Graduation Date

Spring 5-6-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Genetics, Cell Biology & Anatomy

First Advisor

Dr.Jenny Wang

Abstract

TGFβ signaling is an important regulator in colon cancer. miRNAs regulate TGFβ signaling at multiple levels. In this study, through a functional screening, we identified miR-487b-3p and miR-656-3p, which modulate TGFβ effect in colon cancer cells. Further studies revealed that GRM3 and VGLUT3 are their respective targets.

GRM3, a Metabotropic glutamate receptor in glutamatergic pathway is significantly upregulated in majority of human colonic adenocarcinomas tested and colon cancer cell lines. Knockdown of GRM3 expression or inhibition of GRM3 activation in colon cancer cells reduces cell survival and anchorage-independent growth in vitro and inhibits tumor growth in vivo. Mechanistically, GRM3 antagonizes TGFβ-mediated activation of protein kinase A and inhibition of AKT. In addition, TGFβ signaling increases GRM3 protein stability and knockdown of GRM3 enhances TGFβ-mediated tumor suppressor function. Since miR-487b-3p directly targets GRM3, overexpression of miR-487b-3p mimics the effects of GRM3 knockdown in vitro and in vivo. Expression of miR-487b-3p is decreased in colon adenocarcinomas and inversely correlates with GRM3 expression.

VGLUT3, a vesicular glutamate transporter, is also markedly upregulated in human colonic adenocarcinomas and colon cancer cell lines. Knockdown of VGLUT3 expression in colon cancer cells reduces cell survival and anchorage-independent growth in vitro and inhibits tumor growth in vivo. Mechanistically, VGLUT3 antagonizes TGFβ-mediated suppression of cell survival and clonogenicity by maintaining AKT activation. MiR-656-3p represses VGLUT3 expression and mimics the effects of VGLUT3 knockdown in vitro and in vivo. Moreover, expression of miR-656-3p is decreased in colon cancer specimens and inversely correlates with VGLUT3 expression.

This is particularly interesting and important from a therapeutic standpoint because numerous glutamatergic signaling inhibitor, many of which have been found unsuitable for treatment of neuropsychiatric disorders for reasons such as inability to readily penetrate blood brain barriers. Since GRM3 and VGLUT3 are upregulated in colon cancer, but rarely expressed in normal peripheral tissues, targeting GRM3 and VGLUT3 with such agents would not likely cause adverse neurological or peripheral side effects, making them attractive and specific molecular targets for colon cancer treatment.

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