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.

Exploration of individual colorectal cancer cell responses to H2O2 eustress using hopping probe scanning ion conductance microscopy.

Colorectal cancer (CRC), a widespread malignancy, is closely associated with tumor microenvironmental hydrogen peroxide (H2O2) levels. Some clinical trials targeting H2O2 for cancer treatment have revealed its paradoxical role as a promoter of cancer progression. Investigating the dynamics of cancer cell H2O2 eustress at the single-cell level is crucial. In this study, non-contact hopping probe mode scanning ion conductance microscopy (HPICM) with high-sensitive Pt-functionalized nanoelectrodes was employed to measure dynamic extracellular to intracellular H2O2 gradients in individual colorectal cancer Caco-2 cells. We explored the relationship between cellular mechanical properties and H2O2 gradients. Exposure to 0.1 or 1 mmol/L H2O2 eustress increased the extracellular to intracellular H2O2 gradient from 0.3 to 1.91 or 3.04, respectively. Notably, cellular F-actin-dependent stiffness increased at 0.1 mmol/L but decreased at 1 mmol/L H2O2 eustress. This H2O2-induced stiffness modulated AKT activation positively and glutathione peroxidase 2 (GPX2) expression negatively. Our findings unveil the failure of some H2O2-targeted therapies due to their ineffectiveness in generating H2O2, which instead acts eustress to promote cancer cell survival. This research also reveals the complex interplay between physical properties and biochemical signaling in cancer cells' antioxidant defense, illuminating the exploitation of H2O2 eustress for survival at the single-cell level. Inhibiting GPX and/or catalase (CAT) enhances the cytotoxic activity of H2O2 eustress against CRC cells, which holds significant promise for developing innovative therapies targeting cancer and other H2O2-related inflammatory diseases.

Measuring Melanoma Nanomechanical Properties in Relation to Metastatic Ability and Anti-Cancer Drug Treatment Using Scanning Ion Conductance Microscopy.

A cell's mechanical properties have been linked to cancer development, motility and metastasis and are therefore an attractive target as a universal, reliable cancer marker. For example, it has been widely published that cancer cells show a lower Young's modulus than their non-cancerous counterparts. Furthermore, the effect of anti-cancer drugs on cellular mechanics may offer a new insight into secondary mechanisms of action and drug efficiency. Scanning ion conductance microscopy (SICM) offers a nanoscale resolution, non-contact method of nanomechanical data acquisition. In this study, we used SICM to measure the nanomechanical properties of melanoma cell lines from different stages with increasing metastatic ability. Young's modulus changes following treatment with the anti-cancer drugs paclitaxel, cisplatin and dacarbazine were also measured, offering a novel perspective through the use of continuous scan mode SICM. We found that Young's modulus was inversely correlated to metastatic ability in melanoma cell lines from radial growth, vertical growth and metastatic phases. However, Young's modulus was found to be highly variable between cells and cell lines. For example, the highly metastatic cell line A375M was found to have a significantly higher Young's modulus, and this was attributed to a higher level of F-actin. Furthermore, our data following nanomechanical changes after 24 hour anti-cancer drug treatment showed that paclitaxel and cisplatin treatment significantly increased Young's modulus, attributed to an increase in microtubules. Treatment with dacarbazine saw a decrease in Young's modulus with a significantly lower F-actin corrected total cell fluorescence. Our data offer a new perspective on nanomechanical changes following drug treatment, which may be an overlooked effect. This work also highlights variations in cell nanomechanical properties between previous studies, cancer cell lines and cancer types and questions the usefulness of using nanomechanics as a diagnostic or prognostic tool.

Isolation, Culture, and Transfection of Melanocytes.

Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in a type of lysosome-related-organelle (LRO) called the melanosome. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally, melanoma. Therefore, melanocytes are a useful model for studying both cellular senescence and melanoma, as well as many other aspects of biology such as pigmentation, organelle biogenesis and transport, and the diseases affecting these mechanisms. Melanocytes for use in basic research can be obtained from a range of sources, including surplus postoperative skin or from congenic murine skin. Here we describe methods to isolate and culture melanocytes from both human and murine skin (including the preparation of mitotically inactive keratinocytes for use as feeder cells). We also describe a high-throughput transfection protocol for human melanocytes and melanoma cells. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Primary explantation of human melanocytic cells Basic Protocol 2: Preparation of keratinocyte feeder cells for use in the primary culture of mouse melanocytes Basic Protocol 3: Primary culture of melanocytes from mouse skin Basic Protocol 4: Transfection of human melanocytes and melanoma cells.

SLC45A2 protein stability and regulation of melanosome pH determine melanocyte pigmentation.

SLC45A2 encodes a putative transporter expressed primarily in pigment cells. SLC45A2 mutations cause oculocutaneous albinism type 4 (OCA4) and polymorphisms are associated with pigmentation variation, but the localization, function, and regulation of SLC45A2 and its variants remain unknown. We show that SLC45A2 localizes to a cohort of mature melanosomes that only partially overlaps with the cohort expressing the chloride channel OCA2. SLC45A2 expressed ectopically in HeLa cells localizes to lysosomes and raises lysosomal pH, suggesting that in melanocytes SLC45A2 expression, like OCA2 expression, results in the deacidification of maturing melanosomes to support melanin synthesis. Interestingly, OCA2 overexpression compensates for loss of SLC45A2 expression in pigmentation. Analyses of SLC45A2- and OCA2-deficient mouse melanocytes show that SLC45A2 likely functions later during melanosome maturation than OCA2. Moreover, the light skin-associated SLC45A2 allelic F374 variant restores only moderate pigmentation to SLC45A2-deficient melanocytes due to rapid proteasome-dependent degradation resulting in lower protein expression levels in melanosomes than the dark skin-associated allelic L374 variant. Our data suggest that SLC45A2 maintains melanosome neutralization that is initially orchestrated by transient OCA2 activity to support melanization at late stages of melanosome maturation, and that a common allelic variant imparts reduced activity due to protein instability.

Loss of Both CDKN2A and CDKN2B Allows for Centrosome Overduplication in Melanoma.

Centrosomes duplicate only once in coordination with the DNA replication cycle and have an important role in segregating genetic material. In contrast, most cancer cells have centrosome aberrations, including supernumerary centrosomes, and this correlates with aneuploidy and genetic instability. The tumor suppressors p16 (CDKN2A) and p15 (CDKN2B) (encoded by the familial melanoma CDKN2 locus) inhibit CDK4/6 activity and have important roles in cellular senescence. p16 is also associated with suppressing centrosomal aberrations in breast cancer; however, the role of p15 in centrosome amplification is unknown. Here, we investigated the relationship between p15 and p16 expression, centrosome number abnormalities, and melanoma progression in cell lines derived from various stages of melanoma progression. We found that normal human melanocyte lines did not exhibit centrosome number abnormalities, whereas those from later stages of melanoma did. Additionally, under conditions of S-phase block, p15 and p16 status determined whether centrosome overduplication would occur. Indeed, removal of p15 from p16-negative cell lines derived from various stages of melanoma progression changed cells that previously would not overduplicate their centrosomes into cells that did. Although this study used cell lines in vitro, it suggests that, during clinical melanoma progression, sequential loss of p15 and p16 provides conditions for centrosome duplication to become deregulated with consequences for genome instability.

High-resolution label-free 3D mapping of extracellular pH of single living cells.

Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.

Mitochondrial NCKX5 regulates melanosomal biogenesis and pigment production.

