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

Ten-Year Longitudinal Study of Thyroid Function in Children with Down's Syndrome

Background/Aims: The natural history of thyroid function in children with Down's syndrome is relatively unknown. We hypothesized that in these patients the occurrence of thyroid dysfunction rises during development. Methods: Thyroid function was assessed yearly in 145 children with Down's syndrome, all followed from birth up to 10 years of age. Heteroskedastic binary and ordinary logistic regression for repeated measures was used to evaluate the relationship of thyroid function with continuous time. Results: Congenital hypothyroidism was detected in 7% of cases. The probability of acquired thyroid dysfunction increased from 30% at birth to 49% at 10 years (p < 0.001). The subclinical hypothyroidism was nearly stable during the follow-up. The probability of hypothyroidism increased from 7 to 24% at 10 years (p < 0.001). Positive anti-thyroglobulin antibodies were associated with higher odds of more severe hypothyroidism (odds ratio 3.6). Positive anti-thyroid peroxidase antibodies were a better predictor of more severe hypothyroidism (odds ratio 6.1). Diffuse hypoechogenicity on thyroid ultrasound was found in 34 out of 145 children. Conclusion: The probability of thyroid dysfunction increasing during development is higher than previously reported. Such children should be carefully monitored annually to early identify thyroid dysfunction

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

Hair(y) Matters in Melanoma Biology

Melanocyte stem cells (MeSCs), one candidate for the cellular origin of melanoma, reside in the bulge region of the hair follicle (HF), an immune-privileged tissue niche with impaired tumor immunosurveillance. Surprisingly, however, primary melanoma is only very rarely associated with HFs. Here, we explore the hypothesis that this profoundly immunoinhibitory signaling environment deprives both MeSCs and melanocytes of the anagen hair matrix of proinflammatory signals required for full oncogenic transformation. Understanding the cellular and molecular mechanisms for generating a putative antimelanoma tissue habitat, namely in the bulge, could help to recreate a similar melanoma-suppressive signaling environment in melanoma high-risk individuals. We further discuss how mimicking the bulge immune privilege may be an effective melanoma prevention strategy. Despite the paramount advances in the management of metastatic melanoma, including the introduction of immunotherapy and targeted small molecule therapy, metastatic melanoma remains one of the deadliest forms of cancer.Although research has tried to identify the cell of origin of melanoma, opposite lines of research point at mature melanocytes or at melanocyte stem cells.Paradoxically, melanoma rarely arises from melanocytes in the hair follicle, a region rich in melanocytes and melanocyte stem cells that reside in a uniquely immune-privileged microenvironment

Blockade of CCR5 in melanoma: An alternative immune checkpoint modulator

Melanoma is a deadly tumor, which in recent years has been successfully treated with immune checkpoint inhibitors as PD-1/PD-L1 and CTLA-4 inhibitors and targeted therapy as BRAF and MEK inhibitors. However, immunotherapy poses deleterious side effects and pursuit of new therapeutic targets is warranted. As knowledge of tumor immunology advances, such targets are being recognized. C-motif chemokine receptor-5 (CCR5) is a receptor found on immune cells whose effects impact the immune response both to induce inflammation and to activate suppressor cells causing an anti-inflammatory effect. CCR5 is well known as a target for HIV therapy where its blockade is efficient and safe, it is also known that its mutation CCR5delta32 is for the most part non-pathological to its carriers. In oncology, activation of the CCR5 receptor has been observed in high-stage disease and CCR5 blockade has been associated with an increased immune response. In this letter, we build up the rationale to utilize CCR5 as a therapeutic target for metastatic melanoma

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