29 cells facilitates the differentiation of colon cancer cell as indicated by the IAP activity and the expression of Lactase and E-cadherin. Accordingly, knockdown of QKI through RNAi reduced the expression of lactase and IAP.⑤F ACS data revealed that either QKI-5 or QKI-6 expression prolonged the G1 phase and facilitated G1 synchronization after serum starvation.⑥I n addition to the pro-differentiation role, overexpression of QKI, especially QKI6 reduced the cell proliferating ability both in HT29 cells and in HCT116 cells as indicated by MTT assay. HCT116 cells infected with Ad-EGFP, Ad-QKI5, Ad-QKI6 were cultured in semisolid medium. Colonies of Ad-QKI6 infected cells are much smaller and fewer, compared with the control. These data implicate a tumor suppressing role of QKI in colon epithelium.⑦As an RNA binding protein, QKI acts through modulating the expression of its targets at post-transcriptional level. Among the thousands of putative QKI targets, p27 andβ-catenin are most fascinating and highly possible to be involved in our model. In line with the confluence induced early differentiation of HT29 cells, the stability of the p27 mRNA increased significantly at high density, which should contribute at least partially to the increased protein level. Consistent with previous finding, we found QKI6 increased the expression of p27 through enhancing the RNA stability in our model too. What's more, QKI5 expression also increased p27 expression only if HT29 cells were induced to differentiation by confluence. RNA-IP assay confirmed the direct interaction between p27 and QKI5. In addition, knock-down of QKI reduced high density induced p27 mRNA stability partially. Our data revealed that QKI5 and 6 are involved in the increased mRNA stability of p27 under confluence.⑧B esides p27 upregulation, we observed the relationship between QKI andβ-catenin. Results from the subcellular fraction revealed that overexpression of QKI reduced the cytosol and nucleus fraction ofβ-catenin. Consistently, overexpression of QKI reduced the luciferase activity of Topflash under such confluent condition, with no significant change to Fopflash reporter. Whereas the mRNA level ofβ-catenin was not affected by forced expression of QKI, implicating the regulation might be at the layer of translational efficiency. In this regard, serial reporter vectors containing both of the two potential QREs, only the proximal QRE, and none of the QREs were constructed. Overexpression of QKI5 or QKI6 decreased the activity ofβ-catenin 3'UTR andΔβ-catenin 3'UTR1, whileΔβ-catenin 3'UTR2 with no QREs did not responded to QKI, suggesting the proximal QRE was the true responsive QRE. In order to test the possibility of direct interaction betweenβ-catenin and QKI, RNA-IP assay was employed. Forced expression of flag-tagged QKI5 or flag-tagged QKI6 co-precipitates with β-catenin mRNA, suggesting a direct interaction between them. In contrast to the reduction of cytosol and nucleusβ-catenin by QKI, both QKI5 and QKI6 increased the membrane boundβ-catenin.【Conclusion】In view of the above data, we hypothesize that QKI might affect the carcinogenesis through coordinately regulating the differentiation and proliferation of intestinal epithelial cells. In summary, our data demonstrate for the first time that RNA binding protein QKI is normally expressed in the colon epithelium in young individuals while absent or downregulated in colon cancers due to aberrant hypermethylation. Furthermore, by increasing the expression of p27 and membrane boundβ-catenin, QKI facilitates the differentiation of colon epithelial cells.