Abstract P6-11-15: Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment
Authors: Pendleton Katherine E. , Baek Mokryun L. , Lee Junegoo , Tan Lin , Johnson Hannah L. , Dobrolecki Lacey E. , Barrish James P. , Lewis Michael T. , Lorenzi Philip L. , Stossi Fabio , Echeverria Gloria V. 
Published: March 2023

Abstract
Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which limited targeted therapies are available. Therefore, conventional chemotherapy remains the backbone of standard neoadjuvant treatment (NACT) for TNBC patients. Unfortunately, ~45% of patients will have substantial residual tumor burden post neoadjuvant chemotherapy, leading to poor prognoses (PMID: 28135148). Recently, it has been demonstrated that mitochondrial oxidative phosphorylation (oxphos) is upregulated and is a therapeutic vulnerability in chemoresistant TNBC (PMID: 30996079; Baek et al., BioRxiv doi.org/10.1101/2022.02.25.481996). However, mechanisms driving increased oxphos in chemoresistant TNBC are not understood. Upregulated fatty acid (FA) metabolism is a common adaptation in tumors, providing an energy source through fatty acid β-oxidation (FAO), and promoting lipid accumulation after fatty acid synthesis (FAS) when energy needs are met. Chemotherapy can induce oxidative stress through the generation of reactive oxygen species. Cancer cells adapt to these damaging molecules by increasing de novo lipogenesis, resulting in the accumulation of lipid droplets (LDs) in the cytosol (PMID: 32782526, 20876798). We hypothesize that TNBC cells metabolically adapt to the stress of NACT by upregulating lipid metabolic pathways, providing highly energetic molecules that can be utilized to drive oxphos in chemoresistant TNBC. Methods: Using orthotopic patient-derived xenograft (PDX) models of TNBC (PIM001-P, PMID: 30996079, HCI-010, PMID: 22019887; WHIM14, PMID:24055055), we are measuring protein levels of fatty acid synthase (FASN) in vehicle tumors vs residual tumors surviving treatment with the standard front-line neoadjuvant chemotherapy regimens (Adriamycin plus cyclophosphamide (AC), docetaxel, carboplatin, or docetaxel+carboplatin) using immunohistochemistry (IHC). Vectra 3 microscopy (Akoya) is being used to quantify tumor cell-specific staining. We complemented our IHC analysis with reverse-phase protein array (RPPA). To assess LD accumulation in residual PDX tumors, we conducted transmission electron microscopy (TEM). To complement these PDX studies, we modeled the residual tumor metabolic state in cultured human TNBC cells. Following treatment with the IC50 of standard chemotherapeutic agents (AC, carboplatin, paclitaxel, docetaxel), we assessed oxphos by measuring oxygen consumption rate (OCR) using a Seahorse Bioanalyzer (Agilent). Further, we tested LD accumulation using LipidTOX staining. In ongoing studies, we are measuring incorporation of 13C palmitate into the tricarboxylic acid cycle (TCA) prior to and following chemotherapy treatments to assess if lipids fuel mitochondrial metabolism in residual TNBC cells. Results/Discussion: IHC in the PIM001-P PDX model after in vivo AC treatment revealed increased levels of FASN in post-AC residual tumors compared to the treatment-naive tumors. Further, key proteins involved in fatty acid synthesis, FASN and Acetyl-CoA carboxylase, were significantly increased in residual PIM001-P cells that survived AC compared to vehicle by RPPA. TEM analysis of the HCI-010 PDX revealed significantly more LDs in carboplatin-treated tumors compared to vehicle. This finding was supported by increased LDs observed in TNBC cell lines treated with NACT compared to vehicle in our LipidTOX analyses. Taken together, these data indicate that NACT induces increased expression of key lipid metabolism proteins and accumulation of cytosolic LDs. Our future experiments will reveal if chemoresistant TNBC cells preferentially utilize and incorporate lipids into the tricarboxylic acid cycle, in turn driving oxphos. These data have the potential to provide rationale for the incorporation of FAO/LD inhibitors in sequential combinations with conventional chemotherapies to more effectively kill TNBC cells that are chemo-refractory.
Citation Format: Katherine E. Pendleton, Mokryun L. Baek, Junegoo Lee, Lin Tan, Hannah L. Johnson, Lacey E. Dobrolecki, James P. Barrish, Michael T. Lewis, Philip L. Lorenzi, Fabio Stossi, Gloria V. Echeverria. Lipid accumulation in residual triple negative breast cancer cells surviving chemotherapy treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-15.