Landscape fire smoke airway exposure impairs respiratory and cardiac function and worsens experimental asthma.

BACKGROUND: Millions of people are exposed to landscape fire smoke (LFS) globally, and inhalation of LFS particulate matter (PM) is associated with poor respiratory and cardiovascular outcomes. However, how LFS affects respiratory and cardiovascular function is less well understood. OBJECTIVE: We aimed to characterize the pathophysiologic effects of representative LFS airway exposure on respiratory and cardiac function and on asthma outcomes. METHODS: LFS was generated using a customized combustion chamber. In 8-week-old female BALB/c mice, low (25 µg/m3, 24-hour equivalent) or moderate (100 µg/m3, 24-hour equivalent) concentrations of LFS PM (10 µm and below [PM10]) were administered daily for 3 (short-term) and 14 (long-term) days in the presence and absence of experimental asthma. Lung inflammation, gene expression, structural changes, and lung function were assessed. In 8-week-old male C57BL/6 mice, low concentrations of LFS PM10 were administered for 3 days. Cardiac function and gene expression were assessed. RESULTS: Short- and long-term LFS PM10 airway exposure increased airway hyperresponsiveness and induced steroid insensitivity in experimental asthma, independent of significant changes in airway inflammation. Long-term LFS PM10 airway exposure also decreased gas diffusion. Short-term LFS PM10 airway exposure decreased cardiac function and expression of gene changes relating to oxidative stress and cardiovascular pathologies. CONCLUSIONS: We characterized significant detrimental effects of physiologically relevant concentrations and durations of LFS PM10 airway exposure on lung and heart function. Our study provides a platform for assessment of mechanisms that underpin LFS PM10 airway exposure on respiratory and cardiovascular disease outcomes.

Sex-based differences in short- and longer-term diet-induced metabolic heart disease.

Sex-based differences in the development of obesity-induced cardiometabolic dysfunction are well documented, however, the specific mechanisms are not completely understood. Obesity has been linked to dysregulation of the epitranscriptome, but the role of N6-methyladenosine (m6A) RNA methylation has not been investigated in relation to the sex differences during obesity-induced cardiac dysfunction. In the current study, male and female C57BL/6J mice were subjected to short- and long-term high-fat/high-sucrose (HFHS) diet to induce obesogenic stress. Cardiac echocardiography showed males developed systolic and diastolic dysfunction after 4 mo of diet, but females maintained normal cardiac function despite both sexes being metabolically dysfunctional. Cardiac m6A machinery gene expression was differentially regulated by duration of HFHS diet in male, but not female mice, and left ventricular ejection fraction correlated with RNA machinery gene levels in a sex- and age-dependent manner. RNA-sequencing of cardiac transcriptome revealed that females, but not males may undergo protective cardiac remodeling early in the course of obesogenic stress. Taken together, our study demonstrates for the first time that cardiac RNA methylation machinery genes are regulated early during obesogenic stress in a sex-dependent manner and may play a role in the sex differences observed in cardiometabolic dysfunction.NEW & NOTEWORTHY Sex differences in obesity-associated cardiomyopathy are well documented but incompletely understood. We show for the first time that RNA methylation machinery genes may be regulated in response to obesogenic diet in a sex- and age-dependent manner and levels may correspond to cardiac systolic function. Our cardiac RNA-seq analysis suggests female, but not male mice may be protected from cardiac dysfunction by a protective cardiac remodeling response early during obesogenic stress.

Cancer Therapies and Cardiomyocyte Viability: Which Drugs are Directly Cardiotoxic?

