PI3K-regulated Glycine N-methyltransferase is required for the development of prostate cancer.

Glycine N-Methyltransferase (GNMT) is a metabolic enzyme that integrates metabolism and epigenetic regulation. The product of GNMT, sarcosine, has been proposed as a prostate cancer biomarker. This enzyme is predominantly expressed in the liver, brain, pancreas, and prostate tissue, where it exhibits distinct regulation. Whereas genetic alterations in GNMT have been associated to prostate cancer risk, its causal contribution to the development of this disease is limited to cell line-based studies and correlative human analyses. Here we integrate human studies, genetic mouse modeling, and cellular systems to characterize the regulation and function of GNMT in prostate cancer. We report that this enzyme is repressed upon activation of the oncogenic Phosphoinositide-3-kinase (PI3K) pathway, which adds complexity to its reported dependency on androgen signaling. Importantly, we demonstrate that expression of GNMT is required for the onset of invasive prostate cancer in a genetic mouse model. Altogether, our results provide further support of the heavy oncogenic signal-dependent regulation of GNMT in prostate cancer.

The dystrophia myotonica WD repeat-containing protein DMWD and WDR20 differentially regulate USP12 deubiquitinase.

Despite its potential clinical relevance, the product of the DMWD (dystrophia myotonica, WD repeat containing) gene is a largely uncharacterized protein. The DMWD amino acid sequence is similar to that of WDR20, a known regulator of the USP12 and USP46 deubiquitinases (DUBs). Here, we apply a combination of in silico and experimental methods to investigate several aspects of DMWD biology. Molecular evolution and phylogenetic analyses reveal that WDR20 and DMWD, similar to USP12 and USP46, arose by duplication of a common ancestor during the whole genome duplication event in the vertebrate ancestor lineage. The analysis of public human gene expression datasets suggests that DMWD expression is positively correlated with USP12 expression in normal tissues and negatively correlated with WDR20 expression in tumors. Strikingly, a survey of the annotated interactome for DMWD and WDR20 reveals a largely nonoverlapping set of interactors for these proteins. Experimentally, we first confirmed that DMWD binds both USP12 and USP46 through direct coimmunoprecipitation of epitope-tagged proteins. We found that DMWD and WDR20 share the same binding interface in USP12, suggesting that their interaction with the DUB may be mutually exclusive. Finally, we show that both DMWD and WDR20 promote USP12 enzymatic activity, but they differentially modulate the subcellular localization of the DUB. Altogether, our findings suggest a model whereby mutually exclusive binding of DMWD and WDR20 to USP12 may lead to formation of deubiquitinase complexes with distinct subcellular localization, potentially targeting different substrate repertoires.

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions.

BACKGROUND: The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded. METHODS: In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes. RESULTS: In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction. CONCLUSIONS: These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α over-activation.

SUMOylation regulates LKB1 localization and its oncogenic activity in liver cancer.

