Subcellular compartmentalization of STIM1 for the distinction of Darier disease from Hailey-Hailey disease.

BACKGROUND AND OBJECTIVES: Darier disease (DD) and Hailey-Hailey disease (HHD) are rare disorders caused by mutations in the ATPase, Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 2 (ATP2A2) and ATPase Ca2+ Transporting Type 2C, Member 1 (ATP2C1) gene, respectively, which lead to a disturbance of calcium metabolism in keratinocytes. Clinically, this is reflected by an impairment of keratinization. Histologically, acantholysis with variable degrees of dyskeratosis and parakeratosis is observed. Both diseases can usually be differentiated clinically, histopathologically and genetically. However, their routine distinction might be challenging since some patients do not harbor ATP2A2 or ATP2C1 mutations. To solve this diagnostic challenge, we studied the differential expression of two proteins of store-operated calcium entry (SOCE), stromal interaction molecule 1 (STIM1) and calcium release-activated calcium modulator 1 (ORAI1), by immunohistochemistry. PATIENTS AND METHODS: Five individuals with ambiguous diagnostic findings and eight controls with an unambiguous diagnosis were studied clinically, histologically, genetically, and by immunohistochemistry for STIM1 and ORAI1. RESULTS: DD patients consistently showed a cytoplasmic STIM1 expression while patients with HHD revealed a membrane-associated staining pattern. In contrast, ORAI1 did not show a differential expression pattern. CONCLUSIONS: Our data suggest subcellular compartmentalization of STIM1 as novel biomarker for the distinction of the two disorders.

MCU controls melanoma progression through a redox-controlled phenotype switch.

Melanoma is the deadliest of skin cancers and has a high tendency to metastasize to distant organs. Calcium and metabolic signals contribute to melanoma invasiveness; however, the underlying molecular details are elusive. The MCU complex is a major route for calcium into the mitochondrial matrix but whether MCU affects melanoma pathobiology was not understood. Here, we show that MCUA expression correlates with melanoma patient survival and is decreased in BRAF kinase inhibitor-resistant melanomas. Knockdown (KD) of MCUA suppresses melanoma cell growth and stimulates migration and invasion. In melanoma xenografts, MCUA_KD reduces tumor volumes but promotes lung metastases. Proteomic analyses and protein microarrays identify pathways that link MCUA and melanoma cell phenotype and suggest a major role for redox regulation. Antioxidants enhance melanoma cell migration, while prooxidants diminish the MCUA_KD -induced invasive phenotype. Furthermore, MCUA_KD increases melanoma cell resistance to immunotherapies and ferroptosis. Collectively, we demonstrate that MCUA controls melanoma aggressive behavior and therapeutic sensitivity. Manipulations of mitochondrial calcium and redox homeostasis, in combination with current therapies, should be considered in treating advanced melanoma.

The Impact of COVID-19 Pandemic on Medical Doctors' Work-Family Balance at German University Clinics.

The measures taken to cope with the COVID-19 pandemic by governments worldwide have vast consequences on all areas of life. To assess the impact of the COVID-19 pandemic on long-term career development, we evaluated the work-family balance of medical doctors at nine German university clinics. The results indicate a severely disturbed work-family balance, which was mostly due to insufficient childcare, based on restrictions in school operations and childcare. Despite the newly created emergency childcare options, aiming to ensure the functioning of the "systematically important" professional groups, medical doctors feel that they are not sufficiently supported by the measures taken by local governments. Women, in particular, see their professional development at risk. Our results underline that proper and flexible childcare is essential for the career advancement of female medical doctors and is particularly important in times of crises such as the current COVID-19 pandemic. At university medicine clinics, increased work time flexibility and optimized schooling and childcare are needed to promote the career development of female as well as male medical doctors in the early stage of their careers.

Protein Signatures of NK Cell-Mediated Melanoma Killing Predict Response to Immunotherapies.

Despite impressive advances in melanoma-directed immunotherapies, resistance is common and many patients still succumb to metastatic disease. In this context, harnessing natural killer (NK) cells, which have thus far been sidelined in the development of melanoma immunotherapy, could provide therapeutic benefits for cancer treatment. To identify molecular determinants of NK cell-mediated melanoma killing (NKmK), we quantified NK-cell cytotoxicity against a panel of genetically diverse melanoma cell lines and observed highly heterogeneous susceptibility. Melanoma protein microarrays revealed a correlation between NKmK and the abundance and activity of a subset of proteins, including several metabolic factors. Oxidative phoshorylation, measured by oxygen consumption rate, negatively correlated with melanoma cell sensitivity toward NKmK, and proteins involved in mitochondrial metabolism and epithelial-mesenchymal transition were confirmed to regulate NKmK. Two- and three-dimensional killing assays and melanoma xenografts established that the PI3K/AKT/mTOR signaling axis controls NKmK via regulation of NK cell-relevant surface proteins. A "protein-killing-signature" based on the protein analysis predicted NKmK of additional melanoma cell lines and the response of patients with melanoma to anti-PD-1 checkpoint therapy. Collectively, these findings identify novel NK cell-related prognostic biomarkers and may contribute to improved and personalized melanoma-directed immunotherapies. SIGNIFICANCE: NK-cell cytotoxicity assays and protein microarrays reveal novel biomarkers of NK cell-mediated melanoma killing and enable development of signatures to predict melanoma patient responsiveness to immunotherapies.