BACKGROUND: Increased cancer survivorship represents a remarkable achievement for modern medicine. Unfortunately, cancer treatments have inadvertently contributed to cardiovascular (CV) damage, significantly threatening the health and quality of life of patients living with, through and beyond cancer. Without understanding the mechanisms, including whether the cardiotoxicity is due to the direct or indirect effects on cardiomyocytes, prevention and management of cardiotoxicity can pose challenges in many patients. To date, the cardiotoxicity profiles of most of the chemotherapy drugs are still poorly understood. AIM: To conduct a pilot study to investigate the direct effects of a range of cancer therapies on cardiomyocyte viability. METHODS: Primary human cardiomyocytes (HCM) were cultured and seeded into 96-well culture plates. A total of 35 different Food and Drug Administration-approved anti-cancer drugs were added to the HCM cells with a concentration of 1uM for 72 hours. The viability of HCMs was determined using CellTitre-Glo. The experiments were repeated at least three times for each drug with HCMs of different passages. RESULTS: We identified 15 anti-cancer agents that significantly reduced HCM viability. These drugs were: (1) anthracyclines (daunorubicin [HCM viability, mean %±standard error, 13.7±3.2%], epirubicin [47.6±5.3%]), (2) antimetabolite (azacitidine [67.1±2.4%]), (3) taxanes (paclitaxel [60.2±3.0%]), (4) protein kinase inhibitors (lapatinib [49.8±7.0%], ponatinib [42.4±9.0%], pemigatinib [68.1±2.3%], sorafenib [52.9±10.6%], nilotinib [64.4±4.5%], dasatinib [38.5±3.6%]), (5) proteasome inhibitors (ixazomib citrate [65.4±7.2%]), (6) non-selective histone-deacetylase inhibitor (panobinostat [19.1±4.1%]), poly adenosine diphosphate-ribose polymerase inhibitor (olaparib [68.2±1.7%]) and (7) vinca alkaloids (vincristine [44.6±7.4%], vinblastine [31.2±3.9%]). CONCLUSIONS: In total, 15 of the 35 commercially available anti-cancer drugs have direct cardiotoxic effects on HCM. Some of those, have not been associated with clinical cardiotoxicity, while others, known to be cardiotoxic do not appear to mediate it via direct effects on cardiomyocytes. More detailed investigations of the effects of cancer therapies on various cardiovascular cells should be performed to comprehensively determine the mechanisms of cardiotoxicity.

Heart Failure in Breast Cancer Survivors: Focus on Early Detection and Novel Biomarkers.

PURPOSE OF REVIEW: Breast cancer survival rate has greatly improved in the last two decades due to the emergence of next-generation anti-cancer agents. However, cardiotoxicity remains a significant adverse effect arising from traditional and emerging chemotherapies as well as targeted therapies for breast cancer patients. In this review, we will discuss cardiotoxicities of both traditional and emerging therapies for breast cancer. We will discuss current practices to detect cardiotoxicity of these therapies with the focus on new and emerging biomarkers. We will then focus on 'omics approaches, especially the use of epigenetics to discover novel biomarkers and therapeutics to mitigate cardiotoxicity. RECENT FINDINGS: Significant cardiotoxicities of conventional chemotherapies remain and new and unpredictable new forms of cardiac and/or vascular toxicity emerge with the surge in novel and targeted therapies. Yet, there is no clear guidance on detection of cardiotoxicity, except for significant left ventricular systolic dysfunction, and even then, there is no uniform definition of what constitutes cardiotoxicity. The gold standard for detection of cardiotoxicity involves a serial echocardiography in conjunction with blood-based biomarkers to detect early subclinical cardiac dysfunction. However, the ability of these tests to detect early disease remains limited and not all forms of toxicity are detectable with these modalities. There is an unprecedented need to discover novel biomarkers that are sensitive and specific for early detection of subclinical cardiotoxicity. In that space, novel echocardiographic techniques, such as strain, are becoming more common-place and new biomarkers, discovered by epigenetic approaches, seem to become promising alternatives or adjuncts to conventional non-specific cardiac biomarkers.

BAG3-dependent expression of Mcl-1 confers resistance of mutant KRAS colon cancer cells to the HSP90 inhibitor AUY922.

Past studies have shown that mutant KRAS colon cancer cells are susceptible to apoptosis induced by the HSP90 inhibitor AUY922. Nevertheless, intrinsic and acquired resistance remains an obstacle for the potential application of the inhibitor in the treatment of the disease. Here we report that Mcl-1 is important for survival of colon cancer cells in the presence of AUY922. Mcl-1 was upregulated in mutant KRAS colon cancer cells selected for resistance to AUY922-induced apoptosis. This was due to its increased stability mediated by Bcl-2-associated athanogene domain 3 (BAG3), which was also increased in resistant colon cancer cells by heat shock factor 1 (HSF1) as a result of chronic endoplasmic reticulum (ER) stress. Functional investigations demonstrated that inhibition of Mcl-1, BAG3, or HSF1 triggered apoptosis in resistant colon cancer cells, and rendered AUY922-naïve colon cancer cells more sensitive to the inhibitor. Together, these results identify that the HSF1-BAG3-Mcl-1 signal axis is critical for protection of mutant KRAS colon cancer cells from AUY922-induced apoptosis, with potential implications for targeting HSF1/BAG3/Mcl-1 to improve the efficacy of AUY922 in the treatment of colon cancer.

