Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1.

Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies.

DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer.

Despite clinical benefits of tyrosine kinase inhibitors (TKIs) in cancer, most tumors can reactivate proliferation under TKI therapy. Here we present transcriptional profiling of HER2+ breast cancer cells transitioning from dormant drug tolerant cells to re-proliferating cells under continuous HER2 inhibitor (HER2i) therapy. Focusing on phosphatases, expression of dual-specificity phosphatase DUSP6 was found inhibited in dormant cells, but strongly induced upon regrowth. DUSP6 expression also selectively associated with poor patient survival in HER2+ breast cancers. DUSP6 overexpression conferred apoptosis resistance, whereas its pharmacological blockade prevented therapy tolerance development under HER2i therapy. DUSP6 targeting also synergized with clinically used HER2i combination therapies. Mechanistically DUSP6 is a positive regulator of HER3 expression, and its impact on HER2i tolerance was mediated by neuregulin-HER3 axis. In vivo, genetic targeting of DUSP6 reduced tumor growth in brain metastasis model, whereas its pharmacological targeting induced synthetic lethal therapeutic effect in combination with HER2i. Collectively this work demonstrates that DUSP6 drives escape from HER2i-induced dormancy, and that DUSP6 is a druggable target to overcome HER3-driven TKI resistance.

RAS and PP2A activities converge on epigenetic gene regulation.

RAS-mediated human cell transformation requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A). However, the phosphoprotein targets and cellular processes in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168, and TP53BP1. We validate RAS- and PP2A-elicited regulation of HDAC1/2 chromatin recruitment, of RNF168-TP53BP1 interaction, and of gene expression. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter derepression and activation of oncogenic transcription. Transcriptional derepression by PP2A inhibition was associated with an increase in euchromatin and a decrease in global DNA methylation. Collectively, the results indicate that epigenetic protein complexes constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Furthermore, the work provides an important resource for future studies focusing on phosphoregulation of epigenetic gene regulation in cancer and in other RAS/PP2A-regulated cellular processes.

System biology analysis of endosulfan biodegradation in bacteria and its effect in other living systems: modeling and simulation studies.

Endosulfan is a broadly applied cyclodiene insecticide which has been in use across 80 countries since last 5 decades. Owing to its recalcitrant nature, endosulfan residues have been reported from air, water and soil causing toxicity to various non-target organisms. Microbial decontamination of endosulfan has been reported previously by several authors. In the current study, we have evaluated the pathways of endosulfan degradation and its hazardous impact on other living beings including insects, humans, plants, aquatic life and environment by in-silico methods. For establishment of the endosulfan metabolism in different ecosystems, cell designer was employed. The established model was thereafter assessed and simulated to understand the biochemical and physiological metabolism of the endosulfan in various systems of the network. Topological investigation analysis of the endosulfan metabolism validated the presence of 207 nodes and 274 edges in the network. We have concluded that biomagnification of the endosulfan generally occurs in the various elements of the ecosystem. Dynamics study of endosulfan degrading enzymes suggested the important role of monooxygenase I, II and hydrolase in endosulfan bioremediation. Endosulfan shows toxicity in human beings, fishes and plants, however it is biodegraded by the microbes. To date, there are no reports of in- silico analysis of bioremediation of endosulfan and its hazardous effects on the environment. Thus, this report can be important in terms of modelling and simulation of biodegradation network of endosulfan and similar compounds and their impact on several other systems.Communicated by Ramaswamy H. Sarma.

UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis.

For maximal oncogenic activity, cellular MYC protein levels need to be tightly controlled so that they do not induce apoptosis. Here, we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC protein expression and regulated MYC target genes. Consistent with the tumor suppressor function of UBR5 (HYD) in Drosophila, HYD suppressed dMYC-dependent overgrowth of wing imaginal discs. In contrast, in cancer cells, UBR5 suppressed MYC-dependent priming to therapy-induced apoptosis. Of direct cancer relevance, MYC and UBR5 genes were coamplified in MYC-driven human cancers. Functionally, UBR5 suppressed MYC-mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC coamplification. Furthermore, single-cell immunofluorescence analysis demonstrated reciprocal expression of UBR5 and MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC activity. In MYC-amplified, and p53-mutant breast cancer cells, UBR5 has an important role in suppressing MYC-mediated apoptosis priming and in protection from drug-induced apoptosis. SIGNIFICANCE: These findings identify UBR5 as a novel MYC regulator, the inactivation of which could be very important for understanding of MYC dysregulation on cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1414/F1.large.jpg.

Akinetes From Late Paleoproterozoic Salkhan Limestone (>1600 Ma) of India: A Proxy for Understanding Life in Extreme Conditions.

