PAX3 and FOXD3 Promote CXCR4 Expression in Melanoma.

Metastatic melanoma is an aggressive and deadly disease. The chemokine receptor CXCR4 is active in melanoma metastasis, although the mechanism for the promotion and maintenance of CXCR4 expression in these cells is mostly unknown. Here, we find melanoma cells express two CXCR4 isoforms, the common version and a variant that is normally restricted to cells during development or to mature blood cells. CXCR4 expression is driven through a highly conserved intronic enhancer element by the transcription factors PAX3 and FOXD3. Inhibition of these transcription factors slows melanoma cell growth, migration, and motility, as well as reduces CXCR4 expression. Overexpression of these transcription factors drives the production of increased CXCR4 levels. Loss of PAX3 and FOXD3 transcription factor activity results in a reduction in cell motility, migration, and chemotaxis, all of which are rescued by CXCR4 overexpression. Here, we discover a molecular pathway wherein PAX3 and FOXD3 promote CXCR4 gene expression in melanoma.

FOXD3 Promotes PAX3 Expression in Melanoma Cells.

Several key transcription factors regulate cell growth, survival, and differentiation during neural crest and melanoblast development in the embryo, and these same pathways may be reactivated in tumors arising from the progenitors of these cells. The transcription factors PAX3 and FOXD3 have essential roles in melanoblasts and melanoma. In this study, we define a regulatory pathway where FOXD3 promotes the expression of PAX3. Both factors are expressed in melanoma cells and there is a positive correlation between the transcript levels of PAX3 and FOXD3. The PAX3 gene contains two FOX binding motifs within highly conserved enhancer regulatory elements that are essential for neural crest development. FOXD3 binds to both of these motifs in vitro but only one of these sites is preferentially utilized in melanoma cells. Overexpression of FOXD3 upregulates PAX3 levels while inhibition of FOXD3 function does not alter PAX3 protein levels, supporting that FOXD3 is sufficient but not necessary to drive PAX3 expression in melanoma cells. Here, we identify a molecular pathway where FOXD3 upregulates PAX3 expression and therefore contributes to melanoma progression.

YAP facilitates melanoma migration through regulation of actin-related protein 2/3 complex subunit 5 (ARPC5).

Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcriptional coactivators that have been implicated in driving metastasis and progression in many cancers, mainly through their transcriptional regulation of downstream targets. Although YAP and TAZ have shown redundancy in many contexts, it is still unknown whether or not this is true in melanoma. Here, we show that while both YAP and TAZ are expressed in a panel of melanoma cell lines, depletion of YAP results in decreased cell numbers, focal adhesions, and the ability to invade matrigel. Using non-biased RNA-sequencing analysis, we find that melanoma cells depleted of YAP, TAZ, or YAP/TAZ exhibit drastically different transcriptomes. We further uncover the ARP2/3 subunit ARPC5 as a specific target of YAP but not TAZ and that ARPC5 is essential for YAP-dependent maintenance of melanoma cell focal adhesion numbers. Our findings suggest that in melanoma, YAP drives melanoma progression, survival, and invasion.

Inactivation of the Hippo tumor suppressor pathway promotes melanoma.

Melanoma is commonly driven by activating mutations in the MAP kinase BRAF; however, oncogenic BRAF alone is insufficient to promote melanomagenesis. Instead, its expression induces a transient proliferative burst that ultimately ceases with the development of benign nevi comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that restrain nevus melanocyte proliferation remain poorly understood. Here we utilize cell and murine models to demonstrate that oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway, both in melanocytes in vitro and nevus melanocytes in vivo. Mechanistically, we show that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome doubling events, which independently reduce RhoA activity to promote Hippo activation. We also demonstrate that functional impairment of the Hippo pathway enables oncogenic BRAF-expressing melanocytes to bypass nevus formation and rapidly form melanomas. Our data reveal that the Hippo pathway enforces the stable arrest of nevus melanocytes and represents a critical barrier to melanoma development.

Distinct enhancers at the Pax3 locus can function redundantly to regulate neural tube and neural crest expressions.

Pax3 is a transcription factor expressed in somitic mesoderm, dorsal neural tube and pre-migratory neural crest during embryonic development. We have previously identified cis-acting enhancer elements within the proximal upstream genomic region of Pax3 that are sufficient to direct functional expression of Pax3 in neural crest. These elements direct expression of a reporter gene to pre-migratory neural crest in transgenic mice, and transgenic expression of a Pax3 cDNA using these elements is sufficient to rescue neural crest development in mice otherwise lacking endogenous Pax3. We show here that deletion of these enhancer sequences by homologous recombination is insufficient to abrogate neural crest expression of Pax3 and results in viable mice. We identify a distinct enhancer in the fourth intron that is also capable of mediating neural crest expression in transgenic mice and zebrafish. Our analysis suggests the existence of functionally redundant neural crest enhancer modules for Pax3.

