UBXN2A, a Ubiquitin-Like Protein, Alters Proteins in mTORC2 Pathway

Introduction: Colorectal cancer (CRC) is the third leading cause of cancer death in the United States causing approximately 50,000 deaths annually. Metastasis is a characteristic feature of CRC tumors, and is dominantly responsible for the high mortality of CRC patients. Thus, an urgent need exists for new therapies for patients with metastatic CRC. Recent studies indicate that the mTORC2 signaling pathway plays a fundamental role in formation and progression of CRC. The mTORC2 complex contains mTOR, mLST8 (GβL), mSIN1, deptor, protor-1, and Rictor. The aim of this study was to investigate whether UBXN2A, a known tumor suppressor protein, regulates protein turnover within the mTORC2 complex and suppresses the mTORC2 downstream signaling cascade.

Methods: A set of biological assays including western blot was used to determine the turnover of proteins in the mTORC2 complex in the presence and absence of overexpressed UBXN2A. Western blot of human colon cancer cells was used to determine the relationship between UBXN2A levels and members of mTORC2 complex, including Rictor. xCELLigence software was used to measure cell migration, an important component of tumor metastasis. Flow cytometry analysis was used to determine the level of colon cancer stem cells in the presence and the absence of veratridine (VTD), a natural plant alkaloid known to upregulate UBXN2A.

Results: This study revealed that overexpression of UBXN2A protein decreases Rictor protein levels in a human metastatic cell line. Consequently, SGK1, a protein located downstream of mTORC2 pathway, decreases in the presence of UBXN2A induced by VTD. VTD was also shown to decrease migration of colon cancer and downregulate CD44+ and LgR5+ cancer stem cell populations. Furthermore, UBXN2A induction increases the turnover of Rictor protein, an effect that is reversed by inhibition of the proteasome complex. These results suggest that upregulation of UBXN2A downregulates a key protein in the mTORC2 complex, resulting in decreased tumorigenic and metastatic functions of CRC cells.

Conclusions: This study demonstrated that VTD-dependent upregulation of UBXN2A targets mTORC2 by targeting Rictor protein, a critical member of mTORC2 complex. By targeting mTORC2 complex, UBXN2A suppresses mTORC2 downstream pathway as well as cancer stem cells essential for tumor metastasis. VTD's anti-migration and anti-cancer stem cell functions can be turned into a potential new-targeted therapy in patients with colon cancer.