Helen Michael

Sporadic naturally occurring melanoma in dogs as a preclinical model for human melanoma

Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun‐exposed sites. Most occur in the oral cavity, with a subset having intra‐epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ‐line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c‐kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model.

Functional Analysis of Prognostic Gene Expression Network Genes in Metastatic Breast Cancer Models

Identification of conserved co-expression networks is a useful tool for clustering groups of genes enriched for common molecular or cellular functions [1]. The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2]. Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer. Here, we validate one of the highly connected genes as a metastasis associated gene. Tpx2, the most highly connected gene within a proliferation network specifically prognostic for estrogen receptor positive (ER+) breast cancers, enhances metastatic disease, but in a tumor autonomous, proliferation-independent manner. Histologic analysis suggests instead that variation of TPX2 levels within disseminated tumor cells may influence the transition between dormant to actively proliferating cells in the secondary site. These results support the co-expression network approach for identification of new metastasis-associated genes to provide new information regarding the etiology of breast cancer progression and metastatic disease.

Mouse models of UV-induced melanoma: genetics, pathology, and clinical relevance

Melanocytes, a neural crest cell derivative, produce pigment to protect keratinocytes from ultraviolet radiation (UVR). Although melanocytic lesions such as nevi and cutaneous malignant melanomas are known to be associated with sun exposure, the role of UVR in oncogenesis is complex and has yet to be clearly elucidated. UVR appears to have a direct mutational role in inducing or promoting melanoma formation as well as an indirect role through microenvironmental changes. Recent advances in the modeling of human melanoma in animals have built platforms upon which prospective studies can begin to investigate these questions. This review will focus exclusively on genetically engineered mouse models of UVR-induced melanoma. The role that UVR has in mouse models depends on multiple factors, including the waveband, timing, and dose of UVR, as well as the nature of the oncogenic agent(s) driving melanomagenesis in the model. Work in the field has examined the role of neonatal and adult UVR, interactions between UVR and common melanoma oncogenes, the role of sunscreen in preventing melanoma, and the effect of UVR on immune function within the skin. Here we describe relevant mouse models and discuss how these models can best be translated to the study of human skin and cutaneous melanoma.

Synergistic targeted inhibition of MEK and dual PI3K/mTOR diminishes viability and inhibits tumor growth of canine melanoma underscoring its utility as a preclinical model for human mucosal melanoma

Human mucosal melanoma (MM), an uncommon, aggressive and diverse subtype, shares characteristics with spontaneous MM in dogs. Although BRAF and N‐RAS mutations are uncommon in MM in both species, the majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Canine MM cell lines, with varying ERK and AKT/mTOR activation levels reflective of naturally occurring differences in dogs, were sensitive to the MEK inhibitor GSK1120212 and dual PI3K/mTOR inhibitor NVP‐BEZ235. The two‐drug combination synergistically decreased cell survival in association with caspase 3/7 activation, as well as altered expression of cell cycle regulatory proteins and Bcl‐2 family proteins. In combination, the two drugs targeted their respective signaling pathways, potentiating reduction of pathway mediators p‐ERK, p‐AKT, p‐S6, and 4E‐BP1 in vitro, and in association with significantly inhibited solid tumor growth in MM xenografts in mice. These findings provide evidence of synergistic therapeutic efficacy when simultaneously targeting multiple mediators in melanoma with Ras/ERK and PI3K/mTOR pathway activation.

Naturally Occurring Canine Melanoma as a Predictive Comparative Oncology Model for Human Mucosal and Other Triple Wild-Type Melanomas

Melanoma remains mostly an untreatable fatal disease despite advances in decoding cancer genomics and developing new therapeutic modalities. Progress in patient care would benefit from additional predictive models germane for human disease mechanisms, tumor heterogeneity, and therapeutic responses. Toward this aim, this review documents comparative aspects of human and naturally occurring canine melanomas. Clinical presentation, pathology, therapies, and genetic alterations are highlighted in the context of current basic and translational research in comparative oncology. Somewhat distinct from sun exposure-related human cutaneous melanomas, there is growing evidence that a variety of gene copy number alterations and protein structure/function mutations play roles in canine melanomas, in circumstances more analogous to human mucosal melanomas and to some extent other melanomas with murine sarcoma viral oncogene homolog B (BRAF), Neuroblastoma RAS Viral (V-Ras) Oncogene Homolog (NRAS), and neurofibromin 1 tumor suppressor NF1 triple wild-type genotype. Gaps in canine genome annotation, as well as an insufficient number and depth of sequences covered, remain considerable barriers to progress and should be collectively addressed. Preclinical approaches can be designed to include canine clinical trials addressing immune modulation as well as combined-targeted inhibition of Rat Sarcoma Superfamily/Mitogen-activated protein kinase (RAS/MAPK) and/or Phosphatidylinositol-3-Kinase/Protein Kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal transduction, pathways frequently activated in both human and canine melanomas. Future investment should be aimed towards improving understanding of canine melanoma as a predictive preclinical surrogate for human melanoma and for mutually benefiting these uniquely co-dependent species.

A Topical Solution to the Sunless Tanning Problem

Skin cancer is the most commonly diagnosed type of cancer and is strongly associated with UV exposure and skin pigmentation. Recent advances in pharmacologic non-UV tanning methods open the possibility of preventing melanoma and non-melanoma skin cancer, especially in people who do not tan in the sun.

A direct link between MITF, innate immunity, and hair graying

Melanocyte stem cells (McSCs) and mouse models of hair graying serve as useful systems to uncover mechanisms involved in stem cell self-renewal and the maintenance of regenerating tissues. Interested in assessing genetic variants that influence McSC maintenance, we found previously that heterozygosity for the melanogenesis associated transcription factor, Mitf, exacerbates McSC differentiation and hair graying in mice that are predisposed for this phenotype. Based on transcriptome and molecular analyses of Mitfmi-vga9/+ mice, we report a novel role for MITF in the regulation of systemic innate immune gene expression. We also demonstrate that the viral mimic poly(I:C) is sufficient to expose genetic susceptibility to hair graying. These observations point to a critical suppressor of innate immunity, the consequences of innate immune dysregulation on pigmentation, both of which may have implications in the autoimmune, depigmenting disease, vitiligo.

Abstract 1037: Progression from melanocytic nevi to melanoma is associated with increased genomic mutations in a UV-induced mouse model of human melanoma

Melanoma is the deadliest form of skin cancer with approximately 132,000 cases worldwide each year. Benign melanocytic nevi are nearly universal, and although progression of nevi to melanoma is very rare, 20-50% of melanoma appear to arise from a pre-existing nevus. UV exposure, particularly childhood sunburn, is believed to play an important role in the development of melanocytic nevi and melanoma, but the exact mechanism is unknown. Alterations in MAPK pathway genes, especially NRAS and BRAF, are common in both benign nevi and melanoma, but approximately 1/3 of melanomas do not have an identified driver mutation. Studying nevus initiation and progression prospectively in the human population is impractical due to the long latency to progression and repeated UV exposures Our laboratory has developed a hepatocyte growth factor (HGF) genetically engineered mouse model with “humanized” junctional distribution of melanocytes on an iDCT-GFP background with melanocyte-specific GFP expression, allowing melanocytic lesions to be tracked through percutaneous GFP imaging. Following a single relevant dose of UV modeling childhood sunburn, HGF iDCT-GFP develop discrete, small melanocytic lesions consistent with nevi. Most nevi remain stable over the lifetime of the mouse, but around 1 in 30 progress to melanoma usually starting at 6-12 months. The melanocytic lesions are histologically similar to human nevi and melanoma, label with melanocyte markers and tumors are transplantable into syngeneic mice. Melanomas that arise in the model are heterogeneous and include radial growth phase and vertical growth phase tumors and sometimes metastasize to liver and lung. Exome sequencing of 28 nevi and melanomas show that vertical growth phase melanomas have approximately 3x more mutations than radial growth phase melanomas or nevi. The increased number of mutations in vertical growth phase tumors is due to an increase in C>T transitions despite the lack of additional UV exposure. Interestingly, melanocytic nevi and melanomas with DNA repair pathway mutations average 3x more mutations than lesions without mutations in these pathways. Melanomas sometime contain mutations in hotspot locations from human melanomas, including GNAQ, but most do not have a previously identified dominant driver. Genes potentially involved in the initiation of melanocytic lesions or progression to aggressive melanomas and relevant to human melanoma have been identified and are being functionally tested using CRISPR to introduce point mutations or knock out genes and in vitro skin reconstitution assays. Identification of novel drivers and pathways involved in non-BRAF, non-NRAS melanoma has the potential to uncover biomarkers and new therapeutic targets to improve clinical outcomes for melanoma patients. Citation Format: Helen Michael, Chi-Ping Day, Howard Yang, Aleksandra Michalowski, Maxwell Lee, Glenn Merlino. Progression from melanocytic nevi to melanoma is associated with increased genomic mutations in a UV-induced mouse model of human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1037. doi:10.1158/1538-7445.AM2017-1037

Abstract 994: Integrated embryonic transcriptome analyses identify key melanoma metastasis regulator

The enhanced ability of melanoma cells to metastasize is reminiscent of the innate propensity of melanoblasts to migrate to distant sites during embryonic development - from the neural crest to their eventual niche in the skin. Once transformed, melanoma cells mimic migratory and growth capabilities similar to those of embryonic melanoblasts. This putative relationship between tumorigenesis and developmental processes was first suggested by Rudolf Virchow more than 150 years ago. While this theory is largely unproven, there are mechanistic links between the processes regulating development and malignancy. Here we investigate this age-old puzzle using a mouse model with melanocyte-specific GFP expression to capture and sequence embryonic melanoblasts. We have, for the first time, isolated and sequenced the transcriptomes of murine embryonic melanoblasts at several key representative developmental stages. To uncover the overall classes of gene expression and to identify and characterize genesets whose expression is common and equally important to melanomagenic and developmental processes, a heat-map of the top 1000 most variable developmental genes was generated, and then shortlisted based on compared levels of expression in human and mouse metastatic melanomas, and on the relationship with melanoma patient survival data. By integrating bioinformatics and functional data we have devised a new cross-species multi-dimensional embryonic-onco-genomics analyses (MEGA) approach and show that late stage melanomas reactivate genes used during embryonic development to achieve a more aggressive metastatic phenotype (which we refer to as metafetal genes). One such identified gene, a KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum (ER) protein retention receptor (KDELR) family member, was expressed in advanced mouse melanomas relative to normal skin or benign nevi. We confirmed this in human melanoma by showing that this metafetal gene was expressed at high levels in metastatic patient samples relative to benign lesions, and also predicted patient survival. Next, we determined the consequences of RNAi-based knockdown on experimental metastasis in mouse models. We validated the functional significance in human and mouse melanomas by showing that this KDELR plays a key role in melanoma metastasis through adaptation to chronic ER stress for survival by modulating the unfolded protein response (UPR), which can be targeted. We anticipate that this approach will identify a variety of key hardwired pathways associated with melanocyte development that can be co-opted by opportunistic metastatic melanoma cells. This approach also offers a novel perspective on melanoma therapeutics and intervention and offers both mechanistic as well as prognostic insights into our understanding of this fatal disease. Citation Format: Pravin J. Mishra, Theresa Guo, Raza Zaidi, Sean Davis, Aleksandra Michalowski, Helen Michael, William Reinhold, Heinz Arnheiter, Paul Meltzer, Glenn Merlino. Integrated embryonic transcriptome analyses identify key melanoma metastasis regulator. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 994. doi:10.1158/1538-7445.AM2014-994