Abstract 1371: Isoprenylcysteine carboxylmethyltransferase (ICMT) function is critical for epithelial malignant transformation by mutant-Ras

Abstract

Abstract Purpose Limited progress has been made in the development of direct inhibitors of Ras. Therefore, targeting isoprenylcysteine carboxylmethyltransferase (ICMT), the final enzyme in the prenylation pathway for Ras and other proteins containing the so-called CaaX-motif, remains an important strategy to inhibit Ras function in cancers. In this study, we investigate the role ICMT plays in the initiation of human epithelial cancers involving constitutively-active mutant Ras using genetic manipulation strategies. Methods Small T antigen and a shRNA against p53 (shp53) were sequentially expressed in human mammary epithelial cells immortalized by hTERT (HME1-hTERT) to create pre-malignant HME1-shp53 cells. All 4 isoforms of wild-type or mutant Ras, namely H-Ras, N-Ras, K-Ras-4A and K-Ras-4B, were independently introduced into HME1-shp53 cells to determine their transforming ability. Using CRISPR-Cas9 genome editing technology, ICMT-null HME1-shp53 cells were generated and mutant Ras isoforms were subsequently introduced; tumor formation abilities of thus generated cells were determined using soft-agar anchorage-independent colony formation assay and subcutaneous xenografts in mice. ICMT knock-out was also performed in MiaPaCa-2 and MDA-MB-231 cells, both expressing mutant K-Ras, and subcutaneous xenograft tumor formation in mice was measured. Results Pre-malignant HME1-shp53 cells can be transformed by all mutant Ras isoforms but not by any wild-type Ras. ICMT loss-of-function dramatically reduced anchorage-independent colony formation caused by all mutant Ras isoforms in HME1-shp53 cells, and in vivo xenograft formation was completely abolished. ICMT loss-of-function also dramatically reduced in vivo xenograft formation for mutant K-Ras-expressing MiaPaCa-2 and MDA-MB-231 naturally-occurring cancer cells. Conclusion ICMT function is essential for the malignant transformation of HME1 cells by all isoforms of Ras. Furthermore, ICMT function is also crucial for the maintenance of the malignant phenotype in mutant K-Ras-expresing cells such as MiaPaCa-2 and MDA-MB-231 cells. Citation Format: Hiu Yeung Lau, Jingyi Tang, Patrick J. Casey, Mei Wang. Isoprenylcysteine carboxylmethyltransferase (ICMT) function is critical for epithelial malignant transformation by mutant-Ras [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1371. doi:10.1158/1538-7445.AM2017-1371