Pax3 functions at a nodal point in melanocyte stem cell differentiation.

Most stem cells are not totipotent. Instead, they are partially committed but remain undifferentiated. Upon appropriate stimulation they are capable of regenerating mature cell types. Little is known about the genetic programmes that maintain the undifferentiated phenotype of lineage-restricted stem cells. Here we describe the molecular details of a nodal point in adult melanocyte stem cell differentiation in which Pax3 simultaneously functions to initiate a melanogenic cascade while acting downstream to prevent terminal differentiation. Pax3 activates expression of Mitf, a transcription factor critical for melanogenesis, while at the same time it competes with Mitf for occupancy of an enhancer required for expression of dopachrome tautomerase, an enzyme that functions in melanin synthesis. Pax3-expressing melanoblasts are thus committed but undifferentiated until Pax3-mediated repression is relieved by activated beta-catenin. Thus, a stem cell transcription factor can both determine cell fate and simultaneously maintain an undifferentiated state, leaving a cell poised to differentiate in response to external stimuli.

YK-4-279 Attenuates Progression of Pre-Existing Pigmented Lesions to Nodular Melanoma in a Mouse Model.

More options are needed for the effective treatment of melanoma. In a previous study, we discovered the small molecule drug YK-4-279 almost completely inhibited tumor progression in the BrafCA;Tyr-CreERT2;Ptenflox/flox transgenic mouse model. YK-4-279 had no effect on tumor initiation but blocked progression of invasive melanoma. Our current study was designed as a treatment model, where YK-4-279 was administered during pigmented lesion formation. The study design included the use of three groups: (1) a control group that received only DMSO without a drug (MOCK), (2) mice following our prior studies with YK-4-279 administered at the time of tumor induction (YK-4-279), and (3) mice treated during tumor initiation (YK-4-279 delay). While the MOCK mice had progression of tumors, both YK-4-279 and YK-4-279 delay groups had a significant block or delay of progression. The majority of mice in the YK-4-279 groups had a block of progression, while the YK-4-279 delay group had either a partial block (60% in male mice or 29% in females) or a delay in disease progression in females (28 days in controls to 50 days in YK-4-279 delay group). Here, we demonstrate that YK-4-279 has a significant impact on blocking or delaying tumor progression in a pre-clinical treatment model of melanoma.

Targeting Pan-ETS Factors Inhibits Melanoma Progression.

The failure of once promising target-specific therapeutic strategies often arises from redundancies in gene expression pathways. Even with new melanoma treatments, many patients are not responsive or develop resistance, leading to disease progression in terms of growth and metastasis. We previously discovered that the transcription factors ETS1 and PAX3 drive melanoma growth and metastasis by promoting the expression of the MET receptor. Here, we find that there are multiple ETS family members expressed in melanoma and that these factors have redundant functions. The small molecule YK-4-279, initially developed to target the ETS gene-containing translocation product EWS-FLI1, significantly inhibited cellular growth, invasion, and ETS factor function in melanoma cell lines and a clinically relevant transgenic mouse model, BrafCA;Tyr-CreERT2;Ptenf/f. One of the antitumor effects of YK-4-279 in melanoma is achieved via interference of multiple ETS family members with PAX3 and the expression of the PAX3-ETS downstream gene MET. Expression of exogenous MET provided partial rescue of the effects of YK-4-279, further supporting that MET loss is a significant contributor to the antitumor effects of the drug. This is the first study identifying multiple overlapping functions of the ETS family promoting melanoma. In addition, targeting all factors, rather than individual members, demonstrated impactful deleterious consequences in melanoma progression. Given that multiple ETS factors are known to have oncogenic functions in other malignancies, these findings have a high therapeutic impact. SIGNIFICANCE: These findings identify YK-4-279 as a promising therapeutic agent against melanoma by targeting multiple ETS family members and blocking their ability to act as transcription factors.

PAX6 is expressed in pancreatic cancer and actively participates in cancer progression through activation of the MET tyrosine kinase receptor gene.

