Tumour extracellular vesicles and particles induce liver metabolic dysfunction.

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.

Soluble adenylyl cyclase contributes to imiquimod-mediated inflammation and is a potential therapeutic target in psoriasis.

Cyclic AMP (cAMP) has a key role in psoriasis pathogenesis, as indicated by the therapeutic efficacy of phosphodiesterase inhibitors that prevent the degradation of cAMP. However, whether soluble adenylate cyclase (sAC) (encoded by the ADCY10 gene), which is an important source for cAMP, is involved in Th17 cell-mediated inflammation or could be an alternative therapeutic target in psoriasis is unknown. We have utilized the imiquimod model of murine psoriasiform dermatitis to address this question. Adcy10-/- mice had reduced erythema, scaling and swelling in the skin and reduced CD4+ IL17+ cell numbers in the draining lymph nodes, compared with wild-type mice after induction of psoriasiform dermatitis with imiquimod. Keratinocyte-specific knock out of Adcy10 had no effect on imiquimod-induced ear swelling suggesting keratinocyte sAC has no role in imiquimod-induced inflammation. During Th17 polarization in vitro, naive T cells from Adcy10-/- mice exhibited reduced IL17 secretion and IL-17+ T-cell proliferation suggesting that differentiation into Th17 cells is suppressed without sAC activity. Interestingly, loss of sAC did not impact the expression of Th17 lineage-defining transcription factors (such as Rorc and cMaf) but rather was required for CREB-dependent gene expression, which is known to support Th17 cell gene expression. Finally, topical application of small molecule sAC inhibitors (sACi) reduced imiquimod-induced psoriasiform dermatitis and Il17 gene expression in the skin. Collectively, these findings demonstrate that sAC is important for psoriasiform dermatitis in mouse skin. sACi may provide an alternative class of topical therapeutics for Th17-mediated skin diseases.

Computational Investigation of the pH Dependence of Stability of Melanosome Proteins: Implication for Melanosome formation and Disease.

Intravesicular pH plays a crucial role in melanosome maturation and function. Melanosomal pH changes during maturation from very acidic in the early stages to neutral in late stages. Neutral pH is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin-synthesizing enzyme tyrosinase (TYR). This dramatic change in pH is thought to result from the activity of several proteins that control melanosomal pH. Here, we computationally investigated the pH-dependent stability of several melanosomal membrane proteins and compared them to the pH dependence of the stability of TYR. We confirmed that the pH optimum of TYR is neutral, and we also found that proteins that are negative regulators of melanosomal pH are predicted to function optimally at neutral pH. In contrast, positive pH regulators were predicted to have an acidic pH optimum. We propose a competitive mechanism among positive and negative regulators that results in pH equilibrium. Our findings are consistent with previous work that demonstrated a correlation between the pH optima of stability and activity, and they are consistent with the expected activity of positive and negative regulators of melanosomal pH. Furthermore, our data suggest that disease-causing variants impact the pH dependence of melanosomal proteins; this is particularly prominent for the OCA2 protein. In conclusion, melanosomal pH appears to affect the activity of multiple melanosomal proteins.

Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase.

The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.

Stoma care products represent a common and previously underreported source of peristomal contact dermatitis.

BACKGROUND: Peristomal dermatitis is a common complication for the >700 000 patients in the United States with an ostomy. The role of stoma skin care products in peristomal dermatitis is poorly understood. OBJECTIVE: To evaluate stoma skin care products as a cause of peristomal dermatitis. METHODS: A retrospective chart review of patients with peristomal dermatitis at four academic hospitals from January 2010 to March 2014 was performed. Patient demographics, clinical information and use test and patch test results were documented. RESULTS: Eighteen patients identified as having peristomal dermatitis were tested. Twelve of these had peristomal contact dermatitis. We identified numerous stoma skin care products as triggers of irritant and/or allergic contact dermatitis. The most common stoma skin care product used and/or involved in dermatitis was Cavilon™ No Sting Barrier Film. CONCLUSIONS: Our data support a paradigm shift whereby healthcare workers treating patients with peristomal dermatitis, which is currently considered to be a reaction mainly to bodily fluids, must consider those products used to protect the skin as potential triggers for this disease. Therefore, patients with peristomal dermatitis should be tested with their stoma skin care agents to determine the need for removal or change of these products. Additionally, full ingredient labelling by manufacturers would help identify new allergens and irritants.

Investigation of cAMP microdomains as a path to novel cancer diagnostics.

