Malignant Transformation Of Melanocytes Research Articles

Cutaneous pH landscape as a facilitator of melanoma initiation and progression.

Melanoma incidence is on the rise and currently causes the majority of skin cancer-related deaths. Yet, therapies for metastatic melanoma are still insufficient so that new concepts are essential. Malignant transformation of melanocytes and melanoma progression are intimately linked to the cutaneous pH landscape and its dysregulation in tumour lesions. The pH landscape of normal skin is characterized by a large pH gradient of up to 3 pH units between surface and dermis. The Na+ /H+ exchanger NHE1 is one of the major contributors of acidity in superficial skin layers. It is also activated by the most frequent mutation in melanoma, BRAFV600E , thereby causing pH dysregulation during melanoma initiation. Melanoma progression is supported by an extracellular acidification and/or NHE1 activity which promote the escape of single melanoma cells from the primary tumour, migration and metastatic spreading. We propose that viewing melanoma against the background of the acid-base physiology of the skin provides a better understanding of the pathophysiology of this disease and allows the development of novel therapeutic concepts.

A 3D Organotypic Melanoma Spheroid Skin Model.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Prognostic significance of RBP2-H1 variant of JARID1B in melanoma

BackgroundHistone demethylase JARID1B plays several context dependent roles in epigenetic regulation of cellular differentiation in normal development and is highly expressed in multiple human cancers. The protein is a strong transcriptional repressor capable of downregulating numerous genes. There are three splicing isoforms of JARID1B, however the links between the protein structure and function are not clear. The expression pattern of JARID1B in human melanoma seems to be different from observed in other cancers. Moreover, up to now no data on the impact of JARID1B expression in cutaneous melanoma on the patients’ prognosis have been reported.MethodsWe investigated immunohistochemically the association of intratumoral expression of total JARID1B protein and its RBP2-H1 isoform in primary and metastatic melanomas with prognosis for the patients.ResultsExpression of both total JARID1B protein and its RBP2-H1 variant was found in all the melanomas investigated. Our results indicate, however, that only high (above 90% of the cells) intratumoral expression of RBP2-H1 can be considered prognostic factor associated with worse overall survival of the patients.ConclusionsSuch results if considered together with data demonstrating a switch to enhanced expression of RBP2-H1 at early stages of malignant transformation of melanocytes are in agreement with hypothetical crucial role of JARID1B in the course of melanoma development and progression and suggest that altered splicing of JARID1B may be important factor increasing melanoma aggressiveness.

Melanoma: Genetic Abnormalities, Tumor Progression, Clonal Evolution and Tumor Initiating Cells.

Melanoma is an aggressive neoplasia issued from the malignant transformation of melanocytes, the pigment-generating cells of the skin. It is responsible for about 75% of deaths due to skin cancers. Melanoma is a phenotypically and molecularly heterogeneous disease: cutaneous, uveal, acral, and mucosal melanomas have different clinical courses, are associated with different mutational profiles, and possess distinct risk factors. The discovery of the molecular abnormalities underlying melanomas has led to the promising improvement of therapy, and further progress is expected in the near future. The study of melanoma precursor lesions has led to the suggestion that the pathway of tumor evolution implies the progression from benign naevi, to dysplastic naevi, to melanoma in situ and then to invasive and metastatic melanoma. The gene alterations characterizing melanomas tend to accumulate in these precursor lesions in a sequential order. Studies carried out in recent years have, in part, elucidated the great tumorigenic potential of melanoma tumor cells. These findings have led to speculation that the cancer stem cell model cannot be applied to melanoma because, in this malignancy, tumor cells possess an intrinsic plasticity, conferring the capacity to initiate and maintain the neoplastic process to phenotypically different tumor cells.

