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The TGF-β associated growth factor Nodal is highly expressed in aggressive metastatic melanoma. Determining the risk for melanomagenesis from Nodal expression in nevi prior to the development of melanoma may be useful for both the screening and prevention of melanoma. Tissue sections of human adult nevi with or without a history of melanoma were stained by immunohistochemistry (IHC) for Nodal, the Nodal co-receptor Cripto-1, and Notch4, which have previously been shown to be associated with Nodal expression in melanoma. The degree of Nodal, Cripto-1 and Notch4 staining was scored and correlated with available clinical data. Median IHC scores for Nodal, Cripto-1 and Notch4 expression were significantly higher in nevi removed from patients who eventually developed melanoma compared with nevi from patients with no history of melanoma. In addition, the degree of Nodal expression in nevi from patients who eventually developed melanoma correlated significantly with the Breslow depth of the melanoma. Expression of Nodal and components of its signaling pathway in nevi may represent a biomarker for selecting a unique subset of patients requiring increased surveillance for screening and prevention of melanoma. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 6 (IRF6) is a non-canonical member of the interferon regulatory factor family of transcription factors. We recently identified IRF6 as a novel Maspin-interacting protein in mammary epithelial cells. Maspin is a tumor suppressor in the breast and has also been implicated in mammary gland morphogenesis. To explore a possible role for IRF6 in conjunction with Maspin during mammary gland growth and differentiation, we examined the expression of IRF6 and Maspin during post-utero mammary gland development using a combination of in vitro and in vivo approaches. The data revealed that the expression of IRF6 and Maspin is temporally and spatially regulated throughout mammary gland development, with maximal expression of both proteins occurring in fully differentiated, lactating lobuloalveolar cells. We further show that IRF6 adopts a lumenal localization pattern following complete epithelial cell polarization and present new evidence for the secretion of IRF6 into the milk. These results support the hypothesis that IRF6 and Maspin are important for mammary epithelial cell differentiation, and advance our understanding of the Maspin-IRF6 partnership during normal mammary gland development. [ABSTRACT FROM AUTHOR]

The incidence of melanoma has increased dramatically over the last 50 yr, and although melanoma accounts for only 10% of all skin cancers, it is responsible for over 80% of skin cancer deaths. Recent studies have uncovered critical molecular events underlying melanocytic transformation and melanomagenesis. Among these noteworthy observations are the acquisition of stem cell-associated proteins, such as the Notch receptors and Nodal, which have also been implicated in melanoma progression. For example, we have demonstrated that Nodal expression is limited to invasive vertical growth phase and metastatic melanoma lesions, and that inhibition of Nodal signaling promotes the reversion of metastatic melanoma cells toward a more differentiated, less invasive non-tumorigenic phenotype. In addition, molecular cross-talk exists between the Notch and Nodal signaling pathways. Interestingly, the acquisition of stem cell-associated plasticity is often acquired via epigenetic mechanisms, and is therefore receptive to reprogramming in response to embryonic microenvironments. Here, we review the concept of melanoma plasticity, with an emphasis on the emerging role of Nodal as a regulator of melanoma tumorigenesis and progression, and present findings related to epigenetic reprogramming. [ABSTRACT FROM AUTHOR]

Embryonic stem cells sustain a microenvironment that facilitates a balance of self-renewal and differentiation. Aggressive cancer cells, expressing a multipotent, embryonic cell-like phenotype, engage in a dynamic reciprocity with a microenvironment that promotes plasticity and tumorigenicity. However, the cancer-associated milieu lacks the appropriate regulatory mechanisms to maintain a normal cellular phenotype. Previous work from our laboratory reported that aggressive melanoma and breast carcinoma express the embryonic morphogen Nodal, which is essential for human embryonic stem cell (hESC) pluripotency. Based on the aberrant expression of this embryonic plasticity gene by tumor cells, this current study tested whether these cells could respond to regulatory cues controlling the Nodal signaling pathway, which might be sequestered within the microenvironment of hESCs, resulting in the suppression of the tumorigenic phenotype. Specifically, we discovered that metastatic tumor cells do not express the inhibitor to Nodal, Lefty, allowing them to overexpress this embryonic morphogen in an unregulated manner. However, exposure of the tumor cells to a hESC microenvironment (containing Lefty) leads to a dramatic down-regulation in their Nodal expression concomitant with a reduction in clonogenicity and tumorigenesis accompanied by an increase in apoptosis. Furthermore, this ability to suppress the tumorigenic phenotype is directly associated with the secretion of Lefty, exclusive to hESCs, because it is not detected in other stem cell types, normal cell types, or trophoblasts. The tumor-suppressive effects of the hESC microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored therapeutic modalities for cancer treatment. [ABSTRACT FROM AUTHOR]