Understanding of cAMP signaling has greatly improved over the past decade. The advent of live cell imaging techniques and more specific pharmacologic modulators has led to an improved understanding of the intricacies by which cAMP is able to modulate such a wide variety of cellular pathways. It is now appreciated that cAMP is able to activate multiple effector proteins at distinct areas in the cell leading to the activation of very different downstream targets. The investigation of signaling proteins in cancer is a common route to the development of diagnostic tools, prognostic tools, and/or therapeutic targets, and in this review we highlight how investigation of cAMP signaling microdomains driven by the soluble adenylyl cyclase in different cancers has led to the development of a novel cancer biomarker. Antibodies directed against the soluble adenylyl cyclase (sAC) are highly specific markers for melanoma especially for lentigo maligna melanoma and are being described as "second generation" cancer diagnostics, which are diagnostics that determine the 'state' of a cell and not just identify the cell type. Due to the wide presence of cAMP signaling pathways in cancer, we predict that further investigation of both sAC and other cAMP microdomains will lead to additional cancer biomarkers. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

Type 10 soluble adenylyl cyclase is overexpressed in prostate carcinoma and controls proliferation of prostate cancer cells.

cAMP signaling plays an essential role in modulating the proliferation of different cell types, including cancer cells. Until now, the regulation of this pathway was restricted to the transmembrane class of adenylyl cyclases. In this study, significant overexpression of soluble adenylyl cyclase (sAC), an alternative source of cAMP, was found in human prostate carcinoma, and therefore, the contribution of this cyclase was investigated in the prostate carcinoma cell lines LNCaP and PC3. Suppression of sAC activity by treatment with the sAC-specific inhibitor KH7 or by sAC-specific knockdown mediated by siRNA or shRNA transfection prevented the proliferation of prostate carcinoma cells, led to lactate dehydrogenase release, and induced apoptosis. Cell cycle analysis revealed a significant rise in the G(2) phase population 12 h after sAC inhibition, which was accompanied by the down-regulation of cyclin B(1) and CDK1. sAC-dependent regulation of proliferation involves the EPAC/Rap1/B-Raf signaling pathway. In contrast, protein kinase A does not play a role. In conclusion, this study suggests a novel sAC-dependent signaling pathway that controls the proliferation of prostate carcinoma cells.

CO2/HCO3(-)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In ß cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.

A randomized trial of a wearable UV dosimeter for skin cancer prevention.

Background: Non-melanoma skin cancer (NMSC) is the most prevalent cancer in the United States. Despite guidelines on ultraviolet (UV) avoidance, it remains difficult for people to assess their exposure, as UV is invisible and the onset of UV-induced symptoms is delayed. Methods: In a prospective randomized trial, 97 elderly patients with a history of actinic keratoses (AK) were followed over 6 months. Fifty patients received UV counseling from a dermatologist and a wearable UV dosimeter that provided real-time and cumulative UV exposure. Forty-seven patients received only UV counseling from a dermatologist. Results: Over 75% of participants recorded UV exposure at least once a week during the summer. After 6 months of intervention, when comparing the device group to the control group, we observed a non-significant 20% lower ratio of incidence rates of AKs (95% CI = [-41, 55%], p-value = 0.44) and a significant 95% lower ratio of incidence rates of NMSCs (95% CI = [33, 99.6%], p-value = 0.024). Surveys demonstrated that the control group's score in self-perceived ability to participate in social activities significantly increased by 1.2 (p-value = 0.04), while in the device group, this score non-significantly decreased by 0.9 (p-value = 0.1). We did not observe changes, or between-group differences, in anxiety and depression surveys. Conclusion: This pilot clinical trial has a short duration and a small sample size. However, device adherence and quality of life questionnaires suggest a smartphone-connected wearable UV dosimeter is well accepted by an elderly population. This trial also indicates that a wearable UV dosimeter may be an effective behavioral change tool to reduce NMSC incidence in an elderly population with a prior history of AKs.Clinical trial registration: clinicaltrials.gov, identifier NCT03315286.

Two-pore channel 2 is required for soluble adenylyl cyclase-dependent regulation of melanosomal pH and melanin synthesis.

Melanosomal pH is important for the synthesis of melanin as the rate-limiting enzyme, tyrosinase, is very pH-sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC-dependent regulation of melanosomal pH were undefined. We now systematically examine four well-characterized melanosomal membrane proteins to determine whether any of them are required for sAC-dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC-dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC-dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP-AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC-dependent regulation of melanosomal pH and pigmentation.

