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.

Summary This study investigates the mechanism of action behind the long-term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. Although single agent MK-2206 inhibited phospho-AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin was cytotoxic in long-term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase-dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti-oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs. Significance Approximately 30% of all cutaneous melanomas are wild-type for both BRAF and NRAS. As yet, no targeted therapy strategies exist for this sub-set of tumors. Constitutive signaling through the PI3K/AKT pathway is a common occurrence in cutaneous melanoma, irrespective of the driver mutation. Here we report durable responses to the AKT inhibitor MK-2206 in combination with carboplatin and paclitaxel in two patients with BRAF wild-type melanoma. Through mechanistic studies, we demonstrate a role for autophagy induction in the response to the AKT inhibitor/chemotherapy combination and suggest that autophagy inhibitors may be one strategy to enhance efficacy in the clinical setting.

Evidence sustains a role for the acute-phase protein serum amyloid A (SAA) in carcinogenesis and metastasis, and the protein has been suggested as a marker for tumor progression. Nevertheless, the demonstration of a direct activity of SAA on tumor cells is still incipient. We have investigated the effect of human recombinant SAA (rSAA) on two human glioma cell lines, A172 and T98G. rSAA stimulated the [3H]-thymidine incorporation of both lines, but had dual effects on migration and invasiveness which varied according to the cell line. In T98G, the rSAA increased migration and invasion behaviors whereas in A172 it decreased these behaviors. These findings agree with the effect triggered by rSAA on matrix metalloproteinases (MMPs) activities measured in a gelatinolytic assay. rSAA inhibited activity of both MMPs in A172 cells while increasing them in T98G cells. rSAA also affected the production of compounds present in the tumor microenvironment that orchestrate tumor progression, such as IL-8, the production of reactive oxygen species (ROS) and nitric oxide (NO). We also observed that both lines expressed all three of the isoforms of SAA: SAA1, SAA2, and SAA4. These data suggest that some tumor cells are responsive to SAA and, in these cases, SAA may have a role in cancer progression that varies according to the cell type.

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Summary Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half‐life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4‐Nerolidylcatechol (4‐NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4‐NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl‐1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4‐NC is time‐dependent upon the pro‐apoptotic protein Noxa, which is able to bind and neutralize Mcl‐1. We demonstrate the role of 4‐NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4‐NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.

RECK is an anti-tumoral gene whose activity has been associated with its inhibitory effects regulating MMP-2, MMP-9, and MT1-MMP. RECK level decreases as gliobastoma progresses, varying from less invasive grade II gliomas to very invasive human glioblastoma multiforme (GBM). Since RECK expression and glioma invasiveness show an inverse correlation, the aim of the present study is to investigate whether RECK expression would inhibit glioma invasive behavior. We conducted this study to explore forced RECK expression in the highly invasive T98G human GBM cell line. Expression levels as well as protein levels of RECK, MMP-2, MMP-9, and MT1-MMP were assessed by qPCR and immunoblotting in T98G/RECK+ cells. The invasion and migration capacity of RECK+ cells was inhibited in transwell and wound assays. Dramatic cytoskeleton modifications were observed in the T98G/RECK+ cells, when compared to control cells, such as the abundance of stress fibers (contractile actin-myosin II bundles) and alteration of lamellipodia. T98G/RECK+ cells also displayed phosphorylated focal adhesion kinase (P-FAK) in mature focal adhesions associated with stress fibers; whereas P-FAK in control cells was mostly associated with immature focal complexes. Interestingly, the RECK protein was predominantly localized at the leading edge of migrating cells, associated with membrane ruffles. Unexpectedly, introduced expression of RECK effectively inhibited the invasive process through rearrangement of actin filaments, promoting a decrease in migratory ability. This work has associated RECK tumor-suppressing activity with the inhibition of motility and invasion in this GBM model, which are two glioma characteristics responsible for the inefficiency of current available treatments.

Despite the recent advances in the clinical management of melanoma, there remains a need for new pharmacological approaches to treat this cancer. 2-methoxyestradiol (2ME) is a metabolite of estrogen that has shown anti-tumor effects in many cancer types. In this study we show that 2ME treatment leads to growth inhibition in melanoma cells, an effect associated with entry into senescence, decreased pRb and CyclinB1 expression, increased p21/Cip1 expression and G2/M cell cycle arrest. 2ME treatment also inhibits melanoma cell growth in colony formation assays, including cell lines with acquired resistance to BRAF and BRAF+MEK inhibitors. We further show that 2ME is effective against melanoma with different BRAF and NRAS mutational status. Moreover, 2ME induced the retraction of cytoplasmic projections in a 3D spheroid model and significantly decreased cell proliferation in a 3D skin reconstruct model. Together our studies bring new insights into the mechanism of action of 2ME allowing melanoma targeted therapy to be further refined. Continued progress in this area is expected to lead to improved anti-cancer treatments and the development of new and more effective clinical analogues.

Among skin cancers, melanoma has the highest mortality rate. The heterogeneous genetic melanoma background leads to a tumor-propagating capacity particularly important in maintaining therapeutic resistance, and tumor recurrence. The identification of efficient molecules able to control melanoma progress represents an important opportunity for new therapeutic strategies, particularly in combination with the current standard-of-care treatments. In this context, several studies have reported the antitumor effects of melatonin against different types of cancer, including melanoma. Here, we describe the underlying mechanisms associated with melatonin’s activity in human melanoma cell lines, focusing on cell cycle and cytoskeleton remodeling. Interestingly, while melatonin induced melanocyte DNA replication, melanoma cells exhibited cell cycle arrest in the G1-phase. This phenomenon was associated with cyclin-D1 downregulation or p21 overexpression. The efficacy of melatonin on melanoma cells survival and proliferation was detected using the clonogenic assay, with a decrease in both the number and size of colonies. Additionally, melatonin induced a dramatic cytoskeleton remodeling in all melanoma cell lines, leading to a star-like morphology or cell swelling. The role of melatonin on melanoma cytoskeleton was associated with the actin disruption, with thinning and/or broken actin fibers, and weak and/or loss of paxillin along stress fibers. These data support the observed findings that melatonin impairs melanoma invasion in skin reconstructed models. Together, our results suggest that melatonin could be used to control melanoma growth and support basic and clinical studies on melatonin as a promising immunometabolic adjuvant for melanoma therapy.