Tumors of the exocrine pancreas have a poor prognosis. Several proteins are overexpressed in this cancer type, including the MET tyrosine kinase receptor and the transcription factor PAX6. In this report, we find that PAX6(5a), an alternately spliced variant form of PAX6, is expressed in pancreatic carcinoma cell lines at higher levels than the canonical PAX6 protein. Both protein forms of PAX6 bind directly to an enhancer element in the MET promoter and activate the expression of the MET gene. In addition, inhibition of PAX6 transcripts leads to a decline in cell growth and survival, differentiation, and a concurrent reduction of MET protein expression. These data support a model for a neoplastic pathway, where expression of a transcription factor from development activates the MET receptor, a protein that has been directly linked to protumorigenic processes of resisting apoptosis, tumor growth, invasion, and metastasis.

PAX5 is expressed in small-cell lung cancer and positively regulates c-Met transcription.

PAX5 is a nuclear transcription factor required for B cell development, and its expression was evaluated in upper aerodigestive malignancies and pancreatic cancer by immunoblotting. The PAX5 protein expression was relatively strong in small-cell lung cancer (SCLC, 11/12); however, its expression was not detected in non-SCLC (NSCLC, n=13), mesothelioma (n=7), pancreatic (n=6), esophageal (n=6) and head and neck cancer cell lines (n=12). In comparison, PAX8 and PAX3 expressions were absent or non-detectable in SCLC cell lines; however, PAX8 was expressed in most of the tested NSCLC cell lines (13/13) and also frequently in all the other cell lines. We also detected frequent expressions of PAX2 and PAX9 protein in the various cell lines. Utilizing neuroendocrine tumor samples, we found that the frequency as well as the average intensity of the expression of PAX5 increased from pulmonary carcinoid (9%, moderate and strong PAX5 expression, n=44), to large-cell neuroendocrine carcinoma (LCNC, 27%, n=11) to SCLC (33%, n=76). FISH analysis revealed no translocations of the PAX5 gene, but polyploidy in some SCLC tumor tissues (6/37). We determined that PAX5 could regulate the transcription of c-Met using luciferase-coupled reporter and chromatin immunoprecipitation analysis. In addition, the phospho-c-Met (active form) and PAX5 were both localized to the same intra-nuclear compartment in hepatocyte growth factor treated SCLC cells and interacted with each other. Finally, we determined the therapeutic translational potential of PAX5 using PAX5 knockdown SCLC cells in conjunction with Topoisomerase 1 (SN38) and c-Met (SU11274) inhibitors. Loss of endogenous PAX5 significantly decreased the viability of SCLC cells, especially when combined with SN38 or SU11274, and maximum effect was seen when both inhibitors were used. Therefore, we propose that PAX5 could be an important regulator of c-Met transcription and a potential target for therapy in SCLC.

Alternative promoter and GATA5 transcripts in mouse.

GATA5 is a member of the GATA zinc finger transcription factor family involved in tissue-specific transcriptional regulation during cell differentiation and embryogenesis. Previous reports indicate that null mutation of the zebrafish GATA5 gene results in embryonic lethality, whereas deletion of exon 1 from the mouse GATA5 gene causes only derangement of female urogenital development. Here, we have identified an alternate promoter within intron 1 of the mouse GATA5 gene that transcribes a 2.5-kb mRNA that lacks exon 1 entirely but includes 82 bp from intron 1 and all of exons 2-6. The alternative promoter was active during transient transfection in cultured airway myocytes and bronchial epithelial cells, and it drove reporter gene expression in gastric epithelial cells in transgenic mice. The 2.5-kb alternative transcript encodes an NH(2)-terminally truncated "short GATA5" comprising aa 226-404 with a single zinc finger, which retains ability to transactivate the atrial natriuretic factor promoter (albeit less efficiently than full-length GATA5). Another new GATA5 transcript contains all of exons 1-5 and the 5' portion of exon 6 but lacks the terminal 1143 bp of the 3'-untranslated region from exon 6. These findings extend current understanding of the tissue distribution of GATA5 expression and suggests that GATA5 expression and function are more complex than previously appreciated.

The Efficiency of Verteporfin as a Therapeutic Option in Pre-Clinical Models of Melanoma.

