D. S. Hill

Real‐time cell cycle imaging during melanoma growth, invasion, and drug response

Solid cancers are composed of heterogeneous zones containing proliferating and quiescent cells. Despite considerable insight into the molecular mechanisms underlying aberrant cell cycle progression, there is limited understanding of the relationship between the cell cycle on the one side, and melanoma cell motility, invasion, and drug sensitivity on the other side. Utilizing the fluorescent ubiquitination‐based cell cycle indicator (FUCCI) to longitudinally monitor proliferation and migration of melanoma cells in 3D culture and in vivo, we found that invading melanoma cells cycle actively, while G1‐arrested cells showed decreased invasion. Melanoma cells in a hypoxic environment or treated with mitogen‐activated protein kinase pathway inhibitors remained G1‐arrested for extended periods of time, with proliferation and invasion resuming after re‐exposure to a more favorable environment. We challenge the idea that the invasive and proliferative capacity of melanoma cells are mutually exclusive and further demonstrate that a reversibly G1‐arrested subpopulation survives in the presence of targeted therapies.

Abstract 314: Cell cycle phase-specific drug resistance as an escape mechanism of melanoma cells

The tumor microenvironment is characterized by cancer cell subpopulations with heterogeneous cell cycle profiles. For example, hypoxic areas within tumors contain clusters of cancer cells that arrest in the G1 cell cycle phase. It is conceivable that cancer cells exhibit differential drug sensitivity based on their residence in specific cell cycle phases. Here, we have used two-dimensional and organotypic melanoma culture models in combination with fluorescent cell cycle indicators to investigate the effects of G1-arrest on clinically used melanoma drugs. We demonstrate that G1-arrested melanoma cells, irrespective of the underlying cause mediating G1-arrest, are resistant to induced cell death by the proteasome inhibitor bortezomib and the alkylating agent temozolomide. In contrast, G1-arrested cells were more sensitive to MAPK pathway inhibition. Of clinical relevance, pre-treatment of melanoma cells with a MAPK pathway inhibitor resulted in resistance to temozolomide or bortezomib, while in contrast pre-treatment with temozolomide did not result in resistance to the MAPK pathway inhibitor. In summary, we have established a model to study the effects of the cell cycle on drug sensitivity. Cell cycle phase-specific drug resistance is an escape mechanism of melanoma cells that has implications on the choice and timing of drug combination therapies. Citation Format: Kimberley A. Beaumont, David S. Hill, Sheena M. Daignault, Danae M. Sharp, Brian Gabrielli, Wolfgang Weninger, Nikolas K. Haass. Cell cycle phase-specific drug resistance as an escape mechanism of melanoma cells. [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 314.

Melanoma tumor sub-populations are defined by MITF expression, and exhibit enhanced proliferation and characteristics of an EMT

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.

Melanoma cells in G1 phase escape proteasome inhibitor cytotoxicity

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.

