LIN28B induces a differentiation program through CDX2 in colon cancer
Kensuke Suzuki, Yasunori Masuike, Rei Mizuno, Uma M. Sachdeva, Priya Chatterji, Sarah F. Andres, Wenping Sun, Andres J. Klein-Szanto, Sepideh Besharati, Helen E. Remotti, Michael P. Verzi, Anil K. Rustgi
Kensuke Suzuki, Yasunori Masuike, Rei Mizuno, Uma M. Sachdeva, Priya Chatterji, Sarah F. Andres, Wenping Sun, Andres J. Klein-Szanto, Sepideh Besharati, Helen E. Remotti, Michael P. Verzi, Anil K. Rustgi
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Research Article Cell biology Gastroenterology

LIN28B induces a differentiation program through CDX2 in colon cancer

Abstract

Most colorectal cancers (CRCs) are moderately differentiated or well differentiated, a status that is preserved even in metastatic tumors. However, the molecular mechanisms underlying CRC differentiation remain to be elucidated. Herein, we unravel a potentially novel posttranscriptional regulatory mechanism via a LIN28B/CDX2 signaling axis that plays a critical role in mediating CRC differentiation. Owing to a large number of mRNA targets, the mRNA-binding protein LIN28B has diverse functions in development, metabolism, tissue regeneration, and tumorigenesis. Our RNA-binding protein IP (RIP) assay revealed that LIN28B directly binds CDX2 mRNA, which is a pivotal homeobox transcription factor in normal intestinal epithelial cell identity and differentiation. Furthermore, LIN28B overexpression resulted in enhanced CDX2 expression to promote differentiation in subcutaneous xenograft tumors generated from CRC cells and metastatic tumor colonization through mesenchymal-epithelial transition in CRC liver metastasis mouse models. A ChIP sequence for CDX2 identified α-methylacyl-CoA racemase (AMACR) as a potentially novel transcriptional target of CDX2 in the context of LIN28B overexpression. We also found that AMACR enhanced intestinal alkaline phosphatase activity, which is known as a key component of intestinal differentiation, through the upregulation of butyric acid. Overall, we demonstrated that LIN28B promotes CRC differentiation through the CDX2/AMACR axis.

Authors

Kensuke Suzuki, Yasunori Masuike, Rei Mizuno, Uma M. Sachdeva, Priya Chatterji, Sarah F. Andres, Wenping Sun, Andres J. Klein-Szanto, Sepideh Besharati, Helen E. Remotti, Michael P. Verzi, Anil K. Rustgi

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Figure 1

LIN28B upregulates CDX2 expression in colorectal cancer.

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LIN28B upregulates CDX2 expression in colorectal cancer. (A) LIN28B and ...
(A) LIN28B and CDX2 expression in human colorectal cancer (CRC) cell lines by Western blotting (WB) analysis. Lower: the fold change of band intensity compared with the expression of target gene in Caco-2 cells (n = 3). (B) WB analysis of LIN28B and CDX2 in Caco-2 control and LIN28B knockdown (KD) cells. Lower: band intensities were normalized by densitometry to GAPDH (n = 3). (C) WB analysis of LIN28B and CDX2 in Caco-2 LIN28B KD and LIN28B long isoform expression (LIN28B o/e) cells. Lower: band intensities were normalized by densitometry to GAPDH (n = 3). (D) Representative IHC staining for LIN28B (left) and CDX2 (right) in the subcutaneous xenograft tumor of Caco-2 cells with control or LIN28B KD. Scale bars: 100 μm. (E) Upper: WB analysis of CK20 and CDX2 expression in Caco-2 cells with control or sh-LIN28B at the confluence time point. Lower: densitometry analysis. The value for CK20 or CDX2 at day –2 for the sh-control samples was referred to as 1. (F) Upper: representative image for dome formation in Caco-2 cell with control/LIN28B KD at postconfluence day 3. Scale bars: 500 μm. Lower: the graph indicates the number of domes. A dome was defined as greater than 100 μm diameter (n = 5). (G) ALP activity assay in Caco-2 control and LIN28B KD cells at postconfluence day 3 (n = 3). Data are presented as mean ± SEM. Unpaired, 2-tailed Student’s t tests were performed. *P < 0.05, **P < 0.01.