Melanoma Tumorigenesis Research Articles

Abstract A04: Cross-species oncogenomics approach identifies PTPN11 as an oncogene and potential therapeutic target in melanoma

Abstract Melanoma is a deadly disease carrying many genetic mutations. A major challenge to the development of effective targeted therapies in melanoma is the identification of true “driver” mutations among numerous “passenger” alterations. Several previous studies support using cross-species comparative oncogenomic approaches for cancer gene discovery. Specifically, it has been shown that mice and humans share several genetic events in the development of cancer and that these events that are conserved across different species may point to functionally important and evolutionary conserved alterations targeting “driver” genes. Recently, we analyzed melanoma genomes from a mouse model driven by the loss of PTEN and CDKN2A (INK4A/ARF), commonly observed alterations in human melanoma patients, by whole-exome sequencing. This study identified several conserved cross-species orthologous mutations in Kras, Erbb3, and Ptpn11. In this study, we addressed the functional roles of PTPN11 in melanoma tumorigenesis and tumor maintenance, its effect on RAS/RAF/MEK/ERK signaling pathway, and its activation status in human melanoma. Melanoma displays frequent activation of the RAS/RAF/MEK/ERK signaling pathway, which is intricately regulated by multiple proteins including PTPN11 (Tyrosine-Protein Phosphatase Non-Receptor Type 11, encoding SHP2). Although implicated as an oncogene in multiple cancer types, the oncogenic role of PTPN11 has not been fully established in melanoma. PTPN11 can be activated by receptor tyrosine kinases (RTKs) and/or by point mutations. Although the mutation rate is low (1~3%), we observed activating phosphorylation on Tyr 542 of PTPN11 in 40% (n=15/38) of melanoma specimens and the majority of human melanoma cell lines (n=14), indicating the potential frequent activation of PTPN11 in human melanoma. PTPN11 knock-down suppressed ERK activation in NRAS mutant (WM1361A, 1366, 1346) and BRAF/NRAS wt (WM3211, MeWo, CHL1) melanoma cells, but not in BRAF mutant (1205Lu, IGR1, 983C) cells. Moreover, we have shown that the expression of active PTPN11 E76K mutant drives soft-agar colony growth in vitro, tumor growth in nude mice, RAS/RAF/MEK/ERK activation, and resistance to MEK inhibition, whereas knock-down of Ptpn11 reduces colony growth and ERK activation. We generated a tet-inducible, melanocyte-specific, PTPN11 E76K transgenic mouse model in a Pten and Cdkn2a null background and observed melanoma formation. Implantation of melanoma cells derived from this model showed doxycycline-dependent tumor growth in nude mice; additionally, withdrawal of doxycycline and subsequent extinction of PTPN11 E76K caused regression of established tumors, supporting a tumor-maintenance role of PTPN11. These data support the oncogenic roles of PTPN11 in melanoma by regulating RAS/RAF/MAPK pathway activation and the value of PTPN11 as a novel and actionable therapeutic target. Citation Format: Kristen S. Hill, Xue Wang, Evan R. Roberts, Ellen M. Marin, Jamie K. Teer, Youngchul Kim, Jane Messina, Jie Wu, Minjung Kim. Cross-species oncogenomics approach identifies PTPN11 as an oncogene and potential therapeutic target in melanoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A04.

SUV39H1/DNMT3A‐dependent methylation of the RB1 promoter stimulates PIN1 expression and melanoma development