Redox signals at the ER-mitochondria interface control melanoma progression.

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease.

The role of the mitochondrial calcium uniporter (MCU) complex in cancer.

The important role of mitochondria in cancer biology is gaining momentum. With their regulation of cell survival, metabolism, basic cell building blocks, and immunity, among other functions, mitochondria affect not only cancer progression but also the response and resistance to current treatments. Calcium ions are constantly shuttled in and out of mitochondria; thus, playing an important role in the regulation of various cellular processes. The mitochondrial calcium uniporter (MCU) channel and its associated regulators transport calcium across the inner mitochondrial membrane to the mitochondrial matrix. Due to this central role and the capacity to affect cell behavior and fate, the MCU complex is being investigated in different cancers and cancer-related conditions. Here, we review current knowledge on the role of the MCU complex in multiple cancer types and models; we also provide a perspective for future research and clinical considerations.

A calcium-redox feedback loop controls human monocyte immune responses: The role of ORAI Ca2+ channels.

In phagocytes, pathogen recognition is followed by Ca(2+) mobilization and NADPH oxidase 2 (NOX2)-mediated "oxidative burst," which involves the rapid production of large amounts of reactive oxygen species (ROS). We showed that ORAI Ca(2+) channels control store-operated Ca(2+) entry, ROS production, and bacterial killing in primary human monocytes. ROS inactivate ORAI channels that lack an ORAI3 subunit. Staphylococcal infection of mice reduced the expression of the gene encoding the redox-sensitive Orai1 and increased the expression of the gene encoding the redox-insensitive Orai3 in the lungs or in bronchoalveolar lavages. A similar switch from ORAI1 to ORAI3 occurred in primary human monocytes exposed to bacterial peptides in culture. These alterations in ORAI1 and ORAI3 abundance shifted the channel assembly toward a more redox-insensitive configuration. Accordingly, silencing ORAI3 increased the redox sensitivity of the channel and enhanced oxidation-induced inhibition of NOX2. We generated a mathematical model that predicted additional features of the Ca(2+)-redox interplay. Our results identified the ORAI-NOX2 feedback loop as a determinant of monocyte immune responses.

The role of Orai-STIM calcium channels in melanocytes and melanoma.

Calcium signalling within normal and cancer cells regulates many important cellular functions such as migration, proliferation, differentiation and cytokine secretion. Store operated Ca(2+) entry (SOCE) via the Ca(2+) release activated Ca(2+) (CRAC) channels, which are composed of the plasma membrane based Orai channels and the endoplasmic reticulum stromal interaction molecules (STIMs), is a major Ca(2+) entry route in many cell types. Orai and STIM have been implicated in the growth and metastasis of multiple cancers; however, while their involvement in cancer is presently indisputable, how Orai-STIM-controlled Ca(2+) signals affect malignant transformation, tumour growth and invasion is not fully understood. Here, we review recent studies linking Orai-STIM Ca(2+) channels with cancer, with a particular focus on melanoma. We highlight and examine key molecular players and the signalling pathways regulated by Orai and STIM in normal and malignant cells, we expose discrepancies, and we reflect on the potential of Orai-STIMs as anticancer drug targets. Finally, we discuss the functional implications of future discoveries in the field of Ca(2+) signalling.

Inverse regulation of melanoma growth and migration by Orai1/STIM2-dependent calcium entry.

Spontaneous melanoma phenotype switching is controlled by unknown environmental factors and may determine melanoma outcome and responsiveness to anticancer therapy. We show that Orai1 and STIM2 are highly expressed and control store-operated Ca(2+) entry in human melanoma. Lower extracellular Ca(2+) or silencing of Orai1/STIM2 caused a decrease in intracellular Ca(2+) , which correlated with enhanced proliferation and increased expression of microphthalmia-associated transcription factor, a marker for proliferative melanoma phenotype. In contrast, the invasive and migratory potential of melanoma cells was reduced upon silencing of Orai1 and/or STIM2. Accordingly, markers for a non-proliferative, tumor-maintaining phenotype such as JARID1B and Brn2 decreased. Immunohistochemical staining of primary melanomas and lymph node metastases revealed a heterogeneous distribution of Orai1 and STIM2 with elevated expression in the invasive rim of the tumor. In summary, our results support a dynamic model in which Orai1 and STIM2 inversely control melanoma growth and invasion. Pharmacological tuning of Orai1 and particularly STIM2 might thus prevent metastatic spread and render melanomas more susceptible to conventional therapy.

