A Translational Zebrafish Model to Study Breast Cancer Inflammation and Metastasis

Though modern medicine has greatly improved detection and treatment of breast cancer and overall survival rates are around 85%, this disease is still the second most fatal cancer in Canada. Once the cancer becomes metastatic, it is considered incurable and treatment strategies are directed towards maintenance of the disease rather than curing it. Another hurdle to breast cancer treatment is the severity of side effects from chemotherapeutics, for example cardiotoxicity and hepatotoxicity, that are sometimes fatal. Dexamethasone (Dex) is a synthetic glucocorticoid (GC) that has been shown to be effective at reducing the less severe side effects of chemotherapeutics, such as nausea and inflammation. There is growing concern that Dex interferes with the effectiveness of anti-cancer drugs because of chronic suppression of the immune system, which has been implicated in cancer progression in some inflammatory diseases. The use of natural health products (NHPs) to treat inflammation is a growing field of research to find alternatives to synthetic GCs and many are already on the market. To study the toxicity of drug combinations there needs to be an efficient model that accurately incorporates immune response and organ toxicity. Zebrafish have become an increasingly used animal model to study human cancer and drug toxicity because they are cost effective and can be used for high throughput assays. Using a zebrafish model optimized for this work, we show that Dex increases the metastatic potential of breast cancer cells and accentuates the cardiotoxicity and hepatotoxicity of embryos when treated in combination with cyclophosphamide but not with paclitaxel. We also show that the NHP, Nutria plus, has anti-inflammatory and antioxidant properties and may be a beneficial supplement for treating inflammatory diseases and preventing cancer drug toxicity. Together these results show that the ubiquitous use of Dex in clinics should be re-evaluated. We also studied cell cycle regulation of mammary acini development and cancer metastasis. We show, for the first time, that increased expression of the cell cycle regulator, Spy1, leads to multi-acinar mammary alveolar structures in vitro, and leads to increased metastasis of breast cells in an in vivo zebrafish model, introducing Spy1 as a potential target for treatment of metastatic breast cancer

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis

Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis

Frequently used antiemetic agent dexamethasone enhances the metastatic behaviour of select breast cancer cells

Glucocorticoids, such as dexamethasone (Dex), are used to prevent common side effects induced by chemotherapy and are heavily prescribed for solid cancers such as breast cancer. There is substantial pre-clinical data to support that Dex activation of the glucocorticoid receptor overrides chemotherapy-induced apoptosis in breast cancer cell lines. These findings are compounded by a recent study demonstrating that increased glucocorticoid receptor activation by endogenous stress hormones increased breast cancer heterogeneity and metastasis. Our study is the first to use both in vitro and in vivo models to thoroughly compare the Dex response on the migration of multiple estrogen receptor negative (ER-) and ER+ cancer cell lines. ER+ and ER-breast cancer cell lines were studied to compare their endogenous glucocorticoid activity as well as their metastatic ability in response to Dex treatment. We show that in the ER-breast cancer lines, Dex increases cell numbers, invasiveness, and migration, while decreasing apoptotic ability. Furthermore, we show that following Dex treatment, ER-breast cancer lines migrate further in an in vivo zebrafish model in comparison to ER+ cell lines. The use of ROR1 antibody to block WNT signaling diminished the metastatic properties of ER-cells, however recombinant WNT5A alone was not sufficient to induce migration. Taken together, we demonstrate that Dex treatment exacerbates the metastatic potential of ER-but not ER+ cells. These findings add to the growing body of data stressing the potential adverse role of endogenous and synthetic glucocorticoids in breast cancer biology

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

The Effects of Dexamethasone on Breast Cancer and Chemotherapeutic Treatment

Breast cancer is the most common cancer among Canadian women and is the second leading cause of death by cancer in Canadian Women. Triple-negative breast cancer (TNBC) accounts for approximately 15% of all breast cancer diagnoses. Unlike other breast cancer subtypes, it does not express well-defined molecular targets, such as hormone receptors, that allow targeted treatment. TNBCs are therefore treated with cocktails of potent cytotoxic chemical therapies. Chemotherapy treatment often causes adverse side effects such as hypersensitive reactions, nausea, and vomiting, which are combatted with synthetic glucocorticoids, such as Dexamethasone (Dex). It is of clinical significance to understand the impact Dex has on breast cancer behavior and how it influences chemotherapy treatment response. This literature review compiles data that investigates the impact of Dex on breast cancer through experimental design and clinical studies. It was found that administration of Dex promotes cell survival and metastasis in some breast cancer subtypes. It is important to understand the effects of glucocorticoids on breast cancer cell biology and the interaction with chemotherapeutic agents, which can help lead to alternative therapeutic strategies and improved patient outcomes

The Efficacy of Targeting Cell Cycle Regulators in the Treatment of Medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumour diagnosed in children. This type of brain tumour is comprised of four molecular subgroups: WNT, Sonic Hedgehog (SHH), Group 3, and Group 4. Previous research has indicated that these four subgroups have a diverse set of clinical features, genetics, and cell subpopulations driving the tumour progression and resistance to treatment. Currently, a combination of surgery, radiotherapy, and chemotherapy are used to treat MB. Standard of care chemotherapy is a very aggressive protocol characterized by high general cytotoxicity. The search for new drugs efficiently targeting MB at the source of its origin, both on the genetic as well as cellular level is of high importance. In our lab, we study a cell cycle regulatory protein called Speedy (Spy1) which is able to override select cell cycle checkpoints thereby providing an avenue for cells to over-proliferate and become cancerous. Speedy is implicated in the maintenance of the aggressive and expansive stem-like populations of tumour initiating cells in the most aggressive brain tumour known, glioblastoma multiforme. It is our hypothesis that Spy1 drives specific populations of tumour initiating cells in MB and the reduction of Spy1 levels will increase sensitivity to synthetic CKIs (cyclin-dependent kinase inhibitors). To address our hypothesis we have sorted select populations of patient-derived MB initiating cells and have manipulated levels of Spy1 and/or Cyclin E1 using a lentiviral system. We use a high throughput platform where manipulated human cancer cells are injected into Zebrafish prior to establishment of the acquired immune system. This allows us to determine the effect of specific factors on tumour foci formation and cell characteristics in the presence of an intact immune system. We also study the effect of specific drugs on these in vivo tumours. This work addressed the role of Spy1 and other cyclins on MB aggressiveness and response CKI treatment. The results from our study will not only aid in better understanding of MB biology and dissecting its molecular drivers, but will potentially contribute to improved design of therapy regimens and successful clinical outcome

pH Effects on Bio-Permeability of Natural Rubber Latex Polymers

Barrier materials used in medical gloves and condoms to prevent the penetration of infectious agents are of utmost importance for the preservation of public health. Surrogate viruses have proven effective as conservative measures of infectious potential. Previous work has tested the integrity of barrier materials exposed to a host of pressures and stresses, however none have tested the effects of pH exposure mimicking that prevailing through coitus. We optimized the use of bacteriophage ФX174 and PCR detection to conduct a pilot study on the effects of pH on the viral penetration of latex condoms. Our results suggest that exposure of condoms to acidic pH ranges of physiological significance increases the frequency of condom failure rate. This proof of principal work supports the need for a larger study to investigate the significance of these effects over a wide range of condom brands

The role of cell cycle regulators in neuroendocrine prostate cancer transdifferentiation and resistance in therapy

Prostate adenocarcinoma (AdPC) can transdifferentiate to an aggressive treatment-resistant subtype termed neuroendocrine prostate cancer (NEPC). The majority of NEPC tumors are androgen receptor (AR)-suppressed and they express some of NE markers. Determining how AdPC tumors progress to become resistant to AR-inhibition is a high priority. Our lab has characterized a novel cell cycle regulator, coined Spy1, which is found in many aggressive and stem-like forms of cancer and functions to override senescent barriers in the cell cycle. We have preliminary data to support that elevated Spy1 levels correlate with suppressed AR expression, N-myc elevation, increasing stemness in NE-like and high-grade prostate cancers with a lower survival rate. Also, our in vitro and zebrafish xenograft model indicates a direct correlation between Spy1 overexpression and migration ability of AdPC cell lines. Herein, we hypothesize that Spy1 compensates for AR loss in AdPC and facilitates progression to a proliferative, NE-like and stem-like phenotype. In this work, we evaluate the role of different cell cycle regulators including Spy1 in AdPC progression and address whether this could represent a novel therapeutic direction for NEPC patients. This research could lead to higher success rates of AR-suppressed diagnosis and survival of patients with NEPC