Type-I and -III interferons play a central role in immune rejection of pathogens and tumors, thus promoting immunogenicity and suppressing tumor recurrence. Double strand RNA is an important ligand that stimulates tumor immunity via interferon responses. Differentiation of embryonic stem cells to pluripotent epithelial cells activates the interferon response during development, raising the question of whether epithelial vs. mesenchymal gene signatures in cancer potentially regulate the interferon pathway as well. Here, using genomics and signaling approaches, we show that Grainyhead-like-2 (GRHL2), a master programmer of epithelial cell identity, promotes type-I and -III interferon responses to double-strand RNA. GRHL2 enhanced the activation of IRF3 and relA/NF-kB and the expression of IRF1; a functional GRHL2 binding site in the IFNL1 promoter was also identified. Moreover, time to recurrence in breast cancer correlated positively with GRHL2 protein expression, indicating that GRHL2 is a tumor recurrence suppressor, consistent with its enhancement of interferon responses. These observations demonstrate that epithelial cell identity supports interferon responses in the context of cancer. • This manuscript demonstrates that Grainyhead-like-2, a core master programmer of epithelial cell identity, promotes type-I/III interferon responses to double strand RNA and suppresses tumor recurrence. • The work is significant because it connects the epithelial gene expression program with interferon response capability. • These observations have important inplications for tumor immune surveillance and development [ABSTRACT FROM AUTHOR]

Aggressive cancer cells are characterized by their capacity to proliferate indefinitely and to propagate a heterogeneous tumor comprised of subpopulations with varying degrees of metastatic propensity and drug resistance properties. Particularly daunting is the challenge we face in the field of oncology of effectively targeting heterogeneous tumor cells expressing a variety of markers, especially those associated with a stem cell phenotype. This dilemma is especially relevant in breast cancer, where therapy is based on traditional classification schemes, including histological criteria, differentiation status, and classical receptor markers. However, not all patients respond in a similar manner to standard-of-care therapy, thereby necessitating the need to identify and evaluate novel biomarkers associated with the difficult-to-target stem cell phenotype and drug resistance. Findings related to the convergence of embryonic and tumorigenic signaling pathways have identified the embryonic morphogen Nodal as a promising new oncofetal target that is reactivated only in aggressive cancers, but not in normal tissues. The work presented in this paper confirms previous studies demonstrating the importance of Nodal as a cancer stem cell molecule associated with aggressive breast cancer, and advances the field by providing new findings showing that Nodal is not targeted by standard-of-care therapy in breast cancer patients. Most noteworthy is the linkage found between Nodal expression and the drug resistance marker ATP-binding cassette member 1 (ABCA1), which may provide new insights into developing combinatorial approaches to overcome drug resistance and disease recurrence. [ABSTRACT FROM AUTHOR]

The Ras-ERK pathway is deregulated in approximately a third of human cancers, particularly those of epithelial origin. In aggressive, triple-negative, basal-like breast cancers, most tumors display increased MEK and ERK phosphorylation and exhibit a gene expression profile characteristic of Kras or EGFR mutant tumors; however, Ras family genetic mutations are uncommon in triple-negative breast cancer and EGFR mutations account for only a subset of these tumors. Therefore, the upstream events that activate MAPK signaling and promote tumor aggression in triple-negative breast cancers remain poorly defined. We have previously shown that a secreted TGF-β family signaling ligand, Nodal, is expressed in breast cancer in correlation with disease progression. Here we highlight key findings demonstrating that Nodal is required in aggressive human breast cancer cells to activate ERK signaling and downstream tumorigenic phenotypes both in vitro and in vivo . Experimental knockdown of Nodal signaling downregulates ERK activity, resulting in loss of c-myc, upregulation of p27, G1 cell cycle arrest, increased apoptosis and decreased tumorigenicity. The data suggest that ERK activation by Nodal signaling regulates c-myc and p27 proteins post-translationally and that this cascade is essential for aggressive breast tumor behavior in vivo. As the MAPK pathway is an important target for treating triple-negative breast cancers, upstream Nodal signaling may represent a promising target for breast cancer diagnosis and combined therapies aimed at blocking ERK pathway activation. [ABSTRACT FROM AUTHOR]