Functional identification of a novel transcript variant of INPP4B in human colon and breast cancer cells.

The 4-phosphatase Inositol polyphosphate 4-phosphatase II (INPP4B) is a regulator of the PI3K signalling pathway and functions to suppress or promote activation of downstream kinases depending on cell type and context. Here we report the identification of a novel small transcript variant of INPP4B (INPP4B-S) that has a role in promoting proliferation of colon and breast cancer cells. INPP4B-S differed from full length INPP4B (INPP4B-FL) by the insertion of a small exon between exons 15 and 16 and the deletion of exons 20-24. Nevertheless, INPP4B-S retained all the functional domains of INPP4B-FL and was similarly located to the cytoplasm. Overexpression of INPP4B-S increased, whereas selective knockdown of INPP4B-S reduced the rate of proliferation in HCT116 and MCF-7 cells. These results warrant further investigation of the role INPP4B-S in activation of downstream kinases and in regulation of cancer pathogenesis.

2014 Rural Clinical School Training and Support Program Snapshot survey.

INTRODUCTION: The Rural Clinical Training and Support (RCTS) program is an Australian Government initiative to address the shortage of medical practitioners within rural and remote Australia. There is a large amount of published information about the RCTS program and rural medical student cohorts who have undertaken short- and long-term rotations. However, very little is known about the academic and professional staff involved in the program, a knowledge gap that may impact workforce and succession planning. To address this, the Federation of Rural Australian Medical Educators (FRAME) initiated the pilot 2014 RCTS Snapshot survey to obtain data on the current RCTS workforce. METHODS: All professional, academic and clinical academic staff (fixed-term and continuing, regardless of fraction) employed through the RCTS program were invited to complete a short, web-based survey. The survey was conducted from March to June 2014. The quantitative variables in the survey included demographics (age and gender), rural background and exposure, employment history in rural/regional areas and at rural clinical schools (RCS), experience and expertise, reasons for working at RCS, and future employment intentions. The last three questions also were of a qualitative open-ended format to allow respondents to provide additional details regarding their reasons for working at RCSs and their future intentions. RESULTS: The estimated total RCTS workforce was 970. A total of 413 responses were received and 316 (40.9%) complete responses analysed. The majority of respondents were female (71%), the 40-60-year age group was predominant (28%), and professional staff constituted the majority (62%). The below 40-year age group had more professionals than academics (21% vs 12%) and more than 62% of academics were aged above 50 years. Notably, there were no academics aged less than 30 years. The percentage of professional staff with a rural background was higher (62%) than that of academics with a rural background (42%). However, more than 70% of academics had previous exposure to a rural area as an adult and 32% had an exposure as a part of university or the TAFE (technical and further education) system. More than half (62%) of RCTS academics were aged more than 50 years and thus approaching retirement age. The implementation of a FRAME-sponsored leadership and succession program was considered by most staff (84%) as one strategy that could be used to prevent a future shortage of academics. Lifestyle reasons for working at an RCS were common to both academic (54%) and professional (63%) staff. A passion for rural health and building capacity within the rural health workforce were other central themes to emerge from the qualitative data. Uncertainty around contract renewal and future funding were dominant themes to emerge from respondents regarding their future employment intentions within the RCTS program. CONCLUSIONS: This study has provided valuable insights into the professional and academic staff's views and aspirations about the RCTS program. These data on the current RCTS workforce provide a benchmark to which future surveys of the workforce can be compared to monitor trends in turnover or predict future shortages due to cohort ageing.

Medical graduates becoming rural doctors: rural background versus extended rural placement.

