miR-138-5p induces aggressive traits by targeting Trp53 expression in murine melanoma cells, and correlates with poor prognosis of melanoma patients.

Deregulation of miRNAs contributes to the development of distinct cancer types, including melanoma, an aggressive form of skin cancer characterized by high metastatic potential and poor prognosis. The expression of a set of 580 miRNAs was investigated in a model of murine melanoma progression, comprising non-metastatic (4C11-) and metastatic melanoma (4C11+) cells. A significant increase in miR-138-5p expression was found in the metastatic 4C11+ melanoma cells compared to 4C11-, which prompted us to investigate its role in melanoma aggressiveness. Functional assays, including anoikis resistance, colony formation, collective migration, serum-deprived growth capacity, as well as in vivo tumor growth and experimental metastasis were performed in 4C11- cells stably overexpressing miR-138-5p. miR-138-5p induced an aggressive phenotype in mouse melanoma cell lines leading to increased proliferation, migration and cell viability under stress conditions. Moreover, by overexpressing miR-138-5p, low-growing and non-metastatic 4C11- cells became highly proliferative and metastatic in vivo, similar to the metastatic 4C11+ cells. Luciferase reporter analysis identified the tumor suppressor Trp53 as a direct target of miR-138-5p. Using data sets from independent melanoma cohorts, miR-138-5p and P53 expression were also found deregulated in human melanoma samples, with their levels negatively and positively correlated with prognosis, respectively. Our data shows that the overexpression of miR-138-5p contributes to melanoma metastasis through the direct suppression of Trp53.

In vivo antitumoral effect of 4-nerolidylcatechol (4-NC) in NRAS-mutant human melanoma.

NRAS-mutations arise in 15-20% of all melanomas and are associated with aggressive disease and poor prognosis. Besides, the treatment for NRAS-mutant melanoma are not very efficient and is currently limited to immune checkpoints inhibitors or aggressive chemotherapy. 4-nerolidylcathecol (4-NC), a natural product extracted from Pothomorphe umbellata, induces apoptosis in melanoma cells by ROS production, DNA damage and increased p53 expression, in addition to inhibiting invasion in reconstructed skin. Moreover, 4-NC showed cytotoxicity in BRAF/MEKi-resistant and naive melanoma cells by Endoplasmic Reticulum (ER) stress induction in vitro. We evaluated the in vivo efficacy and the systemic toxicity of 4-NC in a NRAS-mutant melanoma model. 4-NC was able to significantly suppress tumor growth 4-fold compared to controls. Cleaved PARP and p53 expression were increased indicating cell death. As a proof of concept, MMP-2 and MMP-14 gene expression were decreased, demonstrating a possible role of 4-NC in melanoma invasion inhibition. Toxicological analysis indicated minor changes in the liver and bone marrow, but this toxicity was very mild when compared to other proteasome inhibitors and ER stress inductors already described. Our data indicate that 4-NC can counteract melanoma growth in vivo with minor adverse effects, suggesting further investigation as a potential NRAS-mutant melanoma treatment.

The Role of SIRT1 on DNA Damage Response and Epigenetic Alterations in Cancer.

Sirtuin-1 (SIRT1) is a class-III histone deacetylase (HDAC), an NAD+-dependent enzyme deeply involved in gene regulation, genome stability maintenance, apoptosis, autophagy, senescence, proliferation, aging, and tumorigenesis. It also has a key role in the epigenetic regulation of tissue homeostasis and many diseases by deacetylating both histone and non-histone targets. Different studies have shown ambiguous implications of SIRT1 as both a tumor suppressor and tumor promoter. However, this contradictory role seems to be determined by the cell type and SIRT1 localization. SIRT1 upregulation has already been demonstrated in some cancer cells, such as acute myeloid leukemia (AML) and primary colon, prostate, melanoma, and non-melanoma skin cancers, while SIRT1 downregulation was described in breast cancer and hepatic cell carcinomas. Even though new functions of SIRT1 have been characterized, the underlying mechanisms that define its precise role on DNA damage and repair and their contribution to cancer development remains underexplored. Here, we discuss the recent findings on the interplay among SIRT1, oxidative stress, and DNA repair machinery and its impact on normal and cancer cells.

ER stress promotes antitumor effects in BRAFi/MEKi resistant human melanoma induced by natural compound 4-nerolidylcathecol (4-NC).

