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 3

CDX2 regulates CRC tumor differentiation in the context of LIN28B overexpression.

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CDX2 regulates CRC tumor differentiation in the context of LIN28B overex...
(A) WB showing CDX2 KD using shCDX2 in Caco2 cells. Lower graph shows the densitometry normalized to GAPDH (n = 3). (B) CDX2 and ALPi expression (qPCR) in Caco-2 control/CDX2 KD cells (n = 3). (C) ALP activity assay in Caco-2 control/CDX2 KD cells (n = 3). (D) Upper: representative image for dome formation in Caco-2 control/CDX2 KD cells at postconfluence day 3. Scale bars: 500 μm. Right: the graph indicates the number of domes. A dome was defined as greater than 100 μm diameter (n = 5). (E) Upper: WB analysis of CK20 and CDX2 expression in Caco-2 cells with sh-control or sh-CDX2 at the confluence time point. Lower: densitometry. The value for CK20 at day 2 for the sh-control samples was referred to as 1 (n = 3). (F) Representative IHC in the subcutaneous xenograft tumor of Caco-2 cells with control or CDX2 KD for H&E (first panel), IHC of CDX2 (second and third panels), and IHC of LIN28B (fourth panel). (G) Cumulative ratio of differentiation status in subcutaneous xenograft tumors (n = 6). (H) Representative IHC of CK20 in the subcutaneous xenograft tumor of Caco-2 cells with control or CDX2 KD. (I) Representative ALP staining in the subcutaneous xenograft tumor of Caco-2 cells with control or CDX2 KD. Scale bars: 100 μm. Data are presented as mean ± SEM. Unpaired, 2-tailed Student’s t test (A, D, E, and G) or 1-way ANOVA followed by Dunnett’s multiple-comparison test as post hoc analysis (B and C) were performed. *P < 0.05, **P < 0.01.