OBJECTIVES: To determine whether recruitment of rural students and uptake of extended rural placements are associated with students' expressed intentions to undertake rural internships and students' acceptance of rural internships after finishing medical school, and to compare any associations. DESIGN, SETTING AND PARTICIPANTS: Longitudinal study of three successive cohorts (commencing 2005, 2006, 2007) of medical students in the Sydney Medical Program (SMP), University of Sydney, New South Wales, using responses to self-administered questionnaires upon entry to and exit from the Sydney Medical School and data recorded in rolls. MAIN OUTCOME MEASURES: Students' expressed intentions to undertake rural internships, and their acceptance of rural internships after finishing medical school. RESULTS: Data from 448 students were included. The proportion of students preferring a rural career dropped from 20.7% (79/382) to 12.5% (54/433) between entry into and exit from the SMP. A total of 98 students took extended rural placements. Ultimately, 8.1% (35/434) accepted a rural internship, although 14.5% (60/415) had indicated a first preference for a rural post. Students who had undertaken an extended rural placement were more than three times as likely as those with rural backgrounds to express a first preference for a rural internship (23.9% v 7.7%; χ(2) = 7.04; P = 0.008) and more than twice as likely to accept a rural internship (21.3% v 9.9%; χ(2) = 3.85; P = 0.05). CONCLUSION: For the three cohorts studied, rural clinical training through extended placements in rural clinical schools had a stronger association than rural background with a preference for, and acceptance of, rural internship.

Overexpression of Mitochondrial Catalase within Adipose Tissue Does Not Confer Systemic Metabolic Protection against Diet-Induced Obesity.

Obesity is associated with significant metabolic co-morbidities, such as diabetes, hypertension, and dyslipidaemia, as well as a range of cardiovascular diseases, all of which lead to increased hospitalisations, morbidity, and mortality. Adipose tissue dysfunction caused by chronic nutrient stress can result in oxidative stress, mitochondrial dysfunction, inflammation, hypoxia, and insulin resistance. Thus, we hypothesised that reducing adipose tissue oxidative stress via adipose tissue-targeted overexpression of the antioxidant mitochondrial catalase (mCAT) may improve systemic metabolic function. We crossed mCAT (floxed) and Adipoq-Cre mice to generate mice overexpressing catalase with a mitochondrial targeting sequence predominantly in adipose tissue, designated AdipoQ-mCAT. Under normal diet conditions, the AdipoQ-mCAT transgenic mice demonstrated increased weight gain, adipocyte remodelling, and metabolic dysfunction compared to the wild-type mice. Under obesogenic dietary conditions (16 weeks of high fat/high sucrose feeding), the AdipoQ-mCAT mice did not result in incremental impairment of adipose structure and function but in fact, were protected from further metabolic impairment compared to the obese wild-type mice. While AdipoQ-mCAT overexpression was unable to improve systemic metabolic function per se, our results highlight the critical role of physiological H2O2 signalling in metabolism and adipose tissue function.

Association of Circulating Plasma Secreted Frizzled-Related Protein 5 (Sfrp5) Levels with Cardiac Function.

Secreted frizzled-related protein 5 (SFRP5) is a novel anti-inflammatory adipokine that may play a role in cardiovascular development and disease. However, there is yet to be a comprehensive investigation into whether circulating SFRP5 can be a biomarker for cardiac function. Plasma SFRP5 levels were measured via ELISA in 262 patients admitted to a cardiology unit. Plasma SFRP5 levels were significantly lower in patients with a history of heart failure (HF), coronary artery disease (CAD), and atrial fibrillation (AF; p = 0.001). In univariate analyses, SFRP5 levels were also significantly positively correlated with left ventricular ejection fraction (LVEF) (r = 0.52, p < 0.001) and negatively correlated with E/E' (r = -0.30, p < 0.001). Patients with HF, CAD, low LVEF, low triglycerides, high CRP, and high eGFR were associated with lower SFRP5 levels independent of age, BMI, or diabetes after multivariate analysis (overall model r = 0.729, SE = 0.638). Our results show that low plasma SFRP5 levels are independently associated with the presence of HF, CAD, and, importantly, impaired LV function. These results suggest a potential role of SFRP5 as a biomarker, as well as a mediator of cardiac dysfunction independent of obesity and metabolic regulation.