Oculocutaneous albinism (OCA) is a heterogeneous and autosomal recessive hypopigmentation disorder, which is caused by mutations of genes involved in pigment biosynthesis or melanosome biogenesis. We have previously identified NCKX5 (also known as SLC24A5) as a causative gene for OCA type 6 (OCA6). However, the pathogenesis of OCA6 is unknown. We found that NCKX5 is localized to mitochondria, not to melanosomes. Pharmacological inhibition of mitochondrial function or NCKX exchanger activity reduced pigment production. Loss of NCKX5 attenuated Ca2+ enrichment in melanosomes, which compromised PMEL fibril formation, melanosome maturation and pigment production. Thus, we have defined a new class of hypopigmentation attributable to dysfunctional mitochondria and an impairment of mitochondrial Ca2+ transfer into melanosomes. Thus, it is possible that mitochondrial function could have a role in the graying of hair in older people and formation of hypopigmented lesions in vitiligo patients.

Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis.

Nanometric field-effect-transistor (FET) sensors are made on the tip of spear-shaped dual carbon nanoelectrodes derived from carbon deposition inside double-barrel nanopipettes. The easy fabrication route allows deposition of semiconductors or conducting polymers to comprise the transistor channel. A channel from electrodeposited poly pyrrole (PPy) exhibits high sensitivity toward pH changes. This property is exploited by immobilizing hexokinase on PPy nano-FETs to give rise to a selective ATP biosensor. Extracellular pH and ATP gradients are key biochemical constituents in the microenvironment of living cells; we monitor their real-time changes in relation to cancer cells and cardiomyocytes. The highly localized detection is possible because of the high aspect ratio and the spear-like design of the nano-FET probes. The accurately positioned nano-FET sensors can detect concentration gradients in three-dimensional space, identify biochemical properties of a single living cell, and after cell membrane penetration perform intracellular measurements.

The E3 ligase APC/C(Cdh1) promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth.

The anaphase-promoting complex or cyclosome with the subunit Cdh1 (APC/C(Cdh1)) is an E3 ubiquitin ligase involved in the control of the cell cycle. Here, we identified sporadic mutations occurring in the genes encoding APC components, including Cdh1, in human melanoma samples and found that loss of APC/C(Cdh1) may promote melanoma development and progression, but not by affecting cell cycle regulatory targets of APC/C. Most of the mutations we found in CDH1 were those associated with ultraviolet light (UV)-induced melanomagenesis. Compared with normal human skin tissue and human or mouse melanocytes, the abundance of Cdh1 was decreased and that of the transcription factor PAX3 was increased in human melanoma tissue and human or mouse melanoma cell lines, respectively; Cdh1 abundance was further decreased with advanced stages of human melanoma. PAX3 was a substrate of APC/C(Cdh1) in melanocytes, and APC/C(Cdh1)-mediated ubiquitylation marked PAX3 for proteolytic degradation in a manner dependent on the D-box motif in PAX3. Either mutating the D-box in PAX3 or knocking down Cdh1 prevented the ubiquitylation and degradation of PAX3 and increased proliferation and melanin production in melanocytes. Knocking down Cdh1 in melanoma cells in culture or before implantation in mice promoted doxorubicin resistance, whereas reexpressing wild-type Cdh1, but not E3 ligase-deficient Cdh1 or a mutant that could not interact with PAX3, restored doxorubicin sensitivity in melanoma cells both in culture and in xenografts. Thus, our findings suggest a tumor suppressor role for APC/C(Cdh1) in melanocytes and that targeting PAX3 may be a strategy for treating melanoma.

Genomic analysis reveals distinct mechanisms and functional classes of SOX10-regulated genes in melanocytes.