Melanoma is among the most aggressive and treatment-resistant human cancers. Aberrant histone H3 methylation at Lys 9 (H3K9) correlates with carcinogenic gene silencing, but the significance of suppressor of variegation 3-9 homolog 1 (SUV39H1), an H3K9-specific methyltransferase, in melanoma initiation and progression remains unclear. Here, we show that SUV39H1-mediated H3K9 trimethylation facilitates retinoblastoma ( RB) 1 promoter CpG island methylation by interacting with DNA methyltransferase 3A and decreasing RB mRNA and protein in melanoma cells. Reduced RB abundance, in turn, impairs E2F1 transcriptional inhibition, leading to increased peptidyl-prolyl cis-trans isomerase never-in-mitosis A (NIMA)-interacting 1 (PIN1) levels, human keratinocyte neoplastic cell transformation, and melanoma tumorigenesis via enhanced rapidly accelerated fibrosarcoma 1(RAF1)-MEK-ERK signaling pathway activation. In a synergistic model with B16-F1 murine melanoma cells, SUV39H1 and PIN1 overexpression increased melanoma growth, which was abrogated by their inhibition in SUV39H1-overexpressing B16-F1 mice. SUV39H1 also positively correlated with PIN1 expression in human melanoma. Our studies establish SUV39H1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.-Kim, G., Kim, J.-Y., Lim, S.-C., Lee, K. Y., Kim, O., Choi, H. S. SUV39H1/DNMT3A-dependent methylation of the RB1 promoter stimulates PIN1 expression and melanoma development.

1207 RNF4 ubiquitin ligase drives melanoma progression

Deciphering new molecular targets that are crucial for melanoma progression is of utmost importance. One such potential target is RNF4, a SUMO-targeted ubiquitin ligase. In most cases, RNF4-dependent ubiquitination tags SUMOylated proteins for degradation. Additionally, RNF4 enhances the stability & transcriptional activity of selected phospho-oncoproteins, hence promoting tumorigenesis. We aimed to evaluate the contribution of RNF4 to human melanoma progression in culture and in-vivo. We found that RNF4 is essential for melanoma progression and survival. Knockdown of RNF4 attenuated melanoma cell proliferation, migration and clonogenicity (p<0.05). Likewise, Conditional expression of RNF4, but not the inactive mutant, promoted tumor growth, resulting in larger and highly vascular xenigrafts (p<0.001). RNA-seq analysis of RNF4-expressing tumors identified a distinct set of 102 genes that were upregulated by RNF4. These genes regulate angiogenesis, migration, and cell growth. Pathway analysis identified the translation machinery as a key node controled by RNF4 in these tumors. Thus, RNF4 directly links translation with protein dynamics during melanoma tumorigenesis. In addition, the potential implications to melanoma patients will be discussed. In conclusion, our results establish a critical role for RNF4 in melanoma tumorigenesis.

Understanding Gαq/11 localization and trafficking in uveal melanoma

Constitutively activating mutations in Gαq and Gα11 (Gαq/11) have been reported in up to 93% of uveal melanomas. Although constitutively active Gαq/11 promote uveal melanoma tumorigenesis through activation of multiple downstream pathways, no therapies inhibit constitutively active Gαq/11. Studies suggest that upon activation of some Gα subunits increased palmitate turnover and increased cytoplasmic localization occur. The purpose of our studies is to understand the role of palmitoylation in trafficking and signaling of constitutively active Gαq/11 in uveal melanoma. Using live cell imaging and cellular fractionation of HEK 293 cells and uveal melanoma cells, constitutively active GαqQ209L and GαqQ209P show decreased localization at membranes compared to wild type Gαq, suggesting increased turnover of attached palmitate in constitutively active mutants. Moreover, immunofluorescence microscopy revealed that a palmitoylation‐deficient constitutively active mutant displays a complete loss of plasma membrane localization and an inability to signal as measured by Rho‐ and Rac‐dependent YAP translocation into the nucleus, TEAD‐dependent luciferase activity, and ERK phosphorylation. These studies demonstrate that palmitoylation of mutationally activated Gαq/11 is required for its signaling functions. Although live cell imaging and cellular fractionation experiments reveal decreased membrane localization of constitutively active Gαq compared to wild‐type Gαq, our studies suggest that complete loss of membrane localization abolishes constitutively active Gαqdependent signaling. Disruption of constitutively active Gαq/11 palmitoylation and localization may be an effective strategy for inhibiting constitutively active Gαq/11 oncogenic signaling in uveal melanoma.Support or Funding InformationThis research was funded by NIH grants R01GM056444‐16, R01GM056444‐16S1 (Research Supplements to Promote Diversity in Health‐Related Research) and T32 GM100836.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The lncRNA CCAT1 Upregulates Proliferation and Invasion in Melanoma Cells via Suppressing miR-33a.

