Endothelial nitric oxide synthase uncoupling as a key mediator of melanocyte malignant transformation associated with sustained stress conditions

Melanoma cell lines and cells corresponding to premalignant melanocytes were established by our group after subjecting a nontumorigenic murine melanocyte lineage, melan-a, to sequential cycles of anchorage blockade. Previous results showed that in melan-a cells the superoxide level increases after such procedure. Superoxide production during melanocyte de-adhesion was inhibited by L-sepiapterin, the precursor of eNOS cofactor BH4, and increased by the inhibitor of BH4 synthesis, DAHP, hence indicating a partial uncoupling state of eNOS. The eNOS uncoupling seems to be maintained in cells derived from melan-a, because they present decreased nitric oxide and increased superoxide levels. The inhibition of superoxide production in Tm5 melanoma cells with L-sepiapterin reinforces their eNOS-uncoupled state. The maintenance of oxidative stress seems to be important in melanoma apoptosis resistance because Mn(III)TBAP, a superoxide scavenger, or L-sepiapterin renders Tm5 cells more sensitive to anoikis and chemotherapy. More importantly, eNOS uncoupling seems to play a pivotal role in melanocyte malignant transformation induced by sustained anchorage impediment, because no malignant transformation was observed when L-NAME-treated melanocytes were subjected to sequential cycles of de-adhesion. Our results show that uncoupled eNOS contributes to superoxide production during melanocyte anchorage impediment, contributing to anoikis resistance and malignant transformation.     

Epigenetic reprogramming as a key contributor to melanocyte malignant transformation

Melanoma progression requires deregulation of gene expression by currently uncharacterized epigenetic mechanisms. A mouse model based on changes in cell microenvironment was developed by our group to study melanocyte malignant transformation. Melanoma cell lines (4C11- and 4C11+) were obtained as result of 5 sequential anchorage blockades of non-tumorigenic melan-a melanocytes. Melan-a cells submitted to 4 de-adhesion cycles were also established (4C), are non-tumorigenic and represent an intermediary phase of tumor progression. The aim of this work was to identify factors contributing to epigenetic modifications in early and later phases of malignant transformation induced by anchorage impediment. Epigenetic alterations occur early in tumorigenesis; 4C cell line shows changes in global and gene-specific DNA methylation and histone marks. Many histone modifications differ between melan-a, 4C, 4C11- (non-metastatic melanoma cell line) and 4C11+ (metastatic melanoma cell line) which could be associated with changes in gene and microRNA expression. These epigenetic alterations seem to play a key role in malignant transformation since melanocytes treated with 5-Aza-2'-deoxycytidine before each anchorage blockade do not transform. Some epigenetic changes seem to be also responsible for the maintenance of malignant phenotype, since melanoma cell lines (4C11- and 4C11+) treated in vitro with 5-Aza-2'-deoxycytidine or Trichostatin A showed reduction of tumor growth in vivo. Changes in gene expression reflecting cell adaptation to new environment were also observed. We propose a model in which sustained microenvironmental stress in melanocytes results in epigenetic reprogramming. Thus, after adaptation, cells may acquire epigenetic marks that could contribute to the establishment of a malignant phenotype.     

Epigenetic reprogramming as a key contributor to melanocyte malignant transformation

