Metabolic determinants of therapy resistance in estrogen receptor positive breast cancer

The majority of breast cancers are estrogen receptor positive (ER+) and epidermal growth factor receptor two negative (HER2-) and are dependent on estrogens for their growth and survival. Endocrine therapy (ET), which acts by targeting the ER signaling pathway, is the standard of care for these tumors. Unfortunately, ~40% of women relapse with ET resistant disease and understanding the metabolic reprogramming underlying such resistance is an important need. In the first part of this thesis, we performed a global gene expression analysis in ET resistant compared to parental cells revealing a downregulation of the neutral and basic amino acid transporter SLC6A14 governed by enhanced miR-23b-3p expression, resulting in impaired amino acid uptake. Biochemical and biological assays showed that this deregulation of the amino acid metabolism is supported by autophagy activation and increased import of acidic amino acids (i.e., aspartate and glutamate) mediated by the cognate SLC1A2 transporter in ET resistant cells. We then analyzed aspartate and glutamate destiny by radioactive tracing assay and LC-MS, and we observed that both amino acids (i) fuel lipid, protein, and nucleotide biosynthesis and (ii) enhance 13C-labelled TCA cycle intermediates (e.g., citrate, α-ketoglutarate, succinate, fumarate, and malate) together with uridine-5'-triphosphate (UTP, DNA synthesis) and glutamine (protein synthesis) levels, indicating that both glutamate and aspartate boost TCA and interrelated anaplerotic pathways in ET resistant cells compared to the parental counterpart. Interestingly, Seahorse analysis showed that the concomitant deprivation of aspartate and glutamate in ET resistant cells significantly impaired oxygen consumption rate and subsequent oxidative potential, whereas the withdrawal of each single amino acid has no effect, suggesting that the mitochondrial-dependent catabolism is sustained by either one or the other amino acid. The clinical relevance of these findings is validated by multiple orthogonal approaches in large cohorts of ET treated patients and in patient-derived xenografts (PDX). Targeting amino acid metabolic reprogramming re-sensitizes ET resistant cells to the therapy and impairs their aggressive features (e.g., proliferation, invasion, clonogenicity/stemness), including their metastatic ability in in vivo experiments. In the second part of the thesis, we decided to broaden the investigation of therapy resistance in ER+ breast cancer, based on the notion that, recent clinical trial have shown that a superior clinical outcome is achieved in a subset of ER+/HER2- metastatic breast cancer patients receiving a combination of a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor (e.g., palbociclib, PD) together with the standard ET. Moreover, CDK4/6 inhibitors have also been tested in ER+/HER2+ preclinical breast cancer models and reported encouraging results. Despite the clinical advances of a combinatorial therapy using ET plus CDK4/6 inhibitors, potential limitations (i.e., PD resistance) could emerge and investigating the metabolic adaptations underlying such resistance warrants further elucidations. Thus, we subjected a panel of ER+ breast cancer cells sensitive to PD (PDS) and their resistant derivatives (PDR) to a metabolic profiling using an array of complementary high-end techniques including 14C-radioactive glucose tracing, western blotting, and qRT-PCR analysis of key metabolic enzymes, together with Seahorse analysis coupled to gas chromatography-mass spectrometry (GC-MS). This approach revealed a differential metabolic behavior of PDR cells when compared to PDS, independently of their proliferative status. Moreover, the metabolic phenotype of the PDR cells showed significant differences between cells that are HER2+ and HER2-. Specifically, ER+/HER2+ PDR cells are characterized by enhanced glucose dependency in both basal and under metabolic stress conditions compared to PDS cells. Conversely, ER+/HER2- PDR cells exhibit a decreased glycolytic phenotype compared to their parental counterpart. We have therefore targeted these glucose dependencies using 2-deoxyglucose, glucose deprivation, galactose-containing medium, and HK2 (hexokinase two) silencing. Crucially, glycolysis inhibition re-sensitizes ER+/HER2+ PDR cells to PD as well as potentiates the response of ER+/HER2- PDS cells to the therapy. Finally, HK2 higher-expressing ER+/HER2+ breast cancers show a worse prognosis when compared to the lowering-expressing patients, even in multivariate analysis, suggesting that HK2 may characterize a subset of tumors more susceptible to therapy resistance and subsequent relapse. In conclusion, our results suggest that the deregulated tumor metabolism could represent a strategic mechanism that sustains therapy resistance and offer a series of predictive biomarkers and potential targetable pathways to be exploited to combat or delay ET resistance in ER+ breast cancer.