Generation of Model Systems for the Study of Novel Cell Cycle Regulation in Development: Implications for Spy1 in Tumour Susceptibility

Cell cycle regulation lies at the heart of all developmental decisions, and aberrant regulation represents an important step in the onset of tumourigenesis. Alterations in cell cycle regulators are known to play a critical role in promoting tumour formation in a wide variety of tissues. The cyclin-like protein Spy1 is capable of binding and activating CDK2 and promoting progression through G1/S phase of the cell cycle. Spy1 can also target the cell cycle inhibitor p27 for degradation, and it has been shown to override cellular checkpoints and apoptotic pathways in response to DNA damage leading to enhanced cell survival. Previous data has shown that Spy1 levels are tightly regulated throughout mammary gland development, and high levels are associated with breast cancer, as well as cancer of the brain and liver. This suggests a role for Spy1 in normal mammary development as well as in the development of tumourigenesis. Transgenic and gene targeted models represent an ideal system in which to study altered protein expression on development and tumour initiation. This work describes the development of three novel model systems to study altered Spy1 expression on normal and abnormal development of the mammary gland. Using the newly generated MMTV-Spy1 mouse we have demonstrated that elevated levels of Spy1 increases mammary tumour susceptibility, and interestingly liver tumour susceptibility. We demonstrate that levels of Spy1 are downregulated in the event of DNA damage and the tumour suppressor p53 may be responsible for mediating this event. If p53 is unable to keep levels of Spy1 in check this can lead to uncontrolled cell proliferation, a hallmark of oncogenesis, and may contribute to tumour initiation. Thus for the first time, we demonstrate a role for Spy1 in mediating tumour susceptibility, highlighting the importance of maintaining proper checkpoint responses. Our work demonstrates that Spy1 could prove to be an attractive diagnostic marker and therapeutic target in the treatment of various forms of cancer, helping to eradicate this deadly disease

The Essentiality of Spy1 in Cooperation with Hepatocellular Carcinoma Drivers to Promote Tumour Formation

Liver cancer is the fourth leading cause of cancer-related deaths worldwide, standing at an estimation of 800,000 deaths annually. Among the various subtypes, hepatocellular carcinoma (HCC) is the most common primary liver malignancy. HCC is known to develop through a series of genetic and epigenetic alterations of proto-oncogenes and tumour suppressor genes in the liver environment. These changes ultimately lead to the malignant transformation of hepatocytes, the primary cells of the liver. Various HCC drivers are known to cause disruption of cellular pathways and promote tumour formation. Importantly, several cell cycle mediators cause misregulation, thereby stimulating tumour formation and progression. The cyclin-like protein Spy1, promotes cell cycle progression and overrides apoptosis. Recent reports have detected increased levels of Spy1 in human HCC, which directly correlates to severity of the disease and poor prognosis. We hypothesize that Spy1 plays a critical role along with hepatocellular carcinoma drivers to advance tumour development.  We will test the essentiality of Spy1 on HCC development by first investigating potential gRNAs to use for Spy1 knockout in vivo in the liver, for hydrodynamic tail vein injections in wildtype mice. Simultaneously, in vitro testing of HCC cells (HepG2) will study the importance of Spy1 in HCC cell characteristics in combination with HCC drivers such as c-myc, p53, and -catenin. This project will assist in understanding the essentiality of Spy1 in HCC, which may reveal insight into the molecular mechanism of the tumour suppressors and proto-oncogenes connected with this subset of liver cancer

The Role of Cell Cycle Mediators in the Progression of Non-alcoholic Steatohepatitis in Male and Female Murine Models

Hepatocellular carcinoma (HCC) is the most prevalent primary cancer of the liver and one of the leading causes of cancer-related deaths worldwide. There are a multitude of risk factors that contribute to the development of HCC including viral infection, obesity, alcoholism, as well as non-alcoholic steatohepatitis (NASH). In the case of these chronic diseases and repeated injury, the liver continuously repairs itself to maintain its structural integrity, resulting in fibrosis, and in more serious cases, cirrhosis– major risk factors in the development of HCC. Men are 3-5x more likely to develop liver cancer than women; however, the exact mechanism for this remains undetermined. Previous work in our lab using a transgenic mouse model overexpressing the cyclin-like protein Spy1 showed an increased incidence of HCC and decrease in rates of fibrosis, suggesting a link between cell cycle regulation and progression to HCC. Spy1 binds and activates CDKs at the G1-S and G2-M checkpoints, leading to cell cycle progression independent of cyclin-based regulation. Using a methionine-choline deficient diet to induce NASH, the role of various cell cycle mediators will be investigated to uncover the link between cell cycle regulation and NASH disease progression. In addition, differences between female and male mouse responses to the diet with respect to cell cycle regulation will be analyzed. A better understanding of the relationship between cell cycle regulation and NASH disease progression in both female and male mouse models will help identify novel diagnostic markers and pathways of therapeutic importance in HCC