Melanoma progression requires deregulation of gene expression by currently uncharacterized epigenetic mechanisms. A mouse model based on changes in cell microenvironment was developed by our group to study melanocyte malignant transformation. Melanoma cell lines (4C11− and 4C11+) were obtained as result of 5 sequential anchorage blockades of non-tumorigenic melan-a melanocytes. Melan-a cells submitted to 4 de-adhesion cycles were also established (4C), are non-tumorigenic and represent an intermediary phase of tumor progression. The aim of this work was to identify factors contributing to epigenetic modifications in early and later phases of malignant transformation induced by anchorage impediment. Epigenetic alterations occur early in tumorigenesis; 4C cell line shows changes in global and gene-specific DNA methylation and histone marks. Many histone modifications differ between melan-a, 4C, 4C11− (non-metastatic melanoma cell line) and 4C11+ (metastatic melanoma cell line) which could be associated with changes in gene and microRNA expression. These epigenetic alterations seem to play a key role in malignant transformation since melanocytes treated with 5-Aza-2′-deoxycytidine before each anchorage blockade do not transform. Some epigenetic changes seem to be also responsible for the maintenance of malignant phenotype, since melanoma cell lines (4C11− and 4C11+) treated in vitro with 5-Aza-2′-deoxycytidine or Trichostatin A showed reduction of tumor growth in vivo. Changes in gene expression reflecting cell adaptation to new environment were also observed. We propose a model in which sustained microenvironmental stress in melanocytes results in epigenetic reprogramming. Thus, after adaptation, cells may acquire epigenetic marks that could contribute to the establishment of a malignant phenotype.Key words: anchorage blockade, sustained stress, pluripotency, epigenetic reprogramming, malignant melanoma     

Tissue Inhibitor of Metalloproteinase 1 Expression Associated with Gene Demethylation Confers Anoikis Resistance in Early Phases of Melanocyte Malignant Transformation

Although anoikis resistance has been considered a hallmark of malignant phenotype, the causal relation between neoplastic transformation and anchorage-independent growth remains undefined. We developed an experimental model of murine melanocyte malignant transformation, where a melanocyte lineage (melan-a) was submitted to sequential cycles of anchorage blockade, resulting in progressive morphologic alterations, and malignant transformation. Throughout this process, cells corresponding to premalignant melanocytes and melanoma cell lines were established and show progressive anoikis resistance and increased expression of Timp1. In melan-a melanocytes, Timp1 expression is suppressed by DNA methylation as indicated by its reexpression after 5-aza-2′-deoxycytidine treatment. Methylation-sensitive single-nucleotide primer extension analysis showed increased demethylation in Timp1 in parallel with its expression along malignant transformation. Interestingly, TIMP1 expression has already been related with negative prognosis in some human cancers. Although described as a MMP inhibitor, this protein has been associated with apoptosis resistance in different cell types. Melan-a cells overexpressing Timp1 showed increased survival in suspension but were unable to form tumors in vivo, whereas Timp1-overexpressing melanoma cells showed reduced latency time for tumor appearance and increased metastatic potential. Here, we demonstrated for the first time an increment in Timp1 expression since the early phases of melanocyte malignant transformation, associated to a progressive gene demethylation, which confers anoikis resistance. In this way, Timp1 might be considered as a valued marker for melanocyte malignant transformation.     

Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation.

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.     

The Notch pathway: hair graying and pigment cell homeostasis

The Notch signaling pathway is an essential cell-cell interaction mechanism, which regulates processes such as cell proliferation, cell fate decisions, differentiation or stem cell maintenance. Pigmentation in mammals is provided by melanocytes, which are derived from the neural crest, and by the retinal pigment epithelium (RPE), which is part of the optic cup and hence orginates from neuroectoderm. The importance of functional Notch signaling in melanocytes has been unveiled recently. Here, the pathway is essential for the maintenance of proper hair pigmentation. Deletion of Notch1 and Notch2 or RBP-Jkappa in the melanocyte lineage resulted in a gene dosage-dependent precocious hair graying, due to the elimination of melanoblasts and melanocyte stem cells. Expression data support the idea that Notch signaling might equally be involved in development of the RPE. Furthermore, recent analyses indicate a possible role of Notch signaling in the development of melanoma. In this review, we address the essential role of Notch signaling in the regeneration of the melanocyte population during hair follicle cycles, and discuss data supporting the implication of this signaling pathway in RPE development and melanoma.     

