Luigi Fattore

Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF/MEK inhibition and is abrogated by anti-ErbB3 antibodies

BackgroundTreatment of advanced melanoma has been improved with the advent of the BRAF inhibitors. However, a limitation to such treatment is the occurrence of resistance. Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent. In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results. However, also in this case resistance inevitably occurs. It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance.MethodsThree melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D). Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array. Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies. The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting. The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays.ResultsIn the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT. Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth. We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells.ConclusionsOn the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response.

Monoclonal antibody-induced ErbB3 receptor internalization and degradation inhibits growth and migration of human melanoma cells

Members of the ErbB receptor family are targets of a growing numbers of small molecules and monoclonal antibodies inhibitors currently under development for the treatment of cancer. Although historical efforts have been directed against ErbB1 (EGFR) and ErbB2 (HER2/neu), emerging evidences have pointed to ErbB3 as a key node in the activation of proliferation/survival pathways from the ErbB receptor family and have fueled enthusiasm toward the clinical development of anti-ErbB3 agents. In this study, we have evaluated the potential therapeutic efficacy of a set of three recently generated anti-human ErbB3 monoclonals, A2, A3 and A4, in human primary melanoma cells. We show that in melanoma cells expressing ErbB1, ErbB3 and ErbB4 but not ErbB2 receptor ligands activate the PI3K/AKT pathway, and this leads to increased cell proliferation and migration. While antibodies A3 and A4 are able to potently inhibit ligand-induced signaling, proliferation and migration, antibody A2 is unable to exert this effect. In attempt to understand the mechanism of action and the basis of this different behavior, we demonstrate, through a series of combined approaches, that antibody efficacy strongly correlates with antibody-induced receptor internalization, degradation and inhibition of receptor recycling to the cell surface. Finally, fine epitope mapping studies through a peptide array show that inhibiting vs. non-inhibiting antibodies have a dramatically different mode of binding to the to the receptor extracellular domain. Our study confirms the key role of ErbB3 and points to exploitation of novel combination therapies for treatment of malignant melanoma.

miR-579-3p controls melanoma progression and resistance to target therapy

Significance In this paper we identify a previously poorly characterized miRNA, namely miR-579-3p, as a master regulator of melanoma progression and drug resistance. Our results underscore the complexity of adaptive mechanisms that help the establishment of resistance to target therapies and the necessity to identify them to develop more effective combination therapies. Therapy of melanoma patients harboring activating mutations in the BRAF (V-raf murine sarcoma viral oncogene homolog B1) oncogene with a combination of BRAF and MEK inhibitors is plagued by the development of drug resistance. Mutational events, as well as adaptive mechanisms, contribute to the development of drug resistance. In this context we uncover here the role of a miRNA, miR-579-3p. We first show that low expression of miR-579-3p is a negative prognostic factor correlating with poor survival. Expression levels of miR-579-3p decrease from nevi to stage III/IV melanoma samples and even further in cell lines resistant to BRAF/MEK inhibitors. Mechanistically, we demonstrate that miR-579-3p acts as an oncosuppressor by targeting the 3′UTR of two oncoproteins: BRAF and an E3 ubiquitin protein ligase, MDM2. Moreover miR-579-3p ectopic expression impairs the establishment of drug resistance in human melanoma cells. Finally, miR-579-3p is strongly down-regulated in matched tumor samples from patients before and after the development of resistance to targeted therapies.

MicroRNAs in melanoma development and resistance to target therapy

microRNAs constitute a complex class of pleiotropic post-transcriptional regulators of gene expression involved in the control of several physiologic and pathologic processes. Their mechanism of action is primarily based on the imperfect matching of a seed region located at the 5′ end of a 21-23 nt sequence with a partially complementary sequence located in the 3′ untranslated region of target mRNAs. This leads to inhibition of mRNA translation and eventually to its degradation. Individual miRNAs are capable of binding to several mRNAs and several miRNAs are capable of influencing the function of the same mRNAs. In recent years networks of miRNAs are emerging as capable of controlling key signaling pathways responsible for the growth and propagation of cancer cells. Furthermore several examples have been provided which highlight the involvement of miRNAs in the development of resistance to targeted drug therapies. In this review we provide an updated overview of the role of miRNAs in the development of melanoma and the identification of the main downstream pathways controlled by these miRNAs. Furthermore we discuss a group of miRNAs capable to influence through their respective up- or down-modulation the development of resistance to BRAF and MEK inhibitors.

