Clinical and therapeutic relevance of the metabolic oncogene fatty acid synthase in HER2+ breast cancer

Abstract

Fatty acid synthase (FASN) is a key lipogenic enzyme for de novo fatty acid biosynthesis and a druggable metabolic oncoprotein that is activated in most human cancers. We evaluated whether the HER2-driven lipogenic phenotype might represent a biomarker for sensitivity to pharmacological FASN blockade. A majority of clinically HER2-positive tumors were scored as FASN overexpressors in a series of almost 200 patients with invasive breast carcinoma. Re-classification of HER2-positive breast tumors based on FASN gene expression predicted a significantly inferior relapse-free and distant metastasis-free survival in HER2+/FASN+ patients. Notably, non-tumorigenic MCF10A breast epithelial cells engineered to overexpress HER2 upregulated FASN gene expression, and the FASN inhibitor C75 abolished HER2-induced anchorage-independent growth and survival. Furthermore, in the presence of high concentrations of C75, HER2-negative MCF-7 breast cancer cells overexpressing HER2 (MCF-7/HER2) had significantly higher levels of apoptosis than HER2-negative cells. Finally, C75 at non-cytotoxic concentrations significantly reduced the capacity of MCF-7/HER2 cells to form mammospheres, an in vitro indicator of cancer stem-like cells. Collectively, our findings strongly suggest that the HER2-FASN lipogenic axis delineates a group of breast cancer patients that might benefit from treatment with therapeutic regimens containing FASN inhibitors.

Figure 1

Immunophenotypic classification of FASN-overexpression in HER2-negative and HER2-positive invasive breast carcinomas.

Figure 2

Kaplan-Meier Relapse-free survival curves of breast cancer subtypes stratified by low/high FASN gene expression.

Figure 3

Kaplan-Meier Distant metastasis-free survival curves of breast cancer subtypes stratified by low/high FASN gene expression.

Figure 4. Pharmacological blockade of FASN activity suppresses the HER2-induced transforming phenotype in breast epithelial cells

Left. Transcriptional activities were expressed as relative (×-fold) changes in luciferase activity of FASN promoter-transfected MCF10A and MCF10/HER2 cells in response to treatments after normalization to pRL-CMV luciferase activity. Each experimental value represents the mean fold increase (columns) ± SD (bars) from at least three independent experiments measured in triplicate. Right. Soft-agar colony formation assay of MCF10A and MCF10A/HER2 cells cultured with/without C75. Each experimental value represents the mean colony number (columns) ± S. D. (bars) from at least three independent experiments measured in triplicate.

Figure 5. HER2-overexpressing breast cancer cells are exquisitely sensitive to FASN inhibition-induced cell death

Top. Cell viability and IC₅₀ values were determined using MTT reduction. Data summarize the mean±SD of five independent experiments measured in triplicate. Bottom. Cell cycle analysis of MCF-7/neo and MCF-7/HER2 cells by flow cytometry after 48 h with/without C75 (10 μg/mL). Representative cell cycle profiles are shown for each cell line and treatment. Cell cycle analyses were repeated at least three times. Dashed boxes highlight the sub-G₁ (apoptotic) cell populations in each cell line and treatment.

Figure 6. HER2 gene-amplified but not HER2-negative breast cancer cells are sensitive to FASN inhibition-induced apoptosis

Top. HER2 protein expression in a panel of human breast cancer cell lines. Bottom. Fold-change in the C75-induced percentage of sub-G₁, G₀/G₁, S, and G₂/M phases relative to untreated MDA-MB-231 and BT-474 cells (=1-fold in each phase). Cell cycle analyses were repeated at least three times. Standard deviations were less 0.1-fold for each experimental condition.

Figure 7. Pharmacological blockade of FASN activity suppresses the HER2-driven tumor-initiating capacity of CSCs

Mammosphere formation of MCF-7 and MCF-7/HER2 cells with/without C75 (left) or (-)-C75 (right). MSFE of vehicle-alone control cells was normalized to one in each cell line.