α‐MSH activates immediate defense responses to UV‐induced oxidative stress in human melanocytes

Exposure of cultured human melanocytes to ultraviolet radiation (UV) results in DNA damage. In melanoma, UV‐signature mutations resulting from unrepaired photoproducts are rare, suggesting the possible involvement of oxidative DNA damage in melanocyte malignant transformation. Here we present data demonstrating immediate dose‐dependent generation of hydrogen peroxide in UV‐irradiated melanocytes, which correlated directly with a decrease in catalase activity. Pretreatment of melanocytes with α‐melanocortin (α‐MSH) reduced the UV‐induced generation of 7,8‐dihydro‐8‐oxyguanine (8‐oxodG), a major form of oxidative DNA damage. Pretreatment with α‐MSH also increased the protein levels of catalase and ferritin. The effect of α‐MSH on 8‐oxodG induction was mediated by activation of the melanocortin 1 receptor (MC1R), as it was absent in melanocytes expressing loss‐of‐function MC1R, and blocked by concomitant treatment with an analog of agouti signaling protein (ASIP), ASIP‐YY. This study provides unequivocal evidence for induction of oxidative DNA damage by UV in human melanocytes and reduction of this damage by α‐MSH. Our data unravel some mechanisms by which α‐MSH protects melanocytes from oxidative DNA damage, which partially explain the strong association of loss‐of‐function MC1R with melanoma.     

Crosstalk in skin: melanocytes,keratinocytes, stem cells,and melanoma

In the vertebrate embryo, melanocytes arise from the neural crest, migrate to and colonize the basal layer within the skin and skin appendages. Post-migratory melanocytes are securely attached to the basement membrane, and their morphology, growth, adhesion, and migration are under control of neighboring keratinocytes. Melanoma is a malignant tumor originated from melanocytes or their progenitor cells. During melanocyte transformation and melanoma progression, melanocytes lose their interactions with keratinocytes, resulting in uncontrolled proliferation and invasion of the malignant cells. Melanoma cells at the advanced stages often lack melanocytic features and resemble multipotent progenitors, which are a potential melanocyte reservoir in human skin. In this mini-review, we will summarize findings on cell-cell interactions that are responsible for normal melanocyte homeostasis, stem cell self-renewal, and differentiation. Our ultimate goal is to define molecules and pathways, which are essential for normal cell-cell interactions but deregulated in melanoma formation and progression.     

Cutaneous photobiology. The melanocyte vs. the sun: who will win the final round?

Solar ultraviolet radiation (UV) is a major environmental factor that dramatically alters the homeostasis of the skin as an organ by affecting the survival, proliferation and differentiation of various cutaneous cell types. The effects of UV on the skin include direct damage to DNA, apoptosis, growth arrest, and stimulation of melanogenesis. Long-term effects of UV include photoaging and photocarcinogenesis. Epidermal melanocytes synthesize two main types of melanin: eumelanin and pheomelanin. Melanin, particularly eumelanin, represents the major photoprotective mechanism in the skin. Melanin limits the extent of UV penetration through the epidermal layers, and scavenges reactive oxygen radicals that may lead to oxidative DNA damage. The extent of UV-induced DNA damage and the incidence of skin cancer are inversely correlated with total melanin content of the skin. Given the importance of the melanocyte in guarding against the adverse effects of UV and the fact that the melanocyte has a low self-renewal capacity, it is critical to maintain its survival and genomic integrity in order to prevent malignant transformation to melanoma, the most fatal form of skin cancer. Melanocyte transformation to melanoma involves the activation of certain oncogenes and the inactivation of specific tumor suppressor genes. This review summarizes the current state of knowledge about the role of melanin and the melanocyte in photoprotection, the responses of melanocytes to UV, the signaling pathways that mediate the biological effects of UV on melanocytes, and the most common genetic alterations that lead to melanoma.     

Cutaneous Photobiology. The Melanocyte vs. the Sun: Who Will Win the Final Round?

