Role of mesenchymal stem cell conditioned medium on wound healing using a developed 3D skin model.

Alternative 3-dimensional (3D) skin models that replicate in vivo human skin are required to investigate important events during wound healing, such as collective cell migration, epidermal layer formation, dermal substrate formation, re-epithelialisation and collagen production. In this study, a matched human 3D skin equivalent model (3D-SEM) was developed from human skin cells (fibroblast and keratinocytes), characterised using haematoxylin and eosin, immunofluorescence staining and microRNA profiling. The 3D-SEM was then functionally tested for its use in wound healing studies. Mesenchymal stem cells (MSCs) were isolated and characterised according to the criteria stipulated by the International Society for Cell Therapy. Cytokine and growth factor secretions were analysed by enzyme-linked immunosorbent assay. MSC-conditioned medium (MSC-CM) was then tested for wound healing capacity using the developed 3D-SEM at different timepoints i.e., at one, two and four weeks. The constructed 3D-SEM showed consistent development of skin-like structures composed of dermal layers and epidermal layers, with the ability to express epidermal differentiation markers and full stratification. They also showed prolonged longevity in culture media, retaining full differentiation and stratification within the four weeks. MicroRNA profiling revealed a strong correlation in microRNA expression between the developed 3D-SEM and the original native skin (p<0.001; R=0.64). Additionally, MSC-CM significantly enhanced migration, proliferation and differentiation of epidermal cells in the wounded models compared to control models at the different timepoints. In conclusion, in this study, the developed 3D-SEM mimicked native skin at the cellular and molecular levels, and clearly showed the important stages of skin regeneration during the healing process. MSC secretome contains growth factors that play a pivotal role in the healing process and could be used as a therapeutic option to accelerate skin healing.

Suppressed basal mitophagy drives cellular aging phenotypes that can be reversed by a p62-targeting small molecule.

Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here, we demonstrated that primary human cells maintain highly active basal mitophagy initiated by mitochondrial superoxide signaling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor (SAR). Importantly, this pathway was suppressed upon the induction of cellular senescence and in naturally aged cells, leading to a robust shutdown of mitophagy. Inhibition of mitophagy in proliferating cells was sufficient to trigger the senescence program, while reactivation of mitophagy was necessary for the anti-senescence effects of NAD precursors or rapamycin. Furthermore, reactivation of mitophagy by a p62-targeting small molecule rescued markers of cellular aging, which establishes mitochondrial quality control as a promising target for anti-aging interventions.

AMBRA1 levels predict resistance to MAPK inhibitors in melanoma.

Intrinsic and acquired resistance to mitogen-activated protein kinase inhibitors (MAPKi) in melanoma remains a major therapeutic challenge. Here, we show that the clinical development of resistance to MAPKi is associated with reduced tumor expression of the melanoma suppressor Autophagy and Beclin 1 Regulator 1 (AMBRA1) and that lower expression levels of AMBRA1 predict a poor response to MAPKi treatment. Functional analyses show that loss of AMBRA1 induces phenotype switching and orchestrates an extracellular signal-regulated kinase (ERK)-independent resistance mechanism by activating focal adhesion kinase 1 (FAK1). In both in vitro and in vivo settings, melanomas with low AMBRA1 expression exhibit intrinsic resistance to MAPKi therapy but higher sensitivity to FAK1 inhibition. Finally, we show that the rapid development of resistance in initially MAPKi-sensitive melanomas can be attributed to preexisting subclones characterized by low AMBRA1 expression and that cotreatment with MAPKi and FAK1 inhibitors (FAKi) effectively prevents the development of resistance in these tumors. In summary, our findings underscore the value of AMBRA1 expression for predicting melanoma response to MAPKi and supporting the therapeutic efficacy of FAKi to overcome MAPKi-induced resistance.

Porcine-derived collagen peptides promote re-epithelialisation through activation of integrin signalling.

Chronic non-healing cutaneous wounds represent a major burden to patients and healthcare providers worldwide, emphasising the continued unmet need for credible and efficacious therapeutic approaches for wound healing. We have recently shown the potential for collagen peptides to promote proliferation and migration during cutaneous wound healing. In the present study, we demonstrate that the application of porcine-derived collagen peptides significantly increases keratinocyte and dermal fibroblast expression of integrin α2ß1 and activation of an extracellular signal-related kinase (ERK)-focal adhesion kinase (FAK) signalling cascade during wound closure in vitro. SiRNA-mediated knockdown of integrin ß1 impaired porcine-derived collagen peptide-induced wound closure and activation of ERK-FAK signalling in keratinocytes but did not impair ERK or FAK signalling in dermal fibroblasts, implying the activation of differing downstream signalling pathways. Studies in ex vivo human 3D skin equivalents subjected to punch biopsy-induced wounding confirmed the ability of porcine-derived collagen peptides to promote wound closure by enhancing re-epithelialisation. Collectively, these data highlight the translational and clinical potential for porcine-derived collagen peptides as a viable therapeutic approach to promote re-epithelialisation of superficial cutaneous wounds.