Melanoma accounts for only 4% of malignant neoplasms of the skin, but is considered the most serious because it is highly deadly. Mutations in the MAPK (Ras-Raf-MEK-ERK) pathway is closely linked to the lack of control of cell proliferation. Especially in melanoma, this pathway has become a target for the development of oncogene-targeted therapies, such as the potent inhibitors of v-Raf murine sarcoma viral oncogene homolog B (BRAFi) and mitogen-activated protein kinase kinase (MEKi). Very high rates of response have been achieved, but most patients are relapsed due to the development of resistance, justifying the constant search for new therapeutic compounds. Early results from our group indicated that 4-nerolidylcatechol (4-NC), a catechol compound extracted from Pothomorphe umbellata, induces DNA damage, ROS production, increased p53 expression culminating in apoptosis in melanoma but with no data regarding the 4-NC effects in cells resistant to BRAFi or MEKi. Therefore, here we evaluated the role of 4-NC alone or in combination with BRAFi/MEKi in resistant melanoma cells. Double-resistant cells were generated and characterized by MAPK pathway reactivation. 4-NC alone or in combination (30 µM) with MAPK inhibitors was cytotoxic, inhibited colony formation and decreased invasiveness in two and three-dimensional cell culture models of treatment-naïve, BRAFi-resistant and BRAF/MEKi double-resistant melanoma cells. Apoptosis induction was demonstrated in resistant and double-resistant melanoma cell lines after 4-NC treatments. 4-NC showed important ability to induce apoptosis via Endoplasmatic Reticulum (ER) stress and specifically BiP and CHOP that had increased protein expression in all melanoma cell lines proving to be part of the ER stress pathway activation. CHOP knockdown slightly but enough increases cellular viability following 4-NC treatment indicating that apoptosis observed is partially dependent on CHOP. In summary, we show that 4-NC is a compound with activity against cutaneous melanoma, including resistant cells to clinically approved therapies.

Toxicogenomic and bioinformatics platforms to identify key molecular mechanisms of a curcumin-analogue DM-1 toxicity in melanoma cells.

Melanoma is a highly invasive and metastatic cancer with high mortality rates and chemoresistance. Around 50% of melanomas are driven by activating mutations in BRAF that has led to the development of potent anti-BRAF inhibitors. However resistance to anti-BRAF therapy usually develops within a few months and consequently there is a need to identify alternative therapies that will bypass BRAF inhibitor resistance. The curcumin analogue DM-1 (sodium 4-[5-(4-hydroxy-3-methoxy-phenyl)-3-oxo-penta-1,4-dienyl]-2-methoxy-phenolate) has substantial anti-tumor activity in melanoma, but its mechanism of action remains unclear. Here we use a synthetic lethal genetic screen in Saccharomyces cerevisiae to identify 211 genes implicated in sensitivity to DM-1 toxicity. From these 211 genes, 74 had close human orthologues implicated in oxidative phosphorylation, insulin signaling and iron and RNA metabolism. Further analysis identified 7 target genes (ADK, ATP6V0B, PEMT, TOP1, ZFP36, ZFP36L1, ZFP36L2) with differential expression during melanoma progression implicated in regulation of tumor progression, cell differentiation, and epithelial-mesenchymal transition. Of these TOP1 and ADK were regulated by DM-1 in treatment-naïve and vemurafenib-resistant melanoma cells respectively. These data reveal that the anticancer effect of curcumin analogues is likely to be mediated via multiple targets and identify several genes that represent candidates for combinatorial targeting in melanoma.

Vemurafenib resistance increases melanoma invasiveness and modulates the tumor microenvironment by MMP-2 upregulation.

The BRAF(V600E) mutation confers constitutive kinase activity and accounts for >90% of BRAF mutations in melanoma. This genetic alteration is a current therapeutic target; however, the antitumorigenic effects of the BRAF(V600E) inhibitor vemurafenib are short-lived and the majority of patients present tumor relapse in a short period after treatment. Characterization of vemurafenib resistance has been essential to the efficacy of next generation therapeutic strategies. Herein, we found that acute BRAF inhibition induced a decrease in active MMP-2, MT1-MMP and MMP-9, but did not modulate the metalloproteinase inhibitors TIMP-2 or RECK in naïve melanoma cells. In vemurafenib-resistant melanoma cells, we observed a lower growth rate and an increase in EGFR phosphorylation followed by the recovery of active MMP-2 expression, a mediator of cancer metastasis. Furthermore, we found a different profile of MMP inhibitor expression, characterized by TIMP-2 downregulation and RECK upregulation. In a 3D spheroid model, the invasion index of vemurafenib-resistant melanoma cells was more evident than in its non-resistant counterpart. We confirmed this pattern in a matrigel invasion assay and demonstrated that use of a matrix metalloproteinase inhibitor reduced the invasion of vemurafenib resistant melanoma cells but not drug naïve cells. Moreover, we did not observe a delimited group of cells invading the dermis in vemurafenib-resistant melanoma cells present in a reconstructed skin model. The same MMP-2 and RECK upregulation profile was found in this 3D skin model containing vemurafenib-resistant melanoma cells. Acute vemurafenib treatment induces the disorganization of collagen fibers and consequently, extracellular matrix remodeling, with this pattern observed even after the acquisition of resistance. Altogether, our data suggest that resistance to vemurafenib induces significant changes in the tumor microenvironment mainly by MMP-2 upregulation, with a corresponding increase in cell invasiveness.