Tandem Deubiquitination and Acetylation of SPRTN Promotes DNA-Protein Crosslink Repair and Protects against Aging.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions that threaten genomic integrity. Recent findings highlight that SPRTN, a specialized DNA-dependent metalloprotease, is a central player in proteolytic cleavage of DPCs. Previous studies suggest that SPRTN deubiquitination is important for its chromatin association and activation. However, the regulation and consequences of SPRTN deubiquitination remain unclear. Here we report that, in response to DPC induction, the deubiquitinase VCPIP1/VCIP135 is phosphorylated and activated by ATM/ATR. VCPIP1, in turn, deubiquitinates SPRTN and promotes its chromatin relocalization. Deubiquitination of SPRTN is required for its subsequent acetylation, which promotes SPRTN relocation to the site of chromatin damage. Furthermore, Vcpip1 knockout mice are prone to genomic instability and premature aging. We propose a model where two sequential post-translational modifications (PTMs) regulate SPRTN chromatin accessibility to repair DPCs and maintain genomic stability and a healthy lifespan.

PD-L1 (B7-H1) Competes with the RNA Exosome to Regulate the DNA Damage Response and Can Be Targeted to Sensitize to Radiation or Chemotherapy.

Programmed death ligand 1 (PD-L1, also called B7-H1) is an immune checkpoint protein that inhibits immune function through its binding of the programmed cell death protein 1 (PD-1) receptor. Clinically approved antibodies block extracellular PD-1 and PD-L1 binding, yet the role of intracellular PD-L1 in cancer remains poorly understood. Here, we discovered that intracellular PD-L1 acts as an RNA binding protein that regulates the mRNA stability of NBS1, BRCA1, and other DNA damage-related genes. Through competition with the RNA exosome, intracellular PD-L1 protects targeted RNAs from degradation, thereby increasing cellular resistance to DNA damage. RNA immunoprecipitation and RNA-seq experiments demonstrated that PD-L1 regulates RNA stability genome-wide. Furthermore, we developed a PD-L1 antibody, H1A, which abrogates the interaction of PD-L1 with CMTM6, thereby promoting PD-L1 degradation. Intracellular PD-L1 may be a potential therapeutic target to enhance the efficacy of radiotherapy and chemotherapy in cancer through the inhibition of DNA damage response and repair.

A phosphorylation-deubiquitination cascade regulates the BRCA2-RAD51 axis in homologous recombination.

Homologous recombination (HR) is one of the major DNA double-strand break (DSB) repair pathways in mammalian cells. Defects in HR trigger genomic instability and result in cancer predisposition. The defining step of HR is homologous strand exchange directed by the protein RAD51, which is recruited to DSBs by BRCA2. However, the regulation of the BRCA2-RAD51 axis remains unclear. Here we report that ubiquitination of RAD51 hinders RAD51-BRCA2 interaction, while deubiquitination of RAD51 facilitates RAD51-BRCA2 binding and RAD51 recruitment and thus is critical for proper HR. Mechanistically, in response to DNA damage, the deubiquitinase UCHL3 is phosphorylated and activated by ATM. UCHL3, in turn, deubiquitinates RAD51 and promotes the binding between RAD51 and BRCA2. Overexpression of UCHL3 renders breast cancer cells resistant to radiation and chemotherapy, while depletion of UCHL3 sensitizes cells to these treatments, suggesting a determinant role of UCHL3 in cancer therapy. Overall, we identify UCHL3 as a novel regulator of DNA repair and reveal a model in which a phosphorylation-deubiquitination cascade dynamically regulates the BRCA2-RAD51 pathway.

USP49 negatively regulates tumorigenesis and chemoresistance through FKBP51-AKT signaling.