Abstract 2413: SIRT1 regulates Mxd1 throughout melanoma progression

Abstract In a murine melanoma model of malignant transformation promoted by sustained stress, increased levels of reactive oxygen species were found to result in DNA damage and were related to massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, γH2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. Mxd1 was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, Mxd1 was found down-regulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the Mxd1 expression. Sirt1 stable silencing increased Mxd1 mRNA expression and led to up-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, the upregulation of which is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. Mxd1 was identified as a new SIRT1 target gene. SIRT1 promoted Mxd1 silencing, which led to increased activity of MYC oncogene contributing to melanoma progression. Citation Format: Fabiana M. Meliso, Danilo Micali, Camila T. Silva, Thais S. Sabedot, Simon G. Coetzee, Adrian Koch, Fabian B. Fahlbusch, Houtan Noushmehr, Regine Schneider-Stock, Miriam G. Jasiulionis. SIRT1 regulates Mxd1 throughout melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2413. doi:10.1158/1538-7445.AM2017-2413

Role of testosterone synthesized by skin melanocytes in preventing malignant transformation of nevi.

e21066 Background: Melanocytes are involved into the synthesis and metabolism of steroid hormones in the skin. Cutaneous melanoma in 75% of cases occurs in congenital or acquired pigmented nevi. There are no studies comparing local hormonogenesis in nevi and melanomas. The purpose of the study was a comparative analysis of hormone levels in tissues of dysplastic nevi and cutaneous melanoma (pТ1-2N0M0). Methods: Levels of prolactin (PRL), progesterone (P4), total and free testosterone (T and fT), estrone (E1) and estriol (E3) and sex steroid-binding globulin (SSBG) were studied by ELISA in 17 samples of рТ1-2N0M0 melanoma and in 23 samples of dysplastic nevi. Intact tissues (n = 15) obtained from non-cancer patients during surgical treatment was used as the control. Results: Levels of T in nevi, compared to intact tissues, were 1.4 times higher, fT – 8.7 times higher, while SSBG content was 1.9 times lower; E3 was 1.6 times higher, PRL – 1.4 times lower, and levels of E1 in nevi were similar to the values in intact tissues. рТ1-2N0M0 melanomas, compared to intact tissues, demonstrated local androgen imbalance: T levels were decreased by 1.8 times, while fT exceeded the norm by 1.8 times, and SSBG – by 6 times. E1 content increased by 1.6 times, E3 decreased by 2.8 times. The E1/E3 ratio in nevi was reduced by 1.6 times, while in melanoma it was increased by 4.4 times. The T/E1 ratio in nevi was increased by 1.4 times, and in melanoma it was decreased by 2.8 times. Levels of PRL and P4 in melanomas, nevi and in intact tissues did not differ significantly. Conclusions: Melanoma was characterized by hyperestrogenia due to increased levels of E1 and low E3 concentrations which caused malignant transformation of melanocytes. Most likely, in melanoma spent SVT estrone synthesis, as evidenced by increase in the E1 / E3 and decrease T / E1.Apparently, fT in melanoma is used for estrogen synthesis, as indicated by the E1/E3 increase and decrease in the T/E1 ratio. Increased E1 synthesis can contribute to the realization of estrogen genotoxic effects. These mechanisms are absent in tissues of nevi. Hyperandrogenism in nevi probably prevents malignant transformation due to low aromatase activity.

Sox2 is not required for melanomagenesis, melanoma growth and melanoma metastasis in vivo

Melanoma is a dangerous form of skin cancer derived from the malignant transformation of melanocytes. The transcription factor SOX2 is not expressed in melanocytes, however, it has been shown to be differentially expressed between benign nevi and malignant melanomas and to be essential for melanoma stem cell maintenance and expansion in vitro and in xenograft models. By using a mouse model in which BRafV600E mutation cooperates with Pten loss to induce the development of metastatic melanoma, we investigated if Sox2 is required during the process of melanomagenesis, melanoma growth and metastasis and in the acquisition of resistance to BRAF inhibitors (BRAFi) treatments. We found that deletion of Sox2 specifically in Pten null and BRafV600E-expressing melanocytes did not prevent tumor formation and did not modify the temporal kinetics of melanoma occurrence compared to Sox2 wt mice. In addition, tumor growth was similar between Sox2 wt and Sox2 deleted (del) melanomas. By querying publicly available databases, we did not find statistically significant differences in SOX2 expression levels between benign nevi and melanomas, and analysis on two melanoma patient cohorts confirmed that Sox2 levels did not significantly change between primary and metastatic melanomas. Melanoma cell lines derived from both Sox2 genotypes showed a similar sensitivity to vemurafenib treatment and the same ability to develop vemurafenib resistance in long-term cultures. Development of vemurafenib resistance was not dependent on SOX2 expression also in human melanoma cell lines in vitro. Our findings exclude an oncogenic function for Sox2 during melanoma development and do not support a role for this transcription factor in the acquisition of resistance to BRAFi treatments.

