Hon Leong

The Insulin-Like Growth Factor I Receptor Is Required for Akt Activation and Suppression of Anoikis in Cells Transformed by the ETV6-NTRK3 Chimeric Tyrosine Kinase

ABSTRACT Signaling through the insulin-like growth factor I receptor (IGF-IR) axis is essential for transformation by many dominantly acting oncoproteins. However, the mechanism by which IGF-IR contributes to oncogenesis remains unknown. To examine this, we compared transformation properties of the oncogenic ETV6-NTRK3 (EN) chimeric tyrosine kinase in IGF-IR-null R− mouse embryo fibroblasts with R− cells engineered to reexpress IGF-IR (R+ cells). We previously showed that R− cells expressing EN (R− EN cells) are resistant to transformation but that transformation is restored in R+ cells. We now show that while R− EN cells have intact Ras-extracellular signal-regulated kinase signaling and cell cycle progression, they are defective in phosphatidylinositol-3-kinase (PI3K)-Akt activation and undergo detachment-induced apoptosis (anoikis) under anchorage-independent conditions. In contrast, R+ cells expressing EN (R+ EN cells) suppress anoikis and are fully transformed. The requirement for IGF-IR in R− EN cells is overcome by ectopic expression of either activated Akt or a membrane-targeted form of EN. Moreover, compared to R− EN cells, R+ EN cells show a dramatic increase in membrane localization of insulin receptor substrate 1 (IRS-1) in association with EN. Since EN is known to bind IRS-1 as an adaptor protein, our findings suggest that IGF-IR may function to localize EN/IRS-1 complexes to cell membranes, in turn facilitating PI3K-Akt activation and suppression of anoikis.

Strategies of Conditional Gene Expression in Myocardium

The use of specialized reporter genes to monitor real-time, tissue-specific transgene expression in animal models offers an opportunity to circumvent current limitations associated with the establishment of transgenic mouse models. The Cre-loxP and the tetracycline (Tet)-inducible systems are useful methods of conditional gene expression that allow spatial (cell-type-specific) and temporal (inducer-dependent) control. Most often, the alpha-myosin heavy chain (alpha-MHC) promoter is used in these inducible systems to restrict expression of reporter genes and transgenes to the myocardium. An overview of each inducible system is described, along with suggested reporter genes for real-time, noninvasive imaging in the myocardium. Effective gene delivery of the inducible gene expression system is carried out by lentiviral vectors, which offer high transduction efficiency, long-term transgene expression, and low immunogenicity. This chapter outlines the packaging of myocardium-specific inducible expression systems into lentiviral vectors, in which a transgene and a reporter gene are transduced into cardiomyocytes. In doing so, transgene and reporter expression can be monitored/tracked with bioluminescence imaging (BLI) and positron emission tomography (PET).

Gender and post-ischemic recovery of hypertrophied rat hearts.

BackgroundGender influences the cardiac response to prolonged increases in workload, with differences at structural, functional, and molecular levels. However, it is unknown if post-ischemic function or metabolism of female hypertrophied hearts differ from male hypertrophied hearts. Thus, we tested the hypothesis that gender influences post-ischemic function of pressure-overload hypertrophied hearts and determined if the effect of gender on post-ischemic outcome could be explained by differences in metabolism, especially the catabolic fate of glucose.MethodsFunction and metabolism of isolated working hearts from sham-operated and aortic-constricted male and female Sprague-Dawley rats before and after 20 min of no-flow ischemia (N = 17 to 27 per group) were compared. Parallel series of hearts were perfused with Krebs-Henseleit solution containing 5.5 mM [5-3H/U-14C]-glucose, 1.2 mM [1-14C]-palmitate, 0.5 mM [U-14C]-lactate, and 100 mU/L insulin to measure glycolysis and glucose oxidation in one series and oxidation of palmitate and lactate in the second. Statistical analysis was performed using two-way analysis of variance. The sequential rejective Bonferroni procedure was used to correct for multiple comparisons and tests.ResultsFemale gender negatively influenced post-ischemic function of non-hypertrophied hearts, but did not significantly influence function of hypertrophied hearts after ischemia such that mass-corrected hypertrophied heart function did not differ between genders. Before ischemia, glycolysis was accelerated in hypertrophied hearts, but to a greater extent in males, and did not differ between male and female non-hypertrophied hearts. Glycolysis fell in all groups after ischemia, except in non-hypertrophied female hearts, with the reduction in glycolysis after ischemia being greatest in males. Post-ischemic glycolytic rates were, therefore, similarly accelerated in hypertrophied male and female hearts and higher in female than male non-hypertrophied hearts. Glucose oxidation was lower in female than male hearts and was unaffected by hypertrophy or ischemia. Consequently, non-oxidative catabolism of glucose after ischemia was lowest in male non-hypertrophied hearts and comparably elevated in hypertrophied hearts of both sexes. These differences in non-oxidative glucose catabolism were inversely related to post-ischemic functional recovery.ConclusionGender does not significantly influence post-ischemic function of hypertrophied hearts, even though female sex is detrimental to post-ischemic function in non-hypertrophied hearts. Differences in glucose catabolism may contribute to hypertrophy-induced and gender-related differences in post-ischemic function.

