Polyomavirus Small T Antigen Induces Apoptosis in Mammalian Cells through the UNC5B Pathway in a PP2A-Dependent Manner

Abstract

UNC5B is a dependence receptor that promotes survival in the presence of its ligand, netrin-1, while inducing cell death in its absence. The receptor has an important role in the development of the nervous and vascular systems. It is also involved in the normal turnover of intestinal epithelium. Netrin-1 and UNC5B are deregulated in multiple cancers, including colorectal, neuroblastoma, and breast tumors. However, the detailed mechanism of UNC5B function is not fully understood. We have utilized the murine polyomavirus small T antigen (PyST) as a tool to study UNC5B-mediated apoptosis. PyST is known to induce mitotic arrest followed by extensive cell death in mammalian cells. Our results show that the expression of PyST increases mRNA levels of UNC5B by approximately 3-fold in osteosarcoma cells (U2OS) and also stabilizes UNC5B at the posttranslational level. Furthermore, UNC5B is upregulated predominantly in those cells that undergo mitotic arrest upon PyST expression. Interestingly, although its expression was previously reported to be regulated by p53, our data show that the increase in UNC5B levels by PyST is p53 independent. The posttranslational stabilization of UNC5B by PyST is regulated by the interaction of PyST with PP2A. We also show that netrin-1 expression, which is known to inhibit UNC5B apoptotic activity, promotes survival of PyST-expressing cells. Our results thus suggest an important role of UNC5B in small-T antigen-induced mitotic catastrophe that also requires PP2A. IMPORTANCE UNC5B, PP2A, and netrin-1 are deregulated in a variety of cancers. UNC5B and PP2A are regarded as tumor suppressors, as they promote apoptosis and are deleted or mutated in many cancers. In contrast, netrin-1 promotes survival by inhibiting dependence receptors, including UNC5B, and is upregulated in many cancers. Here, we show that UNC5B-mediated apoptosis can occur independently of p53 but in a PP2A-dependent manner. A substantial percentage of cancers arise due to p53 mutations and are insensitive to chemotherapeutic treatments that activate p53. Unexpectedly, treatment of cancers having functional p53 with many conventional drugs leads to the upregulation of netrin-1 through activated p53, which is counterintuitive. Therefore, understanding the p53-independent mechanisms of the netrin-UNC5B axis, such as those involving PP2A, assumes greater clinical significance. Anticancer strategies utilizing anti-netrin-1 antibody treatment are already in clinical trials.

Interaction of DBC1 with polyoma small T antigen promotes its degradation and negatively regulates tumorigenesis

“…Addition of doxycycline causes the expression of PyST in these cells and leads to the appearance of a remarkable rounding up phenotype within 16 to 20 h, a feature usually associated with cells undergoing mitosis ( 31 , 32 ). Prolonged induction of small T expression (>24 h) leads to the initiation of apoptosis, which kills most of the cells within 48 h ( 4 ).…”

“…Because the expression of PyST causes down-regulation of DBC1, it meant that PyST expressing cells would have higher amounts of TRB3 mRNA and hence reduced the phosphorylation of AKT1 at S473. We had previously performed whole genome microarray analysis on mRNAs obtained from the inducible PyST expressing cells (pTREX-PyST-HA-Flag), using Human Genome U133 Plus 2 Array ( 4 ). Microarray data revealed that PyST expressing cells (+dox) had about 2.8 fold increase in TRB3 mRNA levels ( Fig.…”

“…For inducing mitotic arrest and apoptosis, interaction of PyST with PP2A is essential. We have recently shown that small T induced apoptosis in host mammalian cells also involves the activation of dependence receptor UNC5B, in a PP2A dependent manner ( 4 ). However, the molecular details of PyST-induced mitotic arrest are not fully understood yet.…”

PP2A Phosphatase as an Emerging Viral Host Factor

“…sT and large T antigen (LT) of SV40, merkell cell polyomavirus (MCV), or Murine polyoma virus (Py) are alternatively-spliced oncoproteins which play a role in the transformation of infected cells ( Rundell and Parakati, 2001 ; Yu et al., 2001 ; Kwun et al., 2009 ). This property of sT, however, is fully reliant on its binding to the scaffold subunit of PP2A ( Yang et al., 1991 ; Ruediger et al., 1992 ), which displaces the regulatory subunit ( Pallas et al., 1990 ; Chen et al., 2004 ) and therefore inhibits PP2A-mediated dephosphorylation of many substrates (with the known exception of histone H1) and promotes viral (DNA) replication ( Scheidtmann et al., 1991 ; Sablina et al., 2007 ; Kwun et al., 2009 ; Bollag et al., 2010 ; Bhat et al., 2020 ). A close dissection of this interaction has been made possible through the crystal structure of the SV40 sT in complex with the Aα subunit of PP2A ( Cho et al., 2007 ).…”

“…Examples include the E7 protein of human papillomaviruses (HPV) ( Pim et al., 2005 ) or the leader protein (EBNA-LP) encoded by the Epstein-Barr virus (EBV) ( Garibal et al., 2007 ). At the same time, other viruses target PP2A to drive apoptosis, such as the E4orf4 protein of adenovirus ( Kleinberger and Shenk, 1993 ) and the Py sT-antigen ( Bhat et al., 2020 ).…”