The Atypical Cell Cycle Regulator Spy1 Suppresses Differentiation of the Neuroblastoma Stem Cell Population

Neuroblastoma is an aggressive pediatric cancer originating embryonically from the neural crest. The heterogeneity of the disease, as most solid tumors, complicates diagnosis and treatment. In neuroblastoma this heterogeneity is well represented in both primary tumours and derived cell lines and has been shown to be driven by a population of stem-like tumour initiating cells. Resolving the molecular mediators driving the division of this population of cells may indicate effective therapeutic options for neuroblastoma patients. This study has determined that the atypical cyclin-like protein Spy1, recently indicated in driving symmetric division of glioma stem cells, is a critical factor in the stem-like properties of neuroblastoma tumor initiating cell populations. Spy1 activates Cyclin Dependent Kinases (CDK) in a manner that is unique from classical cyclins. Hence this discovery may represent an important opportunity to design CDK inhibitor drugs to uniquely target subpopulations of cells within these aggressive neural tumours

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

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

The Novel Role of GABAB and CXCR4 in Medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor and it occurs in 16-25% of diagnosed cases, with a higher incidence in children aged 1 to 9 years old. The current standard of care consists of multiple stages of therapy including surgery, irradiation, and chemotherapy. However, a subset of tumors with a still devastating prognosis remains. Metabotropic receptors are G-protein coupled receptors (GPCRs) that act as second messengers. Γ-aminobutyric acid B receptors (GABAB) and C-X-C chemokine receptor type 4 (CXCR4) are metabotropic receptors that belong to the C-family of GPCRs and are activated by the neurotransmitters, γ-aminobutyrate (GABA) and stromal-cell derived factor; SDF-1 (CXCL12), respectively. GABAB receptors are heterodimers where GABA binds to a B1 subunit, and the B2 subunit is coupled to G-proteins regulating activities of the Ca2+ channels, K+ channels, and adenylyl cyclase (AC). Previous studies showed that CXCR4 is highly expressed by glial and neuronal cells in the central nervous system (CNS) and GABAergic neurons. Evidence shows that CXCR4 is overexpressed in MB and upon administration of a CXCR4 antagonist significantly decreased the cell proliferation rate in Type II MB. Evidence also proved that CXCR4 and GABAB­ can crosstalk and GABAB was found to be highly expressed in Type II – MB showing increased Ca2+ levels and protein receptor localization. Current results show that upon administration of the GABAB agonist; baclofen increased cell proliferation in Type II MB cells. Moreover, immunofluorescence showed increased levels of GABAB during mitotic division. In conclusion, by administering the antagonist; phaclofen would enhance the efficacy of chemotherapeutic treatments on MB patients by decreasing the proliferation rate of the aggressive tumors

Role of Mechanical and structural properties of GBM microenvironment in tumour aggressiveness and therapy resistance

Glioblastoma Multiforme (GBM) is the most common and most aggressive type of brain cancer, accounting for 12 to 15% of all intracranial tumours. With a median survival of three to six months for patients with recurrent GBM, there is an urgent need for advancements in the study, diagnosis, and treatment of GBM. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a method of non-invasive tumour analysis that can create a description of mechanical and fluid stress properties of a tumour. DCE-MRI has the potential to couple static mechanical descriptions of analyzed tumours to the flow and fluxes of the fluids in the tumour, thus generating a complete picture of the local stresses, pressure, and flows in and around an embedded tumour. By studying the expression of Vinculin and Hyaluronic Acid (HA), notable stress response markers, in the brain tumour sections; it is the aim of this project to establish protein signatures that correlate with DCE-MRI readings to provide a tool for more effective and detailed diagnosis of GBM. Furthermore, in order to study the tumour with its stress responses to changes in extracellular matrix pressures in a dynamic, three-dimensional setting, we employed brain tumour organoid (BTO) cultures. BTOs provide an in-vitro modelling method that more accurately represents in vivo tissue organization. Through control of microenvironmental factors which an organoid is suspended in, solid stresses can be manipulated and responses studied. As a result, the organoid model can reinforce characterization of cellular responses found when studying tumours. Preliminary results suggest that varying extracellular matrix stiffness results in quantifiable and correlated changes in invasion, aggressiveness and marker protein levels. Defying the local stress factors and their effects on tumour proliferation, aggressiveness marker expression and drug treatment response can help in designing better diagnostic tools and more effective therapeutic strategies

