Maria L. Wei

Multidrug Resistance Decreases with Mutations of Melanosomal Regulatory Genes

Whereas resistance to chemotherapy has long impeded effective treatment of metastatic melanoma, the mechanistic basis of this resistance remains unknown. One possible mechanism of drug resistance is alteration of intracellular drug distribution either by drug efflux or sequestration into intracellular organelles. Melanomas, as well as primary melanocytes from which they arise, have intracellular organelles, called melanosomes, wherein the synthesis and storage of the pigment melanin takes place. In this study, comparisons of congenic cells with and without functional molecules regulating melanosome formation show that sensitivity to the chemotherapeutic agent cis-diaminedichloroplatinum II (cis-platin) significantly increases with the mutation of genes regulating melanosome formation, concomitant disruption of melanosome morphology, and loss of mature melanosomes. Absence of the melanosomal structural protein gp100/Pmel17 causes increased cis-platin sensitivity. Independent mutations in three separate genes that regulate melanosome biogenesis (Dtnbp1, Pldn, Vps33a) also result in increased cis-platin sensitivity. In addition, a mutation of the gene encoding the integral melanosomal protein tyrosinase, resulting in aberrant melanosome formation, also causes increased cis-platin sensitivity. Furthermore, sensitivity to agents in other chemotherapeutic classes (e.g., vinblastine and etoposide) also increased with the mutation of Pldn. In contrast, a mutation in another melanosomal regulatory gene, Hps1, minimally affects melanosome biogenesis, preserves the formation of mature melanosomes, and has no effect on cis-platin or vinblastine response. Together, these data provide the first direct evidence that melanosomal regulatory genes influence drug sensitivity and that the presence of mature melanosomes likely contributes to melanoma resistance to therapy.

The MADS box transcription factor MEF2C regulates melanocyte development and is a direct transcriptional target and partner of SOX10.

Waardenburg syndromes are characterized by pigmentation and autosensory hearing defects, and mutations in genes encoding transcription factors that control neural crest specification and differentiation are often associated with Waardenburg and related disorders. For example, mutations in SOX10 result in a severe form of Waardenburg syndrome, Type IV, also known as Waardenburg-Hirschsprung disease, characterized by pigmentation and other neural crest defects, including defective innervation of the gut. SOX10 controls neural crest development through interactions with other transcription factors. The MADS box transcription factor MEF2C is an important regulator of brain, skeleton, lymphocyte and cardiovascular development and is required in the neural crest for craniofacial development. Here, we establish a novel role for MEF2C in melanocyte development. Inactivation of Mef2c in the neural crest of mice results in reduced expression of melanocyte genes during development and a significant loss of pigmentation at birth due to defective differentiation and reduced abundance of melanocytes. We identify a transcriptional enhancer of Mef2c that directs expression to the neural crest and its derivatives, including melanocytes, in transgenic mouse embryos. This novel Mef2c neural crest enhancer contains three functional SOX binding sites and a single essential MEF2 site. We demonstrate that Mef2c is a direct transcriptional target of SOX10 and MEF2 via this evolutionarily conserved enhancer. Furthermore, we show that SOX10 and MEF2C physically interact and function cooperatively to activate the Mef2c gene in a feed-forward transcriptional circuit, suggesting that MEF2C might serve as a potentiator of the transcriptional pathways affected in Waardenburg syndromes.

Targeting ALDH1 to decrease tumorigenicity, growth and metastasis of human melanoma.

Cells with aldehyde dehydrogenase activity (ALDH+) are the most tumorigenic cells in many cancers, including melanoma, making ALDH a candidate therapeutic target. We examined the effects of chemical inhibition of ALDH1 on the response of human melanoma xenografts to chemotherapy and the effects of ALDH1A1 RNA silencing on melanoma growth and metastasis. Addition of ALDH1 inhibitors (e.g. diethylaminobenzaldehyde) to dacarbazine chemotherapy, not only reduced tumor growth in vivo, but also resulted in a significant decrease in the number of residual cells capable of tumorigenesis. shRNA depletion of ALDH1A1 in melanoma cells resulted not only in a significant delay in appearance of xenograft melanomas and reduction in growth, but also significantly decreased the number of metastases and metastatic burden after lateral tail vein injections in mice. In summary, ALDH1 inhibition in combinatorial therapy with dacarbazine reduced the number of residual tumorigenic cells post-therapy and ALDH1A1 depletion had marked inhibitory effects on both melanoma growth and metastasis. These findings suggest that ALDH1 inhibition may not only be able to provide a therapeutic advantage in melanoma treatment, but may also prevent rapid relapse after therapy, as residual tumorigenic cells are fewer and metastatic ability is diminished.