SOX10 is required for melanocyte development and maintenance, and has been linked to melanoma initiation and progression. However, the molecular mechanisms by which SOX10 guides the appropriate gene expression programs necessary to promote the melanocyte lineage are not fully understood. Here we employ genetic and epigenomic analysis approaches to uncover novel genomic targets and previously unappreciated molecular roles of SOX10 in melanocytes. Through global analysis of SOX10-binding sites and epigenetic characteristics of chromatin states, we uncover an extensive catalog of SOX10 targets genome-wide. Our findings reveal that SOX10 predominantly engages 'open' chromatin regions and binds to distal regulatory elements, including novel and previously known melanocyte enhancers. Integrated chromatin occupancy and transcriptome analysis suggest a role for SOX10 in both transcriptional activation and repression to regulate functionally distinct classes of genes. We demonstrate that distinct epigenetic signatures and cis-regulatory sequence motifs predicted to bind putative co-regulatory transcription factors define SOX10-activated and SOX10-repressed target genes. Collectively, these findings uncover a central role of SOX10 as a global regulator of gene expression in the melanocyte lineage by targeting diverse regulatory pathways.

Mechanistic studies of anti-hyperpigmentary compounds: elucidating their inhibitory and regulatory actions.

Searching for depigmenting agents from natural sources has become a new direction in the cosmetic industry as natural products are generally perceived as relatively safer. In our previous study, selected Chinese medicines traditionally used to treat hyperpigmentation were tested for anti-hyperpigmentary effects using a melan-a cell culture model. Among the tested chemical compounds, 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were found to possess hypopigmentary effects. Western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay, tyrosinase inhibition assay and lipid peroxidation inhibition assay were performed to reveal the underlying cellular and molecular mechanisms of the hypopigmentary effects. 4-Ethylresorcinol and 4-ethylphenol attenuated mRNA and protein expression of tyrosinase-related protein (TRP)-2, and possessed antioxidative effect by inhibiting lipid peroxidation. 1-Tetradecanol was able to attenuate protein expression of tyrosinase. The hypopigmentary actions of 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were associated with regulating downstream proteins along the PKA pathway. 4-Ethylresorcinol was more effective in inhibiting melanin synthesis when compared to 4-ethylphenol and 1-tetradecanol.

Isolation, culture, and transfection of melanocytes.

Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal-like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells.

Caveolin-1 is a risk factor for postsurgery metastasis in preclinical melanoma models.

Melanomas are highly lethal skin tumours that are frequently treated by surgical resection. However, the efficacy of such procedures is often limited by tumour recurrence and metastasis. Caveolin-1 (CAV1) has been attributed roles as a tumour suppressor, although in late-stage tumours, its presence is associated with enhanced metastasis. The expression of this protein in human melanoma development and particularly how the presence of CAV1 affects metastasis after surgery has not been defined. CAV1 expression in human melanocytes and melanomas increases with disease progression and is highest in metastatic melanomas. The effect of increased CAV1 expression can then be evaluated using B16F10 murine melanoma cells injected into syngenic immunocompetent C57BL/6 mice or human A375 melanoma cells injected into immunodeficient B6Rag1-/- mice. Augmented CAV1 expression suppresses tumour formation upon a subcutaneous injection, but enhances lung metastasis of cells injected into the tail vein in both models. A procedure was initially developed using B16F10 melanoma cells in C57BL/6 mice to mimic better the situation in patients undergoing surgery. Subcutaneous tumours of a defined size were removed surgically and local tumour recurrence and lung metastasis were evaluated after another 14 days. In this postsurgery setting, CAV1 presence in B16F10 melanomas favoured metastasis to the lung, although tumour suppression at the initial site was still evident. Similar results were obtained when evaluating A375 cells in B6Rag1-/- mice. These results implicate CAV1 expression in melanomas as a marker of poor prognosis for patients undergoing surgery as CAV1 expression promotes experimental lung metastasis in two different preclinical models.

Melanocytes are selectively vulnerable to UVA-mediated bystander oxidative signaling.

