Modelling melanoma in mice

Phenotypic and molecular heterogeneity in human melanoma has impaired efforts to explain many of the clinically important features of melanoma. For example, many of the underlying mechanisms that might predict age-of-onset, time to metastasis and other key elements in melanoma progression remain unknown. Furthermore, melanoma staging used to predict outcome and treatment has not yet moved beyond a basic phenotypic classification. While molecularly targeted therapies show great promise for melanoma patients, establishing accurate animal models that recapitulate human cutaneous melanoma progression remains a priority. We examine the relevance of mice as models for human melanoma progression and for key molecular and histopathologic variants of melanoma. These mice may be used as preclinical models to probe the relationships between causative mutations, disease progression and outcome for molecularly targeted therapeutics. We ask how new mouse models, or more detailed histopathologic and molecular analyses of existing mouse models, may be used to advance our understanding of genotype-phenotype correlations in this tumour type. This necessarily involves a consideration of the utility of mice as models for ultraviolet radiation-induced melanoma, and how this might be improved.     

The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations

The remarkable success of immune therapies emphasizes the need for immune‐competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here, we describe the Yale University Mouse Melanoma (YUMM) lines, a comprehensive system of mouse melanoma cell lines that are syngeneic to C57BL/6, have well‐defined human‐relevant driver mutations, and are genomically stable. This will be a useful tool for the study of tumor immunology and genotype‐specific cancer biology.     

ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity

Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1(-) bulk tumor cells. ABCB1(+) cells also exhibited enhanced clonogenicity, anchorage-independent growth, tumorigenicity and mitochondrial activity compared to ABCB1(-) cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1(+) cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma.     

Hic‐5 affects proliferation,migration and invasion of B16 murine melanoma cells

Hic‐5 is a shuttling protein between the cell membrane and the nucleus which functions as a focal adhesion adaptor protein and a nuclear receptor coactivator. Although several studies have shown its involvement in other types of cancer, the role of Hic‐5 in melanoma is unknown. Herein, we show for the first time that Hic‐5 is expressed in B16‐F1 murine melanoma cells. To determine its function in melanoma cells, we used shRNA‐mediated RNA interference and established stable clones with down‐regulated Hic‐5 expression. These clones had impaired growth and metastatic potential compared with controls in vivo, which correlated with decreased proliferation, migration and invasion in vitro. Moreover, silencing of Hic‐5 expression in B16‐F1 activated RhoA with an amoeboid phenotypic change, indicating that Hic‐5 is a key regulator of B16‐F1 metastasis in the context of Rho‐dependent motility. These results provide new evidence that Hic‐5 is a possible molecular target for treatment of melanoma.     

Development of Cutaneous Amelanotic Melanoma in the Absence of a Functional Tyrosinase

Lack of characteristic pigmentation and a wide range of clinical presentations account for the diagnostic challenge associated with amelanotic malignant melanoma. Experimental studies of this important human cancer have been hampered by the lack of an appropriate animal model. We previously described a transgenic mouse line (TG‐3) that spontaneously develops pigmented cutaneous melanoma. F1 crosses were generated with TG‐3 and several albino strains, and backcrosses were then made with the albinos. In the present report, we describe the restricted development and characterization of cutaneous amelanotic melanoma in these albino transgenic backcrosses. The incidence and behavior of melanoma in these mice were monitored. A high incidence (80–100%) of spontaneous amelanotic melanoma was observed in albino transgenic mice derived from backcrosses with A, AKR, FVB, and SJL strains. The lowest incidence (30%) was obtained in BALB/c‐derived crosses. No tumors were observed in non‐transgenic mice. Immunohistochemical and western blot analyses using antibodies against three melanocyte‐specific markers of the tyrosinase family of proteins confirmed that the tumors were composed of amelanotic melanocytes. Furthermore, the presence of numerous premelanosomes observed by electron microscopy further supported the melanocytic origin of these tumors. Previous in vitro studies on human melanoma have suggested that cutaneous amelanotic melanoma was evolving from pre‐existing pigmented cutaneous melanoma. However, our results indicate that it can occur directly, as evidenced by the appearance of cutaneous amelanotic melanoma in the tyrosinase‐deficient albino mice. These mice represent a potentially valuable model for studying the mechanistic, diagnostic, and therapeutic features of this highly malignant neoplasm.     