Sex disparities in melanoma outcomes: The role of biology

Melanoma outcomes differ between men and women even when adjusted for prognostic factors such as age, Breslow thickness, body site, ulceration, lymph node dissection, and for treatment, with men having poorer outcomes compared to women. The mechanisms underlying this disparity are not well understood. Behavioral differences between the sexes such as ultraviolet light exposure and health care services utilization have been suggested as contributing, and differences in endogenous biological processes such as immune function, hormonal regulation, oxidative stress response, vitamin D metabolism and sex chromosome gene expression have also been proposed as mechanisms. This review examines the cumulative evidence for biologically based processes that lead to differences in melanoma biology, including inherent sex-based differences in immune function, oxidative stress response and vitamin D metabolism; the complex interplay between sex hormones, the immune system and oxidative stress response; the effect of non-random X chromosome inactivation on tumorigenesis; and the potential contribution of recently identified oncogenes on the Y chromosome.

Basis for Enhanced Barrier Function of Pigmented Skin

Humans with darkly-pigmented skin display superior permeability barrier function in comparison to humans with lightly-pigmented skin. The reduced pH of the stratum corneum (SC) of darkly-pigmented skin could account for enhanced function, because acidifying lightly-pigmented human SC resets barrier function to darkly-pigmented levels. In SKH1 (non-pigmented) vs. SKH2/J (pigmented) hairless mice, we evaluated how a pigment-dependent reduction in pH could influence epidermal barrier function. Permeability barrier homeostasis is enhanced in SKH2/J vs. SKH1 mice, correlating with a reduced pH in the lower SC that co-localizes with the extrusion of melanin granules. Darkly-pigmented human epidermis also shows substantial melanin extrusion in the outer epidermis. Both acute barrier disruption and topical basic pH challenges accelerate re-acidification of SKH2/J (but not SKH1) SC, while inducing melanin extrusion. SKH2/J mice also display enhanced expression of the SC acidifying enzyme, secretory phospholipase A2f (sPLA2f). Enhanced barrier function of SKH2/J mice could be attributed to enhanced activity of two acidic pH-dependent, ceramide-generating enzymes, β-glucocerebrosidase and acidic sphingomyelinase, leading to accelerated maturation of SC lamellar bilayers. Finally, organotypic cultures of darkly-pigmented-bearing human keratinocytes display enhanced barrier function in comparison to lightly-pigmented cultures. Together, these results suggest that the superior barrier function of pigmented epidermis can be largely attributed to the pH-lowering impact of melanin persistence/extrusion and enhanced sPLA2f expression.

The role of IL-17 in vitiligo: A review

IL-17 is involved in the pathogenesis of several autoimmune diseases; however its role in vitiligo has not been well defined. Emerging human and mouse studies have demonstrated that systemic, tissue, and cellular levels of IL-17 are elevated in vitiligo. Many studies have also shown significant positive correlations between these levels and disease activity, extent, and severity. Treatments that improve vitiligo, such as ultraviolet B phototherapy, also modulate IL-17 levels. This review synthesizes our current understanding of how IL-17 may influence the pathogenesis of autoimmune vitiligo at the molecular level. This has implications for defining new vitiligo biomarkers and treatments.

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.