The AKT pathway is a fundamental signaling pathway that mediates multiple cellular processes, such as cell proliferation and survival, angiogenesis, and glucose metabolism. We recently reported that the immunophilin FKBP51 is a scaffolding protein that can enhance PHLPP-AKT interaction and facilitate PHLPP-mediated dephosphorylation of AKT at Ser473, negatively regulating AKT activation. However, the regulation of FKBP51-PHLPP-AKT pathway remains unclear. Here we report that a deubiquitinase, USP49, is a new regulator of the AKT pathway. Mechanistically, USP49 deubiquitinates and stabilizes FKBP51, which in turn enhances PHLPP's capability to dephosphorylate AKT Furthermore, USP49 inhibited pancreatic cancer cell proliferation and enhanced cellular response to gemcitabine in a FKBP51-AKT-dependent manner. Clinically, decreased expression of USP49 in patients with pancreatic cancer was associated with decreased FKBP51 expression and increased AKT phosphorylation. Overall, our findings establish USP49 as a novel regulator of AKT pathway with a critical role in tumorigenesis and chemo-response in pancreatic cancer.

Use of spironolactone to treat acne in adolescent females.

BACKGROUND/OBJECTIVES: Studies assessing the utility of spironolactone for treating acne in adolescent females are lacking. Thus, we sought to examine spironolactone's role in treating this patient population. METHODS: A retrospective review was performed to determine the efficacy of spironolactone treatment in adolescent females seen at Mayo Clinic in Rochester, Minnesota, from 2007 to 2017. RESULTS: In a cohort of 80 pediatric patients with a median age of 19 years (range, 14-20 years), 64 patients (80%) experienced improvement of acne on treatment with spironolactone (median dose, 100 mg daily) with a favorable side effect profile. Approximately a quarter of patients (22.5%) had a complete response; more than half (58.8%) had a complete response or a partial response greater than 50%. Initial and maximal responses were observed at a median of 3 months and 5 months, respectively. Patients received treatment with spironolactone for a median duration of 7 months (range, 3-45 months) with limited side effects. CONCLUSIONS: Spironolactone demonstrated efficacy in treating acne in adolescent females and is a safe long-term alternative to systemic antibiotics in these patients.

Cancer prevention and therapy through the modulation of the tumor microenvironment.

Cancer arises in the context of an in vivo tumor microenvironment. This microenvironment is both a cause and consequence of tumorigenesis. Tumor and host cells co-evolve dynamically through indirect and direct cellular interactions, eliciting multiscale effects on many biological programs, including cellular proliferation, growth, and metabolism, as well as angiogenesis and hypoxia and innate and adaptive immunity. Here we highlight specific biological processes that could be exploited as targets for the prevention and therapy of cancer. Specifically, we describe how inhibition of targets such as cholesterol synthesis and metabolites, reactive oxygen species and hypoxia, macrophage activation and conversion, indoleamine 2,3-dioxygenase regulation of dendritic cells, vascular endothelial growth factor regulation of angiogenesis, fibrosis inhibition, endoglin, and Janus kinase signaling emerge as examples of important potential nexuses in the regulation of tumorigenesis and the tumor microenvironment that can be targeted. We have also identified therapeutic agents as approaches, in particular natural products such as berberine, resveratrol, onionin A, epigallocatechin gallate, genistein, curcumin, naringenin, desoxyrhapontigenin, piperine, and zerumbone, that may warrant further investigation to target the tumor microenvironment for the treatment and/or prevention of cancer.

Therapeutic targeting of replicative immortality.

One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed "senescence," can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells' heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy.

Broad targeting of angiogenesis for cancer prevention and therapy.

