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 1

LIN28B promotes cell migration and collective cell invasion in colon cancer cells.

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LIN28B promotes cell migration and collective cell invasion in colon can...
(A) A comparison of wound closure in DLD-1 cells with an empty vector versus LIN28B overexpression (left) and in Caco-2 cells with shControl versus shLIN28B for LIN28B knockdown. Scale bar represents 200 μm. (B) The wound area in A is quantified using ImageJ (NIH). The wound-healing rate is defined as (initial wound area – wound area at 24 or 48 hours)/initial wound area. Data were analyzed using a 2-tailed Student’s t test or 1-way ANOVA and are represented as mean ± SEM (n = 7). (C) A schematic model of the tumor spheroid 3D invasion assay. (D) 3D invasion of DLD-1 cells with an empty vector versus LIN28B overexpression and of Caco-2 cells with shControl versus shLIN28B is shown at day 0 and day 7. Scale bar = 500 μm. (E) The area of the tumor cells in D is quantified using ImageJ. Data were analyzed using a 2-tailed Student’s t test or 1-way ANOVA, expressed relative to the corresponding value at day 0, and are represented as mean ± SEM (n = 3). *P < 0.05, ****P < 0.0001.