The interferon-rich skin environment regulates Langerhans cell ADAM17 to promote photosensitivity in lupus.

The autoimmune disease lupus erythematosus (lupus) is characterized by photosensitivity, where even ambient ultraviolet radiation (UVR) exposure can lead to development of inflammatory skin lesions. We have previously shown that Langerhans cells (LCs) limit keratinocyte apoptosis and photosensitivity via a disintegrin and metalloprotease 17 (ADAM17)-mediated release of epidermal growth factor receptor (EGFR) ligands and that LC ADAM17 sheddase activity is reduced in lupus. Here, we sought to understand how the lupus skin environment contributes to LC ADAM17 dysfunction and, in the process, differentiate between effects on LC ADAM17 sheddase function, LC ADAM17 expression, and LC numbers. We show through transcriptomic analysis a shared IFN-rich environment in non-lesional skin across human lupus and three murine models: MRL/lpr, B6.Sle1yaa, and imiquimod (IMQ) mice. IFN-I inhibits LC ADAM17 sheddase activity in murine and human LCs, and IFNAR blockade in lupus model mice restores LC ADAM17 sheddase activity, all without consistent effects on LC ADAM17 protein expression or LC numbers. Anti-IFNAR-mediated LC ADAM17 sheddase function restoration is associated with reduced photosensitive responses that are dependent on EGFR signaling and LC ADAM17. Reactive oxygen species (ROS) is a known mediator of ADAM17 activity; we show that UVR-induced LC ROS production is reduced in lupus model mice, restored by anti-IFNAR, and is cytoplasmic in origin. Our findings suggest that IFN-I promotes photosensitivity at least in part by inhibiting UVR-induced LC ADAM17 sheddase function and raise the possibility that anifrolumab ameliorates lupus skin disease in part by restoring this function. This work provides insight into IFN-I-mediated disease mechanisms, LC regulation, and a potential mechanism of action for anifrolumab in lupus.

Distinct cAMP Signaling Microdomains Differentially Regulate Melanosomal pH and Pigmentation.

cAMP signaling is a well-established regulator of melanin synthesis. Two distinct cAMP signaling pathways-the transmembrane adenylyl cyclase pathway, activated primarily by the MC1R, and the soluble adenylyl cyclase (sAC) pathway-affect melanin synthesis. The sAC pathway affects melanin synthesis by regulating melanosomal pH, and the MC1R pathway affects melanin synthesis by regulating gene expression and post-translational modifications. However, whether MC1R genotype affects melanosomal pH is poorly understood. We now report that loss of function MC1R does not affect melanosomal pH. Thus, sAC signaling appears to be the only cAMP signaling pathway that regulates melanosomal pH. We also addressed whether MC1R genotype affects sAC-dependent regulation of melanin synthesis. Although sAC loss of function in wild-type human melanocytes stimulates melanin synthesis, sAC loss of function has no effect on melanin synthesis in MC1R nonfunctional human and mouse melanocytes or skin and hair melanin in e/e mice. Interestingly, activation of transmembrane adenylyl cyclases, which increases epidermal eumelanin synthesis in e/e mice, leads to enhanced production of eumelanin in sAC-knockout mice relative to that in sAC wild-type mice. Thus, MC1R- and sAC-dependent cAMP signaling pathways define distinct mechanisms that regulate melanosomal pH and pigmentation.

Cell-intrinsic melanin fails to protect melanocytes from ultraviolet-mutagenesis in the absence of epidermal melanin.

Melanin is a free-radical scavenger, antioxidant, and broadband absorber of ultraviolet (UV) radiation which protects the skin from environmental carcinogenesis. However, melanin synthesis and UV-induced reactive melanin species are also implicated in melanocyte genotoxicity. Here, we attempted to reconcile these disparate functions of melanin using a UVB-sensitive, NRAS-mutant mouse model, TpN. We crossed TpN mice heterozygous for an inactivating mutation in Tyrosinase to produce albino and black littermates on a C57BL/6J background. These animals were then exposed to a single UVB dose on postnatal day three when keratinocytes in the skin have yet to be melanized. Approximately one-third (35%) of black mice were protected from UVB-accelerated tumor formation. However, melanoma growth rates, tumor mutational burdens, and gene expression profiles were similar in melanomas from black and albino mice. Skin from albino mice contained more cyclobutane pyrimidine dimer (CPD) positive cells than black mice 1-h post-irradiation. However, this trend gradually reversed over time with CPDs becoming more prominent in black than albino melanocytes at 48 h. These results show that in the absence of epidermal pigmentation, melanocytic melanin limits the tumorigenic effects of acute UV exposure but fails to protect melanocytes from UVB-induced mutagenesis.