Determining the Dependency of Spy1 based on Rb Status

Determining the Dependency of Spy1 based on Rb Status Visconti, T., Philbin, N, Fifield, B, Porter L.A. University of Windsor, Windsor, Ontario. N9B 3P4 Porter Lab, Department of Integrative Biology/Biomedical Sciences Breast cancer is the second most common cancer worldwide and the most common cancer among women. Triple Negative Breast Cancer (TNBC) is a particularly aggressive form of breast cancer with many subtypes based on gene expression profiles. There are currently no targeted treatments for TNBC due to its molecular characteristics, urging the discovery of new therapeutic targets. Potential therapeutic avenues are the cell cycle and its mediators which play an important role in cancer formation and progression. Spy1, a cyclin-like protein, promotes cell proliferation through the G1/S and G2/M checkpoints. Spy1 promotes proliferation even in the presence of DNA damage, overriding checkpoints and increasing cancer susceptibility. While Spy1 has been found to be elevated in breast cancer, its unique binding structure makes for an ideal candidate for cell cycle inhibition therapy. The retinoblastoma tumor suppressor protein (Rb) is known to regulate the DNA damage response system and is key in regulating the cell cycle. However, studies have shown that Rb is often mutated in TNBC inducing deregulated cell cycle progression potentially leading to tumor development. For some breast cancer subtypes the presence or absence of Rb (Rb status) can dictate response to treatment by cell cycle inhibitor drugs. Using in vitroTNBC models (MDA-MB-231 & Bt549 cell lines), this study aims to determine if Spy1 can override checkpoints independently of Rb status, and if elevated levels of Spy1 alter this response. These results could provide further guidance in developing cell cycle inhibition targeted therapies and potentially better TNBC patient outcomes

Spy1 and the Long-Term Effects of Childbearing on the Mammary Gland

The Role of Spy1 in Exacerbating the Long-Term Effects of Parity on the Mammary Gland Khan, A.1, Fifield, B.1, Porter L.A.1. 1. University of Windsor, Windsor, Ontario. N9B 3P4. Porter Lab, Department of Biomedical Sciences Age is a significant variable in cancer development, and approximately 1/3 of breast cancer cases occur in patients older than 70. Another emerging risk factor is parity, or childbearing, which may be linked to cellular changes that affect a woman’s risk of developing breast cancer over the course of her lifetime. These changes are thought to result from the mammary gland not reverting to normal after lactation and involution - a developmental remodeling process where the milk secreting cells are cleared and replaced with adipocytes after weaning. Aberrant expression of the cyclin-like protein Spy1 has been shown to stimulate precocious development, resulting in disrupted morphology and oncogenesis within the mammary gland. Preliminary data suggests that the mammary glands of mice overexpressing Spy1 do not fully regress following lactation and involution, which may predispose them to breast cancer. We hypothesize that the overexpression of Spy1 exacerbates the long-term effects of parity on mammary gland morphology. To investigate this, we performed hematoxylin and eosin (H&E) staining as well as immunohistochemistry (IHC) on paraffin embedded sections, and whole mount staining of MMTV-Spy1 mice, a transgenic mouse model that overexpresses Spy1 within the mammary gland. We then compared the mammary gland morphology of parous MMTV-Spy1 mice to nulliparous MMTV-Spy1 mice, parous control FVB mice, and nulliparous control FVB mice. This research begins to improve our understanding of Spy1’s role in regulating proliferation and apoptosis, contributes to our overall knowledge of breast cancer dynamics, and further solidifies Spy1 as an important target for treatment