Molecular Mechanisms of UVA-Induced Melanoma.

Cutaneous melanoma is a deadly skin cancer, resulting from malignant transformation of melanocytes. Long-wave ultraviolet radiation (315-400 nm) is able to damage DNA, cause mutations, and induce melanoma. However, the exact mechanisms of UVA-induced cutaneous melanoma remain a matter of debate. In this review, we give a brief characterization of the most important elements in the photobiology of UVA in melanomagenesis.

Altered Splicing of JARID1B in Development of Human Cutaneous Melanoma?

Upregulated expression of histone H3K4 demethylase JARID1B has been found in several types of human cancer, but the expression pattern of this protein in benign naevi and human cutaneous melanomas seems to differ from that described for other tumors. We demonstrate that the apparent contradiction may be because of the fact that the malignant transformation of melanocytes is associated not so much with a general enhancement of a total expression of JARID1B but rather with a change in relative expression levels of individual splicing variants of the protein. Our data indicate that parallel immunohistochemical assays of the expression levels of all the isoforms and of the RBP2-H1 variant of JARID1B may be an efficient technique of differentiating between benign naevi and melanomas.

Abstract 2680: Cyclic AMP signaling in melanoma: Paradoxical downregulation of pCREB upon activation of adenylate cyclase confers resistance to MAPK inhibition

Abstract Melanoma, which arises from malignant transformation of melanocytes, has proven to be difficult to treat due its intrinsic and acquired resistance for chemotherapy. Although polymorphisms in the melanocortin receptor 1 (MC1R), which result in impairment of cyclic AMP (cAMP) signaling, are associated with increased risk for melanoma development, the role of cAMP signaling in melanoma, specifically in drug resistance, is not fully understood. Given the importance of cAMP signaling in melanocytes proliferation and differentiation, we investigated the role of cAMP signaling in melanoma cell proliferation, survival and drug resistance. We found that upregulation of cAMP signaling, through activation of adenylate cyclase with forskolin (FSK), has both positive and negative effects on proliferation and survival of melanoma cells. The effect of FSK on melanoma proliferation and survival was not dependent on oncogenic (RAS vs. BRAF) mutation. Moreover, upregulation of cAMP conferred resistance to Selumetinib (AZD6244)-mediated MAPK inhibition by restoring the pERK levels. We also show that FSK-mediated resistance is paradoxically related to a downregulation of phosphorylated cAMP Responsive Element Binding Protein (pCREB), suggesting a CREB-independent mechanism of FSK-induced MAPK reactivation in acquired drug resistance. Citation Format: Carlos I. Rodriguez, Vijayasaradhi Setaluri. Cyclic AMP signaling in melanoma: Paradoxical downregulation of pCREB upon activation of adenylate cyclase confers resistance to MAPK inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2680. doi:10.1158/1538-7445.AM2015-2680

Abstract 4767: DNA sequences differentially associated with Sirt1 and Dnmt3b during melanoma progression

Abstract Melanoma is one of the most worrying tumors due to its high capacity to cause metastasis. Sirt1, a class III histone deacetylase, was shown to be attracted to sites of double strand breaks in DNA, favoring, by recruiting components of the epigenetic machinery, aberrant epigenetic silencing in that region (O’HAGAN et al., 2008). Beyond that, subsequent studies have attributed important roles of Sirt1 and other components of the epigenetic machinery, such as DNA methyltransferases (such as Dnmt3b), in tumor initiation and progression. In this sense, using a murine model of malignant transformation of melanocytes, associated with sustained stress conditions, our goal was to identify genomic sequences differentially associated to Sirt1 and/or Dnmt3B before and after stress condition, represented in our model by anchorage blockade, and analyze if these sequences are targeted by aberrant methylation in cell lines obtained by this process that represent different steps in melanoma progression. We have showed increased DNA damage after 24 hours of anchorage impediment followed by increased Sirt1 expression. In addition, we have showed Sirt1/Dnmt3b association just after this stress condition that leads to malignant transformation. It was identified several DNA sequences differentially associated to Sirt1 and also to Dnmt3b after stress condition which are also associated to genes related to different metabolic processes. Some of these sequences remain associated to Sirt1 and Dnmt3b during melanoma progression, indicating that Dnmt3b remains associated to Sirt1 during malignant transformation. Among the sequences, Tiam1 and Mxd1 are potential targets of Sirt1 and Dnmt3b that have important roles in tumor progression. We have also identified motifs where Sirt1 and Dnmt3b are probably bound before and after stress condition. The main motif related to Sirt1 was coincident with Boris motif. Subsequent analysis are been developed to elucidate the importance of these interesting discoveries. Supported by FAPESP and DAAD. Citation Format: Fabiana Marcelino Meliso, Camila Tainah da Silva, Simon Coetzee, Thaís Sarraf Sabedot, Houtan Noushmehr, Regine Schneider-Stock, Miriam Galvonas Jasiulionis. DNA sequences differentially associated with Sirt1 and Dnmt3b during melanoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4767. doi:10.1158/1538-7445.AM2015-4767