The combined immunohistochemical expression of AMBRA1 and SQSTM1 identifies patients with poorly differentiated cutaneous squamous cell carcinoma at risk of metastasis: A proof of concept study.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) incidence continues to increase globally with, as of yet, an unmet need for reliable prognostic biomarkers to identify patients at increased risk of metastasis. The aim of the present study was to test the prognostic potential of the combined immunohistochemical expression of the autophagy regulatory biomarkers, AMBRA1 and SQSTM1, to identify high-risk patient subsets. METHODS: A retrospective cohort of 68 formalin-fixed paraffin-embedded primary cSCCs with known 5-year metastatic outcomes were subjected to automated immunohistochemical staining for AMBRA1 and SQSTM1. Digital images of stained slides were annotated to define four regions of interest: the normal and peritumoral epidermis, the tumor mass, and the tumor growth front. H-score analysis was used to semi-quantify AMBRA1 or SQSTM1 expression in each region of interest using Aperio ImageScope software, with receiver operator characteristics and Kaplan-Meier analysis used to assess prognostic potential. RESULTS: The combined loss of expression of AMBRA1 in the tumor growth front and SQSTM1 in the peritumoral epidermis identified patients with poorly differentiated cSCCs at risk of metastasis (*p < 0.05). CONCLUSIONS: Collectively, these proof of concept data suggest loss of the combined expression of AMBRA1 in the cSCC growth front and SQSTM1 in the peritumoral epidermis as a putative prognostic biomarker for poorly differentiated cSCC.

Validation of epidermal AMBRA1 and loricrin (AMBLor) as a prognostic biomarker for nonulcerated American Joint Committee on Cancer stage I/II cutaneous melanoma.

BACKGROUND: Combined expression of the autophagy-regulatory protein AMBRA1 (activating molecule in Beclin1-regulated autophagy) and the terminal differentiation marker loricrin in the peritumoral epidermis of stage I melanomas can identify tumour subsets at low risk of -metastasis. OBJECTIVES: To validate the combined expression of peritumoral AMBRA1 and loricrin (AMBLor) as a prognostic biomarker able to identify both stage I and II melanomas at low risk of tumour recurrence. METHODS: Automated immunohistochemistry was used to analyse peritumoral AMBRA1 and loricrin expression in geographically distinct discovery (n = 540) and validation (n = 300) cohorts of nonulcerated American Joint Committee on Cancer (AJCC) stage I and II melanomas. AMBLor status was correlated with clinical outcomes in the discovery and validation cohorts separately and combined. RESULTS: Analysis of AMBLor in the discovery cohort revealed a recurrence-free survival (RFS) rate of 95.5% in the AMBLor low-risk group vs. 81.7% in the AMBLor at-risk group (multivariate log-rank, P < 0.001) and a negative predictive value (NPV) of 96.0%. In the validation cohort, AMBLor analysis revealed a RFS rate of 97.6% in the AMBLor low-risk group vs. 78.3% in the at-risk group (multivariate log-rank, P < 0.001) and a NPV of 97.6%. In a multivariate model considering AMBLor, Breslow thickness, age and sex, analysis of the combined discovery and validation cohorts showed that the estimated effect of AMBLor was statistically significant, with a hazard ratio of 3.469 (95% confidence interval 1.403-8.580, P = 0.007) and an overall NPV of 96.5%. CONCLUSIONS: These data provide further evidence validating AMBLor as a prognostic biomarker to identify nonulcerated AJCC stage I and II melanoma tumours at low risk of disease recurrence.

Predictive accuracy of risk prediction models for recurrence, metastasis and survival for early-stage cutaneous melanoma: a systematic review.