How ORAI and TRP channels interfere with each other: interaction models and examples from the immune system and the skin.

Four types of Ca(2+) selective ion channels are known, ten voltage gated Ca(2+) (CaV) channels, four CatSper channels, three store operated CRAC channels (ORAI channels) and at least two members of the TRPV subfamily (TRPV5, TRPV6). Some of the other TRP channels also show some Ca(2+) selectivity like certain splice variants of TRPM3. In addition to Ca(2+) selective channels, various cation channels play an important role for Ca(2+) entry and furthermore, they may also regulate Ca(2+) entry through other channels by modulating the membrane potential or other means as outlined in this review. Of the different types of cation channels, TRP channels form one of the most prominent families of non-selective cation channels with functional relevance in electrically non-excitable and electrically excitable cell types. Among these, the seven channels of the TRPC subfamily are rather non-selective with very modest Ca(2+) selectivity, whereas in the other subfamilies, cation selectivity ranges from monovalent selectivity (i.e. TRPM4, TRPM5) to divalent selectivity (i.e. TRPM6, TRPM7) or Ca(2+) selectivity (i.e. TRPV5, TRPV6). Rather than discussing the heavily reviewed individual functions of ORAI or TRP channels, we summarize data and present models how TRP and ORAI may functionally interact to guide cellular functions. We focus on T lymphocytes representing a more ORAI-dominated tissue and skin as model system in which both ORAI and TRP channel have been reported to control relevant functions. We present several interaction models how ORAI and TRP may interfere with each other's function.

ORAI1 Ca(2+) channels control endothelin-1-induced mitogenesis and melanogenesis in primary human melanocytes.

UV radiation of the skin triggers keratinocytes to secrete endothelin-1 (ET-1) that binds to endothelin receptors on neighboring melanocytes. Melanocytes respond with a prolonged increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which is necessary for proliferation and melanogenesis. A major fraction of the Ca(2+) signal is caused by entry through Ca(2+)-permeable channels of unknown identity in the plasma membrane. ORAI Ca(2+) channels are molecular determinants of Ca(2+) release-activated Ca(2+) (CRAC) channels and are expressed in many tissues. Here, we show that ORAI1-3 and their activating partners stromal interaction molecules 1 and 2 (STIM1 and STIM2) are expressed in human melanocytes. Although ORAI1 is the predominant ORAI isoform, STIM2 mRNA expression exceeds STIM1. Inhibition of ORAI1 by 2-aminoethoxydiphenyl borate (2-APB) or downregulation of ORAI1 by small interfering RNA (siRNA) reduced Ca(2+) entry and CRAC current amplitudes in activated melanocytes. In addition, suppression of ORAI1 caused reduction in the ET-1-induced cellular viability, melanin synthesis, and tyrosinase activity. Our results imply a role for ORAI1 channels in skin pigmentation and their potential involvement in UV-induced stress responses of the human skin.

Reciprocal responses of fibroblasts and melanocytes to α-MSH depending on MC1R polymorphisms.

The melanocortin 1-receptor (MC1R) exhibits several variants in form of single nucleotide polymorphisms (SNPs) that are known to differentially regulate melanocyte function. However, whether and how MC1R polymorphisms also affect fibroblast function has not been investigated so far.Therefore we measured intracellular cyclic adenosine monophosphate (cAMP) concentrations and cellular proliferation upon stimulation with alpha-melanocyte stimulating hormone (α-MSH) in eight different human fibroblast and melanocyte cell lines with wild type and different MC1R SNPs.We found that fibroblasts, as well as melanocytes, show differences in MC1R function depending on the MC1R genotype. MC1R stimulation with α-MSH in wild type (MC1R(wt)) melanocytes results in an increase of intracellular cAMP and cellular proliferation. In contrast, MC1R(wt) fibroblasts react with a decrease of intracellular cAMP and proliferation. In MC1R polymorphic fibroblasts (R163Q, R151C and V60L) both effects are significantly alleviated. Similar, but inverse effects could be found in MC1R polymorphic melanocytes (R142H and V92M) with a significantly lower cAMP increase and proliferation rate compared to MC1R(wt) melanocytes.Our results indicate that the MC1R displays reciprocal growth responses in melanocytes and fibroblasts, depending on the MC1R genotype. Thus, the MC1R seems to be not solely important for the skin pigmentary system, but also for the fibroblast function, and might influence different processes of the dermal compartment like wound healing, fibrosis and keloid formation.