UBXN2A enhances CHIP-mediated proteasomal degradation of oncoprotein mortalin-2 in cancer cells.

Sanam Sane,John L Slunecka,Daniall Masood,Xuejun Wang,Rekha Srinivasan,Khosrow Rezvani,Yiyang Wang,Andre Hafner,Alex D Hanson,Taylor Kruisselbrink,Jun Yin,Collin J Noldner

doi:10.1002/1878-0261.12372

Abstract

Overexpression of oncoproteins is a major cause of treatment failure using current chemotherapeutic drugs. Drug‐induced degradation of oncoproteins is feasible and can improve clinical outcomes in diverse types of cancers. Mortalin‐2 (mot‐2) is a dominant oncoprotein in several tumors, including colorectal cancer (CRC). In addition to inactivating the p53 tumor suppressor protein, mot‐2 enhances tumor cell invasion and migration. Thus, mot‐2 is considered a potential therapeutic target in several cancer types. The current study investigated the biological role of a ubiquitin‐like protein called UBXN2A in the regulation of mot‐2 turnover. An orthogonal ubiquitin transfer technology followed by immunoprecipitation, in vitro ubiquitination, and Magnetic Beads TUBE2 pull‐down experiments revealed that UBXN2A promotes carboxyl terminus of the HSP70‐interacting protein (CHIP)‐dependent ubiquitination of mot‐2. We subsequently showed that UBXN2A increases proteasomal degradation of mot‐2. A subcellular compartmentalization experiment revealed that induced UBXN2A decreases the level of mot‐2 and its chaperone partner, HSP60. Pharmacological upregulation of UBXN2A using a small molecule, veratridine (VTD), decreases the level of mot‐2 in cancer cells. Consistent with the in vitro results, UBXN2A+/− mice exhibited selective elevation of mot‐2 in colon tissues. An in vitro Anti‐K48 TUBE isolation approach showed that recombinant UBXN2A enhances proteasomal degradation of mot‐2 in mouse colon tissues. Finally, we observed enhanced association of CHIP with the UBXN2A‐mot‐2 complex in tumors in an azoxymethane/dextran sulfate sodium‐induced mouse CRC model. The existence of a multiprotein complex containing UBXN2A, CHIP, and mot‐2 suggests a synergistic tumor suppressor activity of UBXN2A and CHIP in mot‐2‐enriched tumors. This finding validates the UBXN2A‐CHIP axis as a novel and potential therapeutic target in CRC.

Highlights

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States
We applied the orthogonal ubiquitin transfer (OUT) technique (Zhao et al, 2012) to identify the substrate proteins of carboxyl terminus of the HSP70-interacting protein (CHIP) E3 ubiquitin ligase in the cell
OUT is enabled by an engineered UB transfer cascade composed of xE1, xE2, and xE3 enzymes that are free of cross-reactivities with their native partners (‘x’ designates engineered enzymes orthogonal to native enzymes)

Summary

Introduction

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States. Understanding the mechanisms of metastatic programs and identifying their negative regulators in CRC cells are, critical steps in the development of novel therapies that will improve the management of advanced disease. Mot-2’s binding protein network allows this oncoprotein to contribute to several steps of tumor development, including sequestration and inactivation of the tumor suppressor protein p53, inhibition of pro-apoptotic proteins, alteration of the PI3K/AKT signaling pathway, activation of EMT (epithelial–mesenchymal transition), and contribution to cancer cell stemness (Black and Rezvani, 2016; Dundas et al, 2005; Gestl and Anne Bottger, 2012; Lu et al, 2011b; Na et al, 2016; Oki et al, 2011; Rozenberg et al, 2013; Wadhwa et al, 1998, 2006). Inactivation of mot-2 by siRNA as well as small molecules including withaferin A and MKT-007 results in growth arrest and induction of apoptosis in cancer cells (Grover et al, 2012; Wadhwa et al, 2000, 2004)

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Conclusion