Ultraviolet radiation accelerates NRas‐mutant melanomagenesis: A cooperative effect blocked by sunscreen

To mitigate melanoma risk, sunscreen use is widely advocated; yet, the ability of sunscreens to prevent melanoma remains controversial. Here, we test the tenet that sunscreens limit melanoma risk by blocking ultraviolet radiation (UV)‐induced DNA damage using murine models that recapitulate the genetics and spontaneous evolution of human melanoma. We find that a single, non‐erythematous dose of UV dramatically accelerates melanoma onset and increases tumor multiplicity in mice carrying an endogenous, melanocyte‐specific NRas^61R allele. By contrast, transient UV exposure does not alter tumor onset in mice lacking p16^INK4a or harboring an NRas^12D allele. To block the rapid onset of melanoma cooperatively caused by UV and NRas^61R, we employed a variety of aerosol sunscreens. While all sunscreens delayed melanoma formation and blocked UV‐induced DNA damage, differences in aerosol output (i.e., amount applied/cm²) caused variability in the cancer preventative efficacy of products with identical sunburn protection factor (SPF) ratings.     

A novel form of melanoma apoptosis resistance: melanogenesis up-regulation in apoptotic B16-F0 cells delays ursolic acid-triggered cell death

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. The resistance mechanisms are complex and melanoma cells may have diverse possibilities for regulating apoptosis to generate apoptotic deficiencies. In this study, we investigated the relationship between melanogenesis and resistance to apoptosis induced by ursolic acid, a natural chemopreventive agent, in B16-F0 melanoma cells. We demonstrated that cells undergoing apoptosis are able to delay their own death. It appeared that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were clearly implicated in an apoptosis resistance mechanism; while TRP-2, a well known mediator of melanoma resistance to cell death, was repressed. Our results confirm the difficulty of treating melanomas, since, even undergoing apoptosis, cells are nevertheless able to trigger a resistance mechanism to delay death.     

Genetic and environmental melanoma models in fish

Experimental animal models are extremely valuable for the study of human diseases, especially those with underlying genetic components. The exploitation of various animal models, from fruitflies to mice, has led to major advances in our understanding of the etiologies of many diseases, including cancer. Cutaneous malignant melanoma is a form of cancer for which both environmental insult (i.e., UV) and hereditary predisposition are major causative factors. Fish melanoma models have been used in studies of both spontaneous and induced melanoma formation. Genetic hybrids between platyfish and swordtails, different species of the genus Xiphophorus, have been studied since the 1920s to identify genetic determinants of pigmentation and melanoma formation. Recently, transgenesis has been used to develop zebrafish and medaka models for melanoma research. This review will provide a historical perspective on the use of fish models in melanoma research, and an updated summary of current and prospective studies using these unique experimental systems.     

Connecting the dots: Melanoma cell of origin,tumor cell plasticity,trans-differentiation,and drug resistance

Melanoma, a lethal malignancy that arises from melanocytes, exhibits a multiplicity of clinico-pathologically distinct subtypes in sun-exposed and non-sun-exposed areas. Melanocytes are derived from multipotent neural crest cells and are present in diverse anatomical locations, including skin, eyes, and various mucosal membranes. Tissue-resident melanocyte stem cells and melanocyte precursors contribute to melanocyte renewal. Elegant studies using mouse genetic models have shown that melanoma can arise from either melanocyte stem cells or differentiated pigment-producing melanocytes depending on a combination of tissue and anatomical site of origin and activation of oncogenic mutations (or overexpression) and/or the repression in expression or inactivating mutations in tumor suppressors. This variation raises the possibility that different subtypes of human melanomas (even subsets within each subtype) may also be a manifestation of malignancies of distinct cells of origin. Melanoma is known to exhibit phenotypic plasticity and trans-differentiation (defined as a tendency to differentiate into cell lineages other than the original lineage from which the tumor arose) along vascular and neural lineages. Additionally, stem cell-like properties such as pseudo-epithelial-to-mesenchymal (EMT-like) transition and expression of stem cell-related genes have also been associated with the development of melanoma drug resistance. Recent studies that employed reprogramming melanoma cells to induced pluripotent stem cells have uncovered potential relationships between melanoma plasticity, trans-differentiation, and drug resistance and implications for cell or origin of human cutaneous melanoma. This review provides a comprehensive summary of the current state of knowledge on melanoma cell of origin and the relationship between tumor cell plasticity and drug resistance.     

Melanoma invasion – current knowledge and future directions

The acquisition of invasive behaviour is the key transition in the progression of benign melanocyte hyperplasia to life threatening melanoma. Understanding this transition and the mechanisms of invasion are the key to understanding why malignant melanoma is such a devastating disease and will aid treatment strategies. Underlying the invasive behaviour is increased cell motility caused by changes in cytoskeletal organization and altered contacts with the extra‐cellular matrix (ECM). In addition, changes in the interactions of melanoma cells with keratinocytes and fibroblasts enable them to survive and proliferate outside their normal epidermal location. Proteomic and genomic initiatives are greatly increasing our knowledge of which gene products are deregulated in invasive and metastatic melanoma; however, the next challenge is to understand how these genes promote the invasion of melanoma cells. In recent years new models have been developed that more closely recapitulate the conditions of melanoma invasion in vivo. It is hoped that these models will give us a better understanding of how the genes implicated in melanoma progression affect the motility of melanoma cells and their interactions with the ECM, stromal cells and blood vessels. This review will summarise our current understanding of melanoma invasion and focus on the new model systems that can be used to study melanoma.     

Melanoma invasion - current knowledge and future directions

The acquisition of invasive behaviour is the key transition in the progression of benign melanocyte hyperplasia to life threatening melanoma. Understanding this transition and the mechanisms of invasion are the key to understanding why malignant melanoma is such a devastating disease and will aid treatment strategies. Underlying the invasive behaviour is increased cell motility caused by changes in cytoskeletal organization and altered contacts with the extra-cellular matrix (ECM). In addition, changes in the interactions of melanoma cells with keratinocytes and fibroblasts enable them to survive and proliferate outside their normal epidermal location. Proteomic and genomic initiatives are greatly increasing our knowledge of which gene products are deregulated in invasive and metastatic melanoma; however, the next challenge is to understand how these genes promote the invasion of melanoma cells. In recent years new models have been developed that more closely recapitulate the conditions of melanoma invasion in vivo. It is hoped that these models will give us a better understanding of how the genes implicated in melanoma progression affect the motility of melanoma cells and their interactions with the ECM, stromal cells and blood vessels. This review will summarise our current understanding of melanoma invasion and focus on the new model systems that can be used to study melanoma.     

Enhancing the evaluation of PI3K inhibitors through 3D melanoma models

Targeted therapies for mutant BRAF metastatic melanoma are effective but not curative due to acquisition of resistance. PI3K signaling is a common mediator of therapy resistance in melanoma; thus, the need for effective PI3K inhibitors is critical. However, testing PI3K inhibitors in adherent cultures is not always reflective of their potential in vivo. To emphasize this, we compared PI3K inhibitors of different specificity in two‐ and three‐dimensional (2D, 3D) melanoma models and show that drug response predictions gain from evaluation using 3D models. Our results in 3D demonstrate the anti‐invasive potential of PI3K inhibitors and that drugs such as PX‐866 have beneficial activity in physiological models alone and when combined with BRAF inhibition. These assays finally help highlight pathway effectors that could be involved in drug response in different environments (e.g. p4E‐BP1). Our findings show the advantages of 3D melanoma models to enhance our understanding of PI3K inhibitors.     