OBJECTIVES: To identify prognostic models for melanoma survival, recurrence and metastasis among American Joint Committee on Cancer stage I and II patients postsurgery; and evaluate model performance, including overall survival (OS) prediction. DESIGN: Systematic review and narrative synthesis. DATA SOURCES: Searched MEDLINE, Embase, CINAHL, Cochrane Library, Science Citation Index and grey literature sources including cancer and guideline websites from 2000 to September 2021. ELIGIBILITY CRITERIA: Included studies on risk prediction models for stage I and II melanoma in adults ≥18 years. Outcomes included OS, recurrence, metastases and model performance. No language or country of publication restrictions were applied. DATA EXTRACTION AND SYNTHESIS: Two pairs of reviewers independently screened studies, extracted data and assessed the risk of bias using the CHecklist for critical Appraisal and data extraction for systematic Reviews of prediction Modelling Studies checklist and the Prediction study Risk of Bias Assessment Tool. Heterogeneous predictors prevented statistical synthesis. RESULTS: From 28 967 records, 15 studies reporting 20 models were included; 8 (stage I), 2 (stage II), 7 (stages I-II) and 7 (stages not reported), but were clearly applicable to early stages. Clinicopathological predictors per model ranged from 3-10. The most common were: ulceration, Breslow thickness/depth, sociodemographic status and site. Where reported, discriminatory values were ≥0.7. Calibration measures showed good matches between predicted and observed rates. None of the studies assessed clinical usefulness of the models. Risk of bias was high in eight models, unclear in nine and low in three. Seven models were internally and externally cross-validated, six models were externally validated and eight models were internally validated. CONCLUSIONS: All models are effective in their predictive performance, however the low quality of the evidence raises concern as to whether current follow-up recommendations following surgical treatment is adequate. Future models should incorporate biomarkers for improved accuracy. PROSPERO REGISTRATION NUMBER: CRD42018086784.

Programmed cell death-1 receptor-mediated regulation of Tbet+NK1.1- innate lymphoid cells within the tumor microenvironment.

Innate lymphoid cells (ILCs) play a key role in tissue-mediated immunity and can be controlled by coreceptor signaling. Here, we define a subset of ILCs that are Tbet+NK1.1- and are present within the tumor microenvironment (TME). We show programmed death-1 receptor (PD-1) expression on ILCs within TME is found in Tbet+NK1.1- ILCs. PD-1 significantly controlled the proliferation and function of Tbet+NK1.1- ILCs in multiple murine and human tumors. We found tumor-derived lactate enhanced PD-1 expression on Tbet+NK1.1- ILCs within the TME, which resulted in dampened the mammalian target of rapamycin (mTOR) signaling along with increased fatty acid uptake. In line with these metabolic changes, PD-1-deficient Tbet+NK1.1- ILCs expressed significantly increased IFNγ and granzyme B and K. Furthermore, PD-1-deficient Tbet+NK1.1- ILCs contributed toward diminished tumor growth in an experimental murine model of melanoma. These data demonstrate that PD-1 can regulate antitumor responses of Tbet+NK1.1- ILCs within the TME.

Health professional and patient views of a novel prognostic test for melanoma: A theoretically informed qualitative study.

OBJECTIVES: Cutaneous melanoma rates are steadily increasing. Up to 20% of patients diagnosed with AJCC Stage I/II melanomas will develop metastatic disease. To date there are no consistently reliable means to accurately identify truly high versus low-risk patient subpopulations. There is hence an urgent need for more accurate prediction of prognosis to determine appropriate clinical management. Validation of a novel prognostic test based on the immunohistochemical expression of two protein biomarkers in the epidermal microenvironment of primary melanomas was undertaken; loss of these biomarkers had previously been shown to be associated with a higher risk of recurrence or metastasis. A parallel qualitative study exploring secondary care health professional and patient views of the test was undertaken and this paper reports the perceived barriers and enablers to its implementation into the melanoma care pathway. METHODS: Qualitative methods were employed drawing upon the Theoretical Domains Framework (TDF) in the exploration and analysis. An inductive-deductive analysis was performed, with all data coded using a thematic then TDF framework. FINDINGS: 20 dermatologists, plastic surgeons, cancer nurse specialists, oncologists and histopathologists participated. Nine TDF domains were relevant to all health professional groups and the 'Skills' and 'Beliefs about Capabilities' domains were relevant only to histopathologists. 'Optimism' and 'Beliefs about consequences' were strong enablers particularly for clinicians. 'Environmental context and resources' (impact on pathology services) and 'Knowledge' (the need for robust evidence about the test reliability) were the main perceived barriers. 19 patients and one carer were interviewed. For the patients eight domains were relevant. ('Knowledge', 'Emotions', 'Beliefs about consequences', 'Social Role and identity', 'Behavioural regulation', 'Memory, attention and decision processes', 'Reinforcement' and 'Skills'). The consequences of the implementation of the test were reassurance about future risk, changes to the follow-up pathway on which there were mixed views, and the need to ensure they maintained self-surveillance (Beliefs about consequences). The test was acceptable to all patient interviewees but the resultant changes to management would need to be supported by mechanisms for fast-track back into the clinic, further information on self-surveillance and clear management plans at the time the result is conveyed (Behavioural regulation). CONCLUSIONS: Health professionals and patients perceived positive consequences-for patients and for health services-of adopting the test. However, its implementation would require exploration of the resource implications for pathology services, psychological support for patients with a high-risk test result and mechanisms to reassure and support patients should the test lead to reduced frequency or duration of follow-up. Exploring implementation at an early stage with health professionals presented challenges related to the provision of specific details of the test and its validation.

