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 8

LIN28B/CLDN1/NOTCH3 axis positively correlates with metastatic progression of human colorectal tumors.

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LIN28B/CLDN1/NOTCH3 axis positively correlates with metastatic progressi...
(A) Primary colonic tumors and liver metastases were collected from patients with CRC. Upon IHC staining, tumors were quantified based on their high or low expression. Data expressed as a proportion of all tumors (%). (B) Representative images of tumors stained with an anti-LIN28B antibody. Scale bar = 100 μm. (C) Representative images of primary colorectal tumors stained with LIN28B, CLDN1, and NOTCH3. Scale bar = 100 μm. (D) Representative images of corresponding liver metastases stained with LIN28B, CLDN1, and NOTCH3. Scale bar = 100 μm. (E) Graph depicting disease-free survival of all patients with CRC. For all patients, frequency with which they developed liver metastases was tracked over 5 years. Data expressed as the proportion of patients with CRC who did not have metastatic liver tumor (y axis) at the time of analyses (x axis). (F) Graph depicting disease-free survival of patients with CRC who had undergone hepatectomy due to liver metastases. For all patients, frequency with which they developed a new liver tumor was tracked over 5 years. Data expressed as the proportion of patients with CRC who did not develop another metastatic liver tumor (y axis) at the time of analyses (x axis). “Others” group (blue line) includes patients with tumors that do not have high expressions of LIN28B, CLDN1, and NOTCH3. Data in graph in A were analyzed by χ2 test. Data in E and F were analyzed by log-rank test.