The potential impact of melanosomal pH and metabolism on melanoma.

Melanin is synthesized in melanocytes and is transferred into keratinocytes to block the effects of ultraviolet (UV) radiation and is important for preventing skin cancers including melanoma. However, it is known that after melanomagenesis and melanoma invasion or metastases, melanin synthesis still occurs. Since melanoma cells are no longer involved in the sun tanning process, it is unclear why melanocytes would maintain melanin synthesis after melanomagenesis has occurred. Aside from blocking UV-induced DNA mutation, melanin may provide other metabolic functions that could benefit melanoma. In addition, studies have suggested that there may be a selective advantage to melanin synthesis in melanoma; however, mechanisms regulating melanin synthesis outside the epidermis or hair follicle is unknown. We will discuss how melanosomal pH controls melanin synthesis in melanocytes and how melanosomal pH control of melanin synthesis might function in melanoma. We will also discuss potential reasons why melanin synthesis might be beneficial for melanoma cellular metabolism and provide a rationale for why melanin synthesis is not limited to benign melanocytes.

Contact Allergy Cross-reactions and Thresholds: A Review.

ABSTRACT: Consideration of contact allergen concomitant reactivity, which encompasses cross-reactors, co-reactors, and pseudo cross-reactors, is an important aspect of patient care, yet information on how these terms are differentiated and used in clinical practice is lacking. In this review, we provide definitions of cross-reactors, coreactors, and pseudo cross-reactors and discuss the utility of the American Contact Dermatitis Society Contact Allergen Management Program database cross-reactor groupings. We also discuss limitations to the current categorization of cross-reactivity and recommend incorporating new terms, including "apparent cross-reactor" and "derivative cross-reactor," when classifying cross-reactors.

Histone 3 Methyltransferases Alter Melanoma Initiation and Progression Through Discrete Mechanisms.

Perturbations to the epigenome are known drivers of tumorigenesis. In melanoma, alterations in histone methyltransferases that catalyze methylation at histone 3 lysine 9 and histone 3 lysine 27-two sites of critical post-translational modification-have been reported. To study the function of these methyltransferases in melanoma, we engineered melanocytes to express histone 3 lysine-to-methionine mutations at lysine 9 and lysine 27, which are known to inhibit the activity of histone methyltransferases, in a zebrafish melanoma model. Using this system, we found that loss of histone 3 lysine 9 methylation dramatically suppressed melanoma formation and that inhibition of histone 3 lysine 9 methyltransferases in human melanoma cells increased innate immune response signatures. In contrast, loss of histone 3 lysine 27 methylation significantly accelerated melanoma formation. We identified FOXD1 as a top target of PRC2 that is silenced in melanocytes and found that aberrant overexpression of FOXD1 accelerated melanoma onset. Collectively, these data demonstrate how histone 3 lysine-to-methionine mutations can be used to uncover critical roles for methyltransferases.

A nuclear cAMP microdomain suppresses tumor growth by Hippo pathway inactivation.

Cyclic AMP (cAMP) signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. Here, we present a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncover a nuclear cAMP microdomain that activates a tumor-suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397 and export of YAP from the nucleus with no change in YAP protein stability. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer; thus, drugs directed at the nuclear cAMP microdomain may provide avenues for the treatment of cancer.

Cyclic adenosine monophosphate (cAMP) signaling in melanocyte pigmentation and melanomagenesis.

The second messenger cyclic adenosine monophosphate (cAMP) regulates numerous functions in both benign melanocytes and melanoma cells. cAMP is generated from two distinct sources, transmembrane and soluble adenylyl cyclases (tmAC and sAC, respectively), and is degraded by a family of proteins called phosphodiesterases (PDEs). cAMP signaling can be regulated in many different ways and can lead to varied effects in melanocytes. It was recently revealed that distinct cAMP signaling pathways regulate pigmentation by either altering pigment gene expression or the pH of melanosomes. In the context of melanoma, many studies report seemingly contradictory roles for cAMP in tumorigenesis. For example, cAMP signaling has been implicated in both cancer promotion and suppression, as well as both therapy resistance and sensitization. This conundrum in the field may be explained by the fact that cAMP signals in discrete microdomains and each microdomain can mediate differential cellular functions. Here, we review the role of cAMP signaling microdomains in benign melanocyte biology, focusing on pigmentation, and in melanomagenesis.