Dinoop Ravindran Menon

Notch4 signaling induces a mesenchymal-epithelial-like transition in melanoma cells to suppress malignant behaviors

The effects of Notch signaling are context-dependent and both oncogenic and tumor-suppressive functions have been described. Notch signaling in melanoma is considered oncogenic, but clinical trials testing Notch inhibition in this malignancy have not proved successful. Here, we report that expression of the constitutively active intracellular domain of Notch4 (N4ICD) in melanoma cells triggered a switch from a mesenchymal-like parental phenotype to an epithelial-like phenotype. The epithelial-like morphology was accompanied by strongly reduced invasive, migratory, and proliferative properties concomitant with the downregulation of epithelial-mesenchymal transition markers Snail2 (SNAI2), Twist1, vimentin (VIM), and MMP2 and the reexpression of E-cadherin (CDH1). The N4ICD-induced phenotypic switch also resulted in significantly reduced tumor growth in vivo Immunohistochemical analysis of primary human melanomas and cutaneous metastases revealed a significant correlation between Notch4 and E-cadherin expression. Mechanistically, we demonstrate that N4ICD induced the expression of the transcription factors Hey1 and Hey2, which bound directly to the promoter regions of Snail2 and Twist1 and repressed gene transcription, as determined by EMSA and luciferase assays. Taken together, our findings indicate a role for Notch4 as a tumor suppressor in melanoma, uncovering a potential explanation for the poor clinical efficacy of Notch inhibitors observed in this setting. Cancer Res; 76(7); 1690-7. ©2016 AACR.

Microenvironment-Driven Resistance to BRAF Inhibition Comes of Age

Increasingly comprehensive observations indicate that the tumor microenvironment contributes to drug resistance toward small molecule inhibitors. Fedorenko et al. describe a role for fibroblasts in creating a favorable niche for melanoma cell survival if treated with the BRAF inhibitor vemurafenib. TGF-β released by vemurafenib-treated melanoma cells stimulated fibroblasts for increased α-smooth muscle actin, neuregulin (NRG), and fibronectin expression. Off-target effects of vemurafenib led to paradoxical secretion of hepatocyte growth factor (HGF) by fibroblasts. Combined inhibition of BRAF/MET/HER kinases was insufficient to reverse the protective effect of the fibroblasts, whereas reversal was achieved by combined BRAF/PI3K inhibition. A thorough understanding of the complex spatiotemporal interactions in tumor microenvironments holds promise for improved targeting using combination therapies in patients with melanoma.

Distinct histone modifications denote early stress-induced drug tolerance in cancer

Besides somatic mutations or drug efflux, epigenetic reprogramming can lead to acquired drug resistance. We recently have identified early stress-induced multi-drug tolerant cancer cells termed induced drug-tolerant cells (IDTCs). Here, IDTCs were generated using different types of cancer cell lines; melanoma, lung, breast and colon cancer. A common loss of the H3K4me3 and H3K27me3 and gain of H3K9me3 mark was observed as a significant response to drug exposure or nutrient starvation in IDTCs. These epigenetic changes were reversible upon drug holidays. Microarray, qRT-PCR and protein expression data confirmed the up-regulation of histone methyltransferases (SETDB1 and SETDB2) which contribute to the accumulation of H3K9me3 concomitantly in the different cancer types. Genome-wide studies suggest that transcriptional repression of genes is due to concordant loss of H3K4me3 and regional increment of H3K9me3. Conversely, genome-wide CpG site-specific DNA methylation showed no common changes at the IDTC state. This suggests that distinct histone methylation patterns rather than DNA methylation are driving the transition from parental to IDTCs. In addition, silencing of SETDB1/2 reversed multi drug tolerance. Alterations of histone marks in early multi-drug tolerance with an increment in H3K9me3 and loss of H3K4me3/H3K27me3 is neither exclusive for any particular stress response nor cancer type specific but rather a generic response.

Acetylsalicylic acid governs the effect of sorafenib in RAS- mutant cancers

Purpose: Identify and characterize novel combinations of sorafenib with anti-inflammatory painkillers to target difficult-to-treat RAS-mutant cancer. Experimental Design: The cytotoxicity of acetylsalicylic acid (aspirin) in combination with the multikinase inhibitor sorafenib (Nexavar) was assessed in RAS-mutant cell lines in vitro. The underlying mechanism for the increased cytotoxicity was investigated using selective inhibitors and shRNA-mediated gene knockdown. In vitro results were confirmed in RAS-mutant xenograft mouse models in vivo. Results: The addition of aspirin but not isobutylphenylpropanoic acid (ibruprofen) or celecoxib (Celebrex) significantly increased the in vitro cytotoxicity of sorafenib. Mechanistically, combined exposure resulted in increased BRAF/CRAF dimerization and the simultaneous hyperactivation of the AMPK and ERK pathways. Combining sorafenib with other AMPK activators, such as metformin or A769662, was not sufficient to decrease cell viability due to sole activation of the AMPK pathway. The cytotoxicity of sorafenib and aspirin was blocked by inhibition of the AMPK or ERK pathways through shRNA or via pharmacologic inhibitors of RAF (LY3009120), MEK (trametinib), or AMPK (compound C). The combination was found to be specific for RAS/RAF–mutant cells and had no significant effect in RAS/RAF–wild-type keratinocytes or melanoma cells. In vivo treatment of human xenografts in NSG mice with sorafenib and aspirin significantly reduced tumor volume compared with each single-agent treatment. Conclusions: Combination sorafenib and aspirin exerts cytotoxicity against RAS/RAF–mutant cells by simultaneously affecting two independent pathways and represents a promising novel strategy for the treatment of RAS-mutant cancers. Clin Cancer Res; 24(5); 1090–102. ©2017 AACR.