It is increasingly evident that various long noncoding RNAs (lncRNAs) participate in the tumorigenesis of multiple tumors, including melanoma. lncRNAs have been validated as oncogenic factors in various tumors; however, the potential regulatory mechanism of CCAT1 in melanoma is still unclear. The purpose of this study was to investigate the regulation of CCAT1 on melanoma genesis. The expression of CCAT1 in melanoma tissue and cell lines was measured using qRT-PCR. Interference oligonucleotide or mimic sequences were applied to up- or downregulate RNA expression. CCK-8 and colony formation assays were performed to detect the proliferation capability. Transwell assay was used to assess the migration and invasion capacities. Bioinformatics analysis was performed to predict the target miRNAs of CCAT1. Expression of CCAT1 was significantly upregulated in melanoma tissue and cell lines. CCAT1 knockdown observably suppressed the proliferation, migration, and invasion abilities. Bioinformatics analysis predicted that miR-33a acted as a target of CCAT1, which was confirmed by dual-luciferase reporter assay. CCAT1 knockdown reversed the tumor-promoting ability of the miR-33a inhibitor. CCAT1 acts as an oncogenic factor in the genesis of melanoma and exerts tumor-promoting roles via sponging miR-33a, providing a novel insight for competing endogenous RNA (ceRNA) in the tumorigenesis of melanoma.

Overexpression of microRNA-612 Restrains the Growth, Invasion, and Tumorigenesis of Melanoma Cells by Targeting Espin.

microRNA (miR)-612 shows anticancer activity in several types of cancers, yet its function in melanoma is still unclear. This study was undertaken to investigate the expression of miR-612 and its biological relevance in melanoma cell growth, invasion, and tumorigenesis. The expression and prognostic significance of miR-612 in melanoma were examined. The effects of miR-612 overexpression on cell proliferation, colony formation, tumorigenesis, and invasion were determined. Rescue experiments were conducted to identify the functional target gene(s) of miR-612. miR-612 was significantly downregulated in melanoma tissues compared to adjacent normal tissues. Low miR-612 expression was significantly associated with melanoma thickness, lymph node metastasis, and shorter overall, and disease-free survival of patients. Overexpression of miR-612 significantly decreased cell proliferation, colony formation, and invasion of SK-MEL-28 and A375 melanoma cells. In vivo tumorigenic studies confirmed that miR-612 overexpression retarded the growth of A375 xenograft tumors, which was coupled with a decline in the percentage of Ki-67-positive proliferating cells. Mechanistically, miR-612 targeted Espin in melanoma cells. Overexpression of Espin counteracted the suppressive effects of miR-612 on melanoma cell proliferation, invasion, and tumorigenesis. A significant inverse correlation (r = −0.376, P = 0.018) was observed between miR-612 and Espin protein expression in melanoma tissues. In addition, overexpression of miR-612 and knockdown of Espin significantly increased the sensitivity of melanoma cells to doxorubicin. Collectively, miR-612 suppresses the aggressive phenotype of melanoma cells through downregulation of Espin. Delivery of miR-612 may represent a novel therapeutic strategy against melanoma.

