Abstract ED02-03: Metformin to target energy imbalance: Clinical experience in women with early breast cancer
Authors: Bonanni Bernardo , Cazzaniga Massimiliano , Serrano Davide , Puntoni Matteo , Gennari Alessandra , De Censi Andrea 
Published: May 2019

Abstract
Breast cancer burden is largely attributable to western life-style, with insulin resistance playing a major role in its development and progression. Hyperinsulinemia induces proliferative tissue abnormalities both in normal and neoplastic tissue and promotes breast tumor growth in animal models. At the molecular level, the binding of insulin to its receptor results in upregulation of the insulin receptor substrate-2, leading to downstream activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K-Akt) pathways, thus increasing breast cell proliferation. This effect may also be explained by the cross-activation of the insulin-like growth factor (IGF) receptor family. It has also been recently suggested that genetic polymorphisms in genes of the insulin and IGF pathway may influence breast cancer risk and progression. With these premises, interventions aimed at lowering plasma insulin levels, either through lifestyle changes or via pharmacological approaches, are actively being assessed to reduce breast cancer risk. Metformin, the oral insulin-sensitizing drug most widely prescribed for the treatment of type 2 diabetes, is being investigated as cancer preventive and therapeutic agent. Highly cost-effective and generally very well tolerated, with no excess of serious adverse events in a large primary prevention trial of diabetes in healthy subjects, metformin has been shown to have anti-tumor effects in vivo in animal models and in epidemiologic studies, including an increased pathological complete response rate in combination with preoperative chemotherapy in breast cancer patients. A key mechanism of action of metformin is through activation of the AMPK pathway, a key regulator of cellular energy homeostasis. In cancer cells, metformin has shown direct antiproliferative properties through AMPK-dependent and LKB1-dependent mechanisms. Stimulation of AMPK by metformin resulted in a significant inhibition of cell proliferation and activation of MAPK1/2 both in ER negative and ER positive human breast cancer cell lines. In addition, metformin directly affects mitochondrial respiration, compromising the switch to mitochondrial energy generation, a putative mechanism of malignant transformation and cancer progression.
We are currently investigating the anti-proliferative effect and the mechanism of action of metformin, - together with the feasibility and safety of such an approach - in a phase II, window-of-opportunity, randomized, biomarker pre-operative trial of metformin vs. placebo in 300 women with newly diagnosed early breast cancer (stage I and IIA) waiting for surgery at the European Institute of Oncology (IEO), Milan, Italy (trial number S425/408, EudraCT code 2008-004912-10).
Women will receive either metformin 850 mg p.o. once daily for the first 3 days and then twice daily or matching placebo for up to 4 weeks (21 daysα7 days) prior to surgery. This interval of 3 to 4 weeks is ethical in Italy, as the median waiting time from initial diagnosis to surgery is approximately 4 weeks. Baseline core biopsies and fasting blood specimens will be obtained at study entry and at surgery. With a sample size of 150 subjects per arm, the study has 95% power to detect a reduction in post-treatment median Ki67 from 18.4% to 13.6% on metformin versus placebo. With the inclusion of least 60 subjects with ER-negative disease, the study will have 80% power to detect a significant interaction between metformin and ER status, assuming a median reduction on metformin of 4% and 6% in ER positive and ER negative disease, respectively.
Our primary hypothesis is that metformin decreases breast cancer and adjacent normal and dysplastic epithelial cell proliferation (Ki67) in humans by modulating components of signaling pathways critically involved in cell growth (LKB1-AMPK and PI3K pathways) and energy balance shift associated with breast carcinogenesis (Electron Transfer Chain complex I). We also hypothesize a decrease of cyclophilin D and mitochondrial pore formation in epithelial cells isolated from patients on study. Our secondary hypothesis is that metformin is more effective in decreasing normal, dysplastic and malignant epithelial cell proliferation in women with insulin resistance, as measured by HOMA Index &gt; 3.8, and because of the association between obesity and insulin resistance and ER-negative disease, it may be a more active agent in patients with ER-negative breast cancer for which no current preventive agents exist.
We will test these hypotheses with the following specific aims:To determine whether metformin given to patients with early breast cancer will decrease cancer and dysplastic cell proliferation. In our trial we will collect serial tissue and determine Ki67 expression at two different time-points in both arms. Ki67 in a pre-surgical model is considered an established surrogate biomarker to test the antitumor and preventive activity of candidate drugs for breast cancer. We have previously used this model to assess the minimal active dose of tamoxifen in ER-positive breast cancer that led to the implementation of phase IIb and phase III prevention trials of low dose tamoxifen. This model has relevant prevention implications because of the availability of normal and dysplastic cells adjacent to the tumor.To investigate the molecular changes in cancer cell PI3K and energy signaling pathways caused by metformin. We will use Reverse Phase Protein Arrays (RPPA), to identify activated (phosphorilated) proteins and measure signaling pathways at both different time points to evaluate the changes in pathway activation generated by metformin intervention. Also, we will measure the levels of cyclophilin D (protein and mRNA), and assess mitochondrial respiration and permeability transition pore formation in epithelial cells isolated from patients on study.To correlate the proliferation and molecular changes with Single Nucleotide Polymorphisms (SNPs) in the IGF and insulin system. We will study SNPs in the IGFIR, IGFBP3, INSR and PPARgamma genes in a subset of patients to evaluate the interaction between treatment activity and candidate SNPs.To correlate the proliferation and molecular changes with insulin resistance and hormone receptor status. We will employ the homeostasis model assessment (HOMA) and the tumor hormone receptor status to determine whether metformin has a different activity in women with insulin resistance (HOMA Index&gt;3.8) as well as in ER-negative breast cancer.
At the moment, we have blinded preliminary data on the first 89 enrolled patients. Mean±SD age is 52±11 years and mean±SD BMI is 24.4±3.8. Grade 2 nausea and G2 diarrhea occurred in 9% and in 6% of the cases, respectively. Median Ki67 was 18% (range 4%–65%) at baseline biopsy. Compliance is very high. Current data prove our recruitment rate ability (15 subjects per month) and the trial feasibility in this set of patients, with limited toxicity in non-diabetic subjects.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):ED02-03.