Deregulation of angiogenesis--the growth of new blood vessels from an existing vasculature--is a main driving force in many severe human diseases including cancer. As such, tumor angiogenesis is important for delivering oxygen and nutrients to growing tumors, and therefore considered an essential pathologic feature of cancer, while also playing a key role in enabling other aspects of tumor pathology such as metabolic deregulation and tumor dissemination/metastasis. Recently, inhibition of tumor angiogenesis has become a clinical anti-cancer strategy in line with chemotherapy, radiotherapy and surgery, which underscore the critical importance of the angiogenic switch during early tumor development. Unfortunately the clinically approved anti-angiogenic drugs in use today are only effective in a subset of the patients, and many who initially respond develop resistance over time. Also, some of the anti-angiogenic drugs are toxic and it would be of great importance to identify alternative compounds, which could overcome these drawbacks and limitations of the currently available therapy. Finding "the most important target" may, however, prove a very challenging approach as the tumor environment is highly diverse, consisting of many different cell types, all of which may contribute to tumor angiogenesis. Furthermore, the tumor cells themselves are genetically unstable, leading to a progressive increase in the number of different angiogenic factors produced as the cancer progresses to advanced stages. As an alternative approach to targeted therapy, options to broadly interfere with angiogenic signals by a mixture of non-toxic natural compound with pleiotropic actions were viewed by this team as an opportunity to develop a complementary anti-angiogenesis treatment option. As a part of the "Halifax Project" within the "Getting to know cancer" framework, we have here, based on a thorough review of the literature, identified 10 important aspects of tumor angiogenesis and the pathological tumor vasculature which would be well suited as targets for anti-angiogenic therapy: (1) endothelial cell migration/tip cell formation, (2) structural abnormalities of tumor vessels, (3) hypoxia, (4) lymphangiogenesis, (5) elevated interstitial fluid pressure, (6) poor perfusion, (7) disrupted circadian rhythms, (8) tumor promoting inflammation, (9) tumor promoting fibroblasts and (10) tumor cell metabolism/acidosis. Following this analysis, we scrutinized the available literature on broadly acting anti-angiogenic natural products, with a focus on finding qualitative information on phytochemicals which could inhibit these targets and came up with 10 prototypical phytochemical compounds: (1) oleanolic acid, (2) tripterine, (3) silibinin, (4) curcumin, (5) epigallocatechin-gallate, (6) kaempferol, (7) melatonin, (8) enterolactone, (9) withaferin A and (10) resveratrol. We suggest that these plant-derived compounds could be combined to constitute a broader acting and more effective inhibitory cocktail at doses that would not be likely to cause excessive toxicity. All the targets and phytochemical approaches were further cross-validated against their effects on other essential tumorigenic pathways (based on the "hallmarks" of cancer) in order to discover possible synergies or potentially harmful interactions, and were found to generally also have positive involvement in/effects on these other aspects of tumor biology. The aim is that this discussion could lead to the selection of combinations of such anti-angiogenic compounds which could be used in potent anti-tumor cocktails, for enhanced therapeutic efficacy, reduced toxicity and circumvention of single-agent anti-angiogenic resistance, as well as for possible use in primary or secondary cancer prevention strategies.

Sustained proliferation in cancer: Mechanisms and novel therapeutic targets.

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression.

A multi-targeted approach to suppress tumor-promoting inflammation.

Cancers harbor significant genetic heterogeneity and patterns of relapse following many therapies are due to evolved resistance to treatment. While efforts have been made to combine targeted therapies, significant levels of toxicity have stymied efforts to effectively treat cancer with multi-drug combinations using currently approved therapeutics. We discuss the relationship between tumor-promoting inflammation and cancer as part of a larger effort to develop a broad-spectrum therapeutic approach aimed at a wide range of targets to address this heterogeneity. Specifically, macrophage migration inhibitory factor, cyclooxygenase-2, transcription factor nuclear factor-κB, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase B, and CXC chemokines are reviewed as important antiinflammatory targets while curcumin, resveratrol, epigallocatechin gallate, genistein, lycopene, and anthocyanins are reviewed as low-cost, low toxicity means by which these targets might all be reached simultaneously. Future translational work will need to assess the resulting synergies of rationally designed antiinflammatory mixtures (employing low-toxicity constituents), and then combine this with similar approaches targeting the most important pathways across the range of cancer hallmark phenotypes.

Immune evasion in cancer: Mechanistic basis and therapeutic strategies.

Cancer immune evasion is a major stumbling block in designing effective anticancer therapeutic strategies. Although considerable progress has been made in understanding how cancers evade destructive immunity, measures to counteract tumor escape have not kept pace. There are a number of factors that contribute to tumor persistence despite having a normal host immune system. Immune editing is one of the key aspects why tumors evade surveillance causing the tumors to lie dormant in patients for years through "equilibrium" and "senescence" before re-emerging. In addition, tumors exploit several immunological processes such as targeting the regulatory T cell function or their secretions, antigen presentation, modifying the production of immune suppressive mediators, tolerance and immune deviation. Besides these, tumor heterogeneity and metastasis also play a critical role in tumor growth. A number of potential targets like promoting Th1, NK cell, γδ T cell responses, inhibiting Treg functionality, induction of IL-12, use of drugs including phytochemicals have been designed to counter tumor progression with much success. Some natural agents and phytochemicals merit further study. For example, use of certain key polysaccharide components from mushrooms and plants have shown to possess therapeutic impact on tumor-imposed genetic instability, anti-growth signaling, replicative immortality, dysregulated metabolism etc. In this review, we will discuss the advances made toward understanding the basis of cancer immune evasion and summarize the efficacy of various therapeutic measures and targets that have been developed or are being investigated to enhance tumor rejection.

