An unconventional KITENIN/ErbB4-mediated downstream signal of EGF up-regulates c-Jun and the invasiveness of colorectal cancer cells Running title: An unconventional oncogenic signal of EGF

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: EGF-stimulated signaling via EGF receptor (EGFR) is important in colorectal tumorigenesis and drug targeting. However, anti-EGFR therapy is not effective in a subset of colorectal cancer (CRC) patients, suggesting that unidentified EGF-stimulated pathways might play roles in CRC. Previously, we identified KITENIN (KAI1 C-terminal interacting tetraspanin) as a metastasis-enhancing gene and found it to be highly expressed in sporadic CRC tissues. We recently found that EGF further increases KITENIN-induced elevated AP-1 activity. Here we attempted to clarify this novel EGF-stimulated molecular pathway and its roles in CRC. Experimental Design: We analyzed how EGF modulates the downstream signaling pathway of oncogenic KITENIN in CRC cells. Biological alterations following EGF treatment were identified in KITENIN-overexpressed CRC cells with or without alteration of EGFR activity. Results: We identified the KITENIN/ErbB4-Dvl2-c-Jun axis as a novel downstream signal of EGF that is switched on under elevated KITENIN conditions in an EGFR-independent manner. This unconventional EGF signal up-regulates c-Jun and enhances invasion and anchorage-independent growth of CRC cells. Additionally, tumor tissues from metastatic CRC patients who showed initial poor responses to cetuximab/chemotherapy expressed higher levels of KITENIN than did responders to therapy. Conclusions: Our results highlight the role of an EGFR-independent EGF signal in mediating the invasiveness and tumorigenesis of CRC cells. This unconventional pathway might be related to the limited clinical efficacy of anti-EGFR agents in a subset of CRC patients. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. EGFR-targeted therapy is an important approach in CRC therapy; however, up to half of all CRC patients do not respond to this therapy. Our present results indicate the presence of an unconventional EGFR-independent signal of EGF, the KITENIN/ErbB4-Dvl2-c-Jun axis, which mediates increased CRC cell invasiveness and thereby enhances tumor progression. Our results suggest that the KITENIN/ErbB4-c-Jun axis could be a molecular basis for conferring resistance to anti-EGFR agents in CRC tissues in which KITENIN is highly expressed. These findings support a rationale for combined targeting of the KITENIN complex to improve responses to cetuximab-based therapy in EGFR/KITENIN-overexpressing metastatic CRC patients. We propose that, together with " quadruple negative " genotype (tumors lacking alterations in KRAS, BRAF, PIK3CA and PTEN genes), lower KITENIN levels in resected tumor tissues from metastatic CRC patients could be useful for identifying patients who would benefit …