LIN28B promotes cell invasion and colorectal cancer metastasis via CLDN1 and NOTCH3
Kensuke Sugiura, Yasunori Masuike, Kensuke Suzuki, Alice E. Shin, Nozomu Sakai, Hisahiro Matsubara, Masayuki Otsuka, Peter A. Sims, Christopher J. Lengner, Anil K. Rustgi
Kensuke Sugiura, Yasunori Masuike, Kensuke Suzuki, Alice E. Shin, Nozomu Sakai, Hisahiro Matsubara, Masayuki Otsuka, Peter A. Sims, Christopher J. Lengner, Anil K. Rustgi
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Research Article Gastroenterology Oncology

LIN28B promotes cell invasion and colorectal cancer metastasis via CLDN1 and NOTCH3

Abstract

The RNA-binding protein LIN28B is overexpressed in over 30% of patients with colorectal cancer (CRC) and is associated with poor prognosis. In the present study, we unraveled a potentially novel mechanism by which LIN28B regulates colonic epithelial cell-cell junctions and CRC metastasis. Using human CRC cells (DLD-1, Caco-2, and LoVo) with either knockdown or overexpression of LIN28B, we identified claudin 1 (CLDN1) tight junction protein as a direct downstream target and effector of LIN28B. RNA immunoprecipitation revealed that LIN28B directly binds to and posttranscriptionally regulates CLDN1 mRNA. Furthermore, using in vitro assays and a potentially novel murine model of metastatic CRC, we show that LIN28B-mediated CLDN1 expression enhances collective invasion, cell migration, and metastatic liver tumor formation. Bulk RNA sequencing of the metastatic liver tumors identified NOTCH3 as a downstream effector of the LIN28B/CLDN1 axis. Additionally, genetic and pharmacologic manipulation of NOTCH3 signaling revealed that NOTCH3 was necessary for invasion and metastatic liver tumor formation. In summary, our results suggest that LIN28B promotes invasion and liver metastasis of CRC by posttranscriptionally regulating CLDN1 and activating NOTCH3 signaling. This discovery offers a promising new therapeutic option for metastatic CRC to the liver, an area where therapeutic advancements have been relatively scarce.

Authors

Kensuke Sugiura, Yasunori Masuike, Kensuke Suzuki, Alice E. Shin, Nozomu Sakai, Hisahiro Matsubara, Masayuki Otsuka, Peter A. Sims, Christopher J. Lengner, Anil K. Rustgi

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Figure 2

LIN28B directly binds to and stabilizes CLDN1 mRNA.

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LIN28B directly binds to and stabilizes CLDN1 mRNA. (A) Western blot ana...
(A) Western blot analysis of adherens and tight junctions from DLD-1 and Caco-2 cells. (B) Quantification of band densities measured by Western blot in A, normalized to GAPDH. Data were analyzed using a 2-tailed Student’s t test or 1-way ANOVA, expressed relative to the corresponding value in empty vector or control groups, and represented as means ± SEM (n = 3). (C) Immunofluorescence staining of CLDN1 (shown in red) in DLD-1 and Caco-2 cells. Nuclei were stained by DAPI (shown in blue). Scale bar = 100 μm. (D) Representative images of Western blots of samples precipitated using IgG or anti-LIN28B antibodies. (E) qRT-PCR analysis of claudin-1 mRNA in RNA immunoprecipitation samples. Data were analyzed using a 2-tailed Student’s t test, expressed relative to the corresponding value of IgG IP samples, and represented as means ± SEM (n = 3). (F) Quantification of claudin-1 mRNA after actinomycin D treatment. Data were analyzed using a 2-way ANOVA, expressed relative to the corresponding value at 0 hour, and represented as means ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. qRT-PCR, quantitative real-time PCR.