Broad targeting of resistance to apoptosis in cancer.

Apoptosis or programmed cell death is natural way of removing aged cells from the body. Most of the anti-cancer therapies trigger apoptosis induction and related cell death networks to eliminate malignant cells. However, in cancer, de-regulated apoptotic signaling, particularly the activation of an anti-apoptotic systems, allows cancer cells to escape this program leading to uncontrolled proliferation resulting in tumor survival, therapeutic resistance and recurrence of cancer. This resistance is a complicated phenomenon that emanates from the interactions of various molecules and signaling pathways. In this comprehensive review we discuss the various factors contributing to apoptosis resistance in cancers. The key resistance targets that are discussed include (1) Bcl-2 and Mcl-1 proteins; (2) autophagy processes; (3) necrosis and necroptosis; (4) heat shock protein signaling; (5) the proteasome pathway; (6) epigenetic mechanisms; and (7) aberrant nuclear export signaling. The shortcomings of current therapeutic modalities are highlighted and a broad spectrum strategy using approaches including (a) gossypol; (b) epigallocatechin-3-gallate; (c) UMI-77 (d) triptolide and (e) selinexor that can be used to overcome cell death resistance is presented. This review provides a roadmap for the design of successful anti-cancer strategies that overcome resistance to apoptosis for better therapeutic outcome in patients with cancer.

Evasion of anti-growth signaling: A key step in tumorigenesis and potential target for treatment and prophylaxis by natural compounds.

The evasion of anti-growth signaling is an important characteristic of cancer cells. In order to continue to proliferate, cancer cells must somehow uncouple themselves from the many signals that exist to slow down cell growth. Here, we define the anti-growth signaling process, and review several important pathways involved in growth signaling: p53, phosphatase and tensin homolog (PTEN), retinoblastoma protein (Rb), Hippo, growth differentiation factor 15 (GDF15), AT-rich interactive domain 1A (ARID1A), Notch, insulin-like growth factor (IGF), and Krüppel-like factor 5 (KLF5) pathways. Aberrations in these processes in cancer cells involve mutations and thus the suppression of genes that prevent growth, as well as mutation and activation of genes involved in driving cell growth. Using these pathways as examples, we prioritize molecular targets that might be leveraged to promote anti-growth signaling in cancer cells. Interestingly, naturally occurring phytochemicals found in human diets (either singly or as mixtures) may promote anti-growth signaling, and do so without the potentially adverse effects associated with synthetic chemicals. We review examples of naturally occurring phytochemicals that may be applied to prevent cancer by antagonizing growth signaling, and propose one phytochemical for each pathway. These are: epigallocatechin-3-gallate (EGCG) for the Rb pathway, luteolin for p53, curcumin for PTEN, porphyrins for Hippo, genistein for GDF15, resveratrol for ARID1A, withaferin A for Notch and diguelin for the IGF1-receptor pathway. The coordination of anti-growth signaling and natural compound studies will provide insight into the future application of these compounds in the clinical setting.

Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6).

Anoctamin-1 (Ano1) is a widely expressed protein responsible for endogenous Ca(2+)-activated Cl(-) currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next-generation RNA sequencing (RNA-seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1-fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL-4, increased promoter activity by 1.6 ± 0.02-fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL-4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12-fold increase in binding of STAT6 to P0 after IL-4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases.

An ex vivo assay of XRT-induced Rad51 foci formation predicts response to PARP-inhibition in ovarian cancer.