Exendin-4 stimulates autophagy in pancreatic ß-cells via the RAPGEF/EPAC-Ca2+-PPP3/calcineurin-TFEB axis.

Macroautophagy/autophagy is critical for the regulation of pancreatic ß-cell mass and its deregulation has been implicated in the pathogenesis of type 2 diabetes (T2D). We have previously shown that treatment of pancreatic ß-cells with the GLP1R (glucagon like peptide 1 receptor) agonist exendin-4 stimulates autophagic flux in a setting of chronic nutrient excess. The aim of this study was to identify the underlying pathways contributing to enhanced autophagic flux.Pancreatic ß-cells (INS-1E),mouse and human islets were treated with glucolipotoxic stress (0.5 mM palmitate and 25 mM glucose) in the presence of exendin-4. Consistent with our previous work, exendin-4 stimulated autophagic flux. Using chemical inhibitors and siRNA knockdown, we identified RAPGEF4/EPAC2 (Rap guanine nucleotide exchange factor 4) and downstream calcium signaling to be essential for regulation of autophagic flux by exendin-4. This pathway was independent of AMPK and MTOR signaling. Further analysis identified PPP3/calcineurin and its downstream regulator TFEB (transcription factor EB) as key proteins mediating exendin-4 induced autophagy. Importantly, inhibition of this pathway prevented exendin-4-mediated cell survival and overexpression of TFEB mimicked the cell protective effects of exendin-4 in INS-1E and human islets. Moreover, treatment of db/db mice with exendin-4 for 21 days increased the expression of lysosomal markers within the pancreatic islets. Collectively our data identify the RAPGEF4/EPAC2-calcium-PPP3/calcineurin-TFEB axis as a key mediator of autophagic flux, lysosomal function and cell survival in pancreatic ß-cells. Pharmacological modulation of this axis may offer a novel therapeutic target for the treatment of T2D.Abbreviations: AKT1/protein kinase B: AKT serine/threonine kinase 1; AMPK: 5' AMP-activated protein kinase; CAMKK: calcium/calmodulin-dependent protein kinase kinase; cAMP: cyclic adenosine monophosphate; CASP3: caspase 3; CREB: cAMP response element-binding protein; CTSD: cathepsin D; Ex4: exendin-4(1-39); GLP-1: glucagon like peptide 1; GLP1R: glucagon like peptide 1 receptor; GLT: glucolipotoxicity; INS: insulin; MTOR: mechanistic target of rapamycin kinase; NFAT: nuclear factor of activated T-cells; PPP3/calcineurin: protein phosphatase 3; PRKA/PKA: protein kinase cAMP activated; RAPGEF3/EPAC1: Rap guanine nucleotide exchange factor 3; RAPGEF4/EPAC2: Rap guanine nucleotide exchange factor 4; SQSTM1/p62: sequestosome 1; T2D: type 2 diabetes; TFEB: transcription factor EB.

Optimal surveillance strategies for patients with stage 1 cutaneous melanoma post primary tumour excision: three systematic reviews and an economic model.