Yes Associated Protein 1 (YAP) and Transcriptional coactivator with PDZ-Binding Motif (TAZ) have gained notoriety for their ability to drive tumor initiation and progression in a wide variety of cancers, including melanoma. YAP and TAZ act as drivers of melanoma through its interaction with the TEAD family of transcription factors. Verteporfin is a benzoporphyrin derivative that is used clinically for photodynamic treatment of macular degeneration. Recently it has emerged as a potential inhibitor of YAP/TAZ-TEAD interaction independent of light activation. In this study we determine if verteporfin has clinical potential by testing this compound on human melanoma cell cultures and in a clinically significant mouse model, BrafCA; Tyr-CreERT2; Ptenf/f, which parallels human melanoma in terms of disease progression, genetics, and histopathology. In culture, Verteporfin treatment induces a rapid drop in YAP and TAZ protein levels and cell numbers. In the transgenic model, utilizing drug levels that correspond to previously determined safe doses in human patients and with a dosing regimen calculated in this study, Verteporfin did not inhibit melanoma initiation or progression in comparison to mock treated controls. Taken together, our study suggests that although Verteporfin induces YAP/TAZ degradation in melanoma cell lines, Verteporfin was not effective as a YAP/TAZ-TEAD specific inhibitor of melanoma in our studies that aimed to mimic conditions found in clinic in terms of treatment regimen and disease model.

PAX6 is expressed in pancreatic adenocarcinoma and is downregulated during induction of terminal differentiation.

Tumors of the exocrine pancreas are a major cause of cancer death and have among the poorest prognosis of any malignancy. Following the "cancer stem cell hypothesis," where tumors are believed to originate in tissue specific stem cells, we screened primary ductal pancreatic carcinomas and cell lines for the expression of possible stem cell factors. We find 32/46 (70%) of primary tumors and 9/10 (90%) of cell lines express PAX6. PAX6 is a transcription factor expressed throughout the pancreatic bud during embryogenesis but not in the mature exocrine pancreas. PAX proteins have also been implicated in maintaining stem cells in a committed but undifferentiated state but a role for PAX proteins in putative pancreas stem cells is not known. We induced a pancreatic carcinoma cell line, Panc-1, to differentiate by transfecting wild-type p53 and treating the cells with differentiation agents gastrin or butyrate. This treatment induces cells to terminally differentiate into a growth-arrested cell with neurite-like processes, express the terminal differentiation marker somatostatin and downregulate PAX6. This phenotype can be replicated by directly inhibiting PAX6 expression. These data support a model where PAX proteins are aberrantly expressed in tumors and downregulation leads to differentiation.

PAX genes: roles in development, pathophysiology, and cancer.

PAX proteins function as transcription factors and play an essential role in organogenesis during embryonic development in regulating cell proliferation and self-renewal, resistance to apoptosis, migration of embryonic precursor cells, and the coordination of specific differentiation programs. Recent studies have also discovered a role for PAX proteins in specific stem cell or progenitor cell populations, including melanocytes, muscle, and B-cells. The normal functions of the PAX proteins, including apoptosis resistance and repression of terminal differentiation, may be subverted during the progression of a number of specific malignancies. This is supported by the fact that expression of PAX proteins is dysregulated in several different types of tumors, although the precise roles for PAX proteins in cancer are not clearly understood. An emerging hypothesis is that PAX proteins play an essential role in maintaining tissue specific stem cells by inhibiting terminal differentiation and apoptosis and that these functional characteristics may facilitate the development and progression of specific cancers. In this review, we provide a general background to the PAX protein family and focus on specific cells and tissues and the role PAX proteins play within these tissues in terms of development, mature tissue maintenance, and expression in tumors. Understanding the normal developmental pathways regulated by PAX proteins may shed light on potentially parallel pathways shared in tumors, and ultimately result in defining new molecular targets and signaling pathways for the development of novel anti-cancer therapies.

Canonical and alternative transcript expression of PAX6 and CXCR4 in pancreatic cancer.

Pancreatic cancer is a lethal disease with a propensity for invading and metastasizing into the surrounding tissues, including the liver and intestines. A number of factors are aberrantly overexpressed in this tumor type and actively promote cancer progression and metastasis. The present study demonstrates that paired box transcription factor 6 (PAX6) and C-X-C chemokine receptor 4 (CXCR4) are frequently co-expressed in primary pancreatic adenocarcinoma tumors and established cell lines. Expression analysis methods used in the present study included evaluation of protein expression by western blot analysis and immunofluorescence, transcript expression levels by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and luciferase assays utilizing regulatory elements from the CXCR4 gene locus. Canonical PAX6 and alternative splice variant PAX6(5a) proteins are expressed in pancreatic cancer and can drive gene expression through a conserved enhancer element within the first intron of the CXCR4 gene. As demonstrated by the introduction of an exogenous reporter construct with or without the intronic enhancer, loss of this element inhibited gene expression within numerous pancreatic cancer cell lines including Panc1, MIA-PaCa2 and BxPC3. All of the pancreatic cancer cell lines expressed the canonical CXCR4B transcript in addition to the alternatively spliced variant CXCR4A as determined by RT-qPCR experiments. The discovery of variant transcripts in pancreatic cancer cells may provide new candidates for future targeted therapies.

