Role of hypoxia and hypoxia-inducible factor-1 in tumour immune escape

Previous studies revealed that, upon exposure to hypoxia, tumour cells acquire resistance to the cytolytic activity of IL-2-activated lymphocytes. The MHC class I chain-related (MIC) molecules – comprised of MICA and MICB – are ligands for the activating NKG2D receptor on Natural Killer (NK) and CD8+ T cells. MIC-NKG2D interactions lead to the activation of NK and CD8+ T cells and the subsequent lysis of the tumour cells. The study also showed that the mechanism of the hypoxia-mediated immune escape involves the shedding of MIC, specifically MICA, from the tumour cell surface. The objective of the present study was to determine whether the shedding of MICA requires the expression of hypoxia inducible factor-1 (HIF-1), a transcription factor that regulates cellular adaptations to hypoxia. Exposure to hypoxia (0.5% O2 vs. 20% O2) led to the shedding of MIC from the surface of MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells as determined by flow cytometry. Knockdown of HIF-1α mRNA using siRNA technology resulted in inhibition of HIF-1α accumulation under hypoxic conditions as determined by Western blot analysis. Parallel study revealed that knockdown of HIF-1α also blocked the shedding of MICA from the surface of MDA-MB-231 cells exposed to hypoxia. These results indicate that HIF-1 is required for the hypoxia-mediated shedding of MICA and, consequently, that HIF-1 may play an important role in tumour immune escape. Ongoing studies aim to determine the HIF-1 target genes involved in the shedding of MICA under hypoxia.

Role of Stromal Cell-Derived Factor-1 in Neoangiogenesis in Endometriosis Lesions

Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Treatment for endometriosis primarily focuses on symptom relief, is short term with severe side effects and often leads to recurrence of the condition. Establishing new blood supply is a fundamental requirement for endometriosis lesions growth. This has led to the idea that antiangiogenic therapy may be a successful approach for inhibiting endometriosis. Recent evidence indicates that endothelial progenitor cells (EPCs) contribute to neoangiogenesis of endometriotic lesions. These EPCs are recruited to the lesion site by stromal cell-derived factor-1 (SDF-1). We hypothesize that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions and that administration of SDF-1 blocking antibody will inhibit lesion growth by inhibiting angiogenesis in a murine model of endometriosis. Immunohistochemistry for SDF-1 and CD34 was performed on human endometriosis and normal endometrial samples. Quantification of SDF-1 and EPCs was performed in the blood of endometriosis patients and controls using ELISA and flow cytometry, respectively. A new mouse model of endometriosis was developed using BALB/c-Rag2-/-/IL2rg-/- mice to investigate role of SDF-1 in neoangiogenesis. Either SDF-1 blocking antibody or an isotype control was administered on a weekly basis for four weeks. Weekly samples of peripheral blood from mice were analyzed for SDF-1, other cytokines of interest and EPCs. Mice were euthanized at seven weeks to observe lesion growth and blood vessel development. Our results indicate overabundance of SDF-1 and CD34+ progenitor cells in human endometriotic lesions compared to eutopic endometrium. In the mouse model, SDF-1 and circulating EPC levels decreased from pre-treatment levels after one week, and remained constant over the course of the treatment in both SDF-1 blocking antibody and isotype control groups. In the SDF-1 blocking group, reduced vascularity of lesions, identified by immunofluorescence staining for CD31, was revealed compared to isotype controls. These findings suggest that SDF-1 may be responsible for CD34+ progenitor cell recruitment to the neoangiogenic sites in endometriosis. Blocking of SDF-1 reduces neovascularization of human endometriotic lesions in a mouse model. Further studies on blocking SDF-1 in combination with other antiangiogenic agents are needed.