Solar ultraviolet radiation (UV) is a major environmental factor that dramatically alters the homeostasis of the skin as an organ by affecting the survival, proliferation and differentiation of various cutaneous cell types. The effects of UV on the skin include direct damage to DNA, apoptosis, growth arrest, and stimulation of melanogenesis. Long‐term effects of UV include photoaging and photocarcinogenesis. Epidermal melanocytes synthesize two main types of melanin: eumelanin and pheomelanin. Melanin, particularly eumelanin, represents the major photoprotective mechanism in the skin. Melanin limits the extent of UV penetration through the epidermal layers, and scavenges reactive oxygen radicals that may lead to oxidative DNA damage. The extent of UV‐induced DNA damage and the incidence of skin cancer are inversely correlated with total melanin content of the skin. Given the importance of the melanocyte in guarding against the adverse effects of UV and the fact that the melanocyte has a low self‐renewal capacity, it is critical to maintain its survival and genomic integrity in order to prevent malignant transformation to melanoma, the most fatal form of skin cancer. Melanocyte transformation to melanoma involves the activation of certain oncogenes and the inactivation of specific tumor suppressor genes. This review summarizes the current state of knowledge about the role of melanin and the melanocyte in photoprotection, the responses of melanocytes to UV, the signaling pathways that mediate the biological effects of UV on melanocytes, and the most common genetic alterations that lead to melanoma.     

Roles of endothelin signaling in melanocyte development and melanoma

Endothelin (Edn) signaling via the G-coupled, Edn receptor type B (Ednrb) is essential for the development of melanocytes from the neural crest (NC) and has been associated with melanoma progression. Edn3 plays varying roles during melanocyte development, promoting the proliferation and self-renewal of NC-derived multi- and bi-potential precursors as well as the survival, proliferation, differentiation and migration of committed melanocyte precursors. Melanocyte differentiation is achieved via the interaction of Ednrb and Kit signaling, with Ednrb being specifically required in the final differentiation step, rather than in the initial specification of melanocytic fate. Ednrb has also been implicated in the de-differentiation of mature melanocytes, a process that takes place during the malignant transformation of these cells. Ednrb was found to be upregulated in melanoma metastases and was shown to alter tumor–host interactions leading to melanoma progression. Antagonists to this receptor were shown to inhibit melanoma cell growth and increase the apoptotic rate of these cells, and to lead to disease stabilization in melanoma patients. Thus, Edn signaling inhibition may prove useful in the treatment of certain types of melanoma.     

The cAMP signaling pathway has opposing effects on Rac and Rho in B16F10 cells: implications for dendrite formation in melanocytic cells

A hallmark of melanocytic cells is their ability to form dendrites in response to growth factors and to ultraviolet irradiation. It is known that the cyclic adenosine monophosphate (cAMP) second messenger pathway stimulates melanocyte dendrite formation because agents that increase cAMP such as forskolin and dibutyrl cAMP induce dendrite formation in normal human and murine melanocytes and melanoma cell lines. The Rho family of guanosine triphosphate (GTP)-binding proteins regulates cytoskeletal reorganization in all cells tested and Rac and Rho have both been shown to regulate melanocyte dendrite formation. In this report, we analyzed the effect of cAMP on the activation of Rac and Rho and show that elevation of cAMP stimulates Rac and inhibits Rho in B16F10 cells. The Rho GTP-binding proteins have also been shown to either cross-activate or inhibit each other and in this report we show that Rac activates Rho in B16F10 cells. Microinjection of C3 botulinum exoenzyme toxin, an agent that specifically inactivates Rho or microinjection of constitutively active mutant Rac protein-induced dendricity in human melanocytes and in B16F10 and B16F1 murine melanoma cell lines. We conclude that cAMP-mediated dendrite formation in melanocytic cells is mediated through upregulation of Rac activity and downregulation of Rho activity.     