Primary Oral Mucosal Melanoma: a Short Review

Primary Oral Mucosal Melanoma (POMM) is a very rare entity and it develops from malignant transformation of melanocytes from mucosal epithelium. POMM has a very poor prognosis and aggressive behavior. It represents only 0.5% of all oral malignancies, accounting 0.2 to 8% of all melanomas, with an incidence of 1.2 cases per 10 million per year. POMM is asymptomatic in its early stages and usually not noticed by the patients, resulting in the delay in diagnosis. Immunoreactivity of melanoma cells to antibodies against: S-100 protein; Melan-A (MART-1) and HMB-45 (gp100) can be very useful to distinguish POMM from other malignancies. Others markers may indicate variations in its biologic behavior and prognosis, such as Ki-67; P53 and P16, and maybe useful for prognostic prediction.

Amelanotic melanoma of the nasal mucosa: A rare case report

Melanomas develop due to malignant transformation of melanocytes, which are derived from the neuroectoderm. Although the majority of melanomas are cutaneous, they occasionally arise from extra-cutaneous tissues as well which contain melanocytes, such as uvea, leptomeninges, or the mucosa. Melanomas can be melanotic or amelanotic. Approximately, 15-20% of all malignant melanomas arise in head and neck region and over 80% of these involve the skin. Mucosal malignant melanomas are very rare and may affect the upper aero-digestive tract (UADT). Their incidence is approximately as low as 0.5-3% of all malignant melanomas. Majority of noncutaneous head and neck malignant melanomas affect the ocular origin and only 6-8% originate in the mucous membranes of the UADT. Nasal mucosal melanomas account for

Metastatic Malign Melanoma of Supraspinatus Muscle

Malignant melanoma, which occurs by malignant transformation of melanocytes that originate from the neural crest, is one of the most aggressive neoplasms of the skin, and it constitutes 4% of skin cancers (1). Malignant melanoma may spread to all organs, like skin, lungs, distant lymph nodes, pleura, liver, central nervous system, skeleton, subcutaneous sites, gastrointestinal tract, heart, adrenal glands, kidney, and thyroid gland, with different frequencies (2). The frequency of metastasis to the skeletal muscles in patients with malignant melanoma is very low (3). Here, we present a case of malignant melanoma that metastasized to the supraspinatus muscle who presented with shoulder pain. A 78-year-old female patient presented with unrelenting shoulder pain mimicking rotator cuff pathology. She had a 4-month history of pain, which was localized to the left shoulder and increased gradually. On physical examination, her shoulder movement was limited due to pain. The patient had a positive Neer sign. There was tenderness with palpation of the supraspinatus. A scar on the back of the patient was inspected. When her medical history had been questioned in more detail, she told us that she had a history of malignant melanoma on her back 30 years ago that was excised and caused no symptoms until that time. The patient also complained about exhaustion with daily activities. No lymph node enlargement in the cervical or axillary region was detected. Erythrocyte sedimentation rate was 24 mm/h (normal <20 mm/h), and other blood tests were normal. Shoulder magnetic resonance imaging (MRI) was performed. It revealed a mass showing contrast uptake in the supraspinatus muscle and contrast uptake in multiple lymph nodes in the axillary and supraclavicular regions (Figure 1). Thereafter, a positron emission tomography (PET) image was taken. The PET was consistent with multiple metastases to the subacromial region and axillary lymph nodes. The biopsy of the lesion confirmed the diagnosis of malignant melanoma. She was referred to an orthopedic surgeon. Malignant melanoma, which could have a large variation of aspects, can spread anywhere in the body, and it mostly often spreads loco-regionally into the skin, subcutaneous tissue, and lymph nodes, and then it goes into distant organs in the order of subcutaneous fat, lungs, liver, brain, bones, and intestines (3,4). Neighboring muscle tissue is also a target for malignant melanoma metastases. Malignant melanoma may

