ErbB3 Phosphorylation as Central Event in Adaptive Resistance to Targeted Therapy in Metastatic Melanoma. Early Detection in CTCs during Therapy and Insights into Regulation by Autocrine Neuregulin

In recent years the introduction of target therapies with BRAF and MEK inhibitors (MAPKi) and of immunotherapy with anti-CTLA-4 and anti-PD-1 monoclonal antibodies have dramatically improved survival of metastatic melanoma patients. Despite these changes drug resistance remains a major hurdle. Several mechanisms are at the basis of drug resistance. Particular attention has been devoted over the last years to unravel mechanisms at the basis of adaptive/non genetic resistance occurring in BRAF mutated melanomas upon treatment with to MAPKi. In this paper we focus on the involvement of activation of ErbB3 receptor following early exposure of melanoma cells to BRAF or MEK inhibitors, and the following induction of PI3K/AKT pathway. Although different mechanisms have been invoked in the past at the basis of this activation we show here with a combination of approaches that autocrine production of neuregulin by melanoma cells is a major factor responsible for ErbB3 phosphorylation and downstream AKT activation. Interestingly the kinetic of neuregulin production and of the ensuing ErbB3 phosphorylation is different in different melanoma cell lines which underscores the high degree of tumor heterogeneity. Moreover, heterogeneity is further highlighted by the evidence that in different cell lines neuregulin upregulation can occur at the transcriptional or at the post-transcritpional level. Finally we complement our study by showing with a liquid biopsy assay that circulating tumor cells (CTCs) from melanoma patients undergo upregulation of ErbB3 phosphorylation in vivo shortly after initiation of therapy

Abstract LB-9: Intersection of Notch and ERBB signalling in melanoma

Abstract Malignant melanoma is an aggressive malignancy of the melanocytes. The incidence rates for melanoma are steadily increasing and melanoma has now become the most common form of cancer among young adults between the ages of 25 and 29 years old. Because melanomas are highly resistant to the majority of therapies, the survival rate for patients with metastatic disease is less than 15%. There is a clear need for novel more effective therapies. Identification of the signaling pathways that are altered in melanoma provides opportunities for the development of novel targeted therapies. The Notch pathway is an evolutionary conserved signaling cascade that has an essential role in embryonic development but is inappropriately active in various types of cancers. Studies from our group and others have shown that hyper activation of Notch1 is an early event in melanocyte transformation and modulates both growth and metastasis in melanoma. We have found a new interaction between Notch1 and neuregulin1 (NRG1) signaling that plays a role in melanoma. Neuregulin1 is the ligand for ERBB3, a member of the Epidermal Growth Factor family of receptors that are involved in the genesis and progression of a number of cancers. Notch1 binds to the NRG1 promoter thereby modulating NRG1 expression and consequently the activation of the ERBB3 pathway. Inhibition of either NRG1 or ERBB3 activity in melanoma cells lead to cell growth inhibition and tumor growth delay similar to Notch1 inhibition. Mechanistically, these effects are dependent on the accumulation of p27 following either NRG1 or Notch1 down regulation. In addition, we find that NRG1/ERBB3 signaling influences Notch1 activation likely through the modulation of the Notch ligands Jaggged-1 (JAG-1) and Delta like 3 (Dll3), thus providing a feed forward regulatory loop linking the two pathways. Taken together, our findings underline a new, previously undescribed interaction between Notch1 and NRG1/ERBB3 in melanoma. Our goal is to investigate the mechanisms by which these two oncogenic signaling pathways intersect to promote a highly aggressive disease phenotype and to provide experimental evidence that the targeting of Notch and ERBB signaling is a tractable and effective approach to treat forms of melanoma that may be resistant to current available therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-9. doi:10.1158/1538-7445.AM2011-LB-

The membrane tethered matrix metalloproteinase MT1-MMP at the forefront of melanoma cell invasion and metastasis

The Extracellular Matrix (ECM) plays an important role in normal physiological development and functioning of cells, tissues and organs [1]. Under normal physiological conditions degradation of the ECM is a finely regulated process, and altered homeostasis of ECM degradation (excessive or insufficient) is associated with many diseases [2-5] such as cancer, fibrosis, arthritis, nephritis, encephalomyelitis and chronic ulcers. The remodeling of the ECM is carried out by a family of enzymes known as matrix metalloproteinases (MMP). MMPs constitute a large group of multidomain, zinc dependent endopeptidases capable of hydrolyzing all protein components of the ECM [6]. Additional functions of MMPs have also been identified. MMPs, and in particular MT1-MMP, the prototypic membrane-tethered matrix metalloproteinase, are no longer only ECM remodeling enzymes but rather regulators of several cellular functions including growth, migration, invasion and gene expression. Here we will focus on the role of the membrane bound MT1-MMP in melanoma growth, invasion and metastasis. MT1-MMP has in fact emerged as a multifaceted protease capable of influencing melanoma metastasis by canonical means, i.e. ECM degradation, but also via regulation of genes involved in several pro-tumorigenic functions including tumor cell growth and motility

Abstract B27: MT1-MMP targeting in melanoma brain metastasis

Abstract Background: Melanoma brain metastases contribute to morbidity and mortality of melanoma, and the treatment is limited due to its accessibility for drugs and also for surgical approaches. Radiotherapy is a widely used approach for disseminated metastatic foci in the brain. However, there is a gap in therapeutics since there are few to no alternatives to melanoma brain metastasis after the radiotherapy dose has been maximized. MT1-MMP is a metalloproteinase that has shown a direct correlation with metastasis and whose inhibition slows down the occurrence of metastasis and tumor growth. By microarray analysis, it has been found that inhibition of MT1-MMP downregulates the expression of DNA-damage response genes. Objective/Hypothesis: We hypothesize that the inhibition of MT1-MMP in combination with radiotherapy will enhance the tumor cell killing efficacy by means of preventing cells from responding to the DNA damage that is caused by radiation. Further, we hypothesize that the Integrin beta 1 pathway downstream of MT1-MMP is the mediator in the downregulation of DNA-damage response genes. Methods: We utilized melanoma cell lines, A375, WM-266-4-Luciferase, and K457, in which we introduced shMT1-MMP lentivirus with control shGFP. Cell culture assays compared the response of shMT1-MMP cells and shGFP cells in the presence of radiotherapy and the assays included comet assay, foci of gammaH2AX staining, clonogenic assay, and evaluation of radiotherapy response by Western blot. Furthermore, a nude mouse model was utilized in which WM-266-4-luciferase shMT1-MMP and control shGFP cells were inoculated by stereotactic injection. In vivo imaging system (IVIS) was utilized to follow up the tumors. Results: Ongoing experiments aim to look at the response of shMT1-MMP cells to radiotherapy compared to shGFP cells by DNA damage evaluation assays (Comet assay, gammaH2AX foci, DNA-damage response proteins by Western blot), downstream signaling of Integrin beta 1 pathway, and the survival of nude mice in shMT1-MMP inoculated tumors and their response to radiotherapy. Impact: This project has the potential to advance the current treatment of melanoma brain metastasis and also has the potential of advancing the understanding of the DNA-damage response in melanoma. Citation Format: Julia Escandon, Varsha Thakur, Barbara Bedogni. MT1-MMP targeting in melanoma brain metastasis [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B27

Wound Healing Assay for Melanoma Cell Migration

Cell migration is a critical process involved in morphogenesis, inflammation, and cancer metastasis. Wound healing assay is a simple, non-expensive, and highly reproducible method to study cancer cell migration in vitro. It is based on the observation that cells growing in a monolayer migrate to re-establish cell contacts after the development of an artificial wound. The assay involves creation of a wound in a monolayer, image acquisition during wound closure, and comparison of migrated area at initial and final time points

An ERBB3/ERBB2 oncogenic unit plays a key role in NRG1 signaling and melanoma cell growth and survival

Summary We recently identified neuregulin‐1 (NRG1) as a novel target of Notch1 required in Notch‐dependent melanoma growth. ERBB3 and ERBB4, tyrosine kinase receptors specifically activated by NRG1, have been shown to be either elevated in melanoma cell lines and tumors or to be mutated in 20% of melanomas, respectively. While these data support key roles of NRG1 and its receptors in the pathogenesis of melanoma, whether ERBB3 and ERBB4 display redundant or exclusive functions is not known. Here, we show that ERBB3 and ERBB4 inhibition results in distinct outcomes. ERBB3 inhibition ablates the cellular responses to NRG1, results in AKT inactivation and leads to cell growth arrest and apoptotic cell death. In contrast, ERBB4 knockdown mildly affects cell growth, has no effects on cell survival and, importantly, does not alter the responses to NRG1. Finally, we identified ERBB2 as a key coreceptor in NRG1‐dependent ERBB3 signaling. ERBB2 forms a complex with ERBB3, and its inhibition recapitulates the phenotypes observed upon ERBB3 ablation. We propose that an NRG1‐ERBB3‐ERBB2 signaling unit operates in melanoma cells where it promotes growth and survival