Elevated Soluble Suppressor of Tumorigenicity 2 Predict Hospital Admissions Due to Major Adverse Cardiovascular Events (MACE).

The role of soluble suppression of tumorigenicity (sST2) as a biomarker in predicting clinical outcomes in patients with cardiovascular diseases (CVD) has not been fully elucidated. In this study, we sought to determine the relationship between sST2 levels and any unplanned hospital readmissions due to a major adverse cardiovascular event (MACE) within 1 year of first admission. Patients (n = 250) admitted to the cardiology unit at John Hunter Hospital were recruited. Occurrences of MACE, defined as the composite of total death, myocardial infarction (MI), stroke, readmissions for heart failure (HF), or coronary revascularization, were recorded after 30, 90, 180, and 365 days of first admission. On univariate analysis, patients with atrial fibrillation (AF) and HF had significantly higher sST2 levels vs. those who did not. Increasing levels of sST2 by quartiles were significantly associated with AF, HF, older age, low hemoglobin, low eGFR, and high CRP levels. On multivariate analysis: high sST2 levels and diabetes remained as risk predictors of any MACE occurrence; an sST2 level in the highest quartile (Q4: >28.4 ng/mL) was independently associated with older age, use of beta-blockers, and number of MACE events within a 1 year period. In this patient cohort, elevated sST2 levels are associated with unplanned hospital admission due to MACE within 1 year, independent of the nature of the index cardiovascular admission.

P53 in human melanoma fails to regulate target genes associated with apoptosis and the cell cycle and may contribute to proliferation.

BACKGROUND: Metastatic melanoma represents a major clinical problem. Its incidence continues to rise in western countries and there are currently no curative treatments. While mutation of the P53 tumour suppressor gene is a common feature of many types of cancer, mutational inactivation of P53 in melanoma is uncommon; however, its function often appears abnormal. METHODS: In this study whole genome bead arrays were used to examine the transcript expression of P53 target genes in extracts from 82 melanoma metastases and 6 melanoma cell lines, to provide a global assessment of aberrant P53 function. The expression of these genes was also examined in extracts derived from diploid human melanocytes and fibroblasts. RESULTS: The results indicated that P53 target transcripts involved in apoptosis were under-expressed in melanoma metastases and melanoma cell lines, while those involved in the cell cycle were over-expressed in melanoma cell lines. There was little difference in the transcript expression of P53 target genes between cell lines with null/mutant P53 compared to those with wild-type P53, suggesting that altered expression in melanoma was not related to P53 status. Similarly, down-regulation of P53 by short-hairpin RNA (shRNA) had limited effect on P53 target gene expression in melanoma cells, whereas there were a large number of P53 target genes whose mRNA expression was significantly altered by P53 inhibition in melanocytes. Analysis of whole genome gene expression profiles indicated that the ability of P53 to regulate genes involved in the cell cycle was significantly reduced in melanoma cells. Moreover, inhibition of P53 in melanocytes induced changes in gene expression profiles that were characteristic of melanoma cells and resulted in increased proliferation. Conversely, knockdown of P53 in melanoma cells resulted in decreased proliferation. CONCLUSIONS: These results indicate that P53 target genes involved in apoptosis and cell cycle regulation are aberrantly expressed in melanoma and that this aberrant functional activity of P53 may contribute to the proliferation of melanoma.

Nuclear matrix binding is critical for progesterone receptor movement into nuclear foci.

The ovarian hormone progesterone is essential for normal breast development, and progesterone analogues are implicated in increasing breast cancer risk. The progesterone receptor (PR) is a transcription factor that, when ligand activated, moves rapidly into nuclear foci associated with transcriptional activity. However, the role of intranuclear trafficking signals in the focal location of PR is unknown. We have identified a mutation in PR that ablates its binding to the nuclear matrix and prevents PR movement into nuclear foci. Nuclear matrix binding mutants lack transcriptional activity and inhibit dimerization, demonstrating the critical role of matrix binding for PR dynamics and activity. DNA binding of PR is required for fidelity of location in foci, as DNA binding domain (DBD) mutants form aberrant foci with reduced mobility and altered tethering to the nucleus. Mutations in either the nuclear matrix targeting sequence or DBD domains were dominant in preventing wild-type receptor from moving to appropriate nuclear locations, demonstrating that both partner proteins in a PR dimer must have intact intranuclear trafficking signals for correct receptor positioning within the nucleus. This study has demonstrated that positioning of PR in foci within the nucleus is critically regulated by intranuclear trafficking signals, which play a key role in transcriptional activity and are relevant to its action in normal and malignant breast cells.