OBJECTIVE: BRCA-positive ovarian cancer patients derive benefit PARP inhibitors. Approximately 50% of ovarian cancer tumors have homologous recombination (HR) deficiencies and are therefore "BRCA-like," possibly rendering them sensitive to PARP inhibition. However, no predictive assay exists to identify these patients. We sought to determine if irradiation-induced Rad51 foci formation, a known marker of HR, correlated to PARP inhibitor response in an ovarian cancer model. METHODS: Ovarian cancer cell lines were exposed to PARP-inhibitor ABT-888 to determine effect on growth. Rad51 protein expression prior to irradiation was determined via Western blot. Cultured cells and patient-derived xenograft tumors (PDX) were irradiated and probed for Rad51 foci. In vivo PDX tumors were treated with ABT-888 and carboplatin; these results were correlated with the ex vivo ionizing radiation assay. RESULTS: Three of seven cell lines were sensitive to ABT-888. Sensitive lines had the lowest Rad51 foci formation rate after irradiation, indicating functional HR deficiency. Approximately 50% of the PDX samples had decreased Rad51 foci formation. Total Rad51 protein levels were consistently low, suggesting that DNA damage induction is required to characterize HR status. The ex vivo IR assay accurately predicted which PDX models were sensitive to PARP inhibition in vitro and in vivo. ABT-888 alone reduced orthotopic tumor growth by 51% in A2780ip2 cell line, predicted to respond by the ex vivo assay. Three PDX models' response also correlated with the assay. CONCLUSIONS: The ex vivo IR assay correlates with response to PARP inhibition. Analysis of total Rad51 protein is not a reliable substitute.

Regulation of AMPK activation by extracellular matrix stiffness in pancreatic cancer.

The adenosine monophosphate (AMP)-activated protein kinase (AMPK) sits at a central node in the regulation of energy metabolism and tumor progression. AMPK is best known to sense high cellular ADP or AMP levels, which indicate the depletion of energy stores. Previous studies have shown that the low expression of phosphorylated AMPK is associated with a poor prognosis of pancreatic cancer. In this study, we report that AMPK is also highly sensitive to extracellular matrix (ECM) stiffness. We found that AMPK is activated in cells when cultured under low ECM stiffness conditions and is functionally required for the metabolic switch induced by ECM stiffness. This regulation of AMPK requires the Hippo kinases but not LKB1/CaMKKß. Hippo kinases directly phosphorylate AMPKα at Thr172 to activate AMPK at low ECM stiffness. Furthermore, we found AMPK activity is inhibited in patients with pancreatic ductal adenocarcinoma (PDAC) with high ECM stiffness and is associated with a poor survival outcome. The activation of Hippo kinases by ROCK inhibitor Y-27632 in combination with the mitochondrial inhibitor metformin synergistically activates AMPK and dramatically inhibits PDAC growth. Together, these findings establish a novel model for AMPK regulation by the mechanical properties of ECMs and provide a rationale for simultaneously targeting the ECM stiffness-Hippo kinases-AMPK signaling and low glucose-LKB1-AMPK signaling pathways as an effective therapeutic strategy against PDAC.

Tumors induce complex DNA damage in distant proliferative tissues in vivo.

That tumors cause changes in surrounding tissues is well documented, but whether they also affect distant tissues is uncertain. Such knowledge may be important in understanding the relationship between cancer and overall patient health. To address this question, we examined tissues distant to sites of implanted tumors for genomic damage using cohorts of C57BL/6 and BALB/c mice with early-stage subcutaneous syngeneic grafts, specifically, B16 melanoma, MO5076 sarcoma, and COLON26 carcinoma. Here we report that levels of two serious types of DNA damage, double-strand breaks (DSBs) measured by γ-H2AX focus formation and oxidatively induced non-DSB clustered DNA lesions (OCDLs), were elevated in tissues distant from the tumor site in tumor-bearing mice compared with their age- and sex-matched controls. Most affected were crypts in the gastrointestinal tract organs and skin, both highly proliferative tissues. Further investigation revealed that, compared with controls, tumor-bearing mice contained elevated amounts of activated macrophages in the distant gastrointestinal tissues, as well as elevated serum levels of several cytokines. One of these cytokines, CCL2/MCP-1, has been linked to several inflammation-related conditions and macrophage recruitment, and strikingly, CCL2-deficient mice lacked increased levels of DSBs and OCDLs in tissues distant from implanted tumors. Thus, this study is unique in being a direct demonstration that the presence of a tumor may induce a chronic inflammatory response in vivo, leading to increased systemic levels of DNA damage. Importantly, these findings suggest that tumors may have more profound effects on their hosts than heretofore expected.