Exploring a Link Between Spy1 and Hepatocellular Carcinoma Progression

Primary liver cancer represents one of the fastest rising cancers in Canada. The increasing incidence of this pluralistic and multi-faceted malignancy make having a full understanding of the disease an essential step when considering treatment options. The most common type of liver cancer, hepatocellular carcinoma (HCC), may stem from a variety of lifestyle factors and circumstances. Cirrhosis and advanced fibrosis of the liver are two common causes. These chronic disease states are often associated with alcoholism, but can also arise due to non-alcoholic steatohepatitis (NASH) and fatty liver disease. Hepatitis and fatty liver elicit liver cell injury, inflammation and oxidative damage, which in turn activate the liver’s regenerative processes. Regeneration, especially when continually called upon, represents a proliferative process that may render patients susceptible to HCC. Spy 1 is a cyclin-like protein that promotes cell cycle progression and drives cell growth during select regenerative processes. A serendipitous finding in mice developed to study the role of Spy1 in the mammary gland revealed that the male Spy1 mice had significantly more primary HCC than their littermate controls. To further explore the role of Spy1 in HCC, NASH has been initiated in wild-type mice using a methionine-choline deficient diet. Spy1 levels and inflammatory, proliferative and regenerative responses are being monitored by analyzing gene expression, protein levels, fat accumulation and morphological changes. This data will determine the physiological processes that correlate with elevated Spy1 levels during liver injury and regeneration. The resulting information will aid in establishing a causal link in the progression of HCC. Future work will determine whether this mechanism represents a target for treatment for patients with this aggressive form of cancer

The Role of Spy1 in the Development of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is among the most aggressive and prevalent forms of primary liver cancer. With an increasing incidence in Canada and only a five-year survival rate of 19%, a more comprehensive understanding of this debilitating disease is imperative in order to consider future treatment options. A variety of lifestyle factors may underlie HCC, including chronic alcoholism, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). These factors are known to elicit liver injury, inflammatory immune responses, and oxidative damage thereby imparting a regenerative response in this visceral organ. This regeneration is manifested as either fibrosis, in order to maintain structural integrity of the organ, or through hepatocyte proliferation, in order to restore functional mass. Progression through the cell cycle by the normally quiescent hepatocytes is thought to contribute to the proliferative response. Spy1 is a novel, cyclin-like protein, which binds and activates CDKs at the G1-S and G2-M checkpoints, leading to cell cycle progression independent of cyclin-based regulation. A serendipitous discovery in the transgenic MMTV-Spy1 murine model demonstrates that Spy1 significantly increases the incidence of fatty liver disease and HCC. Combined with a methionine-choline deficient (MCD) diet in order to elicit liver injury, the MMTV-Spy1 murine livers response to these factors through inflammatory, proliferative and fibrotic responses is analyzed. Preliminary evidence demonstrates Spy1’s role in favouring hepatic regeneration through a proliferative response rather than a fibrotic one, demonstrating a potential mechanism in the development of HCC. Through primary cultures of THLE-2 and Hep G2 cell lines, similar assays are conducted in order to determine Spy1’s role in causing liver cancer. These results will shed light on Spy1’s role in HCC, potentially allowing for determination of a diagnostic markers and pathways of therapeutic importance

Differential expression of glucose transporters and hexokinases in prostate cancer with a neuroendocrine gene signature: A mechanistic perspective for 18 F-FDG imaging of PSMA-suppressed tumors