The post-lactational regression of mammary gland is a complex multi-step process designed to conserve the biological function of the gland for next pregnancy. This developmental stage is a biological intrigue with great relevance to breast cancer research, and thus has been the subject of intensive scrutiny. Multipronged studies (microarray, proteomics profiling, animal knock-out models) have provided a repertoire of genes critical to involution. However, the caveat of these approaches remains in their failure to reveal post-translational modification(s), an emerging and critical aspect of gene regulation in developmental processes and mammary gland remodeling. The massive surge in the lysosomal enzymes concurrent with the onset of involution has been known for decades, and considered essential for “clearance” purposes. However, functional significance of these enzymes in diverse biological processes distinct from their proteolytic activity is just emerging. Studies from our laboratory had indicated specific post-translational modifications of the aspartyl endopeptidase Cathepsin D (CatD) at distinct stages mammary gland development. This study addresses the biological significance of these modifications in the involution process, and reveals that post-translational modifications drive CatD into the nucleus to cleave Histone 3. The cleavage of Histone 3 has been associated with cellular differentiation and could be critical instigator of involution process. From functional perspective, deregulated expression and increased secretion of CatD are associated with aggressive and metastatic phenotype of breast cancer. Thus unraveling CatD’s physiological functions in mammary gland development will bridge the present gap in understanding its pro-tumorigenic/metastatic functions, and assist in the generation of tailored therapeutic approaches. [ABSTRACT FROM AUTHOR]

Breast carcinoma cells and embryonic progenitors similarly implement stem cell-associated signaling pathways to sustain continued growth and plasticity. Indeed, recent studies have implicated signaling pathways, including those associated with the Notch, and Transforming Growth Factor-Beta (TGF-β) superfamilies, as instrumental to both embryological development and breast cancer progression. In particular, Nodal, an embryonic morphogen belonging to the TGF-β superfamily, and its co-receptor, Cripto-1, are requisite to both embryogenesis and mammary gland maturation. Moreover, these developmental proteins have been shown to promote breast cancer progression. Here, we review the role of Nodal and its co-receptor Cripto-1 during development and we describe how this signaling pathway may be involved in breast cancer tumorigenesis. Moreover, we emphasize the potential utility of this signaling pathway as a novel target for the treatment and diagnosis of breast cancer. [ABSTRACT FROM AUTHOR]

Bidirectional cellular communication is integral to both cancer progression and embryological development. In addition, aggressive tumor cells are phenotypically plastic, sharing many properties with embryonic cells. Owing to the similarities between these two types of cells, the developing zebrafish can be used as a biosensor for tumor-derived signals. Using this system, we show that aggressive melanoma cells secrete Nodal (a potent embryonic morphogen) and consequently can induce ectopic formation of the embryonic axis. We further show that Nodal is present in human metastatic tumors, but not in normal skin, and thus may be involved in melanoma pathogenesis. Inhibition of Nodal signaling reduces melanoma cell invasiveness, colony formation and tumorigenicity. Nodal inhibition also promotes the reversion of melanoma cells toward a melanocytic phenotype. These data suggest that Nodal signaling has a key role in melanoma cell plasticity and tumorigenicity, thereby providing a previously unknown molecular target for regulating tumor progression. [ABSTRACT FROM AUTHOR]