Abstract 1781: G1 phase melanoma cells escape proteasome inhibitor cytotoxicity

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Utilising the fluorescent ubiquitination-based cell cycle indicator (FUCCI), which facilitates real-time cell cycle tracking, we have demonstrated in vitro and in vivo that melanomas are composed of differentially cycling tumour cells in a subcompartment-specific distribution. Further, we have shown that targeting the endoplasmic reticulum with fenretinide (synthetic retinoid) or bortezomib (26S proteasome inhibitor) induces cell cycle arrest and apoptosis of metastatic melanoma cells in vitro and in vivo. This study aims to investigate the effect of ER stress-inducing agents on the dynamics of cell division and cell death of individual melanoma cells within the complex tumor microenvironment, and to develop combination strategies that increase the efficacy of ER stress-inducing agents for melanoma therapy. FUCCI-melanoma cells were grown as 3D spheroids and implanted into a collagen matrix to mimic tumor architecture and microenvironment. Utilising the F-XBP1ΔDBD-venus reporter construct, which labels the cytoplasm in response to ER stress, we found that bortezomib induced ER stress, delayed cell cycle progression, and combination with fenretinide increased cell death in 2D and 3D culture. Flow cytometry and confocal microscopy indicated that treatment of FUCCI-melanoma cells with bortezomib induced G2 accumulation in 2D and 3D culture over the course of 24 h. In contrast, by 72 h the majority of cells were in G1 phase. Analysis of 2D and 3D real-time cell cycle imaging movies revealed that bortezomib induced both G1- and G2 arrest, but preferentially killed G2-phase cells. Consequently, pretreatment with temozolomide or fenretinide, leading to G2-arrest, sensitised melanoma cells to bortezomib cytotoxicity. In contrast, pretreatment with MEK inhibitors, leading to G1 arrest, inhibited bortezomib cytotoxicity in all melanoma cells, as did selective BRAF inhibitors in BRAF mutant melanoma cells. Our data suggest that bortezomib combined with fenretinide or temozolamide is a strategy worth exploring for the treatment of BRAF-inhibitor insensitive or resistant melanoma. Importantly, melanoma cells arrested in G1 are protected from bortezomib cytotoxicity, which excludes MAPK pathway inhibitors as combination partners. Citation Format: Sheena M. Daignault, David S. Hill, Kimberley A. Beaumont, Andrea Anfosso, Penny E. Lovat, Wolfgang Weninger, Nikolas K. Haass. G1 phase melanoma cells escape proteasome inhibitor cytotoxicity. [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 1781. doi:10.1158/1538-7445.AM2015-1781

MITF regulates proliferative subpopulation tumor architecture and modifies invasion and characteristics of the epithelial to mesenchymal transition within melanoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Melanoma is the leading cause of skin cancer-related death. Survival rates are high if the disease is diagnosed early, but drop precipitously at later stages. Small molecule inhibitor therapy has given robust responses in the clinic, but relapse is almost certain. This relapse may be due, in part, to tumor heterogeneity. Not only are there multiple cell types within a tumor, but cancer cells themselves can exhibit various phenotypes. This can be due to genotype variation or nutrient availability, and result in populations with different proliferative and invasive capabilities. As these cells display various behaviors, they could also respond to therapies uniquely. Understanding the molecular signature influencing different sub-populations is therefore crucial to design the most effective therapeutic regimen. The fluorescence ubiquitination cell cycle indicator (FUCCI) system, which delineates phases of the cell cycle by visual means, was employed to better understand melanoma tumor heterogeneity. Using this model, it was found that tumor xenografts grown in mice produce two cohorts. One that contained distinct clusters of either arrested or proliferating cells, and another that displayed a homogenous dispersion of proliferating cells throughout the breadth of the tumor. These cohorts were subsequently discovered to display either low or high levels of microphthalmia-associated transcription factor (MITF) expression, respectively. Additionally, loss of MITF by shRNA treatment resulted in conversion of the ability of melanoma cells to give rise to a homogenous xenograft, to instead produce a clustered tumor phenotype. Furthermore, in a 3D in vitro tumor spheroid model, MITF expression was predominantly found in the periphery of the spheroid, which corresponds with the region of highly proliferative cells. Forced over-expression of MITF within these spheroids results in loss of the distinct proliferative ring, and instead a homogenous growth pattern. Not only do spheroids express MITF around the perimeter, but also markers of the Epithelial to Mesenchymal Transition (EMT). These markers, such as Vimentin and Slug, also switch to become expressed homogenously upon high MITF expression. Surprisingly, the increased levels of EMT marker expression by MITF do not correlate to increased migration, and these spheroids in fact show reduced invasion into collagen. We are currently exploring what other means of cancer cell migration could trump an enhanced EMT phenotype and slow invasion in our model. These data outline how tumor heterogeneity, including proliferative and invasive potential, is tightly intertwined with MITF expression, making it an important marker for therapy design. Citation Format: Crystal A. Tonnessen, Kimberley A. Beaumont, David S. Hill, Sheena M. Daignault, Andrea Anfosso, Russell J. Jurek, Wolfgang Weninger, Nikolas K. Haass. MITF regulates proliferative subpopulation tumor architecture and modifies invasion and characteristics of the epithelial to mesenchymal transition within 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 1420. doi:10.1158/1538-7445.AM2015-1420