2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2.

BACKGROUND: Past studies have shown that sensitivity of melanoma cells to TRAIL-induced apoptosis is largely correlated with the expression levels of TRAIL death receptors on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express generally low levels of death receptors for TRAIL. The clinical potential of TRAIL in the treatment of melanoma may therefore be limited unless given with agents that increase the cell surface expression of TRAIL death receptors. 2-Deoxy-D-glucose (2-DG) is a synthetic glucose analogue that inhibits glycolysis and glycosylation and blocks cell growth. It has been in clinical evaluation for its potential use as an anticancer agent. In this study, we have examined whether 2-DG and TRAIL interact to enhance their cytotoxicity towards melanoma cells. RESULTS: 2-DG did not kill melanoma cells, but enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. This was associated with increased activation of the caspase cascade and mitochondrial apoptotic pathway, and was blocked by inhibition of TRAIL-R2, and to a lesser extent, inhibition of TRAIL-R1. Treatment with 2-DG up-regulated TRAIL death receptors, in particular, TRAIL-R2, on the melanoma cell surface. Up-regulation of TRAIL-R2 was due to increased transcription that was not dependent on the transcription factors, p53 and CHOP. Instead, the IRE1 alpha and ATF6 pathways of the unfolded protein response that were activated by 2-DG appeared to be involved. Moreover, XBP-1, which is known to be transcriptionally regulated by ATF6 and functionally activated by IRE1 alpha, was found to play an important role in 2-DG-mediated transcriptional up-regulation of TRAIL-R2 in melanoma cells. CONCLUSION: These results indicate that 2-DG sensitizes human melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-2 via the ATF6/IRE1 alpha/XBP-1 axis of the unfolded protein response. They suggest that 2-DG is a promising agent to increase the therapeutic response to TRAIL in melanoma.

Establishing an advanced care academy and its role in advanced practitioner development.

Advanced clinical practice (ACP) roles evolved to fill gaps in healthcare provision and improve consistency in standards of care. Historically, variance in service specific requirements, combined with inconsistent approaches to role governance and education, resulted in a multitude of ACP roles and remits. Definitions of roles and titles vary, but there is agreement on academic level and role competency assessment. Irrespective of these definitions, staff and services need support to operationalise guidance. In Scotland, development of advanced practice roles is supported through regional NHS Advanced Practice Academies. One Scottish Board has initiated a local board level advanced care academy to fulfil the three key components of advanced practitioner development: master's level theoretical knowledge, effective supervision and robust clinical competency assessment in practice. This development model, known as the 'triangle of capability', has allowed rapid progress with the advanced practice agenda, which is a central component of sustainable healthcare provision.

Cooperativity of HOXA5 and STAT3 Is Critical for HDAC8 Inhibition-Mediated Transcriptional Activation of PD-L1 in Human Melanoma Cells.

Although the expression of programmed death-ligand 1 (PD-L1) is an important mechanism by which cancer cells evade the immune system, PD-L1 expression in cancer cells is commonly associated with patients' responses to treatment with anti-programmed death 1/PD-L1 antibodies. However, how PD-L1 expression is regulated in melanoma cells remains to be fully elucidated. Here we report that the class I histone deacetylase (HDAC) HDAC8 controls transcriptional activation of PD-L1 by a transcription complex consisting of transcription factors homeobox A5 and signal transducer and activator of transcription 3. Inhibition of HDAC8 upregulated PD-L1 in melanoma cells. This was due to an increase in the activity of a fragment of the PD-L1 gene promoter that is enriched with binding sites for both homeobox A5 and signal transducer and activator of transcription 3. Indeed, knockdown of homeobox A5 or signal transducer and activator of transcription 3 abolished upregulation of PD-L1 by HDAC8 inhibition. Moreover, homeobox A5 and signal transducer and activator of transcription 3 were physically associated and appeared interdependent in activating PD-L1 transcription. Functional studies showed that HDAC8-mediated regulation of PD-L1 expression participated in modulating anti-melanoma T-cell responses. Collectively, these results identify HDAC8 as an important epigenetic regulator of PD-L1 expression, with implications for better understanding of the interaction between melanoma cells and the immune system.