The development and progression of cancer is controlled by gene expression, often regulated through chromatin packaging. Heterochromatin protein 1Hsα (HP1Hsα), one of three human HP1 family members, participates in heterochromatin formation and gene regulation. HP1Hsα possesses an amino-terminal chromodomain, which binds methylated lysine 9 of histone H3 (meK9 H3), and a carboxyl-terminal chromoshadow domain (CSD) that is required for dimerization and interaction with partner proteins. HP1Hsα is down-regulated in invasive metastatic breast cancer cells compared with poorly invasive nonmetastatic breast cancer cells. Expression of EGFP-HP1Hsα in highly invasive MDA-MB-231 cells causes a reduction in in vitro invasion, without affecting cell growth. Conversely, knock-down of HP1Hsα levels in the poorly invasive breast cancer cell line MCF-7 increased invasion, without affecting cell growth. To determine whether functions of the CSD were required for the regulation of invasion, mutant forms of HP1Hsα were expressed in MDA-MB-231 cells. A W174A mutation that disrupts interactions between HP1Hsα and PXVXL-containing partner proteins reduced invasion similar to that of the wild type protein. In contrast, an 1165E mutation that disrupts dimerization of HP1Hsα did not decrease invasion. No gross changes in localization and abundance of HP1Hsβ, HP1Hsγ, and meK9 H3 were observed upon expression of wild type and mutant forms of HP1Hsα in MDA-MB-231 cells. Taken together, these data demonstrate that modulation of HP1Hsα alters the invasive potential of breast cancer cells through mechanisms requiring HP1 dimerization, but not interactions with PXVXL-containing proteins. [ABSTRACT FROM AUTHOR]

Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice’s brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients’ PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network. [ABSTRACT FROM AUTHOR]

There is growing evidence and a consensus in the field that most pediatric brain tumors originate from stem cells, of which radial glial cells constitute a subtype. Here we show that orthotopic transplantation of human radial glial (RG) cells to the subventricular zone of the 3rd ventricle - but not to other transplantation sites - of the brain in immunocompromised NOD-SCID mice, gives rise to tumors that have the hallmarks of CNS primitive neuroectodermal tumors (PNETs). The resulting mouse model strikingly recapitulates the phenotype of PNETs. Importantly, the observed tumorigenic transformation was accompanied by aspects of an epithelial to mesenchymal transition (EMT)-like process. It is also noteworthy that the tumors are highly invasive, and that they effectively recruit mouse endothelial cells for angiogenesis. These results are significant for several reasons. First, they show that malignant transformation of radial glial cells can occur in the absence of specific mutations or inherited genomic alterations. Second, they demonstrate that the same radial glial cells may either give rise to brain tumors or differentiate normally depending upon the microenvironment of the specific region of the brain to which the cells are transplanted. In addition to providing a prospect for drug screening and development of new therapeutic strategies, the resulting mouse model of PNETs offers an unprecedented opportunity to identify the cancer driving molecular alterations and the microenvironmental factors that are responsible for committing otherwise normal radial glial cells to a malignant phenotype. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 6 (IRF6) is a novel and unique member of the IRF family of transcription factors. IRF6 has not been linked to the regulatory pathways or functions associated with other IRF family members, and the regulation and function of IRF6 remain unknown. We recently identified a protein interaction between IRF6 and the tumor suppressor maspin. To gain insight into the biological significance of the maspin-IRF6 interaction, we examined the regulation and function of IRF6 in relation to maspin in normal mammary epithelial cells. Our results demonstrate that in quiescent cells, IRF6 exists primarily in a nonphosphorylated state. However, cellular proliferation leads to rapid IRF6 phosphorylation, resulting in proteasome-dependent IRF6 degradation. These data are supported in situ by the increased expression of IRF6 in quiescent, differentiated lobuloalveolar cells of the lactating mammary gland compared to its expression in proliferating ductal and glandular epithelial cells during pregnancy. Furthermore, the reexpression of IRF6 in breast cancer cells results in cell cycle arrest, and the presence of maspin augments this response. These data support a model in which IRF6, in collaboration with maspin, promotes mammary epithelial cell differentiation by facilitating entry into the G0 phase of the cell cycle. [ABSTRACT FROM AUTHOR]

A letter to the editor in response to the article "Melanoma pathogenesis and Nodal: a partial picture?" by Topczewska and colleagues published in the 2006 issue.