Outcomes of Melanoma In Situ Treated With Mohs Micrographic Surgery Compared With Wide Local Excision

Importance Melanoma in situ (MIS) is increasing in incidence, and expert consensus opinion recommends surgical excision for therapeutic management. Currently, wide local excision (WLE) is the standard of care. However, Mohs micrographic surgery (MMS) is now used to treat a growing subset of individuals with MIS. During MMS, unlike WLE, the entire cutaneous surgical margin is evaluated intraoperatively for tumor cells. Objective To assess the outcomes of patients with MIS treated with MMS compared with those treated with WLE. Design, Setting, and Participants Retrospective review of a prospective database. The study cohort consisted of 662 patients with MIS treated with MMS or WLE per standard of care in dermatology and surgery (general surgery, otolaryngology, plastics, oculoplastics, surgical oncology) at an academic tertiary care referral center from January 1, 1978, to December 31, 2013, with follow-up through 2015. Exposure Mohs micrographic surgery or WLE. Main Outcomes and Measures Recurrence, overall survival, and melanoma-specific survival. Results There were 277 patients treated with MMS (mean [SD] age, 64.0 [13.1] years; 62.1% male) and 385 treated with WLE (mean [SD] age, 58.5 [15.6] years; P < .001 for age; 54.8% male). Median follow-up was 8.6 (range, 0.2-37) years. Compared with WLE, MMS was used more frequently on the face (222 [80.2%] vs 141 [36.7%]) and scalp and neck (23 [8.3%] vs 26 [6.8%]; P < .001). The median (range) year of diagnosis was 2008 (1986-2013) for the MMS group vs 2003 (1978-2013) for the WLE group (P < .001). Overall recurrence rates were 5 (1.8%) in the MMS group and 22 (5.7%) in the WLE group (P = .07). Mean (SD) time to recurrence after MMS was 3.91 (4.4) years, and after WLE, 4.45 (2.7) years (P = .73). The 5-year recurrence rate was 1.1% in the MMS group and 4.1% in the WLE group (P = .07). For WLE-treated tumors, the surgical margin taken was greater for tumors that recurred compared with tumors that did not recur (P = .003). Five-year overall survival for MMS was 92% and for WLE was 94% (P = .28). Melanoma-specific mortality for the MMS group was 2 vs 13 patients for the WLE group, with mean (SD) survival of 6.5 (4.8) and 6.1 (0.8) years, respectively (P = .77). Conclusions and Relevance No significant differences were found in the recurrence rate, overall survival, or melanoma-specific survival of patients with MIS treated with MMS compared with WLE.

Chemoprevention agents for melanoma: a path forward into phase 3 clinical trials

Recent progress in the treatment of advanced melanoma has led to unprecedented improvements in overall survival and, as these new melanoma treatments have been developed and deployed in the clinic, much has been learned about the natural history of the disease. Now is the time to apply that knowledge toward the design and clinical evaluation of new chemoprevention agents. Melanoma chemoprevention has the potential to reduce dramatically both the morbidity and the high costs associated with treating patients who have metastatic disease. In this work, scientific and clinical melanoma experts from the national Melanoma Prevention Working Group, composed of National Cancer Trials Network investigators, discuss research aimed at discovering and developing (or repurposing) drugs and natural products for the prevention of melanoma and propose an updated pipeline for translating the most promising agents into the clinic. The mechanism of action, preclinical data, epidemiological evidence, and results from available clinical trials are discussed for each class of compounds. Selected keratinocyte carcinoma chemoprevention studies also are considered, and a rationale for their inclusion is presented. These data are summarized in a table that lists the type and level of evidence available for each class of agents. Also included in the discussion is an assessment of additional research necessary and the likelihood that a given compound may be a suitable candidate for a phase 3 clinical trial within the next 5 years.

Biology and Sex Disparities in Melanoma Outcomes

The incidence of malignant melanoma in most developed countries has risen faster than any other cancer type; In the United States in 2016, women accounted for 39% of the 76,380 new cases of invasive cutaneous melanoma, and only 33% of the 10,130 were melanoma-related deaths. Gender differences in melanoma incidence and outcomes have been consistently observed even when adjusting for prognostic factors such as age, Breslow thickness, body site, ulceration, lymph node dissection, and for treatment. The mechanisms underlying this disparity are not well understood but likely related to both behavioral and biological etiologies. This chapter examines the cumulative evidence for biologically based processes that lead to differences in melanoma biology in men and women, including inherent sex-based differences in immune function, oxidative stress response and vitamin D metabolism; the complex interplay between sex hormones, the immune system and oxidative stress response; the effect of non-random X chromosome inactivation on tumorigenesis; and the potential contribution of recently identified oncogenes on the Y chromosome.