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.
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.
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).
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.
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 α2β1 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 β1 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.
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
does not translate to their use as a prognostic biomarker for melanoma
. 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.
Studies in animal models of type 2 diabetes have shown that glucagon-like peptide 1 (GLP-1) receptor agonists prevent β-cell loss. Whether GLP-1 mediates β-cell survival via the key ...lysosomal-mediated process of autophagy is unknown. In this study, we report that treatment of INS-1E β-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 β-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.
Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain <10%. Novel treatment strategies are therefore urgently required, ...particularly for patients bearing BRAF/NRAS wild-type tumors. Targeting autophagy is a means to promote cancer cell death in chemotherapy-resistant tumors, and the aim of this study was to test the hypothesis that cannabinoids promote autophagy-dependent apoptosis in melanoma. Treatment with Δ9-Tetrahydrocannabinol (THC) resulted in the activation of autophagy, loss of cell viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.
The neuroectodermal tumors neuroblastoma and melanoma represent biologically aggressive and chemoresistant cancers. The chemotherapeutic agents fenretinide and bortezomib induce apoptosis through ...endoplasmic reticulum (ER) stress in these tumor types. The aim of this study was to test the hypothesis that the early events of ER stress signaling and response pathways induced by fenretinide and bortezomib are mediated by the eukaryotic initiation factor 2α (eIF2α)-ATF4 signaling pathway. Treatment of neuroblastoma and melanoma cell lines with fenretinide, bortezomib, or thapsigargin resulted in induction of eIF2α signaling, characterized by increased expression of phosphorylated eIF2α, ATF4, ATF3, and GADD34. These events correlated with induction of the pro-apoptotic protein Noxa. The cytotoxic response, characterized by up-regulation of Noxa and cell death, was dependent on ATF4, but not the ER-related pro-death signaling pathways involving GADD153 or IRE1. Although PERK-dependent phosphorylation of eIF2α enhanced ATF4 protein levels during ER stress, cell death in response to fenretinide, bortezomib, or thapsigargin was not abrogated by inhibition of eIF2α phosphorylation through PERK knockdown or overexpression of wild-type eIF2α. Furthermore, ATF4 induction in response to ER stress was dependent primarily on transcriptional activation, which occurred in a PERK- and phosphorylated eIF2α-independent manner. These results demonstrate that ATF4 mediates ER stress-induced cell death of neuroectodermal tumor cells in response to fenretinide or bortezomib. Understanding the complex regulation of cell death pathways in response to ER stress-inducing drugs has the potential to reveal novel therapeutic targets, thus allowing the development of improved treatment strategies to overcome chemoresistance.
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.
Macroautophagy/autophagy is critical for the regulation of pancreatic β-cell mass and its deregulation has been implicated in the pathogenesis of type 2 diabetes (T2D). We have previously shown that ...treatment of pancreatic β-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 β-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 β-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
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.
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