TrkB is responsible for EMT transition in malignant pleural effusions derived cultures from adenocarcinoma of the lung

Lung cancer is the leading cause of cancer-related mortality worldwide. Recent evidence indicates that tumors contain a subpopulation of cancer stem cells (CSCs) that are responsible for tumor maintenance and spread. CSCs have recently been linked to the occurrence of epithelial-to-mesenchymal transition (EMT). Neurotrophins (NTs) are growth factors that regulate the biology of embryonic stem cells and cancer cells, but still little is known about the role NTs in the progression of lung cancer. In this work, we investigated the role of the NTs and their receptors using as a study system primary cell cultures derived from malignant pleural effusions (MPEs) of patients with adenocarcinoma of the lung. We assessed the expression of NTs and their receptors in MPE-derived adherent cultures vs. spheroids enriched in CSC markers. We observed in spheroids a selectively enhanced expression of TrkB, both at the mRNA and protein levels. Both K252a, a known inhibitor of Trk activity, and a siRNA against TrkB strongly affected spheroid morphology, induced anoikis and decreased spheroid forming efficiency. Treatment with neurotrophins reversed the inhibitory effect of K252a. Importantly, TrkB inhibition caused loss of vimentin expression as well as that of a set of transcription factors known to be linked to EMT. These ex vivo results nicely correlated with an inverse relationship between TrkB and E-cadherin expression measured by immunohistochemistry in a panel of lung adenocarcinoma samples. We conclude that TrkB is involved in full acquisition of EMT in lung cancer, and that its inhibition results in a less aggressive phenotype.

Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in melanoma

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma.

ErbB3 plays a key role in the early phase of establishment of resistance to BRAF and/or MEK inhibitors.

Background A major issue in the management of cancer is the development of drug resistance. In metastatic melanoma bearing V600 mutations in the BRAF oncogene, all patients undergo disease relapse after combination therapy with BRAF and MEK inhibitors. Hence, understanding the mechanisms at the basis of development of resistance is fundamental to the discovery of new therapeutic approaches. In our group we have spent the last years to identify mechanisms of early adaptation of BRAF mutated melanoma to BRAF and or MEK inhibitors. We have recently shown that the ErbB3 receptor is involved in the activation of an early feedback survival loop upon cell exposure to BRAF and/or MEK inhibitors. Upregulation of pErbB3, due to enhanced production of its ligand neuregulin-1 (HRG), causes increased AKT phosphorylation and cell survival. Furthermore, we demonstrated that activation of the ErbB3/AKT axis is abrogated by cotreatment with anti-ErbB3 mAbs previously generated in our laboratory.

Selective targeting of point-mutated KRAS through artificial microRNAs

Significance Recently, small interfering RNAs have been used to specifically target point-mutated KRAS, yet without sufficiently discriminating its wild-type counterpart. Here, we describe an innovative approach based on the development of artificial microRNAs able to efficiently target mutated KRAS, leaving their normal counterpart unaffected and preventing major side effects. Mutated protein-coding genes drive the molecular pathogenesis of many diseases, including cancer. Specifically, mutated KRAS is a documented driver for malignant transformation, occurring early during the pathogenesis of cancers such as lung and pancreatic adenocarcinomas. Therapeutically, the indiscriminate targeting of wild-type and point-mutated transcripts represents an important limitation. Here, we leveraged on the design of miRNA-like artificial molecules (amiRNAs) to specifically target point-mutated genes, such as KRAS, without affecting their wild-type counterparts. Compared with an siRNA-like approach, the requirement of perfect complementarity of the microRNA seed region to a given target sequence in the microRNA/target model has proven to be a more efficient strategy, accomplishing the selective targeting of point-mutated KRAS in vitro and in vivo.

MicroRNA-driven deregulation of cytokine expression helps development of drug resistance in metastatic melanoma

microRNAs are major components of the eukaryotic post-transcriptional machinery and are frequently deregulated during cancer development. Increasing evidence points to them also as key players in the establishment of drug resistance. In this review, we provide an updated overview of the role of miRNAs in melanoma development and drug resistance and postulate that they are able to drive these processes in concert with deregulation of inflammatory and angiogenic cytokine expression. Notably, we have identified by querying the Cancer Genome Atlas database, a defined set of miRNAs which mostly have an impact on the development of melanoma and have recognized the main downstream pathways controlled by them. Most importantly, these miRNAs, which are down-regulated in metastatic melanomas as compared to primary tumors, are also able to predict prognosis of BRAF-mutated melanoma patients. Finally, we discuss the possibility that a common miRNA signature characterizes not only acquired resistance to MAPKi but also innate resistance to anti-PD-1 immunotherapy, since these conditions are both associated with alterations of the same pro-angiogenetic and pro-inflammatory pathways.

Activation of the ErbB3-AKT axis promotes melanoma cell survival and proliferation in response to RAF/MEK inhibition

Background Therapy of advanced melanoma has been improved with the advent of BRAF inhibitors. However, a limitation to such treatment is the occurrence of resistance. Several mechanisms have been implied in the development of resistance, which in most cases lead to downstream MEK reactivation. In order to overcome resistance MEK inhibitors are being clinically developed with promising results. However, also in this case resistance inevitably occurs. It is commonly believed that the establishment of resistance is facilitated by adaptive changes that take place in cancer cells shortly after exposure to kinase inhibitors. Our laboratory is interested in the identification of these early adaptive changes with the intent to discover additional targets for therapeutic intervention. Methods