BACKGROUND: Even though liver kinase B1 (LKB1) is usually described as a tumor suppressor in a wide variety of tissues, it has been shown that LKB1 aberrant expression is associated with bad prognosis in Hepatocellular Carcinoma (HCC). METHODS: Herein we have overexpressed LKB1 in human hepatoma cells and by using histidine pull-down assay we have investigated the role of the hypoxia-related post-translational modification of Small Ubiquitin-related Modifier (SUMO)ylation in the regulation of LKB1 oncogenic role. Molecular modelling between LKB1 and its interactors, involved in regulation of LKB1 nucleocytoplasmic shuttling and LKB1 activity, was performed. Finally, high affinity SUMO binding entities-based technology were used to validate our findings in a pre-clinical mouse model and in clinical HCC. FINDINGS: We found that in human hepatoma cells under hypoxic stress, LKB1 overexpression increases cell viability and aggressiveness in association with changes in LKB1 cellular localization. Moreover, by using site-directed mutagenesis, we have shown that LKB1 is SUMOylated by SUMO-2 at Lys178 hampering LKB1 nucleocytoplasmic shuttling and fueling hepatoma cell growth. Molecular modelling of SUMO modified LKB1 further confirmed steric impedance between SUMOylated LKB1 and the STe20-Related ADaptor cofactor (STRADα), involved in LKB1 export from the nucleus. Finally, we provide evidence that endogenous LKB1 is modified by SUMO in pre-clinical mouse models of HCC and clinical HCC, where LKB1 SUMOylation is higher in fast growing tumors. INTERPRETATION: Overall, SUMO-2 modification of LKB1 at Lys178 mediates LKB1 cellular localization and its oncogenic role in liver cancer. FUND: This work was supported by grants from NIH (US Department of Health and Human services)-R01AR001576-11A1 (J.M.M and M.L.M-C.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), MINECO: SAF2017-87301-R and SAF2014-52097-R integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M., respectively), BFU2015-71017/BMC MINECO/FEDER, EU (to A.D.Q. and I.D.M.), BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Instituto de Salud Carlos III:PIE14/00031, integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovacion 2013-2016 cofinanciado con Fondos FEDER (to M.L.M.-C and J.M.M), Asociación Española contra el Cáncer (T.C.D, P·F-T and M.L.M-C), Daniel Alagille award from EASL (to T.C.D), Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M and M.A), La Caixa Foundation Program (to M.L.M), Programma di Ricerca Regione-Università 2007-2009 and 2011-2012, Regione Emilia-Romagna (to E.V.), Ramón Areces Foundation and the Andalusian Government (BIO-198) (A.D.Q. and I.D.M.), ayudas para apoyar grupos de investigación del sistema Universitario Vasco IT971-16 (P.A.), MINECO:SAF2015-64352-R (P.A.), Institut National du Cancer, FRANCE, INCa grant PLBIO16-251 (M.S.R.), MINECO - BFU2016-76872-R to (E.B.). Work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (M.V-R). Finally, Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644). Funding sources had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Distinct breast cancer stem/progenitor cell populations require either HIF1α or loss of PHD3 to expand under hypoxic conditions.

The heterogeneous nature of breast cancer is a result of intrinsic tumor complexity and also of the tumor microenvironment, which is known to be hypoxic. We found that hypoxia expands different breast stem/progenitor cell populations (cells with increased aldehyde dehydrogenase activity (Aldefluor+), high mammosphere formation capacity and CD44+CD24-/low cells) both in primary normal epithelial and tumor cells. The presence of the estrogen receptor (ER) limits hypoxia-dependent CD44+CD24-/low cell expansion.We further show that the hypoxia-driven cancer stem-like cell enrichment results from a dedifferentiation process. The enhanced mammosphere formation and Aldefluor+ cell content observed in breast cancer cells relies on hypoxia-inducible factor 1α (HIF1α). In contrast, the CD44+CD24-/low population expansion is HIF1α independent and requires prolyl hydroxylase 3 (PHD3) downregulation, which mimics hypoxic conditions, leading to reduced CD24 expression through activation of NFkB signaling. These studies show that hypoxic conditions expand CSC populations through distinct molecular mechanisms. Thus, potential therapies that combine current treatments for breast cancer with drugs that target CSC should take into account the heterogeneity of the CSC subpopulations.

PHD3-SUMO conjugation represses HIF1 transcriptional activity independently of PHD3 catalytic activity.

By controlling HIFα hydroxylation and stability, the prolyl hydroxylase domain (PHD)-containing proteins are essential to the maintenance of oxygen homeostasis; therefore these enzymes are tightly regulated. Small ubiquitin-like modifier (SUMO) is a 10-kDa protein readily conjugated to lysine residues of the targeted proteins in a process termed SUMOylation. In this study, we introduce SUMO conjugation as a novel regulator of PHD3 (also known as EGLN3). PHD3 SUMOylation occurs at a cluster of four lysines at the C-terminal end of the protein. Furthermore, PHD3 SUMOylation by SUMO2 or SUMO3 contributes to PHD3-mediated repression of HIF1-dependent transcriptional activity. Interestingly, PHD3-SUMO conjugation does not affect PHD3 hydroxylase activity or HIF1α stability, providing new evidence for a dual role of PHD3 in HIF1 regulation. Moreover, we show that hypoxia modulates PHD3-SUMO conjugation and that this modification inversely correlates with HIF1 activation. PHD3 SUMOylation highlights a new and additional layer of regulation that is likely required to fine-tune HIF function.