Daniel Coit MD None Michael A Postow MD Bristol

Daniel Coit, MD: None. Michael A. Postow, MD: Bristol Myers Squibb (Consulting or Advisory Role, Travel, Research Funding), Amgen (Consulting or Advisory Role), Novartis (Consulting or Advisory Role). Charlotte Ariyan, MD PhD: None.
Author Contributions
Introduction Colorectal cancer (CRC) is the third most common cancer worldwide and accounts for 14% of all new cancer diagnoses(Fitzmaurice et al., 2015). Tumor angiogenesis that is required for cancer growth and metastasis by nourishing cancer cells and helping spread of metastatic cells to distant tissues has been considered as a potential target for CRC treatment (Hanahan and Weinberg, 2011). Understanding the molecular mechanism by which CRC cells promote angiogenesis is required to develop effective antiangiogenesis treatment for CRC. Angiogenesis is a tightly regulated multistep process that includes endothelial cells breaking through the basement membrane, migrating toward angiogenic stimuli released from tumor cells, proliferating to provide sufficient cells for making a new vessel, and forming tubular structures (Bergers and Benjamin, 2003). The angiogenic switch depends on the balance of pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is one of the key pro-angiogenic stimuli produced within the tumor microenvironment in pathological states (Carmeliet and Jain, 2011; Conway et al., 2001; Yancopoulos et al., 2000). In CRC, the level of VEGF is elevated and correlate with a poor clinical outcome (Takahashi et al., 1995; Ellis et al., 2000). VEGF has, therefore, been a major focus for the development of anti-cancer drug and is considered a negative prognostic indicator for CRC. Many signaling pathways are interconnected to activate VEGF expression during tumorigenesis and progression (Des Guetz et al., 2006; Kerbel, 2008; Grothey and Galanis, 2009). Transcription of VEGF is regulated by multiple external factors. The VEGFA promoter contains In our study for numerous transcription factors, including SP1, AP2, c-JUN, EGR-1, HIF-1, and TCF (Pages and Pouyssegur, 2005; Liu et al., 2016). Recent work reveals that activated β-catenin translocates to the nucleus and complexes with TCF to promote transcription of VEGFA (Easwaran et al., 2003; Clifford et al., 2008), indicating that Wnt/β-catenin signaling regulates vessel formation. Therefore, targeting Wnt/β-catenin mediated VEGFA expression can be a new strategy for inhibiting angiogenesis. RNA helicases are members of the DEAD/H-box family, which are characterized by the presence of a helicase domain and are involved in RNA posttranscriptional procession. In addition to their roles in RNA procession, multiple members of RNA helicases are also implicated in transcription regulations. Aberrant expression of these proteins have been reported in various solid and hematologic malignancies (Wilson and Giguere, 2007; Causevic et al., 2001; Schlegel et al., 2003; Abdelhaleem, 2004a). We reported previously that DHX32, a novel member of the DEAH family, is up-regulated in CRC and contributes to CRC proliferation, apoptosis, migration, and invasion. Array analyses revealed that depleting DHX32 in CRC cells suppressed expression of VEGFA, indicating that DHX32 is involved in tumor angiogenesis (Huang et al., 2009; Lin et al., 2015). However, the mechanism by which DHX32 upregulates expression of VEGFA remains unknown.
Materials and Methods
Results
Discussion As a pro-oncogenic factor, β-catenin is one of the most important regulators that trigger the onset of tumor formation. In normal cells, signals of β-catenin are mainly activated by the canonical Wnt signaling pathway. Activation of the Wnt pathway leads to the dissociation of β-catenin from the APC/axin/GSK-3β complex. The released β-catenin then translocates to the nucleus and activates gene expressions. About 80% of CRC has loss of function mutations in APC. The mutations compromise the degradation of β-catenin, resulting in translocation of β-catenin to the nucleus where it dimerizes with the TCF/LEF factor, activates gene transcriptions, and contributes to angiogenesis in CRC (Clevers and Nusse, 2012; Pendas-Franco et al., 2008; Kiewisz et al., 2015). Several studies demonstrate that targeting the oncogenic activity of β-catenin has potent anti-tumor effects (Bienz and Clevers, 2000). In this study, we identified that DHX32 directly interacted with β-catenin in CRC cells. Binding of DHX32 to β-catenin prevented β-catenin from ubiquitination and degradation, and therefore, enhanced the activity of β-catenin. Previous studies showed that phosphorylation at N-terminal of β-catenin result in β-TrCP-mediated degradation (Yost et al., 1996). To address how DHX32 is stabilizing β-catenin, we tested whether DHX32 influenced N-terminal phosphorylation of β-catenin and whether it regulates β-TRCP protein level. However, neither over-expression nor depletion of DHX32 has significant effect on phosphorylation level of β-catenin and protein level of β-TrCP (Supplementary Fig. S7), indicating that DHX32-dependent regulation of β-catenin is not through inhibiting N-terminal phosphorylation of β-catenin or expression of β-TrCP. The mechanism by which DHX32 stabilizes β-catenin remains unknown, and is the next challenge for us to understand the function of DHX32.