Hormonal factors and insulin-like growth factor-1 in North American postmenopausal women

Background: Mechanisms underlying the effect of estrogen exposure on breast cancer risk remain unclear. Insulin-like growth factor-1 (IGF-1) levels have been positively associated with breast cancer and are a potential mechanism. Objectives: The objectives of this thesis are: 1) to explore whether the reproductive risk factors and the lifetime cumulative number of menstrual cycles (LCMC), as measures for long-term estrogen exposure, are associated with IGF-1 levels, and 2) to examine the effect of an aromatase inhibitor (AI) on IGF-1 levels, and the potential interaction with BMI. Methods: A cross sectional study and a randomized controlled trial nested with the MAP.3 chemoprevention trial were used to address objective 1 and 2, respectively. 567 postmenopausal women were selected. Anthropometric measurements, lifestyle factors, reproductive characteristics and serum IGF-1 concentrations were collected at baseline and one year. Objective 1. The LCMC was computed as a composite measure of the reproductive characteristics. Multivariable linear regression models were used to assess the association between IGF-1 levels and LCMC and the hormonal risk factors, while adjusting for potential covariates. Objective 2. Changes in IGF-1 were compared between the exemestane and placebo, and effect modification by BMI was tested with an interaction term. Results: Objective 1. Women aged 55 years or older at menopause had 16.26 ng/mL (95% CI: 1.76, 30.75) higher IGF-1 compared to women aged less than 50 years at menopause. Women in the highest category of menstrual cycles (≥500 cycles) had an average 19.00 ng/mL (95%CI: 5.86, 32.14) higher concentration of IGF-1 compared to women in the lowest category (<350). Exogenous hormones had no effect on postmenopausal IGF-1 levels. Objective 2. Exemestane significantly increased IGF-1 levels by 18% (95% CI: 14%-22%); while, placebo had no effect on IGF-1. The changes in IGF-1 were significantly different between the treatment arms (P<0.0001) and no significant interaction was observed between treatment and BMI on IGF-1 changes (P=0.1327). Conclusion: Objective 1. Larger number of menstrual cycles and a later age at menopause are positively associated with IGF-1. IGF-1 may be one mechanism by which prolonged estrogen exposure increases cancer risk. Objective 2. We conclude that the reduced cancer risk observed with AI therapy likely occurs in an IGF-1 independent mechanism. Further studies exploring the clinical consequences of increased IGF-1 on AI therapy are needed.

Heightened maternal inflammation is linked to placental oxidative and nitrosative stress associated with fetal growth restriction in the rat

Deficient trophoblast invasion and spiral artery remodeling are associated with pregnancy complications such as pre-eclampsia (PE) and fetal growth restriction (FGR). Using a model in which pregnant Wistar rats are given daily, low-dose, injections of bacterial lipopolysaccharide (LPS; 10 – 40 µg/kg) on gestational days (GD) 13.5 – 16.5, our group has shown that abnormal maternal inflammation is causally linked to shallow trophoblast invasion, deficient spiral artery remodeling, and altered utero-placental hemodynamics leading to FGR/PE; these alterations were shown to be mediated by TNF-a. The present research evaluated certain consequences of decreased placental perfusion; this was accomplished by examining placental alterations indicative of decreased placental perfusion. Additionally, the role of glyceryl trinitrate (GTN) was determined as a potential therapeutic to prevent the consequences of decreased placental perfusion. Results indicated that dams experiencing heightened maternal inflammation showed significantly greater expression of hypoxia-inducible factor-1a (HIF-1a) and nitrotyrosine, both of which are markers of decreased perfusion and oxidative/nitrosative stress. Contrary to expectations, inflammation did not appear to affect nitric oxide (NO) bioavailability, as revealed by a lack of change in placental or plasma levels of cyclic guanosine monophosphate (cGMP). However, continuous transdermal administration of GTN (25 µg/hr) on GD 12.5 – 16.5 prevented the accumulation of HIF-1a and nitrotyrosine in placentas from LPS-treated rats. These results support the concept that maternal inflammation contributes to placental hypoxia and oxidative/nitrosative stress. Additionally, they indicate that GTN has potential applications in the treatment and/or prevention of pregnancy complications associated with abnormal maternal inflammation.