The Regulation Of Cellular Senescence By NOTCH Signaling Pathway In Melanocyte Malignant Transformation And Melanoma Treatment

Background Melanoma is a skin cancer arising from melanocytes or benign melanocytic lesions. Although its incidence is around 5% in new skin cancer cases every year, its highest mortality makes it the leading cause of death of skin cancers, accounting for 75% of all skin-cancer-caused dead cases. Every year around 20,000 new melanoma cases were diagnosed in China. What makes the situation worse is the incidence keeps growing quickly. Unfortunately, the management of melanoma is now facing many challenges: the various players contributing to the malignancy of melanoma including plenty oncogenes and aberrant signaling pathways as well as the complicated cross talks between themmakes it a difficult task to identify a proper target in therapy; although recent years brought a vast improvement of therapy for advanced melanoma patients, eg. BRAF inhibitors posed promising survival rate with sound response rate. Unfortunately, however, most patients develop acquired resistance inevitably. It was reported that 42% melanomas were arising from a pre-existing naevus. Oncogene- induced senescence(OIS) is considered as a critical barrier during melanoma development. According to this classical theory, oncogene first induces a transient proliferation, provoking activation of tumor suppressing pathway p53/p21 and/or p16/p RB in response, which leading the cells into a stable senescence status. However, in the absence of appropriate defense mechanisms, continued growth with additional genetic events may lead to a malignant lesion. In melanoma cells, followed the activation by oncogenic BRAF/ NRAS, MAPK signaling pathway promotes the development of melanoma in many ways including, but not limited to, provoking proliferation, invasion, angiogenesis and anti-apoptosis ability, making it the most crucial pathway in melanoma. In this regard, BRAF inhibitors such as vemurafenib and dabrafenib are fast acting, with response rate of up to 53% in eligible patients. Unfortunately, however, most patients develop acquired resistance against BRAF inhibitors. In melanoma, NOTCH signaling contributes to many malignancy- associated events including, enhancement of cell growth and adhesion, promotion of metastasis in vivo, increase of cell survival and invasion overall supporting an oncogenic role. Based on this multitude of pro- tumorigenic functions, inhibition of the NOTCH pathway has been proposed as a potential therapeutic approach in melanoma. Previous reports have also linked NOTCH signaling with senescence: similar to what we observe in melanocytes, the NOTCH target gene HES1 prevents senescence and differentiation in fibroblasts possibly by affecting chromatin modifications. Moreover, in a recent study NOTCH1 activation was reported to confer drug resistance in melanoma. Therefore, the current preclinical study was undertaken to investigate the effect of combined GSI and BRAF inhibitor(BRAFi) exposure on BRAF mutant melanoma. To sum up, based on previous studies, we hypothesize that NOTCH signaling exert its oncogenic role by preventing oncogene- induced senescence in melanocytes; moreover, NOTCH inhibition prolong treatment efficacy of BRAF inhibitor in BRAF-mutated melanoma cells by senescence induction. Therefore, one aim of the current study was to analyze the impact of NOTCH on OIS in melanocytes. In addition, the current preclinical study was undertaken to investigate the effect of combined GSI and BRAF inhibitor(BRAFi) exposure on BRAF mutant melanoma.Objectives 1. To investigate the effect of NOTCH activation exerts to oncogene- induced senescence in melanocytes. 2. To analyze the effect of NOTCH inhibitor in melanoma growth and elucidate the underlying mechanism. 3. To explore the synergistic effect of combination with NOTCH inhibitor and BRAF inhibitor in BRAF mutant melanoma and reveal the underlying mechanism.Methods 1. The expression of NOTCH1 was investigated by immunohistochemical staining in benign and malignant human melanocytic lesions; the activation of NOTCH signaling was compared between normal human melanocytes and melanoma cell lines by the expression of NOTCH1 intracellular domain protein and NOTCH target downstream genes via western blot and Real- time PCR respectively. 2. The murine intracellular domain NOTCH1(N1ICD) was subcloned from N1IC-V5-p MP195 into a lentiviral c DNA expression vector p CDH- CMV- MCSEF1- Puro. p CDH based BRAFV600 E or NRASQ61 R co-expression was infected using lentivirus. The senescence phenotypes were identified by growth curve, cell cycle analysis and SA- β- gal staining. 3. Cell growth was evaluated in melanoma cells with elevated NOTCH activity after treated with NOTCH blocker DAPT(gamma- secretase inhibitor).4. The effect of combination treatment of DAPT plus vemurafenib in BRAFV600 E mutant melanoma cells was evaluated after short-term and long-term exposure by MTS assay, apoptosis assay, SA- β- gal staining. Westen blot was employed to confirm the candidate regulator. 5. The effect of combination treatment of DAPT plus vemurafenib in BRAFV600 E mutant melanoma cells with constitutive activated NOTCH was evaluated after short-term and long-term exposure.Results 1. The activity of NOTCH in benign and malignant human melanocytic lesions/ cell lines: Immunohistochemical staining confirmed NOTCH activity is elevated in melanoma tissues than in nevi. Western blot and Real- time PCR revealed the increased activity of NOTCH in melanoma cell lines. 2. Constitutive activated NOTCH prevents oncogene- induced senescence in melanocytes: constitutive NOTCH1 activation provokes malignant transformation and prevents oncogene- induced senescence in normal human melanocytes. 3. NOTCH inhibitor induces senescence in melanoma: DAPT induces a senescent- like cell cycle arrest in melanoma cells. 4. The synergistic inhibition of NOTCH inhibitor combined with BRAF inhibitor: combining vemurafenib with DAPT increases the inhibition efficacy in short- term exposure and delays the resistant. 5. Underlying mechanism and target molecules: apoptosis and senescence assay revealed that suppression of resistance by combination of DAPT with BRAF inhibition is associated with the induction of a senescent- like arrest other than apoptosis. Western blot identified CDK6 as the target regulator of DAPT in senescence induction via preventing the phosphorylation of protein RB.Conclusion Our results demonstrate that NOTCH activation in melanocytes might contribute to melanoma tumorigenesis by preventing oncogene-induced senescence. Moreover, weprovide in vitro evidence supporting the potential use of GSI in combination with BRAFi for treatment of BRAF mutant melanoma cells. The enhancement of BRAFi efficacy by addition of GSI is associated with senescent-like arrest induction, which is, at least partially, mediated by the downregulation of CDK6 expression and RB phosphorylation.