Itch, an E3 ubiquitin ligase, influences downstream receptor tyrosine kinase signalling in the colorectal cancer cell line, HCT116

Receptor tyrosine kinase signalling is an important driver of cancer cell development. Deregulation of the receptor tyrosine kinases EGFR and Met is associated with cancer formation and progression to metastasis. Recently, mutations in Met that affect endocytic trafficking have been shown to promote cell migration, metastasis and tumorigenesis due to increased endosomal signalling. Thus, Met endocytic trafficking regulates signalling pathways; such as ERK1/2, STAT3, Gab1 and Rac1; that are implicated in cell survival, invasion and metastasis. Advancements in our understanding of regulation of EGFR and Met signalling is essential in order to understand their role in cancer progression. Ubiquitination regulates both receptor degradation and membrane trafficking. Here we investigated the role of the ubiquitin E3 ligase Itch in endocytosis and EGFR and Met downstream signalling in the colorectal cancer cell line HCT116.
Analysis of the TCGA colorectal adenocarcinoma data set showed that the expression of Itch was altered in 36% of the patient samples, and Met in 7% of the samples. The prognosis for patients carrying changes in both was worse than for either one alone (p=0.066, N=193). Overexpression of Itch in HCT116 cells did not affect ERK1/2 signalling downstream of the EGFR or Met receptor. However, expression of a dominant negative ligase mutant reduced ERK1/2 signalling from the Met receptor but not the EGF receptor. Thus, suggesting that Itch is important for Met signalling. Further supporting this notion, our knockdown experiments showed a significant effect on HGF-induced but not EGF-induced ERK signalling.
Knockdown of Itch in cultured cells showed aberrant endocytic protein levels, as assessed by Western blot. These proteins are involved in clathrin-mediated endocytosis and their expression levels regulate the speed and efficiency of receptor internalisation, thus implicating a novel Itch-mediated regulatory mechanism for membrane trafficking. Our study highlights the importance of assessing the regulation of receptor signalling and membrane trafficking in colorectal cancer.