The role of the ShcD and RET interaction in neuroblastoma survival and migration

Preliminary screening data showed that the ShcD adaptor protein associates with the proto-oncogene RET receptor tyrosine kinase. In the present study, we aimed to investigate the molecular interaction between ShcD and RET in human neuroblastoma cells and study the functional impact of this interaction. We were able to show that ShcD immunoprecipitated with RET from SK-N-AS neuroblastoma cell lysates upon GDNF treatment. This result was validated by ShcD-RET co-localization, which was visualized using a fluorescence microscope. ShcD-RET coexpression promoted ShcD and RET endosomal localization, resulting in unexpected inhibition of the downstream ERK and AKT pathways. Interestingly, ShcD-RET association reduced the viability and migration of SK-N-AS cells. Although ShcD was previously shown to trigger melanoma cell migration and tumorigenesis, our data showed an opposite role for ShcD in neuroblastoma SK-N-AS cells via its association with RET in GDNF-treated cells. In conclusion, ShcD acts as a switch molecule that promotes contrasting biological responses depending on the stimulus ad cell type.

LncRNA-MEG3 Suppresses the Proliferation and Invasion of Melanoma by Regulating CYLD Expression Mediated by Sponging miR-499-5p.

The abnormal expression of long noncoding RNA- (lncRNA-) MEG3 was clearly identified in a number of malignant tumors, but the specific function of MEG3 remains unknown in malignant melanoma until now. The research attempts to explore the effects of MEG3 on the growth and metastasis of malignant melanoma. MEG3 and miR-499-5p expression were determined by qRT-PCR method. Western blotting assay was applied to detect protein expression. Luciferase reporter assay was used to assess the correlation between MEG3 and miR-499-5p and between CYLD and miR-499-5p. Cell growth, cell cycle, and cell apoptosis were examined by CCK-8 assay, EdU assay, and flow cytometry assay, respectively. The invasion ability of melanoma cells was investigated by wound-healing and Transwell assays. The effect of MEG3 on growth of melanoma in vivo and cell chemosensitivity was detected by xenograft animal model and CCK-8 assay. As a result, the expression of MEG3 was decreased in melanoma tissues and cell lines. The level of MEG3 was significantly associated with poor prognosis. MEG3 could bind to miR-499-5p and CYLD mRNA contained a binding site of miR-499-5p. The expression of CYLD was reduced and the level of miR-499-5p was elevated in melanoma tissues and cell lines. Luciferase reporter assay and western blot assay confirmed that MEG3 regulated the expression of CYLD by sponging miR-499-5p. Functionally, upregulation of MEG3 inhibited melanoma cell proliferation, invasion, and migration, enhanced melanoma cell apoptosis, arrested melanoma cell cycle, and regulated the expression of E-cadherin, N-cadherin, and cyclin D1 by regulating CYLD expression mediated by sponging miR-499-5p. Importantly, overexpression of MEG3 suppressed the growth of xenograft tumor and improved chemotherapy sensitivity of A375 cells to cisplatin and 5-FU treatment. In conclusion, MEG3 has a crucial function in the tumorigenesis of melanoma, and MEG3 may be a potential therapeutic target in the treatment of melanoma.

The Dynamics of ERK Signaling in Melanoma, and the Response to BRAF or MEK Inhibition, Are Cell Cycle Dependent

Activating BRAF mutations are thought to drive melanoma tumorigenesis and metastasis by constitutively activating MEK and ERK. Small molecule inhibitors (SMIs) of BRAF or MEK have shown promise as melanoma therapeutics. However, the development of resistance to these inhibitors in both the short- and long-term is common, warranting investigation into how these SMIs influence ERK signaling dynamics. Quantitative single cell imaging of ERK activity in living cells reveals both intra- and inter- cell heterogeneity in this activity in isogenic melanoma populations harboring a BRAFV600E mutation. This heterogeneity is largely due to a cell-cycle dependent bifurcation of ERK activity. Moreover, we show there are also cell-cycle dependent responses in ERK activity following BRAF or MEK inhibition. Prior to, but not following, CDK4/6-mediated passage through the Restriction Point (RP) ERK activity is sensitive to BRAF and MEK inhibitors. In contrast, for cells that have passed the RP, ERK activity will remain elevated in the presence of BRAF or MEK inhibition until mitosis. Our results show that ERK activity - even in the presence of activating BRAF mutations - is regulated by both positive and negative feedback loops that are engaged in cell-cycle dependent fashions. CDK4/6 inhibition sensitizes ERK activity to BRAF or MEK inhibition by preventing passage the transition from a BRAF/MEK dependent to independent state. Our results have implications for the use of MEK and BRAF inhibitors as melanoma therapeutics, and offer a rational basis for the use of these inhibitors in combination with CDK4/6 inhibition during cancer therapy.

Abstract A180: The histone methyltransferase SETDB1 contributes to melanoma tumorigenesis

Abstract Histone modifications play a significant role in the development and progression of various cancers. The histone methyltransferase SETDB1 (SET domain, bifurcated 1) catalyzes the addition of methyl groups to histone H3 at lysine 9 (H3K9). In the present study, we demonstrate that SETDB1 contributes to melanoma tumorigenesis. We show SETDB1 to be highly amplified in melanoma tissues of patients with poor prognosis. The amplification of SETDB1 correlates with the increased expression of the protein. Functional studies show an increased SETDB1 expression to be associated with a more proliferative, invasive and migratory phenotype and give rise to larger tumor masses in xenograft mouse models. In human primary melanomas, SETDB1 expression is elevated at the invasive borders. The activity of SETDB1 can be blocked by small molecules, which leads to reduced cell growth even in melanoma cell lines that are resistant towards BRAF and MEK inhibitors. In conclusion, our findings support SETDB1 as a major driver of melanoma development and progression. We therefore suggest SETB1 as a potential future target for the treatment of melanoma. Citation Format: Elias Orouji, Aniello Federico, Lionel Larribere, Daniel Novak, Jochen Utikal. The histone methyltransferase SETDB1 contributes to melanoma tumorigenesis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A180.

Effect of long non-coding RNA PVT1 on cell proliferation and migration in melanoma.

The present study aimed to investigate the potential role of the long non-coding RNA (lncRNA) Pvt1 oncogene (non-protein coding) (PVT1) in the progression and metastasis of malignant melanoma, and to reveal its possible molecular mechanisms. The expression of lncRNA PVT1 in melanoma tissues and adjacent normal skin from patients with melanoma, and in the melanoma A-375 and sk-mel-5 cell lines, was analyzed using reverse transcription-quantitative polymerase chain reaction and western blot analyses. The effects of PVT1 expression on cell proliferation, the cell cycle, cell migration and cell invasion were analyzed using MTT assay, flow cytometry, Transwell and scratch assays, respectively. The interaction between PVT1 and enhancer of zeste homolog 2 (EZH2) in melanoma cells was analyzed using RNA immunoprecipitation (RIP) assay. The effect of PVT1 on microRNA-200c (miR-200c) expression was analyzed by chromatin immunoprecipitation (ChIP) assay. PVT1 was highly expressed in the melanoma tissues and cells. Silencing of PVT1 significantly inhibited cell proliferation, migration and invasion, and arrested the cell cycle at the G0/G1 stage. Additionally, PVT1 silencing significantly decreased the cyclin D1 expression in the melanoma cells. The expression of E-cadherin was significantly increased and the expression of N-cadherin and vimentin was significantly decreased in the PVT1-silenced group. The RIP assay found that endogenous PVT1 was highly enriched by EZH2 RIP compared with that of the negative control. The ChIP assay revealed that the expression of miR-200c was decreased significantly in the PVT1-silenced group compared with the controls. Overall, the present study demonstrated that the lncRNA PVT1 may contribute to the tumorigenesis and metastasis of melanoma through binding to EZH2 and regulating the expression of miR-200c. lncRNA PVT1 may serve as a potential target for the therapy of melanoma.

Combining molecular and immunohistochemical analyses of key drivers in primary melanomas: interplay between germline and somatic variations.

