Investigating Mechanisms through which Calorie Restriction and Pharmacological Mimetics Attenuates Breast Cancer Growth
Creators: name: Ho, Alyssa
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2020

description: Background: Among US women, basal-like and triple-negative breast cancer subtypes confer worse patient survival due to a lack of targetable receptors. Furthermore, obesity is a major public health concern in the US with a prevalence of 41.9% in women aged >20. Obesity significantly increases the risk of developing and dying from breast cancer by impacting its initiation, progression, and metastasis through IGF-1/Akt/mTOR, and inflammation/immune signaling pathways. A promising area in nutrition and cancer research is caloric restriction (CR) which has been shown to reduce tumor incidence and progression; however, in a nutrient-starved state, cancer cells can break down damaged cellular components to maintain viability through autophagy. This study aimed to determine the effectivity of CR regimens reversing the adverse impact of obesity on the tumor transcriptional profile. We also conducted in vitro studies to test the hypothesis that the addition of an autophagy inhibitor would enhance the anticancer activity of pharmacological agents which mimic the metabolic reprogramming effects of CR.

Methods: Young mice were placed on a diet-induced obesity (DIO) regimen for 15 weeks before being randomized to remain on DIO ad libitum or change to a low-fat control diet ad libitum, a 30% chronic calorie-restricted diet (CCR), or an intermittent calorie-restricted (ICR) diet for 10 weeks. Mice were orthotopically transplanted with E0771 (basal-like subtype) breast cancer cells and tumors were excised at endpoint. Differential tumor gene expression was profiled via Affymetrix microarray and analyzed using transcriptome analysis console and Gene Set Enrichment Analysis. In-vitro, E0771 and MDA-MB-468 cells (triple-negative subtype) were treated with BMS-754807, a dual IGF-1R/INSR inhibitor, and Everolimus, an mTORC1 inhibitor, separately and in combination with the autophagy inhibitor, chloroquine (CQ). Cell viability was determined using MTT assays, and target inhibition and potential induction of autophagy was investigated using Western blots. The potential synergistic effect of Everolimus or BMS-754807 in combination with CQ was analyzed using SynergyFinder software.

Results: Calorie-restricted mice displayed reduced tumor mass compared to DIO and control mice. Gene expression analyses demonstrated that tumors from DIO mice have enrichment of gene sets related to tumor progression (e.g. epithelial-to-mesenchymal transition, hypoxia) and underrepresentation of immune-related antitumor gene markers (e.g. interferon response alpha/gamma, Cd8 T-cells). Both CR interventions reversed DIO-related enrichment of pro-tumorigenic and underrepresentation of immune signaling gene sets. In E0771s and MDA-MB-468s, this study confirmed the target inhibition of Everolimus on mTOR. From combination treatments, it was found that BMS-754807 + CQ synergistically inhibited E0771 viability and Everolimus + CQ synergistically inhibited MDA-MB-468 viability.

Conclusion: This study confirms that obesity induces gene expression alterations within the tumor microenvironment that support tumor growth. Our results demonstrate that weight loss by CCR and ICR interventions effectively reverse pro-tumorigenic effects of obesity, reducing angiogenic signaling and increasing the activation/abundance of immune cell markers within the tumor microenvironment. Our findings show that pharmacological metabolic-reprogramming interventions, mimicking CR and autophagy inhibition as treatments for triple-negative/basal-like BC, has the potential for combination treatments to work synergistically against cell growth.