Long-wave UVA is the major component of terrestrial UV radiation and is also the predominant constituent of indoor sunlamps, both of which have been shown to increase cutaneous melanoma risk. Using a two-chamber model, we show that UVA-exposed target cells induce intercellular oxidative signaling to non-irradiated bystander cells. This UVA-mediated bystander stress is observed between all three cutaneous cell types (i.e., keratinocytes, melanocytes, and fibroblasts). Significantly, melanocytes appear to be more resistant to direct UVA effects compared with keratinocytes and fibroblasts, although melanocytes are also more susceptible to bystander oxidative signaling. The extensive intercellular flux of oxidative species has not been previously appreciated and could possibly contribute to the observed cancer risk associated with prolonged UVA exposure.

Restoring p53 function in human melanoma cells by inhibiting MDM2 and cyclin B1/CDK1-phosphorylated nuclear iASPP.

Nearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy.

Targeting protein-trafficking pathways alters melanoma treatment sensitivity.

Protein-trafficking pathways are targeted here in human melanoma cells using methods independent of oncogene mutational status, and the ability to up-regulate and down-regulate tumor treatment sensitivity is demonstrated. Sensitivity of melanoma cells to cis-diaminedichloroplatinum II (cDDP, cis-platin), carboplatin, dacarbazine, or temozolomide together with velaparib, an inhibitor of poly (ADP ribose) polymerase 1, is increased by up to 10-fold by targeting genes that regulate both protein trafficking and the formation of melanosomes, intracellular organelles unique to melanocytes and melanoma cells. Melanoma cells depleted of either of the protein-trafficking regulators vacuolar protein sorting 33A protein (VPS33A) or cappuccino protein (CNO) have increased nuclear localization of cDDP, increased nuclear DNA damage by platination, and increased apoptosis, resulting in increased treatment sensitivity. Depleted cells also exhibit a decreased proportion of intracellular, mature melanosomes compared with undepleted cells. Modulation of protein trafficking via cell-surface signaling by binding the melanocortin 1 receptor with the antagonist agouti-signaling protein decreased the proportion of mature melanosomes formed and increased cDDP sensitivity, whereas receptor binding with the agonist melanocyte-stimulating hormone resulted in an increased proportion of mature melanosomes formed and in decreased sensitivity (i.e., increased resistance) to cDDP. Mutation of the protein-trafficking gene Hps6, known to impair the formation of mature melanosomes, also increased cDDP sensitivity. Together, these results indicate that targeting protein-trafficking molecules markedly increases melanoma treatment sensitivity and influences the degree of melanosomes available for sequestration of therapeutic agents.

Agouti protein, mahogunin, and attractin in pheomelanogenesis and melanoblast-like alteration of melanocytes: a cAMP-independent pathway.

Melanocortin-1 receptor (MC1R) and its ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signaling protein (ASIP), regulate switching between eumelanin and pheomelanin synthesis in melanocytes. Here we investigated biological effects and signaling pathways of ASIP. Melan-a non agouti (a/a) mouse melanocytes produce mainly eumelanin, but ASIP combined with phenylthiourea and extra cysteine could induce over 200-fold increases in the pheomelanin to eumelanin ratio, and a tan-yellow color in pelletted cells. Moreover, ASIP-treated cells showed reduced proliferation and a melanoblast-like appearance, seen also in melanocyte lines from yellow (A(y)/a and Mc1r(e)/ Mc1r(e)) mice. However ASIP-YY, a C-terminal fragment of ASIP, induced neither biological nor pigmentary changes. As, like ASIP, ASIP-YY inhibited the cAMP rise induced by alphaMSH analog NDP-MSH, and reduced cAMP level without added MSH, the morphological changes and depigmentation seemed independent of cAMP signaling. Melanocytes genetically null for ASIP mediators attractin or mahogunin (Atrn(mg-3J/mg-3J) or Mgrn1(md-nc/md-nc)) also responded to both ASIP and ASIP-YY in cAMP level, while only ASIP altered their proliferation and (in part) shape. Thus, ASIP-MC1R signaling includes a cAMP-independent pathway through attractin and mahogunin, while the known cAMP-dependent component requires neither attractin nor mahogunin.

Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.