Due to the high mutational somatic burden of Cutaneous Malignant Melanoma (CMM) a thorough profiling of the driver mutations and their interplay is necessary to explain the timing of tumorigenesis or for the identification of actionable genetic events. The aim of this study was to establish the mutation rate of some of the key drivers in melanoma tumorigenesis combining molecular analyses and/or immunohistochemistry in 93 primary CMMs from an Italian cohort also characterized for germline status, and to investigate an interplay between germline and somatic variants. BRAF mutations were present in 68% of cases, while CDKN2A germline mutations were found in 16 % and p16 loss in tissue was found in 63%. TERT promoter somatic mutations were detected in 38% of cases while the TERT –245T>C polymorphism was found in 51% of cases. NRAS mutations were found in 39% of BRAF negative or undetermined cases. NF1 was expressed in all cases analysed. MC1R variations were both considered as a dichotomous variable or scored. While a positive, although not significant association between CDKN2A germline mutations, but not MC1R variants, and BRAF somatic mutation was found, we did not observe other associations between germline and somatic events. A yet undescribed inverse correlation between TERT –245T>C polymorphism and the presence of BRAF mutation was found. It is possible to hypothesize that –245T>C polymorphism could be included in those genotypes which may influence the occurrence of BRAF mutations. Further studies are needed to investigate the role of –245T>C polymorphism as a germline predictor of BRAF somatic mutation status.

Yes-Associated Protein (YAP) Promotes Tumorigenesis in Melanoma Cells Through Stimulation of Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1)

YAP is a critical protein in cancer development and can induce transformative phenotypes in mammary epithelial cells. Previous studies have provided evidence that YAP can contribute to the metastatic behavior of melanoma, since specific knockdown of YAP leads to reduced metastatic and invasive capacity in vitro. However, the mechanism by which YAP regulates the function of melanoma is unknown. Here, we identified that YAP has a positive impact on the expression of LRP1, which also plays critical roles in cancer. Mechanically, knockdown of YAP resulted in a significant down-regulation of LRP1 at both the protein and mRNA levels. Tissue microarray analysis (TMA) also showed a positive correlation between YAP and LRP1 expression. In addition, reduction of YAP-impaired pro-carcinogenic phenotypes could be partially reversed by simultaneous overexpression of LRP1, suggesting that LRP1 is functionally important in YAP-induced melanoma tumorigenesis. Furthermore, we found that LRP1 was regulated by YAP through a transcription- and promoter-dependent mechanism. Taken together, our results suggest that YAP regulates LRP1 through stimulation of the LRP1 promoter and that LRP1 may be an important target for influencing YAP in melanoma.

Metabolic strategies of melanoma cells: Mechanisms, interactions with the tumor microenvironment, and therapeutic implications.

Melanomas are metabolically heterogeneous, and they are able to adapt in order to utilize a variety of fuels that facilitate tumor progression and metastasis. The significance of metabolism in melanoma is supported by growing evidence of impact on the efficacy of contemporary therapies for this disease. There are also data to support that the metabolic phenotypes of melanoma cells depend upon contributions from both intrinsic oncogenic pathways and extrinsic factors in the tumor microenvironment. This review summarizes current understanding of the metabolic processes that promote cutaneous melanoma tumorigenesis and progression, the regulation of cancer cell metabolism by the tumor microenvironment, and the impact of metabolic pathways on targeted and immune therapies.

STAP-2 protein promotes prostate cancer growth by enhancing epidermal growth factor receptor stabilization

Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signaling pathways and promotes tumorigenesis in melanoma and breast cancer cells. However, the contribution of STAP-2 to the behavior of other types of cancer cells is unclear. Here, we show that STAP-2 promotes tumorigenesis of prostate cancer cells through up-regulation of EGF receptor (EGFR) signaling. Tumor growth of a prostate cancer cell line, DU145, was strongly decreased by STAP-2 knockdown. EGF-induced gene expression and phosphorylation of AKT, ERK, and STAT3 were significantly decreased in STAP-2-knockdown DU145 cells. Mechanistically, we found that STAP-2 interacted with EGFR and enhanced its stability by inhibiting c-CBL-mediated EGFR ubiquitination. Our results indicate that STAP-2 promotes prostate cancer progression via facilitating EGFR activation.

