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 7

Pharmacologic inhibition of NOTCH3 reduces LIN28B-induced liver metastasis.

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Pharmacologic inhibition of NOTCH3 reduces LIN28B-induced liver metastas...
(A) qRT-PCR analysis of NOTCH3 and HES1 mRNA in DLD-1 LIN28Bhi cells treated with vehicle or 10 μg/mL MOR20350 for 48 hours. (B) Representative images and quantification of the wound closure scratch assay performed using DLD-1 LIN28Bhi cells treated with vehicle or MOR20350. Scale bar = 200 μm. Area of the wound was measured by using ImageJ. (C) Representative images and quantification of the 3D aggregation assay performed using DLD-1 LIN28Bhi cells treated with the vehicle or MOR20350. The number of cell aggregates was counted using Keyence BZ-X810. (D) Representative images and quantification of 2D invasion assay. Cells that have invaded through the 8 μm pore and the ECM were counted using Keyence BZ-X810. (E) Representative images and quantification of 2D migration assay. Cells that have migrated through the 8 μm pore were counted using Keyence BZ-X810. (F) Representative images and quantification of the 3D invasion assay performed using DLD-1 LIN28Bhi cells treated with the vehicle or MOR20350. Scale bar = 500 μm. (G) Representative images of RFP expressed by DLD-1 LIN28Bhi tumors in the liver. Images were taken using Keyence BZ-X810. Scale bar = 500 μm. Data in graphs A–F are represented as means ± SEM and were analyzed by 1-way ANOVA followed by Tukey’s multiple-comparison test. Data in G were analyzed by Fisher’s exact test. *P < 0.05, ***P < 0.001.