BACKGROUND: Malignant melanoma is the fifth most common cancer in the UK, with rates continuing to rise, resulting in considerable burden to patients and the NHS. OBJECTIVES: The objectives were to evaluate the effectiveness and cost-effectiveness of current and alternative follow-up strategies for stage IA and IB melanoma. REVIEW METHODS: Three systematic reviews were conducted. (1) The effectiveness of surveillance strategies. Outcomes were detection of new primaries, recurrences, metastases and survival. Risk of bias was assessed using the Cochrane Collaboration's Risk-of-Bias 2.0 tool. (2) Prediction models to stratify by risk of recurrence, metastases and survival. Model performance was assessed by study-reported measures of discrimination (e.g. D-statistic, Harrel's c-statistic), calibration (e.g. the Hosmer-Lemeshow 'goodness-of-fit' test) or overall performance (e.g. Brier score, R2). Risk of bias was assessed using the Prediction model Risk Of Bias ASsessment Tool (PROBAST). (3) Diagnostic test accuracy of fine-needle biopsy and ultrasonography. Outcomes were detection of new primaries, recurrences, metastases and overall survival. Risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. Review data and data from elsewhere were used to model the cost-effectiveness of alternative surveillance strategies and the value of further research. RESULTS: (1) The surveillance review included one randomised controlled trial. There was no evidence of a difference in new primary or recurrence detected (risk ratio 0.75, 95% confidence interval 0.43 to 1.31). Risk of bias was considered to be of some concern. Certainty of the evidence was low. (2) Eleven risk prediction models were identified. Discrimination measures were reported for six models, with the area under the operating curve ranging from 0.59 to 0.88. Three models reported calibration measures, with coefficients of ≥ 0.88. Overall performance was reported by two models. In one, the Brier score was slightly better than the American Joint Committee on Cancer scheme score. The other reported an R2 of 0.47 (95% confidence interval 0.45 to 0.49). All studies were judged to have a high risk of bias. (3) The diagnostic test accuracy review identified two studies. One study considered fine-needle biopsy and the other considered ultrasonography. The sensitivity and specificity for fine-needle biopsy were 0.94 (95% confidence interval 0.90 to 0.97) and 0.95 (95% confidence interval 0.90 to 0.97), respectively. For ultrasonography, sensitivity and specificity were 1.00 (95% confidence interval 0.03 to 1.00) and 0.99 (95% confidence interval 0.96 to 0.99), respectively. For the reference standards and flow and timing domains, the risk of bias was rated as being high for both studies. The cost-effectiveness results suggest that, over a lifetime, less intensive surveillance than recommended by the National Institute for Health and Care Excellence might be worthwhile. There was considerable uncertainty. Improving the diagnostic performance of cancer nurse specialists and introducing a risk prediction tool could be promising. Further research on transition probabilities between different stages of melanoma and on improving diagnostic accuracy would be of most value. LIMITATIONS: Overall, few data of limited quality were available, and these related to earlier versions of the American Joint Committee on Cancer staging. Consequently, there was considerable uncertainty in the economic evaluation. CONCLUSIONS: Despite adoption of rigorous methods, too few data are available to justify changes to the National Institute for Health and Care Excellence recommendations on surveillance. However, alternative strategies warrant further research, specifically on improving estimates of incidence, progression of recurrent disease; diagnostic accuracy and health-related quality of life; developing and evaluating risk stratification tools; and understanding patient preferences. STUDY REGISTRATION: This study is registered as PROSPERO CRD42018086784. FUNDING: This project was funded by the National Institute for Health Research Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol 25, No. 64. See the NIHR Journals Library website for further project information.

Malignant melanoma is the deadliest of skin cancers; in the UK, > 2500 people die from it every year. Initially, the cancer is removed surgically, which cures it for most people, but, for some, the cancer returns. For this reason, after a melanoma is removed, patients are followed up to see if the melanoma reoccurs or if new melanomas have developed. It is felt that early cancer detection improves the chance of future treatment working. A key question is how best to follow up patients after initial melanoma surgery. This study concentrates on the earliest stage of melanoma (American Joint Committee on Cancer stage I), which accounts for more than 7 out of 10 of all melanoma diagnoses. The study also investigates if new ways of follow-up could be at least as good as current practice and a better use of NHS money. We systematically reviewed studies comparing different ways of organising follow-up, and then methods to identify those patients at high risk of developing a further melanoma and how good different tests are at detecting this cancer. We then compared different possible follow-up strategies. For each strategy, we considered its impact on quality and length of life, and how well it used NHS resources. We found little evidence to support a change in how follow-up should be organised currently. There were some ways of organising follow-up that might be better than current care, but further research is needed. We found that new research on whether or not follow-up should be performed by a cancer nurse specialist, rather than a dermatologist or surgeon, would be worthwhile. We also found that more research could be worthwhile on how frequently melanoma recurs and spreads, as well as how accurately a diagnosis of further cancer is made and how to identify those most at risk of further melanoma spread.

HPV sensitizes OPSCC cells to cisplatin-induced apoptosis by inhibiting autophagy through E7-mediated degradation of AMBRA1.