Lung regeneration after toxic injury is improved in absence of dioxin receptor

Recent experimental evidences from cellular systems and from mammalian and non-mammalian animal models highlight novel functions for the aryl hydrocarbon/dioxin receptor (AhR) in maintaining cell differentiation and tissue homeostasis. Notably, AhR depletion stimulates an undifferentiated and pluripotent phenotype likely associated to a mesenchymal transition in epithelial cells and to increased primary tumorigenesis and metastasis in melanoma. In this work, we have used a lung model of epithelial regeneration to investigate whether AhR regulates proper tissue repair by adjusting the expansion of undifferentiated stem-like cells. AhR-null mice developed a faster and more efficient repair of the lung bronchiolar epithelium upon naphthalene injury that required increased cell proliferation and the earlier activation of stem-like Clara, Basal and neuroepithelial cells precursors. Increased basal content in multipotent Sca1+/CD31−/CD4− cells and in cells expressing pluripotency factors NANOG and OCT4 could also improve re-epithelialization in AhR-null lungs. The reduced response of AhR-deficient lungs to Sonic Hedgehog (Shh) repression shortly after injury may also help their improved bronchiolar epithelium repair. These results support a role for AhR in the regenerative response against toxins, and open the possibility of modulating its activation level to favor recovery from lesions caused by environmental contaminants.

547 FOXD1 is overexpressed in melanoma but not in melanocytic nevi, and associated with melanoma cells proliferation

FOXD1 is a member of forkhead family transcription factors, which plays important roles in the development of the retina, optic chiasm and kidney. Recent studies have demonstrated that FOXD1 is overexpressed in non-small cell lung cancer and breast cancer, correlating with poor prognosis. Since the role of FOXD1 in malignant melanoma is not clarified yet, we carried out the immunohistochemical staining of FOXD1 and evaluated the correlation between the FOXD1 expression and clinicopahtological parameters. Total of 87 cases of melanoma including 24 in situ lesions were investigated. Stage, tumor thickness nor distant metastasis during follow-up period as well as clinical classification was correlated with the expression of FOXD1, unlike lung and breast cancer. On the other hand, we identified two specimens contained concomitant melanocytic nevi in the same section (one is acquired nevus and the other is congenital nevus), which FOXD1 was overexpressed in both primary and metastatic lesions compared to melanocytic nevi. So we carried out knockdown assay using human melanoma cell lines MNT-1 and HMY-1. RNA interference of FOXD1 suppressed the proliferation of both cell lines in alamar blue assay. These findings suggest that FOXD1 may be involved in malignant transformation of nevus cells and proliferation of melanoma cells. We speculate overexpression of FOXD1 seems to be an early event in melanoma tumorigenesis.

Silencing of metallothionein 1A gene in melanoma

BackgroundWhen a CpG island (CGI; a dense cluster of CpGs) located in the 5′ region of a gene is methylated, its transcription is suppressed. Tumorigenesis of melanoma is associated with trace elements. Metallothionein 1A is closely associated with the metabolism of trace elements. However, little is known about the metallothionein 1A gene (MT1A) in melanoma. ObjectiveThe purpose is to reveal the methylation and expression status of MT1A in melanoma. MethodsQuantitative real-time methylation-specific PCR (RT-MSP) and bisulfite sequencing were performed to examine MT1A methylation status. Quantitative real-time reverse transcription-PCR (RT-PCR) was performed to examine MT1A expression. ResultsSome melanoma cell lines exhibited high methylation levels of the CGI located in the 5′ region of MT1A (5′ MT1A CGI) with suppression of MT1A. Other melanoma cell lines and normal cultured melanocytes exhibited low methylation levels of 5′ MT1A CGI with expression of MT1A. Treatment with a demethylating agent resulted in transcriptional induction of MT1A in the melanoma cell lines SK-MEL-5 and G-361 with high methylation levels prior to treatment. The methylation levels of 5′ MT1A CGI ranged widely from 0.0% to 91.4% in 21 clinical melanoma samples but showed a narrow, low range from 0.0% to 6.4% in 23 clinical melanocytic nevus samples. Data of bisulfite sequencing was generally compatible with those of RT-MSP. The methylation levels ranged according to the types of melanoma (Kruskal-Wallis test, P=0.047). ConclusionMT1A is aberrantly silenced by DNA methylation of 5′ MT1A CGI in melanoma.

