KRAS mutation leads to decreased expression of regulator of calcineurin 2, resulting in tumor proliferation in colorectal cancer

H Niitsu,W Yasui,H Ohdan,M Shimomura,T Adachi,Y Saito,M Kochi,T Hinoi,N Oue,H Sada,M Miguchi,Y Sotomaru,K Sentani,Y Kawaguchi,Y Kitadai,R Yuge

doi:10.1038/oncsis.2016.47

Abstract

KRAS mutations occur in 30–40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apcflox/flox; LSL-KrasG12D and CDX2P-G22Cre;Apcflox/flox mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription–PCR (qRT–PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT–PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin–nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin–NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC.

Highlights

It is widely known that mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene occur in the early stages of the adenoma–adenocarcinoma sequence in human colorectal cancer (CRC) development.[1]
Calcineurin, which was first cloned in neural cells as a Ca2+- and calmodulin-dependent serine/threonine protein phosphatase,[10,11,12] dephosphorylates nuclear factor of activated T cells (NFAT)
We identified a novel gene, regulator of calcineurin 2 (RCAN2), that was found to be downregulated in the tumors in Kras-mutated mouse models, which may inhibit calcineurin enzyme activity in CRC and exhibit tumor suppressor function

Summary

Introduction

It is widely known that mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene occur in the early stages of the adenoma–adenocarcinoma sequence in human colorectal cancer (CRC) development.[1]. KRAS and NRAS, which have been shown to exhibit somatic mutations in 30–40% and 2–5% of human CRCs, respectively,[2,3,4] are known predictors for resistance to treatment with anti-epidermal growth factor receptor antibodies.[5,6,7,8] these biomarkers have enabled the development of individualized therapies, especially for the treatment of CRCs with wild-type RAS, specific therapeutic agents against RAS-mutated CRC have not yet been established. RAS mutations cause oncogenic activation independent of epidermal growth factor– epidermal growth factor receptor signal transmission, resulting in a lack of response to anti-epidermal growth factor receptor antibodies.[9] To overcome this resistance mechanism, the identification of downstream molecules that show potential as new therapeutic targets for RAS-mutated CRC has become an important focus. NFAT subsequently translocates into the nucleus, where it acts as a transcription factor that promotes various cellular activities.[13,14,15]

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