Abstract 1679: Notch4 mediates mesenchymal-epithelial-like transition in melanoma

In melanoma Notch signaling is a well-established oncogenic factor and presumably driver of disease progression. Here we report that overexpression of the constitutively active intracellular domain of Notch4 (N4ICD) directs a mesenchymal-epithelial-like transition that results in an epithelial, low migratory, invasive and proliferative phenotype with decreased tumorigenicity in-vivo. Investigating the expression profile of 50 EMT markers in N4ICD overexpressing cells revealed an epithelial-like gene signature with major EMT markers, including Vimentin, MMP2, Snail1, Snail2 and Twist1, significantly decreased and several epithelial markers, including E-cadherin, Desmoplakin and Occludin significantly increased. N4ICD suppresses the expression of Snail2 and Twist1 in a non-canonical fashion by inducing transcription factors Hey-1 and Hey-2 which directly bind to promoter regions of Snail2 and Twist1 as determined by EMSA and luciferase assays. N4ICD overexpression also results in increased beta-catenin protein levels exclusively limited to the cytoplasm. Immunohistochemical stainings of primary melanomas and subcutaneous metastasis showed a correlation between Notch4, E-cadherin and beta-catenin expression. Our results demonstrate that overexpression of Notch4 ICD results in the emergence of an epithelial-like phenotype with increased E-cadherin and cytoplasmic beta catenin potentially steering the reformation of adherens junctions. This indicates that Notch4 supports a mesenchymal-epithelial-like transition trapping cancer cells in a low proliferative, invasive and migratory epithelial state, suggesting Notch4 to be a tumor suppressor in melanoma. Citation Format: Ehsan BonyadiRad, Heinz Hammerlindl, Dinoop Ravindran Menon, Christine Hafner, Meenhard Herlyn, Helmut Schaider. Notch4 mediates mesenchymal-epithelial-like transition in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1679.

Notch4 drives mesenchymal-epithelial transition in melanoma

HUMMER, DOUGLAS ANDREW. Community College Presidential Change from the Department Leader’s Perspective: A Case Study. (Under the direction of Dr. Diane Chapman). Academic department leaders are the least studied level of management in higher education, yet they play a fundamental role in transforming the vision and goals of executive leadership into reality (Gonaim, 2016). Academic department leaders play such a role because as a group, they influence the largest part of the employee population at a college; the faculty (Stringer, 2002; Tierney, 1999). Since the ability to create lasting change is one of the core qualities of a highly effective community college president (The Aspen Institute & Achieving the Dream, 2013), it is important to understand how the strategies, tactics, and actions employed by the executive leadership of an institution of higher learning shape the perceptions academic department leaders have of their workplace. This study took a qualitative approach to understanding these perceptions during the first few months of new president’s administration at a large community college in the southeast US. A transcendental phenomenological (Husserl, 2004) lens and case study methodologies were used to collect and analyze structural and textural data to describe the perceptions and interpretations academic department leaders had of this presidential change phenomenon. The case study itself provided the structural description, or context of the phenomenon, and semi-structured interviews were used to give voice to the academic department leaders that participated in the study. An analysis of the data revealed a number of themes that addressed the research questions asked in this study. These themes led to the following findings in this study. Listening sessions initiated by the new president and the hiring process that brought him to the college were identified as major influencing events in creating a positive impression of this new executive and his vision for the future of the institution. This positive impression is a contributor to creating an organizational climate ready to accept change. Other themes that emerged revolved around executive leadership’s support, communication issues, the hierarchical levels of management, changes that were initiated, and the fear to speak up. The findings from this study can be used by many community colleges as they hire new presidents and prepare for the changes new executives bring. The findings can also be used by any college approaching a major change initiative. The presidential change phenomenon will affect as many as four out of every five community colleges over the next 10 years (Phillippe, 2016). Presidential changes and other major change initiatives that community colleges experience will provide many opportunities to repeat this study and share the findings with others experiencing the same phenomenon.

An early innate stress response precedes acquired drug resistance in melanoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Acquired drug resistance constitutes a major challenge for effective cancer therapies. The dynamics of early drug resistance leading to permanent resistance are poorly understood. Melanoma cell lines were exposed to molecular targeted inhibitors like BRAF or MEK inhibitors or chemotherapy at sublethal drug concentrations for over 90 days. Alternatively melanoma cells were exposed to hypoxic conditions or low glucose media. Cells surviving drug exposure, hypoxia or nutrient starvation were monitored for the expression of CD271, ALDH activity, differentiation markers, ABCB5, chromatin remodeling, histone demethylases and markers for angiogenesis to characterize cells exposed for a minimum of 12 days. Further gene expression analyses, RPPA analyses and in vivo tumorigenicity were performed in these cells. Drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multi-drug resistance, termed induced drug tolerant cells (IDTC). Transition into the IDTC state seems to be an inherent stress reaction for survival towards unfavorable environmental conditions or drug exposure independent of any subpopulation or cancer stem cell. The response comprises chromatin remodeling, activation of signaling cascades, and markers proposed to be stem cell markers with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced ALDH activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Our results suggest a phenotypic shift of parental cells to the induced drug tolerant cell (IDTC) state irrespective of a given subpopulation thus not representing cancer stem cells. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance. Citation Format: Dinoop Ravindran Menon, Suman Das, Clemens Krepler, Adina Vultur, Gao Zhang, Nikolas Haass, Peter H. Soyer, Brian Gabrielli, Rajasekharan Somasundaram, Gerald Hoefler, Meenhard Herlyn, Helmut Schaider. An early innate stress response precedes acquired drug resistance in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2684. doi:10.1158/1538-7445.AM2015-2684