Abstract B07: Targeting an MT1-MMP/MMP2 axis in melanoma by a novel MT1-MMP/MMP2 inhibitor

Abstract Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. Matrix metalloproteases (MMPs) are among the proteolytic enzymes responsible for the degradation/ digestion of the extracellular matrix (ECM) and have been associated with the aggressiveness of a variety of cancers including melanoma (1,2). MT1-MMP is a membrane associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion-promoting, pro-tumorigenic MMP in cancer. We have recently demonstrated that MT1-MMP plays a key role in melanoma metastasis (3). MT1-MMP expression increases with disease progression and is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1-MMP in melanoma pathogenesis. Indeed, we showed that MT1-MMP is required for melanoma cells to metastasize, as cells deprived of MT1-MMP fail to form distant metastasis in an orthotopic mouse melanoma model. Mechanistically, we demonstrated for the first time that MT1-MMP affects cell invasion and motility by activating an MMP2-RAC1 signaling axis in melanoma cells (3). These findings imply MT1-MMP/MMP2 represent good molecular targets to thwart melanoma metastasis. However, the targeting of MMPs has not met with very successful stories mostly because the use of broad-spectrum inhibitors have been accompanied by severe side effects, such as musculoskeletal pain and inflammation (4,5) due to the targeting of both the “good” and the “bad” MMPs (6-8). Here we present new data in support of a novel, specific MT1-MMP/MMP2 inhibitor. ND-322 is a slow binding selective inhibitor of MT1-MMP and MMP2 (9). Data in our laboratory indicate ND-322 inhibits cell growth, migration and invasion of several melanoma cell lines similarly to the knock down of MT1-MMP and/or MMP2; it decreases RAC1 activity; and importantly, significantly reduces in vivo tumor growth and metastasis in an orthotopic mouse melanoma model. Preliminary data in vitro also show ND-322 in combination with vemurafenib decreases melanoma cell viability in a synergistic manner. Given these results and considering that this compound is very well tolerated in vivo (10), we believe ND-322 represent a promising novel, safe addition to the current standard of care for melanoma patients with advanced disease. Literature Cited 1 Bartolome, R.A., Ferreiro, S., Miquilena-Colina, M.E., Martinez-Prats, L., Soto- Montenegro, M.L., Garcia-Bernal, D., Vaquero, J.J., Agami, R., Delgado, R., Desco, M., Sanchez-Mateos, P., & Teixido, J. Am J Pathol 174 (2), 602-612 (2009). 2 Moro, N., Mauch, C., & Zigrino, P. Eur J Cell Biol 93 (1-2), 23-29 (2014). 3 Shaverdashvili k., W.P., Ma J., Zhang K., Osman I., Bedogni B. Pigment Cell and melanoma Res In Press (2013). 4 Skiles, J.W., Gonnella, N.C., & Jeng, A.Y. Curr Med Chem 11 (22), 2911-2977 (2004). 5 Drummond, A.H., Beckett, P., Brown, P.D., Bone, E.A., Davidson, A.H., Galloway, W.A., Gearing, A.J., Huxley, P., Laber, D., McCourt, M., Whittaker, M., Wood, L.M., & Wright, A. Ann N Y Acad Sci 878, 228-235 (1999). 6 Zucker, S. & Cao, J. Cancer Biol Ther 8 (24), 2371-2373 (2009). 7 Hua, H., Li, M., Luo, T., Yin, Y., & Jiang, Y. Cell Mol Life Sci 68 (23), 3853-3868 (2011). 8 Dufour, A. & Overall, C.M. Trends Pharmacol Sci 34 (4), 233-242 (2013). 9 Gooyit, M., Lee, M., Schroeder, V.A., Ikejiri, M., Suckow, M.A., Mobashery, S., & Chang, M. J Med Chem 54 (19), 6676-6690 (2011). 10 Cui, J., Chen, S., Zhang, C., Meng, F., Wu, W., Hu, R., Hadass, O., Lehmidi, T., Blair, G.J., Lee, M., Chang, M., Mobashery, S., Sun, G.Y., & Gu, Z. Mol Neurodegener 7, 21 (2012). Citation Format: Khvaramze Shaverdashvili, Poki Wong, Jun Ma, Keman Zhang, Iman Osman, Barbara Bedogni. Targeting an MT1-MMP/MMP2 axis in melanoma by a novel MT1-MMP/MMP2 inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B07