Oropharyngeal squamous cell carcinoma (OPSCC) is an increasing world health problem with a more favorable prognosis for patients with human papillomavirus (HPV)-positive tumors compared to those with HPV-negative OPSCC. How HPV confers a less aggressive phenotype, however, remains undefined. We demonstrated that HPV-positive OPSCC cells display reduced macroautophagy/autophagy activity, mediated by the ability of HPV-E7 to interact with AMBRA1, to compete with its binding to BECN1 and to trigger its calpain-dependent degradation. Moreover, we have shown that AMBRA1 downregulation and pharmacological inhibition of autophagy sensitized HPV-negative OPSCC cells to the cytotoxic effects of cisplatin. Importantly, semi-quantitative immunohistochemical analysis in primary OPSCCs confirmed that AMBRA1 expression is reduced in HPV-positive compared to HPV-negative tumors. Collectively, these data identify AMBRA1 as a key target of HPV to impair autophagy and propose the targeting of autophagy as a viable therapeutic strategy to improve treatment response of HPV-negative OPSCC.Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; CDDP: cisplatin (CDDP); FFPE: formalin-fixed paraffin-embedded (FFPE); HNC: head and neck cancers (HNC); HPV: human papillomavirus (HPV); hrHPV: high risk human papillomavirus (hrHPV); OCSCC: oral cavity squamous carcinomas (OCSSC); OPSCC: oropharyngeal squamous cell carcinoma (OPSCC); OS: overall survival (OS); qPCR: quantitative polymerase chain reaction; RB1: RB transcriptional corepressor 1; ROC: receiver operating characteristic curve (ROC).

Research Techniques Made Simple: Analysis of Autophagy in the Skin.

Autophagy is required for normal skin homeostasis and its disordered regulation is implicated in a range of cutaneous diseases. Several well-characterized biomarkers of autophagy are used experimentally to quantify autophagic activity or clinically to correlate autophagy with disease progression. This article discusses the advantages and limitations of different approaches for measuring autophagy as well as the techniques for modulating autophagy. These include analysis of endogenous LC3, a central autophagy regulatory protein, and measurement of LC3 flux using a dual-fluorescent reporter, which provides a quantitative readout of autophagy in cell culture systems in vitro and animal models in vivo. Degradation of SQSTM1/p62 during autophagy is proposed as an alternative biomarker allowing the analysis of autophagy both experimentally and clinically. However, the complex regulation of individual autophagy proteins and their involvement in multiple pathways means that several proteins must be analyzed together, preferably over a time course to accurately interpret changes in autophagic activity. Genetic modification of autophagy proteins can be used to better understand basic autophagic mechanisms contributing to health and disease, whereas small molecule inhibitors of autophagy regulatory proteins, lysosomal inhibitors, or activators of cytotoxic autophagy have been explored as potential treatments for skin disorders where autophagy is defective.

TP53 mutant cell lines selected for resistance to MDM2 inhibitors retain growth inhibition by MAPK pathway inhibitors but a reduced apoptotic response.

BACKGROUND: Emergence of resistance to molecular targeted therapy constitutes a limitation to clinical benefits in cancer treatment. Cross-resistance commonly happens with chemotherapeutic agents but might not with targeted agents. METHODS: In the current study, TP53 wild-type cell lines with druggable MAPK pathway mutations [BRAF V600E (WM35) or NRAS Q61K (SJSA-1)] were compared with their TP53 mutant sublines (WM35-R, SN40R2) derived by selection for resistance to MDM2/p53 binding antagonists. RESULTS: The continued presence of the druggable MAPK pathway targets in the TP53 mutant (TP53 MUT) WM35-R and SN40R2 cells was confirmed. Trametinib and vemurafenib were tested on the paired WM35/WM35-R and SJSA-1/SN40R2 cells and similar growth inhibitory effects on the paired cell lines was observed. However, apoptotic responses to trametinib and vemurafenib were greater in WM35 than WM35-R, evidenced by FACS analysis and caspase 3/7 activity, indicating that these MAPK inhibitors acted on the cells partially through p53-regulated pathways. SiRNA mediated p53 knockdown in WM35 replicated the same pattern of response to trametinib and vemurafenib as seen in WM35-R, confirming that p53 plays a role in trametinib and vemurafenib induced apoptosis. In contrast, these differences in apoptotic response between WM35 and WM35-R were not seen with the SJSA-1/SN40R2 cell line pair. This is likely due to p53 suppression by overexpressed MDM2 in SJSA-1. CONCLUSION: The TP53MUT cells selected by resistance to MDM2 inhibitors nevertheless retained growth inhibitory but not apoptotic response to MAPK pathway inhibitors.

ATM Dependent DUSP6 Modulation of p53 Involved in Synergistic Targeting of MAPK and p53 Pathways with Trametinib and MDM2 Inhibitors in Cutaneous Melanoma.

