Dysregulation of netrin-1 receptors in human colon cancer

Abstract

Netrin-1 is a laminin-related secreted protein that performs an important role in intestinal epithelial biology. The netrin-1 pathway may be involved in human tumorigenesis through its interactions with two dependence receptors: deleted in colorectal cancer (DCC) and UNC5C. These 2 receptors induce apoptosis in the absence of netrin-1, but promote cell survival in its presence. Colorectal Cancers (CRCs) frequently lose DCC due to deletions in chromosome 18q, and recent reports indicate that CRCs also exhibit reduced or loss of UNC5C expression. The mechanism for the loss of UNC5C in CRCs is not clearly understood, since mutations have rarely been observed, and allelic losses have been inconsistently reported. Since DCC and UNC5C share the same ligand, and both receptors are frequently down regulated in CRC, a better understanding of underlying molecular processes is critical for appreciating the role of netrin pathway in the colonic tumorigenesis. The hypothesis of this thesis was that UNC5C is epigenetically silenced in CRC, and that there are interactions between losses of UNC5C and DCC in colorectal tumorigenesis. Gene expression and epigenetic analysis of UNC5C was examined in a panel of 8 CRC cell lines, 147 primary sporadic CRCs with corresponding normal mucosa (CNM), and 52 colorectal adenomatous polyps (APs). Allelic imbalances at DCC were determined in CRCs using markers mapping chromosome 18q. The molecular analyses were compared with genetic and clinico-pathological features. All 8 CRC cell lines demonstrated UNC5C methylation and an associated loss of gene expression. Treatment with a demethylating agent resulted in restoration of gene transcription. UNC5C methylation was significantly more frequent in CRCs (76.2%) and APs (63.5%) than in CNM (6%; p<0.001). Allelic imbalance at DCC was observed in 61% of CRCs. Overall, 89.3% of CRCs had alterations of either netrin receptor. Finally, UNC5C methylation occurred predominantly in the earlier lesions (APs and early stage CRC), whereas DCC losses were observed more often in advanced CRCs. I provide first evidence that majority of CRCs harbor defects in netrin-1 receptors, emphasizing the importance of this growth regulatory pathway in cancer. My data also suggest that the timing of the molecular alterations in the two netrin-1 receptors is not random, since UNC5C inactivation occurs early, whereas LOH of DCC occur in later stages of multistep colorectal carcinogenesis.