Ras and Rac1, Frequently Mutated in Melanomas, Are Activated by Superoxide Anion, Modulate Dnmt1 Level and Are Causally Related to Melanocyte Malignant Transformation

A melanocyte malignant transformation model was developed in our laboratory, in which different melanoma cell lines were obtained after submitting the non-tumorigenic melanocyte lineage melan-a to sequential cycles of anchorage impediment. Our group has already showed that increased superoxide level leads to global DNA hypermemethylation as well increased Dnmt1 expression few hours after melanocyte anchorage blockade. Here, we showed that Ras/Rac1/ERK signaling pathway is activated in melanocytes submitted to anchorage impediment, regulating superoxide levels, global DNA methylation, and Dnmt1 expression. Interestingly, Ras and Rac1 activation is not related to codon mutations, but instead regulated by superoxide. Moreover, the malignant transformation was drastically compromised when melan-a melanocytes were submitted to sequential cycles of anchorage blockage in the presence of a superoxide scavenger. This aberrant signaling pathway associated with a sustained stressful condition, which might be similar to conditions such as UV radiation and inflammation, seems to be an early step in malignant transformation and to contribute to an epigenetic reprogramming and the melanoma development.

Metabotropic Glutamate Receptor 1 Expression and Its Polymorphic Variants Associate with Breast Cancer Phenotypes

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.

Obesity and melanoma: Exploring molecular links

Obesity is now a major health problem due to its rapidly increasing incidence worldwide and severe consequences. Among many conditions associated with obesity are some cancers including melanoma. Both genetic defects and environmental risk factors are involved in the carcinogenesis of melanoma. Activation of multiple signal pathways such as the PI3K/Akt and MAPK pathways are necessary for the initiation of melanoma. Activation of the MAPK pathway as a result of activating mutations in BRAF is commonly seen in melanoma though it alone is not sufficient to cause malignant transformation of melanocytes. Obesity can result in the activation of many signal pathways including PI3K/Akt, MAPK, and STAT3. The activation of these pathways may have a synergistic effect with the genetic defects thereby increasing the incidence of melanoma.

Abstract 4043: The role of CD63/Timp1/β1 integrin complex in cell survival.

Abstract Although malignant melanoma is the less frequently diagnosed skin cancer, it shows a poor prognosis due its chemoresistance and metastasis development. One of the acquired abilities of transformed cells is anoikis resistance and this property is closely related to metastasis formation. In our laboratory, we developed a model that allows us to study different steps of melanocyte malignant transformation. Melan-a melanocytes surviving after 1, 2, 3 and 4 deadhesion cycles showed modified morphology and independent PMA growth and have been named 1C, 2C, 3C and 4C cells, respectively. Different melanoma cell lines were established after submitting 4C spheroids to limiting dilution. Previous data from our group demonstrated increased expression of Timp1 and resistance to anoikis during the malignant transformation of melanocytes. Melan-a melanocytes overexpressing Timp1 acquire anoikis-resistant phenotype. In our laboratory, we observed interaction between CD63, Timp1 and β1-integrin in the 4C, 4C11- and 4C11+ cell lines established after cycles of anchorage impediment. This seems to be the first study describing the interaction between Timp1, CD63 and β1-integrin in tumor cells. However, it is unclear how Timp1 modulates resistance to anoikis. Therefore, our aim is to analyze the role of Timp1 in the context of Timp1/CD63/β1-integrin complex and the signaling pathways involved in the acquisition of anoikis resistance. Supported by CAPES and FAPESP. Citation Format: Mariana T. Pinto, Fabiana Henriques Machado de Melo, Miriam Galvonas Jasiulionis. The role of CD63/Timp1/β1 integrin complex in cell survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4043. doi:10.1158/1538-7445.AM2013-4043

Abstract 2977: Malignant transformation associated with repetitive Ras/Rac1 activation, increased production of superoxide anion, and global DNA methylation alterations.