Cyclin A2, a novel regulator of EMT

Our previous work showed that Cyclin A2 deficiency promotes cell invasion in fibroblasts. Given that the majority of cancers emerge from epithelia, we explored novel functions for Cyclin A2 by depleting it in normal mammary epithelial cells. This caused an epithelial to mesenchymal transition (EMT) associated with loss of cell-to-cell contacts, decreased E-Cadherin expression and increased invasive properties characterized by a reciprocal regulation of RhoA and RhoC activities, where RhoA-decreased activity drove cell invasiveness and E-Cadherin delocalization, and RhoC-increased activity only supported cell motility. Phenotypes induced by Cyclin A2 deficiency were exacerbated upon oncogenic activated-Ras expression, which led to an increased expression of EMT-related transcriptional factors. Moreover, Cyclin A2-depleted cells exhibited stem cell-like properties and increased invasion in an in vivo avian embryo model. Our work supports a model where Cyclin A2 downregulation facilitates cancer cell EMT and metastatic dissemination.

Differential Functional Roles of ALDH1A1 and ALDH1A3 in Mediating Metastatic Behavior and Therapy Resistance of Human Breast Cancer Cells

Previous studies indicate that breast cancer cells with high aldehyde dehydrogenase (ALDH) activity and CD44 expression (ALDHhiCD44+) contribute to metastasis and therapy resistance, and that ALDH1 correlates with poor outcome in breast cancer patients. The current study hypothesized that ALDH1 functionally contributes to breast cancer metastatic behavior and therapy resistance. Expression of ALDH1A1 or ALDH1A3 was knocked down in MDA-MB-468 and SUM159 human breast cancer cells using siRNA. Resulting impacts on ALDH activity (Aldefluor® assay); metastatic behavior and therapy response in vitro (proliferation/adhesion/migration/colony formation/chemotherapy and radiation) and extravasation/metastasis in vivo (chick choroiallantoic membrane assay) was assessed. Knockdown of ALDH1A3 but not ALDH1A1 in breast cancer cells decreased ALDH activity, and knockdown of ALDH1A1 reduced breast cancer cell metastatic behavior and therapy resistance relative to control (p < 0.05). In contrast, knockdown of ALDH1A3 did not alter proliferation, extravasation, or therapy resistance, but increased adhesion/migration and decreased colony formation/metastasis relative to control (p < 0.05). This is the first study to systematically examine the function of ALDH1 isozymes in individual breast cancer cell behaviors that contribute to metastasis. Our novel results indicate that ALDH1 mediates breast cancer metastatic behavior and therapy resistance, and that different enzyme isoforms within the ALDH1 family differentially impact these cell behaviors.

Hypoxia promotes tumor cell motility via RhoA and ROCK1 signaling pathways

Blood vasculature provides tissues of the body with oxygen to support their growth. Tumors can outgrow their vascular supply, leading to regions of low oxygen, also known as hypoxia. Tumor cells that are within about 150–200 µm from a functional blood vessel may be hypoxic, and cells more distant are more susceptible to necrosis (1, 2). However, some of these oxygen-deprived tumor cells mobilize and retreat to less hypoxic microenvironments via a molecular axis dependent on activated hypoxia-inducible factors (HIF), including HIF1α. When deprived of oxygen, HIF1α is able to transactivate key promotility genes, such as RHOA and ROCK1 (Rho kinase 1), as described by Gilkes et al. in PNAS (3). During such hypoxia-induced migration, it is conceivable that cancer cells can gain access to the circulation through tumor vasculature, which is often disordered and irregular (4, 5). Hence, hypoxia-induced tumor cell migration could be a major contributor to cancer cell intravasation, the first step of the metastatic cascade. As a result of poor vascularization of the tumor, hypoxia can give rise to significant changes in tumor cell gene-expression patterns, metabolism, and behavior (6), which are believed to work in concert to promote tumor cell motility. However, Gilkes et al. reveal that active HIF1α alone can promote motility by transactivating mRNA expression of RHOA and ROCK1, two major drivers of cell motility (3). The abundance of these two hypoxia-induced promigratory factors has dramatic feed-forward effects on other promotility pathways, resulting in increased focal adhesion kinase (FAK) and myosin light-chain kinase (MLCK) activity.

Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden.

MP73-06 DRUG RESISTANCE CONSEQUENCES OF TUMOR HETEROGENEITY IN METASTATIC RENAL CELL CARCINOMA USING ULTRA-FAST PATIENT DERIVED XENOGRAFTS AND MULTIREGIONAL GENOMIC SEQUENCING

Source of Funding: This study was funded by grants from National Key Projects for Infectious Diseases of China (2012ZX10002012-007, 2016ZX10002018-008), National Natural Science Foundation of China (31100629, 31270863, 81372755, 31470794, 81402082, 81402085, 81471621, 81472227, 81472376, 31570803, 81501999, 81671628 and 81672324), Program for New Century Excellent Talents in University (NCET-13-0146) and Shanghai Municipal Commission of Health and Family Planning Program (20144Y0223). All these study sponsors have no roles in the study design, in the collection, analysis, and interpretation of data.

PD65-02 ONCOSOMES AS A NOVEL LIQUID BIOPSY BIOMARKER FOR QUANTIFYING METASTATIC CANCER DYNAMICS IN REAL-TIME

Laurent Briollais, Hilmi Ozcelik, Jingxiong Xu, Toronto, Canada; Maciej Kwiatkowski, Aarau, Switzerland; Emilie Lalonde, Dorota H Sendorek, Neil E Fleshner, Toronto, Canada; Franz Recker, Aarau, Switzerland; Cynthia Kuk, Ekaterina Olkhov-Mitsel, Toronto, Canada; Sevtap Savas, St. John’s, Canada; Sally Hanna, Tristan Juvet, Geoffrey A Hunter, Matt Friedlander, Hong Li, Karen Chadwick, Ioannis Prassas, Antoninus Soosaipillai, Toronto, Canada; Marco Randazzo, Aarau, Switzerland; John Trachtenberg, Ants Toi, Yu-Jia Shiah, Michael Fraser, Theodorus van der Kwast, Robert G Bristow, Bharati Bapat, Eleftherios P. Diamandis, Paul C Boutros, Alexandre R Zlotta*, Toronto, Canada

PD04-12 CLINICAL CORRELATION OF PATIENT-DERIVED XENOGRAFT MODEL USING THE EX-OVO AVIAN EMBRYO TO PREDICT TARGETED THERAPY TUMOR RESISTANCE IN RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Cytoreductive nephrectomy (CN) remains a mainstay in the treatment of metastatic renal cell carcinoma (mRCC). Prior literature has shown around 2/3rds of patients do not receive timely systemic therapy (ST) after CN, however there is limited understanding of how a delay affects progression and survival. Our aim was to identify whether a delay of the initiation of ST was associated with worse overall survival as well as to further characterize the reasons for a delay. METHODS: From an institutional database of 2,906 patients surgically treated for renal masses between 2005 and 2016, we identified 70 patients who underwent CN for mRCC and who were initiated on ST in the adjuvant setting. Cox regression analysis was used to evaluate whether delays in systemic therapy > 3 months and > 6 months were predictive of worse overall survival. RESULTS: Of the 70 patients, the majority had a favorable ECOG performance status (90% ECOG 0-1, 10% ECOG 2-3) while only 3 patients had brain metastasis at time of CN. Median age at diagnosis was 60 years. Our cohort had a 2-year overall survival of 60.8% from diagnosis and 49.4% after initiation of ST with a median follow-up of 27.1 months. Median time to ST after CN was 3 months (IQR 1.536.77). 94.2% of patients received targeted therapy while the remainder were treated with IL-2. Delays in initiating ST after CN were not associated with worse overall survival > 3 months after CN (HR 1⁄4 0.64, p1⁄4 0.387, 95% CI 0.24-1.75) and from 3 to <6 months (HR 1⁄4 0.5, p 1⁄4 0.208, 95% CI 0.17-1.47). Interestingly, delays in ST > 6 months were associated with improved survival (HR 1⁄4 0.19, p1⁄40.017, 95% CI 0.740.017). Of the patients who experienced unintended delays, 42.3% were awaiting a clinical trial, 30.8% experienced delayed ST due to patient preference or poor follow-up, and 19.2% had a complication from surgical therapy. CONCLUSIONS: A delay in the initiation of ST in patients with mRCC after CN did not appear to be associated with worse overall survival. The improved survival in patients who initiated ST > 6 months and trend towards improved survival at > 3 months after CN is likely related to an immortal time bias. Ongoing randomized controlled trials may provide more evidence regarding the optimal timing of ST after CN and the clinical implications of a delay in ST.