MAPK and p14ARF⁻MDM2⁻p53 pathways are critical in cutaneous melanomas. Here, synergistic combination of the MEK inhibitor, trametinib, with MDM2 inhibitors, nutlin-3/RG7388/HDM201, and the mechanistic basis of responses, for BRAFV600E and p53WT melanoma cells, are reported. The combination treatments induced higher levels of p53 target gene transcripts and protein products, resulting in increased cell cycle arrest and apoptosis compared with MDM2 inhibitors alone, suggesting trametinib synergized with MDM2 inhibitors via upregulation of p53-dependent pathways. In addition, DUSP6 phosphatase involvement was indicated by downregulation of its mRNA and protein following pERK reduction by trametinib. Furthermore, suppression of DUSP6 by siRNA, or inhibition with the small molecule inhibitor, BCI, at a dose without cytotoxicity, potentiated the effect of MDM2 inhibitors through increased ATM-dependent p53 phosphorylation, as demonstrated by complete reversal with the ATM inhibitor, KU55933. Trametinib synergizes with MDM2 inhibitors through a novel DUSP6 mechanism in BRAFV600E and p53WT melanoma cells, in which DUSP6 regulation of p53 phosphorylation is mediated by ATM. This provides a new therapeutic rationale for combination treatments involving activation of the ATM/p53 pathway and MAPK pathway inhibition.

Exposure of Monocytic Cells to Lipopolysaccharide Induces Coordinated Endotoxin Tolerance, Mitochondrial Biogenesis, Mitophagy, and Antioxidant Defenses.

In order to limit the adverse effects of excessive inflammation, anti-inflammatory responses are stimulated at an early stage of an infection, but during sepsis these can lead to deactivation of immune cells including monocytes. In addition, there is emerging evidence that the up-regulation of mitochondrial quality control mechanisms, including mitochondrial biogenesis and mitophagy, is important during the recovery from sepsis and inflammation. We aimed to describe the relationship between the compensatory immune and mitochondrial responses that are triggered following exposure to an inflammatory stimulus in human monocytic cells. Incubation with lipopolysaccharide resulted in a change in the immune phenotype of THP-1 cells consistent with the induction of endotoxin tolerance, similar to that seen in deactivated septic monocytes. After exposure to LPS there was also early evidence of oxidative stress, which resolved in association with the induction of antioxidant defenses and the stimulation of mitochondrial degradation through mitophagy. This was compensated by a parallel up-regulation of mitochondrial biogenesis that resulted in an overall increase in mitochondrial respiratory activity. These observations improve our understanding of the normal homeostatic responses that limit the adverse cellular effects of unregulated inflammation, and which may become ineffective when an infection causes sepsis.

Targeting negative regulation of p53 by MDM2 and WIP1 as a therapeutic strategy in cutaneous melanoma.

BACKGROUND: Cutaneous melanoma is the most serious skin malignancy and new therapeutic strategies are needed for advanced melanoma. TP53 mutations are rare in cutaneous melanoma and hence activation of wild-type p53 is a potential therapeutic strategy in cutaneous melanoma. Here, we investigated the WIP1 inhibitor, GSK2830371, and MDM2-p53 binding antagonists (nutlin-3, RG7388 and HDM201) alone and in combination treatment in cutaneous melanoma cell lines and explored the mechanistic basis of these responses in relation to the genotype and induced gene expression profile of the cells. METHODS: A panel of three p53WT (A375, WM35 and C8161) and three p53MUT (WM164, WM35-R and CHL-1) melanoma cell lines were used. The effects of MDM2 and WIP1 inhibition were evaluated by growth inhibition and clonogenic assays, immunoblotting, qRT-PCR gene expression profiling and flow cytometry. RESULTS: GSK2830371, at doses (⩽10 µM) that alone had no growth-inhibitory or cytotoxic effects on the cells, nevertheless significantly potentiated the growth-inhibitory and clonogenic cell killing effects of MDM2 inhibitors in p53WT but not p53MUT melanoma cells, indicating the potentiation worked in a p53-dependent manner. The siRNA-mediated knockdown of p53 provided further evidence to support the p53 dependence. GSK2830371 increased p53 stabilisation through Ser15 phosphorylation and consequent Lys382 acetylation, and decreased ubiquitination and proteasome-dependent degradation when it was combined with MDM2 inhibitors. These changes were at least partly ATM mediated, shown by reversal with the ATM inhibitor (KU55933). GSK2830371 enhanced the induction of p53 transcriptional target genes, cell cycle arrest and apoptosis. CONCLUSIONS: GSK2830371, a WIP1 inhibitor, at doses with no growth-inhibitory activity alone, potentiated the growth-inhibitory and cytotoxic activity of MDM2 inhibitors by increasing phosphorylation, acetylation and stabilisation of p53 in cutaneous melanoma cells in a functional p53-dependent manner.

Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing.

Mammalian target of rapamycin complex 1 (mTORC1) and cell senescence are intimately linked to each other and to organismal aging. Inhibition of mTORC1 is the best-known intervention to extend lifespan, and recent evidence suggests that clearance of senescent cells can also improve health and lifespan. Enhanced mTORC1 activity drives characteristic phenotypes of senescence, although the underlying mechanisms responsible for increased activity are not well understood. We have identified that in human fibroblasts rendered senescent by stress, replicative exhaustion, or oncogene activation, mTORC1 is constitutively active and resistant to serum and amino acid starvation. This is driven in part by depolarization of senescent cell plasma membrane, which leads to primary cilia defects and a resultant failure to inhibit growth factor signaling. Further, increased autophagy and high levels of intracellular amino acids may act to support mTORC1 activity in starvation conditions. Interventions to correct these phenotypes restore sensitivity to the mTORC1 signaling pathway and cause death, indicating that persistent signaling supports senescent cell survival.

Glucagon-Like Peptide 1 Protects Pancreatic ß-Cells From Death by Increasing Autophagic Flux and Restoring Lysosomal Function.

Studies in animal models of type 2 diabetes have shown that glucagon-like peptide 1 (GLP-1) receptor agonists prevent ß-cell loss. Whether GLP-1 mediates ß-cell survival via the key lysosomal-mediated process of autophagy is unknown. In this study, we report that treatment of INS-1E ß-cells and primary islets with glucolipotoxicity (0.5 mmol/L palmitate and 25 mmol/L glucose) increases LC3 II, a marker of autophagy. Further analysis indicates a blockage in autophagic flux associated with lysosomal dysfunction. Accumulation of defective lysosomes leads to lysosomal membrane permeabilization and release of cathepsin D, which contributes to cell death. Our data further demonstrated defects in autophagic flux and lysosomal staining in human samples of type 2 diabetes. Cotreatment with the GLP-1 receptor agonist exendin-4 reversed the lysosomal dysfunction, relieving the impairment in autophagic flux and further stimulated autophagy. Small interfering RNA knockdown showed the restoration of autophagic flux is also essential for the protective effects of exendin-4. Collectively, our data highlight lysosomal dysfunction as a critical mediator of ß-cell loss and shows that exendin-4 improves cell survival via restoration of lysosomal function and autophagic flux. Modulation of autophagy/lysosomal homeostasis may thus define a novel therapeutic strategy for type 2 diabetes, with the GLP-1 signaling pathway as a potential focus.

Prognostic Impact of Autophagy Biomarkers for Cutaneous Melanoma.

Prognosis and survival for malignant melanoma is highly dependent on early diagnosis and treatment. While the American Joint Committee on Cancer (AJCC) criterion provides a means of staging melanomas and guiding treatment approaches, it is unable to identify the risk of disease progression of early stage tumors or provide reliable stratification for novel adjuvant therapies. The demand for credible prognostic/companion biomarkers able to identify high-risk melanoma subgroups as well as guide more effective personalized/precision-based therapy is therefore of paramount importance. Autophagy, the principle lysosomal-mediated process for the degradation/recycling of cellular debris, is a hot topic in cancer medicine, and observations of its deregulation in melanoma have brought its potential as a prognostic biomarker to the forefront of current research. Key regulatory proteins, including Atg8/microtubule-associated light chain 3 (LC3) and BECN1 (Beclin 1), have been proposed as potential prognostic biomarkers. However, given the dynamic nature of autophagy, their expression in vitro does not translate to their use as a prognostic biomarker for melanoma in vivo. We have recently identified the expression levels of Sequestosome1/SQSTM1 (p62) and activating molecule in Beclin 1-regulated autophagy protein 1 (AMBRA1) as novel independent prognostic biomarkers for early stage melanomas. While increasing followed by subsequent decreasing levels of p62 expression reflects the paradoxical role of autophagy in melanoma, expression levels additionally define a novel prognostic biomarker for AJCC stage II tumors. Conversely, loss of AMBRA1 in the epidermis overlying primary melanomas defines a novel prognostic biomarker for AJCC stage I tumors. Collectively, the definition of AMBRA1 and p62 as prognostic biomarkers for early stage melanomas provides novel and accurate means through which to identify tumors at risk of disease progression, facilitating earlier patient therapeutic intervention and stratification tools for novel personalized therapeutic approaches to improve clinical outcome.