Isolated elongate spore-like cells present in the >1600 Ma-old Salkhan Limestone of the Semri Group, Vindhayan Supergroup, India are considered akinetes of the heterocystous cyanobacteria. Small to large size, and young (single walled) to mature (double walled) akinetes - namely, Archaeoellipsoides bactroformis, A. conjuctivus, A. dolichos, A. elongatus, A. grandis, A. major and A. minor - found in the stromatolitic and bedded cherts have been reported in the present paper. Their role in understanding extreme environmental conditions is a subject matter of this paper. Additionally, the occurrence of doubly-terminated quartz crystals and fan-fabrics in the Salkhan Limestone indicates adverse conditions for the survival of life forms. The depositional environment of the Salkhan Limestone, Vindhyan Supergroup is suggested to be shallow marine intertidal with pulses of the intermittent hypersaline regime during which akinetes, closely resembling those of extant Nostocaceans, were formed by cyanobacteria for survival in the extreme conditions.

Boron Isotopes in the Puga Geothermal System, India, and Their Implications for the Habitat of Early Life.

Boron is associated with several Archean stromatolite deposits, including the tourmaline-rich Barberton stromatolites in South Africa and tourmaline-bearing pyritic laminae associated with stromatolites of the 3.48 Ga Dresser Formation in the Pilbara Craton, Australia. Boron is also a critical element in prebiotic organic chemistry, including in the formation of ribose, a crucial component in RNA. As geological evidence and advances in prebiotic chemistry are now suggesting that hot spring activity may be associated with the origins of life, an understanding of boron and its mobility and isotopic fractionation in geothermal settings may provide important insights into the setting for the origin of life. Here, we report on the boron isotopic compositions and elemental concentrations in a range of fluid, sediment, and mineral samples from the active, boron-rich Puga geothermal system in the Himalayas, India. This includes one of the lowest boron isotope values ever recorded in modern settings: diatom-rich sediments (δ11B = -41.0‰) in a multiphase fractionation system where evaporation is not the dominant form of isotope fractionation. Instead, the extreme boron isotopic fractionation is ascribed to the incorporation of tetrahedral 10B borate anions in precipitating amorphous silica. These findings expand the known limits and drivers of boron isotope fractionation, as well as provide insight into the concentration and fractionation of boron in Archean hot spring environments.

Opposite feedback from mTORC1 to H-ras and K-ras4B downstream of SREBP1.

As a major growth factor transducer, Ras is an upstream activator of mTORC1, which further integrates nutrient and energy inputs. To ensure a contextual coupling of cell division via Ras/MAPK-signalling and growth via mTORC1-signalling, feedback loops from one pathway back to the other are required. Here we describe a novel feedback from mTORC1, which oppositely affects oncogenic H-ras- and K-ras-signalling output, and as a consequence stemness properties of tumourigenic cells. Amino acid stimulation of mTORC1 increases the processed form of SREBP1, a major lipidome regulator. We show that modulation of the SREBP1 levels downstream of S6K1 has opposite effects on oncogenic H-ras and K-ras nanoscale membrane organisation, ensuing signalling output and promotion of mammospheres expressing these oncogenes. Our data suggest that modulation of phosphatidic acid, a major target of SREBP1 controlled lipid metabolism, is sufficient to affect H-ras and K-ras oppositely in the membrane. Thus mTORC1 activation increases H-ras-, but decreases K-ras-signalling output in cells transformed with the respective oncogene. Given the different impact of these two Ras isoforms on stemness, our results could have implications for stem cell biology and inhibition of cancer stem cells.

Rapalogs can promote cancer cell stemness in vitro in a Galectin-1 and H-ras-dependent manner.

Currently several combination treatments of mTor- and Ras-pathway inhibitors are being tested in cancer therapy. While multiple feedback loops render these central signaling pathways robust, they complicate drug targeting.Here, we describe a novel H-ras specific feedback, which leads to an inadvertent rapalog induced activation of tumorigenicity in Ras transformed cells. We find that rapalogs specifically increase nanoscale clustering (nanoclustering) of oncogenic H-ras but not K-ras on the plasma membrane. This increases H-ras signaling output, promotes mammosphere numbers in a H-ras-dependent manner and tumor growth in ovo. Surprisingly, also other FKBP12 binders, but not mTor-inhibitors, robustly decrease FKBP12 levels after prolonged (>2 days) exposure. This leads to an upregulation of the nanocluster scaffold galectin-1 (Gal-1), which is responsible for the rapamycin-induced increase in H-ras nanoclustering and signaling output. We provide evidence that Gal-1 promotes stemness features in tumorigenic cells. Therefore, it may be necessary to block inadvertent induction of stemness traits in H-ras transformed cells by specific Gal-1 inhibitors that abrogate its effect on H-ras nanocluster. On a more general level, our findings may add an important mechanistic explanation to the pleiotropic physiological effects that are observed with rapalogs.