Endothelin‐1 and α‐melanocortin have redundant effects on global genome repair in UV‐irradiated human melanocytes despite distinct signaling pathways

Human melanocyte homeostasis is sustained by paracrine factors that reduce the genotoxic effects of ultraviolet radiation (UV), the major etiological factor for melanoma. The keratinocyte‐derived endothelin‐1 (End‐1) and α‐melanocyte‐stimulating hormone (α‐MSH) regulate human melanocyte function, proliferation and survival, and enhance repair of UV‐induced DNA photoproducts by binding to the G_q‐ and G_i‐protein‐coupled endothelin B receptor (EDNRB), and the G_s‐protein‐coupled melanocortin 1 receptor (MC1R), respectively. We hereby report that End‐1 and α‐MSH regulate common effectors of the DNA damage response to UV, despite distinct signaling pathways. Both factors activate the two DNA damage sensors ataxia telangiectasia and Rad3‐related and ataxia telangiectasia mutated, enhance DNA damage recognition by reducing soluble nuclear and chromatin‐bound DNA damage binding protein 2, and increase total and chromatin‐bound xeroderma pigmentosum (XP) C. Additionally, α‐MSH and End‐1 increase total levels and chromatin localization of the damage verification protein XPA, and the levels of γH2AX, which facilitates recruitment of DNA repair proteins to DNA lesions. Activation of EDNRB compensates for MC1R loss of function, thereby reducing the risk of malignant transformation of these vulnerable melanocytes. Therefore, MC1R and EDNRB signaling pathways represent redundant mechanisms that inhibit the genotoxic effects of UV and melanomagenesis.     

Rac1 protects epithelial cells against anoikis

Rho family members play a critical role in malignant transformation. Anchorage-independent growth and the ability to avoid apoptosis caused by loss of anchorage (anoikis) are important features of transformed cells. Here we show that constitutive activation of Rac1 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. Constitutively active Rac1-V12 decreases DNA fragmentation and caspase activity by 50% in MDCK cells kept in suspension. In addition, expression of Rac1-V12 in MDCK cells in suspension conditions causes an increase in the number of surviving cells. We also investigated the signaling pathways that are activated by Rac1 to stimulate cell survival. We show that expression of Rac1-V12 in MDCK cells in suspension stimulates a number of signaling cascades that have been implicated in the control of cell survival, including the p42/44 ERK, p38, protein kinase B, and nuclear factor kappaB pathways. Using specific chemical or protein inhibitors of these respective pathways, we show that Rac1-mediated cell survival strongly depends on phosphatidylinositol 3-kinase activity and that activation of ERK, p38, and NF-kappaB are largely dispensable for Rac1 survival signaling. In conclusion, these studies demonstrate that Rac1 can suppress apoptosis in epithelial cells in anchorage-independent conditions and suggest a potential role for Rac1-mediated survival signaling in cell transformation.     

小鼠正常黑素细胞和小鼠B16黑素瘤细胞的环状RNAs表达谱

黑素瘤是一种多发于皮肤的恶性肿瘤,因其侵袭性强,预后差等特点一直是科研人员关注的热点。环状RNAs(circRNAs)是一种新型内源性非编码RNA,广泛参与动物生长发育、细胞分化和信号转导等生理过程,但circRNAs在黑素瘤细胞内的分子机制尚未被充分解析。本研究以小鼠(C57BL/6J)正常黑素细胞及B16黑素瘤细胞为研究对象,采用二代测序技术分析两种细胞间circRNAs表达特性。测序结果显示,小鼠正常黑素细胞和黑素瘤细胞中共有851个circRNAs,其中195个差异表达circRNAs(DECs)。GO及KEGG数据库注释发现,DECs的来源基因主要参与细胞周期(cell cycle)、紧密结合(tight junction)、Rap1信号通路(Rap1 signaling pathway)、TGF-beta信号通路(TGF-beta signaling pathway)等与细胞增殖、迁移相关的信号通路;探究发现,黑素瘤细胞中显著性高表达的circE2F5(circ-3:14578602|14606309)通过上调E2F5的表达促进黑素瘤细胞增殖。circRNA靶基因预测发现,...     

Role of Phosphatidylinositol 4,5-Bisphosphate in Ras/Rac-Induced Disruption of the Cortactin-Actomyosin II Complex and Malignant Transformation

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac–phosphatidylinositol 4,5-biphosphate (PIP₂)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP₂ binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP₂ inhibits the actin-EMS1 interaction, HS1 reverses the PIP₂ effect. Thus, we propose that PIP₂, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP₂-binding molecules.