Although the incidence of de novo neuroendocrine prostate cancer (PC) is rare, recent data suggest that low expression of prostatespecific membrane antigen (PSMA) is associated with a spectrum of neuroendocrine hallmarks and androgen receptor (AR) suppression in PC. Previous clinical reports indicate that PCs with a phenotype similar to neuroendocrine tumors can be more amenable to imaging by 18F-FDG than by PSMA-targeting radioligands. In this study, we evaluated the association between neuroendocrine gene signature and 18F-FDG uptake-associated genes including glucose transporters (GLUTs) and hexokinases, with the goal of providing a genomic signature to explain the reported 18F-FDG avidity of PSMA suppressed tumors. Methods: Data-mining approaches, cell lines, and patient-derived xenograft models were used to study the levels of 14 members of the SLC2A family (encoding GLUT proteins), 4 members of the hexokinase family (genes HK1-HK3 and GCK), and PSMA (FOLH1 gene) after AR inhibition and in correlation with neuroendocrine hallmarks. Also, we characterize a neuroendocrine-like PC (NELPC) subset among a cohort of primary and metastatic PC samples with no neuroendocrine histopathology. We measured glucose uptake in a neuroendocrine-induced in vitro model and a zebrafish model by nonradioactive imaging of glucose uptake using a fluorescent glucose bioprobe, GB2-Cy3. Results: This work demonstrated that a neuroendocrine gene signature associates with differential expression of genes encoding GLUT and hexokinase proteins. In NELPC, elevated expression of GCK (encoding glucokinase protein) and decreased expression of SLC2A12 correlated with earlier biochemical recurrence. In tumors treated with AR inhibitors, high expression of GCK and low expression of SLC2A12 correlated with neuroendocrine histopathology and PSMA gene suppression. GLUT12 suppression and upregulation of glucokinase were observed in neuroendocrine- induced PC cell lines and patient-derived xenograft models. A higher glucose uptake was confirmed in low-PSMA tumors using a GB2-Cy3 probe in a zebrafish model. Conclusion: A neuroendocrine gene signature in neuroendocrine PC and NELPC associates with a distinct transcriptional profile of GLUTs and hexokinases. PSMA suppression correlates with GLUT12 suppression and glucokinase upregulation. Alteration of 18F-FDG uptake-associated genes correlated positively with higher glucose uptake in AR- and PSMA-suppressed tumors. Zebrafish xenograft tumor models are an accurate and efficient preclinical method for monitoring nonradioactive glucose uptake

Role of Spy1 in Mammary Development

Breast cancer accounts for 25% of all new cancer cases in women and 13% of all cancer deaths in women. Determining the key mediators that regulate aspects of both normal and abnormal development of the breast is crucial to the development of better diagnostics and treatment options. Proper cell cycle regulation guides cellular changes during the stages of mammary development, and misregulation or mutation/deletion of key cell regulatory genes represents an important step in breast cancer initiation and progression. The cyclin-like protein Spy1 is tightly regulated during normal mammary gland development and has been implicated in several cancers, including breast cancer. Spy1 binds and activates cyclin-dependent kinases (Cdks), promoting progression through the G1/S and G2/M phase of the cell cycle. Elevated levels of Spy1 significantly increases cell proliferation and has been shown to override the DNA damage response. This study seeks to explore the question - Is Spy1 required for normal and abnormal development of the breast? My thesis work involved using the novel genome-editing tool CRISP-Cas9 to knockout Spy1 in the mouse mammary epithelial cell line HC11 and the breast cancer cell line, MDA-MB 231. These knockout cells were tested for effects on mammary cell growth and development such as proliferation, differentiation, stem cell expansion and migration. My results support that this unique family of cell cycle regulators play a critical role in the differentiation and stem cell maintenance in the mammary gland. This work sheds light on the mechanisms of normal mammary development as well as breast cancer initiation and progression and supports further exploration of this mechanism as a therapeutic direction for breast cancer

Identifying Molecular Markers of Progression to Muscle Invasive Bladder Cancer

An estimated 9,000 Canadians are diagnosed with bladder cancer each year making it the 5th most common cancer in Canada, the 12th most common among women and the 4th among men. Most patients are initially diagnosed with non-muscle invasive bladder cancer (NMIBC), which in some cases can progress to muscle invasive bladder cancer (MIBC). MIBC is associated with significantly poorer prognosis than NMIBC, and it is unclear why some progress to MIBC while others do not. . A collaboration with the computer science department found that an increase in discoidin domain-containing receptor 2 (DDR2) is ninety percent specific for muscle invasion in bladder cancer and that DDR2 is overexpressed in MIBC compared to NMIBC. DDR2 is a tyrosine kinase that functions as a cell surface receptor for collagen and regulates cell differentiation, cell migration, invasion and proliferation. It is also involved in the molecular mechanism of disease progression in thirty six percent of MIBC patients. Furthermore, knockdown of DDR2 in vitro in bladder cancer cell lines decreased cell viability, migration and invasion. The mechanism by which DDR2 mediates progression to MIBC and increases invasion in bladder cancer is currently unknown, and may represent a new target to prevent progression from NMIBC to MIBC. This project will seek to demonstrate that DDR2 can be used as a prognostic indicator for the progression to MIBC and can be used as a therapeutic target preventing progression to MIBC