Abstract 1352: The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis

Abstract The Ras family of small GTP binding proteins are frequently activated by mutations in melanoma, as shown for NRAS (20%), KRAS (2%) and HRAS (1%). Ras isoforms can also be activated by inactivation of Ras GTPase activating proteins (RasGAPs), such as NF1, RASA1, and RASA2. In our recent study, we observed that inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform and that R-Ras was required to promote anchorage-independent growth driven by RASA1 inactivation. Moreover, a low level of RASA1 mRNA expression is associated with decreased overall survival in melanoma patients with BRAF mutations. Based on these observations, we hypothesized that, although not mutated, R-Ras is activated in melanoma by inactivation of RasGAPs and that BRAF activation cooperates with this RasGAP/R-Ras pathway activation in melanoma tumorigenesis. In this study, we addressed the importance of R-Ras, a previously less appreciated member of the Ras small GTPases family, in melanoma tumorigenesis. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines. In addition, RNAi-mediated reduced expression of R-Ras suppressed anchorage-independent colony growth and tumor growth. Moreover, among the 3 major RAS effector pathways, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by R-Ras activation downstream of RASA1 inactivation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacological (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a RASA1L/L; BRAFCA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in expression of constitutively activated mutant BRAF and deletion of RASA1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in RASA1L/L BRAFCA/CA mice compared to RASA1+/+ BRAFCA/CA littermates. Tumors generated in this animal model will be analyzed to determine the extent of R-Ras and Ral-A activity in vivo. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways. Citation Format: Kristen Suzanne Hill, Xue Wang, Youngchul Kim, Minjung Kim. The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis [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 1352. doi:10.1158/1538-7445.AM2017-1352

Myeloid-specific genetic ablation of ATP-binding cassette transporter ABCA1 is protective against cancer.

Increased circulating levels of apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), by genetic manipulation or infusion, protects against melanoma growth and metastasis. Herein, we explored potential roles in melanoma tumorigenesis for host scavenger receptor class B, type 1 (SR-B1), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), all mediators of apoA-I and HDL sterol and lipid transport function. In a syngeneic murine melanoma tumor model, B16F10, mice with global deletion of SR-B1 expression exhibited increased plasma HDL cholesterol (HDLc) levels and decreased tumor volume, indicating host SR-B1 does not directly contribute to HDL-associated anti-tumor activity. In mice with myeloid-specific loss of ABCA1 (Abca1−M/−M; A1−M/−M), tumor growth was inhibited by ∼4.8-fold relative to wild type (WT) animals. Abcg1−M/−M (G1−M/−M) animals were also protected by 2.5-fold relative to WT, with no further inhibition of tumor growth in Abca1/Abcg1 myeloid-specific double knockout animals (DKO). Analyses of tumor-infiltrating immune cells revealed a correlation between tumor protection and decreased presence of the immune suppressive myeloid-derived suppressor cell (MDSC) subsets, Ly-6G+Ly-6CLo and Ly-6GnegLy-6CHi cells. The growth of the syngeneic MB49 murine bladder cancer cells was also inhibited in A1−M/−M mice. Collectively, our studies provide further evidence for an immune modulatory role for cholesterol homeostasis pathways in cancer.