Melanocytes, melanocyte stem cells, and melanoma stem cells.

Melanocyte stem cells differ greatly from melanoma stem cells; the former provide pigmented cells during normal tissue homeostasis and repair, and the latter play an active role in a lethal form of cancer. These 2 cell types share several features and can be studied by similar methods. Aspects held in common by both melanocyte stem cells and melanoma stem cells include their expression of shared biochemical markers, a system of similar molecular signals necessary for their maintenance, and a requirement for an ideal niche microenvironment for providing these factors. This review provides a perspective of both these cell types and discusses potential models of stem cell growth and propagation. Recent findings provide a strong foundation for the development of new therapeutics directed at isolating and manipulating melanocyte stem cells for tissue engineering or at targeting and eradicating melanoma specifically, while sparing nontumor cells.

GSK-3 promotes cell survival, growth, and PAX3 levels in human melanoma cells.

GSK-3 is a serine/threonine kinase involved in a diverse range of cellular processes. GSK-3 exists in two isoforms, GSK-3α and GSK-3ß, which possess some functional redundancy but also play distinct roles depending on developmental and cellular context. In this article, we found that GSK-3 actively promoted cell growth and survival in melanoma cells, and blocking this activity with small-molecule inhibitor SB216763 or gene-specific siRNA decreased proliferation, increased apoptosis, and altered cellular morphology. These alterations coincided with loss of PAX3, a transcription factor implicated in proliferation, survival, and migration of developing melanoblasts. We further found that PAX3 directly interacted with and was phosphorylated in vitro on a number of residues by GSK-3ß. In melanoma cells, direct inhibition of PAX3 lead to cellular changes that paralleled the response to GSK-3 inhibition. Maintenance of PAX3 expression protected melanoma cells from the anti-tumor effects of SB216763. These data support a model wherein GSK-3 regulates proliferation and morphology of melanoma through phosphorylation and increased levels of PAX3.

PAX3 and SOX10 activate MET receptor expression in melanoma.

Melanoma is a cancer with a poorly understood molecular pathobiology. We find the transcription factors PAX3, SOX10, MITF, and the tyrosine kinase receptor MET expressed in melanoma cell lines and primary tumors. Analysis for MET expression in primary tumor specimens showed 27/40 (68%) of the samples displayed an increased expression of MET, and this expression was highly correlated with parallel expression of PAX3, SOX10, and MITF. PAX3 and MITF bind to elements in the MET promoter independently, without evidence of either synergistic activation or inhibition. SOX10 does not directly activate the MET gene alone, but can synergistically activate MET expression with either PAX3 or MITF. In melanoma cells, there was evidence of two pathways for PAX3 mediated MET induction: (i) direct activation of the gene, and (ii) indirect regulation through MITF. SK-MEL23 melanoma cells have both of these pathways intact, while SK-MEL28 melanoma cells only have the first pathway. In summary, we find that PAX3, SOX10 and MITF play an active role in melanoma cells by regulating the MET gene. In consequence, MET promotes the melanoma cancer phenotype by promoting migration, invasion, resistance to apoptosis, and tumor cell growth.

Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.

Atrial fibrillation is the most common clinical cardiac arrhythmia. It is often initiated by ectopic beats arising from the pulmonary veins and atrium, but the source and mechanism of these beats remains unclear. The melanin synthesis enzyme dopachrome tautomerase (DCT) is involved in intracellular calcium and reactive species regulation in melanocytes. Given that dysregulation of intracellular calcium and reactive species has been described in patients with atrial fibrillation, we investigated the role of DCT in this process. Here, we characterize a unique DCT-expressing cell population within murine and human hearts that populated the pulmonary veins, atria, and atrioventricular canal. Expression profiling demonstrated that this population expressed adrenergic and muscarinic receptors and displayed transcriptional profiles distinct from dermal melanocytes. Adult mice lacking DCT displayed normal cardiac development but an increased susceptibility to atrial arrhythmias. Cultured primary cardiac melanocyte-like cells were excitable, and those lacking DCT displayed prolonged repolarization with early afterdepolarizations. Furthermore, mice with mutations in the tyrosine kinase receptor Kit lacked cardiac melanocyte-like cells and did not develop atrial arrhythmias in the absence of DCT. These data suggest that dysfunction of melanocyte-like cells in the atrium and pulmonary veins may contribute to atrial arrhythmias.