SN~1991T: Reflections of Past Glory

We have obtained photometry and spectra of SN~1991T which extend more than 1000 days past maximum light, by far the longest a SN~Ia has been followed. Although SN~1991T exhibited nearly normal photometric behavior in the first 400 days following maximum, by 600 days its decline had slowed, and by 950~days the supernova brightness was consistent with a constant apparent magnitude of $m_B=21.30$. Spectra near maximum showed minor variations on the SN~Ia theme which grew less conspicuous during the exponential decline. At 270 days the nebular spectrum was composed of Fe and Co lines common to SNe~Ia. However, by 750 days past maximum light, these lines had shifted in wavelength, and were superimposed on a strong blue continuum. The luminosity of SN~1991T at 950 days is more than $9.0\times10^{38}(D/13~{\rm Mpc})^2$~ergs~s$^{-1}$ with a rate of decline of less than $0.04$ mags/100~days. We show that this emission is likely to be light that was emitted by SN~1991T near maximum light which has reflected from foreground dust, much like the light echos observed around SN~1987A.Comment: 15 pages (includes figures and tables) uuencoded compressed postscript, CfA Preprint - To Appear in ApJ

Targeting the Novel Cell Cycle Regulator, Spy1, for Treatment of Medulloblastoma

Targeting the Novel Cell Cycle Regulator, Spy1, for Treatment of Medulloblastoma Philip Habashy, Rosa M. Ferraiuolo, Dorota Lubanska, Lisa A. Porter Medulloblastoma (MB) is the most common malignant pediatric brain tumour. It occurs in 16-25% of cases, with higher incidence in children between the ages of 1 to 9 years. Current standard of care includes combination radiation, surgery and chemotherapy, this treatment relies on DNA damage to induce death of quickly growing cells. While effective for a small margin of patients the treatment is highly aggressive, is plagued with cytotoxicity and ultimately fails in many patients. One recent approach entering clinical development is the use of synthetic cyclin-dependent kinase inhibitors (CKIs). Finding new drugs and optimizing existing approaches for MB are of high importance. Our lab studies a cell cycle regulatory protein called Speedy (Spy1), which promotes cell proliferation, even during times of DNA damage produced by chemotherapeutic agents. Spy1 has been implicated in the maintenance and expansion of stem-like populations of tumour initiating cells known to be the most chemo-resistant among solid tumours. It is our hypothesis that Spy1 drives tumour initiating cells in MB and reducing the levels of Spy1 will increase sensitivity of aggressive MB to standard of care and CKI therapy. To address this hypothesis we have used patient-derived MB cells and have manipulated the levels of Spy1 using a lentiviral system. Using a high throughput platform these cells are injected into zebrafish prior to the establishment of the acquired immune system. We then determine the effect of CKI treatment on these in vivo tumours. To date our results show promise that this approach may sensitize, at least a subset of MB patients, to therapy. Our work may contribute toward optimizing the design of CKIs and the use in combination therapy. This project holds promise for improving survival and quality of life for MB patients

The Cyclin-dependent Kinase Activator, Spy1A, Is Targeted for Degradation by the Ubiquitin Ligase NEDD4

Spy1A is a cyclin-like protein required for progression through the G(1)/S phase of the cell cycle. Elevated Spy1A protein levels have been implicated in tumorigenesis and are attributed to overriding the DNA damage response and enhancing cell proliferation. Understanding how Spy1A is produced and degraded is essential in resolving how it contributes to normal and abnormal growth processes. Herein, we demonstrate that Spy1A is degraded in a cell cycle-dependent manner during mitosis via the ubiquitin-proteasome system. We have resolved the E3 ligase and essential phosphorylation sites mediating Spy1A degradation. Furthermore, we have determined that non-degradable forms of Spy1A do not trigger cell cycle arrest but, rather, contribute to uncontrolled cell growth. Further investigation into the regulation of Spy1A may reveal novel strategies for understanding the etiology and progression of specific growth disorders