As our understanding of embryonic stem cell biology becomes more sophisticated, the similarities between multipotent cancer cells and these totipotent precursors are increasingly striking. Both multipotent cancer cells and embryonic stem cells possess the ability to self-renew, epigenetically alter their neighboring cellular architecture, and populate a tissue mass with a phenotypically heterogeneous composition of cells. While the molecular signature of these cell types continues to be elucidated, new insights are emerging related to the convergence of embryonic and tumorigenic signaling pathways. Understanding the molecular underpinnings of these two stem cell phenotypes may lead to new therapeutic targets for the elusive cancer cell. While still in its infancy, the potential of adapting embryonic stem cells, and more specifically the factors they produce, is enormous for clinical application. Here we outline evidence that demonstrates the inductive influence of embryonic stem cells and their microenvironment to reprogram cancer cells to exhibit a more benign phenotype, with profound implications for differentiation therapy. [ABSTRACT FROM AUTHOR]

Human metastatic melanoma cells express a dedifferentiated, plastic phenotype, which may serve as a selective advantage, because melanoma cells invade various microenvironments. Over the last three decades, there has been an increased focus on the role of the tumor microenvironment in cancer progression, with the goal of reversing the metastatic phenotype. Here, using an embryonic chick model, we explore the possibility of reverting the metastatic melanoma phenotype to its cell type of origin, the neural-crest-derived melanocyte. GFP-labeled adult human metastatic melanoma cells were transplanted in ovo adjacent to host chick premigratory neural crest cells and analyzed 48 and 96 h after egg reincubation. Interestingly, the transplanted melanoma cells do not form tumors. Instead, we find that transplanted melanoma cells invade surrounding chick tissues in a programmed manner, distributing along host neural-crest-cell migratory pathways. The invading melanoma cells display neural-crest-cell-like morphologies and populate host peripheral structures, including the branchial arches, dorsal root and sympathetic ganglia. Analysis of a melanocyte-specific phenotype marker (MART-1) and a neuronal marker (Tuj1) revealed a subpopulation of melanoma cells that invade the chick periphery and express MART-1 and Tuj1. Our results demonstrate the ability of adult human metastatic melanoma cells to respond to chick embryonic environmental cues, a subset of which may undergo a reprogramming of their metastatic phenotype. This model has the potential to provide insights into the regulation of tumor cell plasticity by an embryonic milieu, which may hold significant therapeutic promise. [ABSTRACT FROM AUTHOR]

Since its reported discovery in 1994, maspin (mammary serine protease inhibitor) has been characterized as a class II tumor suppressor by its ability to promote apoptosis and inhibit cell invasion. Maspin is highly expressed in normal mammary epithelial cells but reduced or absent in aggressive breast carcinomas. However, despite efforts to characterize the mechanism(s) by which maspin functions as a tumor suppressor, its molecular characterization has remained somewhat elusive. Therefore, in an attempt to identify maspin-interacting proteins and thereby gain insight into the functional pathways of maspin, we employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. IRF6 belongs to the IRF family of transcription factors, which is best known for its regulation of interferon and interferon-inducible genes following a pathogenic stimulus. Although many of the IRF family members have been well characterized, IRF6 remains poorly understood. We report that IRF6 is expressed in normal mammary epithelial cells and that it directly associates with maspin in a yeast two-hybrid system and in vitro. The interaction occurs via the conserved IRF protein association domain and is regulated by phosphorylation of IRF6. We have shown that, similar to maspin, IRF6 expression is inversely correlated with breast cancer invasiveness. We further demonstrated that the transient re-expression of IRF6 in breast cancer ceils results in an increase of N-cadherin and a redistribution of vimentin commensurate with changes in cell morphology, suggestive of an epithelial-to-mesenchymal transition event. Concomitantly, we showed that maspin acts as a negative regulator of this process. These findings help to elucidate the molecular mechanisms of maspin and suggest an interactive role between maspin and IRF6 in regulating cellular phenotype, the loss of which can lead to neoplastic transformation. [ABSTRACT FROM AUTHOR]