Reactive Oxygen Species Dictate the Apoptotic Response of Melanoma Cells to TH588.

The effect of MTH1 inhibition on cancer cell survival has been elusive. Here we report that although silencing of MTH1 does not affect survival of melanoma cells, TH588, one of the first-in-class MTH1 inhibitors, kills melanoma cells through apoptosis independently of its inhibitory effect on MTH1. Induction of apoptosis by TH588 was not alleviated by MTH1 overexpression or introduction of the bacterial homolog of MTH1 that has 8-oxodGTPase activity but cannot be inhibited by TH588, indicating that MTH1 inhibition is not the cause of TH588-induced killing of melanoma cells. Although knockdown of MTH1 did not impinge on the viability of melanoma cells, it rendered melanoma cells sensitive to apoptosis induced by the oxidative stress inducer elesclomol. Of note, treatment with elesclomol also enhanced TH588-induced apoptosis, whereas a reactive oxygen species scavenger or an antioxidant attenuated the apoptosis triggered by TH588. Indeed, the sensitivity of melanoma cells to TH588 was correlated with endogenous levels of reactive oxygen species. Collectively, these results indicate that the cytotoxicity of TH588 toward melanoma cells is not associated with its inhibitory effect on MTH1, although it is mediated by cellular production of ROS.

RIP1 Kinase Is an Oncogenic Driver in Melanoma.

Although many studies have uncovered an important role for the receptor-binding protein kinase RIP1 in controlling cell death signaling, its possible contributions to cancer pathogenesis have been little explored. Here, we report that RIP1 functions as an oncogenic driver in human melanoma. Although RIP1 was commonly upregulated in melanoma, RIP1 silencing inhibited melanoma cell proliferation in vitro and retarded the growth of melanoma xenografts in vivo. Conversely, while inducing apoptosis in a small proportion of melanoma cells, RIP1 overexpression enhanced proliferation in the remaining cells. Mechanistic investigations revealed that the proliferative effects of RIP1 overexpression were mediated by NF-κB activation. Strikingly, ectopic expression of RIP1 enhanced the proliferation of primary melanocytes, triggering their anchorage-independent cell growth in an NF-κB-dependent manner. We identified DNA copy-number gain and constitutive ubiquitination by a TNFα autocrine loop mechanism as two mechanisms of RIP1 upregulation in human melanomas. Collectively, our findings define RIP1 as an oncogenic driver in melanoma, with potential implications for targeting its NF-κB-dependent activation mechanism as a novel approach to treat this disease.

INPP4B is upregulated and functions as an oncogenic driver through SGK3 in a subset of melanomas.

Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates PI3K/Akt signalling and has a tumour suppressive role in some types of cancers. However, we have found that it is upregulated in a subset of melanomas. Here we report that INPP4B can function as an oncogenic driver through activation of serum- and glucocorticoid-regulated kinase 3 (SGK3) in melanoma. While INPP4B knockdown inhibited melanoma cell proliferation and retarded melanoma xenograft growth, overexpression of INPP4B enhanced melanoma cell and melanocyte proliferation and triggered anchorage-independent growth of melanocytes. Noticeably, INPP4B-mediated melanoma cell proliferation was not related to activation of Akt, but was mediated by SGK3. Upregulation of INPP4B in melanoma cells was associated with loss of miRNA (miR)-494 and/or miR-599 due to gene copy number reduction. Indeed, overexpression of miR-494 or miR-599 downregulated INPP4B, reduced SGK3 activation, and inhibited melanoma cell proliferation, whereas introduction of anti-miR-494 or anti-miR-599 upregulated INPP4B, enhanced SGK3 activation, and promoted melanoma cell proliferation. Collectively, these results identify upregulation of INPP4B as an oncogenic mechanism through activation of SGK3 in a subset of melanomas, with implications for targeting INPP4B and restoring miR-494 and miR-599 as novel approaches in the treatment of melanomas with high INPP4B expression.