Objective Despite the recent advances in treatment of colon cancer, the prognosis is unfavourable for patients with distant metastases. The aim of this study was to identify targets for prevention and/or therapy of colon cancer metastasis.
Design CMT93 cells, a murine rectal cancer cell line with poor metastasising activity, were transduced with lentiviral shRNA library and transplanted into the rectum of syngeneic C57BL/6 mice. Genomic DNA was collected from metastatic lesions, and the integrated shRNA were retrieved by PCR for sequencing, followed by identification of the candidate genes targeted by the shRNA.
Results The genome-wide shRNA library screen identified Hnrnpll (heterogeneous nuclear ribonucleoprotein L-like) encoding a pre-mRNA splicing factor as a candidate metastasis suppressor gene. Knockdown of Hnrnpll enhanced matrigel invasion activity of colon cancer cells in vitro, as well as their metastatic ability in vivo. An RNA-immunoprecipitation analysis showed Hnrnpll-binding to Cd44 pre-mRNAs, and the level of Cd44 variable exon 6 (Cd44v6), a poor prognosis marker of colorectal cancer, was increased by knocking down Hnrnpll. A neutralising Cd44v6 antibody suppressed the matrigel invasion ability induced by Hnrnpll knockdown. HNRNPLL expression was downregulated when colon cancer cells were induced to undergo epithelial-mesenchymal transition (EMT). Immunohistochemistry of clinical samples indicated that colorectal cancer cells with low E-cadherin expression at the invasion front exhibited decreased HNRNPLL expression.
Conclusions HNRNPLL is a novel metastasis suppressor of colorectal cancer, and modulates alternative splicing of CD44 during EMT.
- COLORECTAL CANCER
- MOLECULAR MECHANISMS
- COLORECTAL METASTASES
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Contributors MA and KS designed the study, analysed and interpreted the data, and wrote the manuscript. KS carried out the experiments. ES, KKi, KKo, YS and YY provided clinical samples. All authors have seen and approved the final version of the manuscript.
Funding This work was supported in part by Grant-in-Aid for Challenging Exploratory Research (24650627) and Grant-in-Aid for Scientific Research B (26290045) from the Japan Society for the Promotion of Science, grants from the Naito Foundation, Suzuken Memorial Foundation, Foundation for Promotion of Cancer Research, and Nagono Medical Foundation, Japan.
Competing interests None declared.
Ethics approval All the animal experiments were conducted according to the protocol approved by the Animal Care and Use Committee of Aichi Cancer Center Research Institute. This study was carried out in accordance with the institutional ethics committee of Aichi Cancer Center.
Provenance and peer review Not commissioned; externally peer reviewed.