Subbareddy Maddika

Interconnections between apoptotic, autophagic and necrotic pathways: implications for cancer therapy development

The rapid accumulation of knowledge on apoptosis regulation in the 1990s was followed by the development of several experimental anticancer‐ and anti‐ischaemia (stroke or myocardial infarction) drugs. Activation of apoptotic pathways or the removal of cellular apoptotic inhibitors has been suggested to aid cancer therapy and the inhibition of apoptosis was thought to limit ischaemia‐induced damage. However, initial clinical studies on apoptosis‐modulating drugs led to unexpected results in different clinical conditions and this may have been due to co‐effects on non‐apoptotic interconnected cell death mechanisms and the ‘yin‐yang’ role of autophagy in survival versus cell death. In this review, we extend the analysis of cell death beyond apoptosis. Upon introduction of molecular pathways governing autophagy and necrosis (also called necroptosis or programmed necrosis), we focus on the interconnected character of cell death signals and on the shared cell death processes involving mitochondria (e.g. mitophagy and mitoptosis) and molecular signals playing prominent roles in multiple pathways (e.g. Bcl2‐family members and p53). We also briefly highlight stress‐induced cell senescence that plays a role not only in organismal ageing but also offers the development of novel anticancer strategies. Finally, we briefly illustrate the interconnected character of cell death forms in clinical settings while discussing irradiation‐induced mitotic catastrophe. The signalling pathways are discussed in their relation to cancer biology and treatment approaches.

WWP2 is an E3 ubiquitin ligase for PTEN

PTEN, a lipid phosphatase, is one of the most frequently mutated tumour suppressors in human cancer. Several recent studies have highlighted the importance of ubiquitylation in regulating PTEN tumour-suppressor function, but the enzymatic machinery required for PTEN ubiquitylation is not clear. In this study, by using a tandem affinity-purification approach, we have identified WWP2 (also known as atrophin-1-interacting protein 2, AIP-2) as a PTEN-interacting protein. WWP2 is an E3 ubiquitin ligase that belongs to the NEDD4-like protein family, which is involved in regulating transcription, embryonic stem-cell fate, cellular transport and T-cell activation processes. We show that WWP2 physically interacts with PTEN and mediates its degradation through a ubiquitylation-dependent pathway. Functionally, we show thatxa0WWP2xa0controls cellular apoptosis and is required for tumorigenicity of cells. Collectively, our results reveal a functional E3 ubiquitin ligase for PTEN that plays a vital role in tumour-cell survival.

Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic β-cells

Glucagon-like peptide-1 receptor (GLP-1R) plays a major role in promoting glucose-stimulated insulin secretion in pancreatic β-cells. In the present study, we synthesized a novel functional analog of GLP-1 conjugated to tetramethyl rhodamine to monitor the internalization of the receptor. Our data show that after being internalized the receptor is sorted to lysosomes. In endosomes, receptor-ligand complex is found to be colocalized with adenylate cyclase. Pharmacological inhibition of endocytosis attenuates GLP-1R-mediated cAMP generation and consequent downstream protein kinase A substrate phosphorylation and glucose-stimulated insulin secretion. Our study underlines a paradigm shift in GLP-1R signaling and trafficking. The receptor ligand complex triggers cAMP generation both in plasma membrane and in endosomes, which has implications for receptor-mediated regulation of insulin secretion.

Deubiquitylation and stabilization of PTEN by USP13

The tumour suppressor PTEN is frequently lost in human cancers. In addition to gene mutations and deletions, recent studies have revealed the importance of post-translational modifications, such as ubiquitylation, in the regulation of PTEN stability, activity and localization. However, the deubiquitylase that regulates PTEN polyubiquitylation and protein stability remains unknown. Here we screened a total of 30 deubiquitylating enzymes (DUBs) and identified five DUBs that physically associate with PTEN. One of them, USP13, stabilizes the PTEN protein through direct binding and deubiquitylation of PTEN. Loss of USP13 in breast cancer cells promotes AKT phosphorylation, cell proliferation, anchorage-independent growth, glycolysis and tumour growth through downregulation of PTEN. Conversely, overexpression of USP13 suppresses tumorigenesis and glycolysis in PTEN-positive but not PTEN-null breast cancer cells. Importantly, USP13 protein is downregulated in human breast tumours and correlates with PTEN protein levels. These findings identify USP13 as a tumour-suppressing protein that functions through deubiquitylation and stabilization of PTEN.