T cell immunotherapy for melanoma from bedside to bench to barn and back: how conceptual advances in experimental mouse models can be translated into clinical benefit for patients

A solid scientific basis now supports the concept that cytotoxic T lymphocytes can specifically recognize and destroy melanoma cells. Over the last decades, clinicians and basic scientists have joined forces to advance our concepts of melanoma immunobiology. This has catalyzed the rational development of therapeutic approaches to enforce melanoma‐specific T cell responses. Preclinical studies in experimental mouse models paved the way for their successful translation into clinical benefit for patients with metastatic melanoma. A more thorough understanding of how melanomas develop resistance to T cell immunotherapy is necessary to extend this success. This requires a continued interdisciplinary effort of melanoma biologists and immunologists that closely connects clinical observations with in vitro investigations and appropriate in vivo mouse models: From bedside to bench to barn and back.     

Understanding the Roles of FAK in Cancer: Inhibitors,Genetic Models,and New Insights

Focal adhesion kinase (FAK) is a protein tyrosine kinase that regulates cellular adhesion, motility, proliferation and survival in various types of cells. Interestingly, FAK is activated and/or overexpressed in advanced cancers, and promotes cancer progression and metastasis. For this reason, FAK became a potential therapeutic target in cancer, and small molecule FAK inhibitors have been developed and are being tested in clinical phase trials. These inhibitors have demonstrated to be effective by inducing tumor cell apoptosis in addition to reducing metastasis and angiogenesis. Furthermore, several genetic FAK mouse models have made advancements in understanding the specific role of FAK both in tumors and in the tumor environment. In this review, we discuss FAK inhibitors as well as genetic mouse models to provide mechanistic insights into FAK signaling and its potential in cancer therapy.     

Lentivirus‐mediated bifunctional cell labeling for in vivo melanoma study

Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long‐term culture and colony formation of several LV‐labeled mouse melanoma cells showed that promoters derived from mammalian house‐keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase–green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP‐labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP‐positive cells can be isolated from the tumors by fluorescence‐activated cell sorter. Pol2‐Luc/GFP labeling, while lower in activity, was more sustainable than FerH‐Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol‐2‐Luc/GFP labeling allows long‐term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models.     

Differential roles of the pRb and Arf/p53 pathways in murine naevus and melanoma genesis

We report on a systematic analysis of genotype-specific melanocyte (MC) UVR responses in transgenic mouse melanoma models along with tumour penetrance and comparative histopathology. pRb or p53 pathway mutations cooperated with Nras^Q61K to transform MCs. We previously reported that MCs migrate from the follicular outer root sheath into the epidermis after neonatal UVR. Here, we found that Arf or p53 loss markedly diminished this response. Despite this, mice carrying these mutations developed melanoma with very early age of onset after neonatal UVR. Cdk4^R24C did not affect the MC migration. Instead, independent of UVR exposure, interfollicular dermal MCs were more prevalent in Cdk4^R24C mice. Subsequently, in adulthood, these mutants developed dermal MC proliferations reminiscent of superficial congenital naevi. Two types of melanoma were observed in this model. The location and growth pattern of the first was consistent with derivation from the naevi, while the second appeared to be of deep dermal origin. In animals carrying the Arf or p53 defects, no naevi were detected, with all tumours ostensibly skipping the benign precursor stage in progression.     

The impact of skin cancer prevention efforts in New South Wales,Australia: Generational trends in melanoma incidence and mortality

BackgroundIn Australia, skin cancer awareness campaigns have focused on raising the awareness and consequences of skin cancer and highlighting the importance of utilising sun protection.MethodsTrends in melanoma incidence and mortality have been explored elsewhere in Australia and this study sought to examine the trends in NSW. Anonymised incidence and mortality data for in situ and invasive melanoma from 1988 to 2014 were obtained from the NSW Cancer Registry. Trends of melanoma incidence and mortality were analysed using segmented regression to allow for changes over time. Birth cohort patterns were assessed using age–period–cohort models.ResultsOver the period, incidence of in situ melanoma increased in all age groups although the rates were lowest in those under 40 years of age. Incidence of invasive melanoma was either stable or decreased in people under 60, while it increased in those aged 60 and above, particularly in men. Age–period–cohort analysis revealed decreasing age-specific incidence of invasive melanoma under 40 years of age. Melanoma mortality over the period was stable or decreased in all groups except in men aged 60 or over. Overall, mortality rates generally declined or remained stable particularly in recent years.ConclusionIt is encouraging that rates of invasive melanoma are declining in the younger age cohorts – which could be attributed to both primary prevention efforts with individuals protecting their skin as well as early detection through self assessment and clinician performed skin checks. In addition, whilst it is important to monitor the increasing rates of in situ melanoma, the increase is likely due to early detection and treatment of melanoma that could have progressed to invasive melanoma and therefore detection whilst still in situ is an improved outcome. Overall, the results demonstrate the need to continue to improve the understanding of and compliance with primary skin cancer prevention measures in order to reduce population UVR exposure and overall melanoma incidence.     

Metastatic risk and resistance to BRAF inhibitors in melanoma defined by selective allelic loss of ATG5

Melanoma is a paradigm of aggressive tumors with a complex and heterogeneous genetic background. Still, melanoma cells frequently retain developmental traits that trace back to lineage specification programs. In particular, lysosome-associated vesicular trafficking is emerging as a melanoma-enriched lineage dependency. However, the contribution of other lysosomal functions such as autophagy to melanoma progression is unclear, particularly in the context of metastasis and resistance to targeted therapy. Here we mined a broad spectrum of cancers for a meta-analysis of mRNA expression, copy number variation and prognostic value of 13 core autophagy genes. This strategy identified heterozygous loss of ATG5 at chromosome band 6q21 as a distinctive feature of advanced melanomas. Importantly, partial ATG5 loss predicted poor overall patient survival in a manner not shared by other autophagy factors and not recapitulated in other tumor types. This prognostic relevance of ATG5 copy number was not evident for other 6q21 neighboring genes. Melanocyte-specific mouse models confirmed that heterozygous (but not homozygous) deletion of Atg5 enhanced melanoma metastasis and compromised the response to targeted therapy (exemplified by dabrafenib, a BRAF inhibitor in clinical use). Collectively, our results support ATG5 as a therapeutically relevant dose-dependent rheostat of melanoma progression. Moreover, these data have important translational implications in drug design, as partial blockade of autophagy genes may worsen (instead of counteracting) the malignant behavior of metastatic melanomas.     

C57BL/6 congenic mouse NRASQ61K melanoma cell lines are highly sensitive to the combination of Mek and Akt inhibitors in vitro and in vivo

RAS is frequently mutated in various tumors and known to be difficult to target. NRAS^Q61K/R are the second most frequent mutations found in human skin melanoma after BRAF^V600E. Aside from surgery, various approaches, including targeted therapies, immunotherapies, and combination therapies, are used to treat patients carrying NRAS mutations, but they are inefficient. Here, we established mouse NRAS^Q61K melanoma cell lines and genetically derived isografts (GDIs) from Tyr::NRAS^Q61K mouse melanoma that can be used in vitro and in vivo in an immune‐competent environment (C57BL/6) to test and discover novel therapies. We characterized these cell lines at the cellular, molecular, and oncogenic levels and show that NRAS^Q61K melanoma is highly sensitive to the combination of Mek and Akt inhibitors. This preclinical model shows much potential for the screening of novel therapeutic strategies for patients harboring NRAS mutations that have limited therapeutic options and resulted in poor prognoses.