Abstract Driver mutations in N-Ras and BRAF, and more recently in Rac1, were described in melanomas. Using a malignant transformation model of murine melanocytes developed in our laboratory, in which melanoma cell lines were obtained after submitting the melanocyte lineage melan-a to sequential cycles of anchorage blockade, we identified dysfunction in both N-Ras and Rac1 during stressful condition. Our group has already showed increased global DNA methylation as well increased DNA methyltransferase 1 (Dnmt1) protein level few hours after anchorage blockade. This increase seems to be related with elevated amounts of superoxide anion. Previous works have showed that both Ras may regulate superoxide level as Ras activation may affect DNA methylation. Therefore, we aimed to study the relation among Ras/MEK/ERK signaling pathway, oxidative stress and DNA methylation during melan-a anchorage blockade. We showed that Ras, Rac1 and ERK are activated few hours after melan-a anchorage blockade and inhibiting this activation abrogates superoxide increase. Furthermore, global DNA methylation levels decrease during anchorage blockade after treating cells with superoxide scavenger or Ras inhibitor (FTI). Dnmt1 protein level is also decreased after treating melan-a cells during anchorage blockade with FTI or ERK inhibitor (U0126). Ras/MEK/ERK pathway seems to be regulated by and regulates superoxide production during melan-a anchorage restriction. In this way, superoxide anion depletion impairs Ras and ERK activation and FTI or U0126 treatment decreases superoxide during adhesion impediment. This aberrant signaling seems to have a significant impact in melanoma genesis since the malignant transformation was drastically compromised when melan-a melanocytes were pre-treated with superoxide scavenger before each anchorage impediment cycle. Elucidating the relation among oxidative stress, epigenetic changes and melanocyte malignant transformation may provide novel avenues to develop alternative therapeutic approaches. Citation Format: Fernanda Molognoni, Fabiana M. Melo, Camila Tainah, Miriam G. Jasiulionis. Malignant transformation associated with repetitive Ras/Rac1 activation, increased production of superoxide anion, and global DNA methylation alterations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2977. doi:10.1158/1538-7445.AM2013-2977

Timp1 interacts with beta-1 integrin and CD63 along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway independently of Akt phosphorylation

BackgroundAnoikis resistance is one of the abilities acquired along tumor progression. This characteristic is associated with metastasis development, since tumorigenic cells must survive independently of cell-matrix interactions in this process. In our laboratory, it was developed a murine melanocyte malignant transformation model associated with a sustained stressful condition. After subjecting melan-a melanocytes to 1, 2, 3 and 4 cycles of anchorage impediment, anoikis resistant cells were established and named 1C, 2C, 3C and 4C, respectively. These cells showed altered morphology and PMA independent cell growth, but were not tumorigenic, corresponding to pre-malignant cells. After limiting dilution of 4C pre-malignant cells, melanoma cell lines with different characteristics were obtained. Previous data from our group showed that increased Timp1 expression correlated with anoikis-resistant phenotype. Timp1 was shown to confer anchorage-independent growth capability to melan-a melanocytes and render melanoma cells more aggressive when injected into mice. However, the mechanisms involved in anoikis regulation by Timp1 in tumorigenic cells are not clear yet.MethodsThe β1-integrin and Timp1 expression were evaluated by Western blotting and CD63 protein expression by flow cytometry using specific antibodies. To analyze the interaction among Timp1, CD63 and β1-integrin, immunoprecipitation assays were performed, anoikis resistance capability was evaluated in the presence or not of the PI3-K inhibitors, Wortmannin and LY294002. Relative expression of TIMP1 and CD63 in human metastatic melanoma cells was analyzed by real time PCR.ResultsDifferential association among Timp1, CD63 and β1-integrins was observed in melan-a melanocytes, 4C pre-malignant melanocytes and 4C11- and 4C11+ melanoma cells. Timp1 present in conditioned medium of melanoma cells rendered melan-a melanocytes anoikis-resistant through PI3-K signaling pathway independently of Akt activation. In human melanoma cell lines, in which TIMP1 and beta-1 integrin were also found to be interacting, TIMP1 and CD63 levels together was shown to correlate significantly with colony formation capacity.ConclusionsOur results show that Timp1 is assembled in a supramolecular complex containing CD63 and β1-integrins along melanoma genesis and confers anoikis resistance by activating PI3-K signaling pathway, independently of Akt phosphorylation. In addition, our data point TIMP1, mainly together with CD63, as a potential biomarker of melanoma.