Mucinous Intestinal Tumorigenesis by Deregulation of CRAD-Controlled Cytoskeleton Dynamics


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Youn-Sang Jung1,Wenqi Wang2,Sohee Jun1,Jie Zhang1,Mrinal Srivastava1,Moon Jong Kim1,Esther Lien1,Joan Shang1,Junjie Chen1,Pierre McCrea1,Songlin Zhang3,Jae-Il Park1
1UT MD Anderson Cancer Center,2University of California, Irvine,3The University of Texas Medical School

Abstract

Background

Epithelial monolayer integrity is maintained via cell-cell adhesion, cytoskeleton, and basement membrane interactions. Cell adhesion proteins and the cytoskeleton are intimately associated, with prime examples being the E-cadherin-catenin complex and F-actin. E-cadherin participates in cell adhesion and contact inhibition, being part of a large complex composed of catenins and additional proteins, that is stabilized by interactions with F-actin. F-actin polymerization is controlled by capping proteins (CPs) and CP regulators. CPs directly bind to and block the barbed (+) end of filaments or to ATP-G-actin, resulting in inhibition of actin assembly. Given that F-actin enhances the stability of the E-cadherin-catenin complex for the maintenance of epithelial cell integrity, we hypothesized that reduced epithelial cell integrity through deregulation of the cytoskeleton and the E-cadherin-catenin complex contributes to intestinal tumorigenesis. Recently, it was reported that CRAD is markedly mutated in small cell lung cancer, having a ranking of third following TP53 and RB1.

Method

CRAD KO mice were generated using CRISPR/Cas9 gene editing system. CRAD KO mice and cancer cells were employed for IHC, In vitro actin polymerization, reporter assays, F-/G-actin fractionation, Xenograft transplantation assays, mass spectrometry, and organoid assays.

Results

i) CRAD is inactivated by mutation or transcriptional downregulation in CRC. 

ii) CRAD enhances actin cytoskeletal dynamics by binding and inhibiting CPs.

iii) CRAD inactivation hyperactivates Wnt signaling by destabilizing CCA complex.

iv) CRAD KO induces and accelerates mucinous intestinal tumorigenesis in mice.

Conclusion

Our study reveals that CRAD is a tumor suppressor and indispensable for the maintenance of epithelial cell integrity through modulation of the cytoskeleton and thereby CCA complex. Furthermore, our results elucidate that, in conjunction with APC inactivation, the deletion of CRAD gene leads to mucinous carcinoma (MC) development in both the small and large intestine, which further reveals a pathologic outcome of CRAD and APC mutations during MC tumorigenesis.