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Triple-negative breast cancer (TNBC) is characterized by a high degree of immune infiltrate in the tumour microenvironment, which may influence the fate of TNBC cells. We reveal that loss of the tumour suppressive transcription factor Elf5 in TNBC cells activates intrinsic interferon-γ (IFN-γ) signalling, promoting tumour progression and metastasis. Mechanistically, we find that loss of the Elf5-regulated ubiquitin ligase FBXW7 ensures stabilization of its putative protein substrate IFN-γ receptor 1 (IFNGR1) at the protein level in TNBC. Elf5(low) tumours show enhanced IFN-γ signalling accompanied by an increase of immunosuppressive neutrophils within the tumour microenvironment and increased programmed death ligand 1 expression. Inactivation of either programmed death ligand 1 or IFNGR1 elicited a robust anti-tumour and/or anti-metastatic effect. A positive correlation between ELF5 and FBXW7 expression and a negative correlation between ELF5, FBXW7 and IFNGR1 expression in the tumours of patients with TNBC strongly suggest that this signalling axis could be exploited for patient stratification and immunotherapeutic treatment strategies for Elf5(low) patients with TNBC.

Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover metastatic melanoma biology. Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival. We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis. In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters. We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation. Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy., Metastatic cells can mimic many of the phenotypic behaviors of embryonic cells. Here, the authors generate a melanoblast-specific transcriptome using a genetically engineered mouse model and identify KDELR3 as a pro-metastasis gene in melanoma.

SUMMARYInterferon-gamma (IFNG) has long been regarded as the flag-bearer for the anti-cancer immunosurveillance mechanisms. However, relatively recent studies have suggested a dual role of IFNG, albeit there is no direct experimental evidence for its potential pro-tumor functions. Here we providein vivoevidence that treatment of mouse melanoma cell lines with physiological levels of Ifng enhances their tumorigenicity and metastasis in lung colonization allograft assays performed in immunocompetent syngeneic host mice, but not in immunocompromised host mice. We also show that this enhancement is dependent on downstream signaling via Stat1 but not Stat3, providing evidence of an oncogenic function of Stat1 in melanoma. The experimental results suggest that melanoma cell-specific Ifng signaling modulates the tumor microenvironment and its pro-tumorigenic effects are dependent on the γδ T cells, as Ifng-enhanced tumorigenesis was inhibited in the TCR-δ knockout mice. Overall, these results show that Ifng signaling may have tumor-promoting effects in melanoma by modulating the immune cell composition of the tumor microenvironment.

Solar ultraviolet radiation (UVR) suppresses skin immunity, which facilitates initiation of skin lesions and establishment of tumors by promoting immune evasion. It is unclear whether immune checkpoints are involved in the modulation of skin immunity by UVR. Here, we report that UVR exposure significantly increased expression of immune checkpoint molecule PD-L1 in melanoma cells. The damage-associated molecular patterns molecule HMGB1 was secreted by melanocytes and keratinocytes upon UVR, which subsequently activated the receptor for advanced glycation endproducts (RAGE) receptor to promote NF-κB– and IRF3-dependent transcription of PD-L1 in melanocytes. UVR exposure significantly reduced the susceptibility of melanoma cells to CD8+ T-cell–dependent cytotoxicity, which was mitigated by inhibiting the HMGB1/TBK1/IRF3/NF-κB cascade or by blocking the PD-1/PD-L1 checkpoint. Taken together, our findings demonstrate that UVR-induced upregulation of PD-L1 contributes to immune suppression in the skin microenvironment, which may promote immune evasion of oncogenic cells and drive melanoma initiation and progression. Significance: These findings identify PD-L1 as a critical component of UV-induced immune suppression in the skin, which facilitates immunoevasion of oncogenic melanocytes and development of melanoma. See related commentary by Sahu, p. 2805

CTLA4 is a cell surface receptor on T cells that functions as an immune checkpoint molecule to enforce tolerance to cognate antigens. Anti–CTLA4 immunotherapy is highly effective at reactivating T-cell responses against melanoma, which is postulated to be due to targeting CTLA4 on T cells. Here, we report that CTLA4 is also highly expressed by most human melanoma cell lines, as well as in normal human melanocytes. Interferon-γ (IFNG) signaling activated the expression of the human CTLA4 gene in a melanocyte and melanoma cell–specific manner. Mechanistically, IFNG activated CTLA4 expression through JAK1/2-dependent phosphorylation of STAT1, which bound a specific gamma-activated sequence site on the CTLA4 promoter, thereby licensing CBP/p300-mediated histone acetylation and local chromatin opening. In melanoma cell lines, elevated baseline expression relied upon constitutive activation of the MAPK pathway. Notably, RNA-seq analyses of melanoma specimens obtained from patients who had received anti–CTLA4 immunotherapy (ipilimumab) showed upregulation of an IFNG-response gene expression signature, including CTLA4 itself, which correlated significantly with durable response. Taken together, our results raise the possibility that CTLA4 targeting on melanoma cells may contribute to the clinical immunobiology of anti–CTLA4 responses. Significance: These findings show that human melanoma cells express high levels of the immune checkpoint molecule CTLA4, with important possible implications for understanding how anti-CTLA4 immunotherapy mediates its therapeutic effects. Cancer Res; 78(2); 436–50. ©2017 AACR.

Melanoma is a complex disease that exhibits highly heterogeneous etiological, histopathological, and genetic features, as well as therapeutic responses. Genetically engineered mouse (GEM) models provide powerful tools to unravel the molecular mechanisms critical for melanoma development and drug resistance. Here, we expound briefly the basis of the mouse modeling design, the available technology for genetic engineering, and the aspects influencing the use of GEMs to model melanoma. Furthermore, we describe in detail the currently available GEM models of melanoma. Cancer 2017;123:2089-103. © 2017 American Cancer Society.

Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posited that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover novel metastatic melanoma biology. We used a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, identified melanoblast-specific genes whose expression contributed to metastatic competence, and derived a 43-gene signature that predicted patient survival. We identified a melanoblast gene,KDELR3, whose loss impaired experimental metastasis. In contrast,KDELR1deficiency enhanced metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters. We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation. Our work demonstrates that the melanoblast transcriptome can be mined to uncover novel targetable pathways for melanoma therapy.

Cutaneous malignant melanoma is one of the few major cancers that continue to exhibit a positive rate of increase in the developed world. A wealth of epidemiological data has undisputedly implicated ultraviolet radiation (UVR) from sunlight and artificial sources as the major risk factor for melanomagenesis. However, the molecular mechanisms of this cause-and-effect relationship remain murky and understudied. Recent efforts on multiple fronts have brought unprecedented expansion of our knowledge base on this subject and it is now clear that melanoma is caused by a complex interaction between genetic predisposition and environmental exposure, primarily to UVR. Here we provide an overview of the effects of the macroenvironment (UVR) on the skin microenvironment and melanocyte-specific intrinsic (mostly genetic) landscape, which conspire to produce one of the deadliest malignancies.

Interferon-gamma (IFNG) has long been implicated as a central orchestrator of antitumor immune responses in the elimination stage of the immunoediting paradigm. However, mounting evidence suggests that IFNG may also have important and significant protumor roles to play in the equilibrium and escape phases through its regulatory effects on immunoevasive functions that promote tumorigenesis. These seemingly contradictory effects of IFNG undoubtedly play profound roles in not only the activation of inflammatory response to cancer but also in the determination of its outcome. In the face of the recent explosion of anticancer immunotherapeutic strategies in the clinic, it is critical that a complete understanding is achieved of the underpinnings of the mechanisms that determine the two faces of IFNG signaling in cancer. Here, the current state of this dichotomy is reviewed.

Macrophages are conventionally classified into M1 and M2 subtypes according to their differentiation status and functional role in the immune system. However, accumulating evidence suggests that this binary classification system is insufficient to account for the remarkable plasticity of macrophages that gives rise to an immense diversity of subtypes. This diverse spectrum of macrophage subtypes play critical roles in various homeostatic and immune functions, but remain far from being fully characterised. In addition to their roles in normal physiological conditions, macrophages also play crucial roles in disease conditions such as cancer. In this review, we discuss the roles tumour-associated macrophages (TAMs) play in regulating different steps of tumour progression and metastasis, and the opportunities to target them in the quest for cancer prevention and treatment.

Solar ultraviolet radiation (UVR) suppresses skin immunity, which facilitates skin lesion initiation and establishment by promoting immune evasion. It is unclear whether immune checkpoints are involved in the modulation of skin immunity by UVR. Here we report that the expression of immune checkpoint molecule PD-L1 was significantly increased in melanocytes and melanoma cells upon UVR exposure. The damage-associated molecular patterns molecule HMGB1 was secreted by melanocytes and keratinocytes upon UVR, which subsequently activated the RAGE (receptor for advanced glycation endproducts) receptor to promote the NF-κB and IRF3-dependent transcription of PD-L1 in melanocytes. We also found that UVR exposure significantly reduced the susceptibility of melanoma cells to CD8+ T cell-dependent cytotoxicity, which was mitigated by inhibiting the HMGB1/TBK1/IRF3/NF-κB cascade or blocking the PD-1/PD-L1 checkpoint. Taken together, our findings demonstrate that UVR-induced PD-L1 upregulation contributes to the immune suppression in the skin microenvironment, which may promote immune evasion of oncogenic cells, and melanoma initiation and progression. Citation Format: Wei Wang, Nicole Chapman, Bo Zhang, Mingqi Li, Meiyun Fan, R. Nicholas Laribee, M. Raza Zaidi, Lawrence Pfeffer, Hongbo Chi, Zhaohui Wu. HMGB1-activated IRF3 and NF-κB contributes to UV radiation-induced immune suppression by upregulating PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 524.

Sequence polymorphisms linked to human diseases and phenotypes in genome-wide association studies often affect non-coding regions. A single nucleotide polymorphism (SNP) within an intron of the gene encoding Interferon Regulatory Factor 4 (IRF4), a transcription factor with no known role in melanocyte biology, is strongly associated with sensitivity of skin to sun exposure, freckles, blue eyes and brown hair color. Here we demonstrate that this SNP lies within an enhancer of IRF4 transcription in melanocytes. The allele associated with this pigmentation phenotype impairs binding of the TFAP2A transcription factor which together with the melanocyte master regulator MITF, regulates activity of the enhancer. Assays in zebrafish and mice reveal that IRF4 cooperates with MITF to activate expression of Tyrosinase (TYR), an essential enzyme in melanin synthesis. Our findings provide a clear example of a non-coding polymorphism that affects a phenotype by modulating a developmental gene regulatory network.

The non-histone chromatin-binding protein HMGA2 is expressed predominantly in the mesenchyme before its differentiation, but it is also expressed in tumors of epithelial origin. Ectopic expression of HMGA2 in epithelial cells induces epithelial–mesenchymal transition (EMT), which has been implicated in the acquisition of metastatic characters in tumor cells. However, little is known about in vivo modulation of HMGA2 and its effector functions in tumor metastasis. Here, we report that HMGA2 loss of function in a mouse model of cancer reduces tumor multiplicity. HMGA2-positive cells were identified at the invasive front of human and mouse tumors. In addition, in a mouse allograft model, HMGA2 overexpression converted nonmetastatic 4TO7 breast cancer cells to metastatic cells that homed specifically to liver. Interestingly, expression of HMGA2 enhanced TGFβ signaling by activating expression of the TGFβ type II receptor, which also localized to the invasive front of tumors. Together our results argued that HMGA2 plays a critical role in EMT by activating the TGFβ signaling pathway, thereby inducing invasion and metastasis of human epithelial cancers. Cancer Res; 73(14); 4289–99. ©2013 AACR.

Alloreactive T cells play a critical role in eliminating hematopoietic malignant cells but are also the mediators of graft-versus-host disease (GVHD), a major complication that subverts the success of allogeneic hematopoietic stem cell transplantation (HSCT). However, induction of alloreactive T cells does not necessarily lead to GVHD. Here we report the development of a cellular programming approach to render alloreactive T cells incapable of causing severe GVHD in both major histocompatibility complex (MHC)-mismatched and MHC-identical but minor histocompatibility antigen-mismatched mouse models. We established a novel platform that produced δ-like ligand 4-positive dendritic cells (Dll4(hi)DCs) from murine bone marrow using Flt3 ligand and Toll-like receptor agonists. Upon allogeneic Dll4(hi)DC stimulation, CD4(+) naive T cells underwent effector differentiation and produced high levels of interferon γ (IFN-γ) and interleukin-17 in vitro, depending on Dll4 activation of Notch signaling. Following transfer, allogeneic Dll4(hi)DC-induced T cells were unable to mediate severe GVHD but preserved antileukemic activity, significantly improving the survival of leukemic mice undergoing allogeneic HSCT. This effect of Dll4(hi)DC-induced T cells was associated with their impaired expansion in GVHD target tissues. IFN-γ was important for Dll4(hi)DC programming to reduce GVHD toxicities of alloreactive T cells. Absence of T-cell IFN-γ led to improved survival and expansion of Dll4(hi)DC-induced CD4(+) T cells in transplant recipients and caused lethal GVHD. Our findings demonstrate that Dll4(hi)DC programming can overcome GVHD toxicity of donor T cells and produce leukemia-reactive T cells for effective immunotherapy.

Cutaneous malignant melanoma is rapidly increasing in the developed world and continues to be a challenge in the clinic. Although extensive epidemiologic evidence points to solar UV as the major risk factor for melanoma, there is a significant gap in our knowledge about how this most ubiquitous environmental carcinogen interacts with the largest organ of the mammalian body (skin) at the microenvironmental and molecular level. We review some recent advances that have started to close this gap. Cancer Res; 72(7); 1591–5. ©2012 AACR.

Interferon-γ is a cytokine whose biological activity is conventionally associated with cytostatic/cytotoxic and antitumor mechanisms during cell-mediated adaptive immune response. It has been used clinically to treat a variety of malignancies, albeit with mixed results and side effects that can be severe. Despite ample evidence implicating a role for IFN-γ in tumor immune surveillance, a steady flow of reports has suggested that it may also have protumorigenic effects under certain circumstances. We propose that, in fact, IFN-γ treatment is a double-edged sword whose anti- and protumorigenic activities are dependent on the cellular, microenvironmental, and/or molecular context. As such, inhibition of the IFN-γ/IFN-γ receptor pathway may prove to be a viable new therapeutic target for a subset of malignancies. Clin Cancer Res; 17(19); 6118–24. ©2011 AACR.

Malignant melanoma of the skin (CMM) is associated with ultraviolet radiation exposure, but the mechanisms and even the wavelengths responsible are unclear. Here we use a mammalian model to investigate melanoma formed in response to precise spectrally defined ultraviolet wavelengths and biologically relevant doses. We show that melanoma induction by ultraviolet A (320–400 nm) requires the presence of melanin pigment and is associated with oxidative DNA damage within melanocytes. In contrast, ultraviolet B radiation (280–320 nm) initiates melanoma in a pigment-independent manner associated with direct ultraviolet B DNA damage. Thus, we identified two ultraviolet wavelength-dependent pathways for the induction of CMM and describe an unexpected and significant role for melanin within the melanocyte in melanomagenesis., Exposure to ultraviolet light is responsible for a large proportion of melanomas but the molecular mechanisms are unknown. In this study, melanoma is found to be induced in mice by UVA and UVB light in a pigment-dependent and -independent manner, respectively, resulting in different types of DNA damage.

Cell type-specific expression of fluorescent proteins allows the purification of rare cells from complex -tissues by flow cytometry. This strategy is especially useful for molecular analysis of cancer cells because these cells can be effectively purified away from the noncancerous tumor stroma. Coexpression of bioluminescence with fluorescence makes further allows in vivo tracking of cancer cells, which can then be purified at specific tumorigenic stages. Here, we describe protocols for purifying rare skin stem cells, and for in vivo monitoring and purification of cancer cells from lung metastases. Also described is a protocol for the isolation of total RNA from the purified cells for the purpose of performing gene expression profiling.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, whose incidence continues to rise. Epidemiological studies reveal that the major etiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood1,2. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor (HGF/SF) transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and etiologic criteria, but only when irradiated as neonatal pups with UVB, not UVA3,4. However, mechanisms underlying UVB-initiated, neonatal-specific melanomagenesis remain largely unknown. Here we introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-γ (IFN-γ), but not type-I interferons. IFN-γ was produced by macrophages recruited to neonatal skin by UVB-induced ligands to the chemokine receptor Ccr2. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-γ blockade abolished macrophage-enhanced melanoma growth and survival. IFN-γ-producing macrophages were also identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-γ in promoting melanocytic cell survival/immunoevasion, and suggest that IFN-γ-R signaling represents a novel therapeutic melanoma target.

Cutaneous malignant melanoma is highly invasive and capable of metastasizing to distant sites where it is typically resistant to available therapy. While striving to prevent or eradicate melanoma, researchers have two significant advantages not shared by those working on many other cancers. The main environmental etiological agent, UV radiation, is known and melanocytic lesions are excisable for molecular analysis from most stages. Yet knowledge about how UV initiates melanoma has been insufficient to achieve prevention, and the understanding of metastatic mechanisms has been inadequate to reduce mortality. Here, we review the value of melanoma mouse models, focusing on these critical early and late stages.

Braf(V600E) induces benign, growth-arrested melanocytic nevus development, but also drives melanoma formation. Cdkn2a loss in Braf(V600E) melanocytes in mice results in rare progression to melanoma, but only after stable growth arrest as nevi. Immediate progression to melanoma is prevented by upregulation of miR-99/100, which downregulates mTOR and IGF1R signaling. mTORC1 activation through Stk11 (Lkb1) loss abrogates growth arrest of Braf(V600E) melanocytic nevi, but is insufficient for complete progression to melanoma. Cdkn2a loss is associated with mTORC2 and Akt activation in human and murine melanocytic neoplasms. Simultaneous Cdkn2a and Lkb1 inactivation in Braf(V600E) melanocytes results in activation of both mTORC1 and mTORC2/Akt, inducing rapid melanoma formation in mice. In this model, activation of both mTORC1/2 is required for Braf-induced melanomagenesis.