Synthesis and evaluation of 3-amino/guanidine substituted phenyl oxazoles as a novel class of LSD1 inhibitors with anti-proliferative properties

A series of functionalized phenyl oxazole derivatives was designed, synthesized and screened in vitro for their activities against LSD1 and for effects on viability of cervical and breast cancer cells, and in vivo for effects using zebrafish embryos. These compounds are likely to act via multiple epigenetic mechanisms specific to cancer cells including LSD1 inhibition.

WD Repeat Protein WDR48 in Complex with Deubiquitinase USP12 Suppresses Akt-dependent Cell Survival Signaling by Stabilizing PH Domain Leucine-rich Repeat Protein Phosphatase 1 (PHLPP1)

Background: PHLPP1 is a tumor suppressor phosphatase. Results: WDR48 in complex with USP12 controls cell survival signaling by deubiquitinating PHLPP1. Conclusion: WDR48 acts as a potential tumor suppressor by positively regulating PHLPP1 stability. Significance: WDR48 was identified as novel PHLPP1 regulator, which is critical in understanding its role as tumor suppressor in human cancers. PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1) is a protein-serine/threonine phosphatase and a negative regulator of the PI3-kinase/Akt pathway. Although its function as a suppressor of tumor cell growth has been established, the mechanism of its regulation is not completely understood. In this study, by utilizing the tandem affinity purification approach we have identified WDR48 and USP12 as novel PHLPP1-associated proteins. The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability. Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis. Functionally, we show that WDR48 and USP12 suppress proliferation of tumor cells. Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression. Together, our results reveal WDR48 and USP12 as novel PHLPP1 regulators and potential suppressors of tumor cell survival.

PNUTS Functions as a Proto-Oncogene by Sequestering PTEN

PTEN is a well-defined tumor suppressor gene that antagonizes the PI3K/Akt pathway to regulate a multitude of cellular processes, such as survival, growth, motility, invasiveness, and angiogenesis. While the functions of PTEN have been studied extensively, the regulation of its activity during normal and disease conditions still remains incompletely understood. In this study, we identified the protein phosphatase-1 nuclear targeting subunit PNUTS (PPP1R10) as a PTEN-associated protein. PNUTS directly interacted with the lipid-binding domain (C2 domain) of PTEN and sequestered it in the nucleus. Depletion of PNUTS leads to increased apoptosis and reduced cellular proliferation in a PTEN-dependent manner. PNUTS expression was elevated in certain cancers compared with matched normal tissues. Collectively, our studies reveal PNUTS as a novel PTEN regulator and a likely oncogene.

TOPK and PTEN participate in CHFR mediated mitotic checkpoint.

Mitotic progression is regulated by co-ordinated action of several proteins and is crucial for the maintenance of genomic stability. CHFR (Check point protein with FHA and RING domains) is an E3 ubiquitin ligase and a checkpoint protein that regulates entry into mitosis. But the molecular players involved in CHFR mediated mitotic checkpoint are not completely understood. In this study, we identified TOPK/PBK, a serine/threonine kinase and PTEN, a lipid phosphatase to play an important role in CHFR mediated mitotic transitions. We demonstrated that CHFR ubiquitinates and regulates TOPK levels, which is essential for its checkpoint function. Moreover, TOPK phosphorylates and inactivates PTEN, which in turn activates Akt that leads to proper G2/M progression. Collectively, our results reveal TOPK and PTEN as new players in CHFR mediated mitotic checkpoint.

HACE1 mediated K27 ubiquitin linkage leads to YB-1 protein secretion

Ubiquitination is an important post-translational modification that is implicated in controlling almost every biological process by targeting cellular proteins to degradation. While the importance of ubiquitination in controlling the fate and the intracellular functions of various proteins was widely studied, its role in extracellular protein secretion has been unexplored so far. In this study, by using YB-1 (Y-box Binding protein 1) as a model protein, we showed that ubiquitination is required for its extracellular secretion. We also identified HACE1 as a specific E3 ligase that polyubiquitinates YB-1 through non-canonical K27 linked ubiquitin chains. Formation of these ubiquitin linkages on YB-1 is necessary for its interaction with Tumor Susceptibility Gene 101 (TSG101), a component of the Multi-Vesicular Body (MVB) pathway, which facilitates its secretion. Finally, we demonstrated that extracellular secreted YB-1 is a functional protein that acts to inhibit Transforming Growth Factor-Beta mediated epithelial to mesenchymal transition. In summary, we identified a novel functional role for non-canonical ubiquitin linkages in mediating protein secretion.

14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein

Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ−/− cells. 14-3-3σ−/− cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene.