M. Raza Zaidi

The Two Faces of Interferon-γ in cancer.

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.

Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry.

Mutually exclusive activating mutations in the GNAQ and GNA11 oncogenes, encoding heterotrimeric Gαq family members, have been identified in ∼ 83% and ∼ 6% of uveal and skin melanomas, respectively. However, the molecular events underlying these GNAQ-driven malignancies are not yet defined, thus limiting the ability to develop cancer-targeted therapies. Here, we focused on the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway that controls organ size. We found that Gαq stimulates YAP through a Trio-Rho/Rac signaling circuitry promoting actin polymerization, independently of phospholipase Cβ and the canonical Hippo pathway. Furthermore, we show that Gαq promotes the YAP-dependent growth of uveal melanoma cells, thereby identifying YAP as a suitable therapeutic target in uveal melanoma, a GNAQ/GNA11-initiated human malignancy.

Interferon-γ links ultraviolet radiation to melanomagenesis in mice.

Cutaneous malignant melanoma is a highly aggressive and frequently chemoresistant cancer, the incidence of which continues to rise. Epidemiological studies show that the major aetiological melanoma risk factor is ultraviolet (UV) solar radiation, with the highest risk associated with intermittent burning doses, especially during childhood. We have experimentally validated these epidemiological findings using the hepatocyte growth factor/scatter factor transgenic mouse model, which develops lesions in stages highly reminiscent of human melanoma with respect to biological, genetic and aetiological criteria, but only when irradiated as neonatal pups with UVB, not UVA. However, the 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, identifying a novel candidate therapeutic target for a subset of melanoma patients.

Melanoma induction by ultraviolet A but not ultraviolet B radiation requires melanin pigment.

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.

HMGA2 is a driver of tumor metastasis.

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.

A polymorphism in IRF4 affects human pigmentation through a tyrosinase-dependent MITF/TFAP2A pathway.

Sequence polymorphisms linked to human diseases and phenotypes in genome-wide association studies often affect noncoding regions. A 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 that, 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 noncoding polymorphism that affects a phenotype by modulating a developmental gene regulatory network.

Misexpression of Full-length HMGA2 Induces Benign Mesenchymal Tumors in Mice

The high-mobility group AT-hook 2 (HMGA2) protein is a member of the high-mobility group family of the DNA-binding architectural factors and participates in the conformational regulation of active chromatin on its specific downstream target genes. HMGA2 is expressed in the undifferentiated mesenchyme and is undetectable in their differentiated counterparts, suggesting its functional importance in mesenchymal cellular proliferation and differentiation. Interestingly, it is a frequent target of chromosomal translocations in several types of human benign differentiated mesenchymal tumors, including lipomas, fibroadenomas of the breast, salivary gland adenomas, and endometrial polyps. The translocations lead to a variety of HMGA2 transcripts, which range from wild-type, truncated, and fusion mRNA species. However, it is not clear whether alteration of the HMGA2 transcript is required for its tumorigenic potential. To determine whether misexpression of HMGA2 in differentiated mesenchymal cells is sufficient to cause tumorigenesis, we produced transgenic mice that misexpressed full-length or truncated human HMGA2 transcript under the control of the differentiated mesenchymal cell (adipocyte)-specific promoter of the adipocyte P2 (Fabp4) gene. Expression of the full-length HMGA2 transgene was observed in a number of tissues, which produced neoplastic phenotype, including fibroadenomas of the breast and salivary gland adenomas. Furthermore, transgenic misexpression of the truncated version of HMGA2, containing only the three DNA-binding domains, produced similar phenotypes. These results show that misexpression of HMGA2 in a differentiated mesenchymal cell is sufficient to cause mesenchymal tumorigenesis and is independent of the nature of the HMGA2 transcript that results from chromosomal translocations observed in humans.

mTORC1 Activation Blocks BrafV600E-Induced Growth Arrest but Is Insufficient for Melanoma Formation

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.

A genetically engineered mouse model with inducible GFP expression in melanocytes

In-depth study of the biology of any cell lineage is best performed while the cells reside in their natural morphological and physiological microenvironment. This task has been difficult to achieve for melanocytes because they make up only about one percent of the cellular milieu of the mammalian skin. The most effective strategy to ‘label’ specific cell types is to express ‘molecular beacons’ under the control of cell lineage-specific gene promoters in transgenic mice. One such transgenic mouse model is Dct-LacZ, which expresses the beta-galactosidase gene under the control of the melanocytespecific promoter of the dopachrome tautomerase (Dct) gene (Mackenzie et al., 1997). Beta-galactosidase cleaves its chromogenic substrate, X-gal, ‘labeling’ the transgene-expressing cells with blue color in visible light, which allows individual melanocytes to be visualized within the skin. The DctLacZ transgenic mouse model has been instrumental in studying the biology of melanocytes; for example, in helping define the existence and location of melanocyte stem cells. However, there are certain desirable features that this model lacks: (i) conditionally inducible labeling and, (ii) fluorescent label, so that it can be used to isolate melanocytes by fluorescenceactivated cell sorting (FACS) following in vivo manipulation. The year 2010 saw publication of three mouse models that expressed fluorescent labels in melanocyte-specific manner. Mort et al. (2010) reported mice that express yellow fluorescent protein (YFP) in conjunction with Tyrosinase-driven Cre recombinase. Monahan et al. (2010) published a similar strategy to express green fluorescent protein (GFP) in melanocytes. However, in both of these models, fluorescence is conditional but not inducible. One of the most effective methods for targeted and inducible gene expression is the expression of the reverse tetracycline-controlled transactivator gene (rtTA) under the control of a tissue or cell-type-specific promoter. rtTA activates any transgene under the control of a tetracycline-responsive element (TRE) in the presence of tetracycline or its analog doxycycline. Expressing rtTA under the control of the Dct promoter would allow inducible expression of virtually any gene in a melanocyte-specific manner. Woods and Bishop (2010) reported the first such mouse last year. They were able to target GFP expression to the melanocytic compartment by crossing their Dct-rtTA mice with the TRE-H2BGFP mice (Tumbar et al., 2004). However, detailed characterization of the inducibility, specificity, and embryonic expression pattern of GFP was not presented. We have now generated a similar mouse model that expresses rtTA under the control of the Dct promoter (Zaidi et al., 2011). We have crossed this mouse model with the TREH2BGFP mice (Figure 1A) to generate mice (iDCT-GFP) that target GFP expression to the melanocytic compartment at all embryonic, neonatal, and adult stages (Figure 1B–E). This allows the study of melanocytes not only at the cellular level but, because the fluorescent cells can also be isolated by FACS, at the molecular level as well; such analyses are otherwise extremely challenging due to the paucity of melanocytes in the skin. The iDct-GFP mouse model is a novel tool that can be used to study melanocytes and diseases originating from them, including melanocyte development and differentiation, melanocyte and melanoma stem cells, and initiation, progression and metastasis of melanoma. In generating the Dct-rtTA transgenic mouse, we have used the rtTA2s-M2 version of the transactivator (Urlinger et al., 2000), which functions at a much lower concentration of doxycycline than the original rtTA, is more stable in eukaryotic cells, and does not cause leaky background expression without doxycycline. The iDct-GFP mice show expression of GFP in cells located in the bulb, the bulge, and the outer root sheath areas of the hair follicles, where melanocytes and melanoblasts reside. We have immunostained these skin sections with anti-Dct (PEP8h) antibody and observed an excellent overlap of GFP and Dct positive signals, indicating that GFP is being exclusively expressed in Dct positive cells (Zaidi et al., 2011). The expression of GFP is both genotype and doxycycline dependent, as there is no leaky background expression in the absence of doxycycline. Furthermore, GFP expression quickly responds, within 12–18 h, to a single intraperitoneal injection of doxycycline (Figure 1F). It should be noted that the TRE-H2BGFP transgenic mice have anomalous expression of H2BGFP in the hematopoietic stem cells of the bone marrow. Although this is not seen in the skin, appropriate controls should be performed when analyzing H2BGFP expression in other anatomic locations (Challen and Goodell, 2008). In order to make breeding simpler, we have established an iDct-GFP double homozygous transgenic mouse line. The following mouse lines are currently available: 1 Dct-rtTA Transgenic mice (line B8): Genetic background: FVB ⁄ N Heterozygous and homozygous genotypes 2 iDct-GFP Transgenic mice (DctrtTA ⁄ TRE-GFP bi-transgenic): Genetic background: FVB ⁄ N Double heterozygous and double homozygous genotypes

Shedding Light on Melanocyte Pathobiology In Vivo

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.