Abstract P3-02-01: Fatty acid uptake as a potentially new resistance mechanism to anti-HER2 treatments in HER2-positive breast cancer
Authors: Vernieri Claudio , Castagnoli Lorenzo , Ligorio Francesca , Belfiore Antonino , Fasano Elena , Corsetto Paola A. , Faraci Simona , Brambilla Marta , Corti Francesca , Triulzi Tiziana , Generali Daniele , Vingiani Andrea , Rizzo Angela M. , Bianchi Giulia V. , Capri Giuseppe , Tagliabue Elda , de Braud Filippo , Pruneri Giancarlo , Pupa Serenella M. 
Published: March 2020

Abstract
Background: Although anti-HER2 treatments, including the monoclonal antibodies trastuzumab (T) and pertuzumab (P) and the tyrosine kinase inhibitor lapatinib (L), have remarkably improved the prognosis of patients with HER2-positive breast cancer (HER2+ BC), acquired resistance to these compounds remains a major clinical issue. HER2-mediated signaling induces fatty acid (FA) de novo biosynthesis, and ongoing clinical trials are investigating FA synthase enzyme (FASN) inhibitors in combination with anti-HER2 treatments. We hypothesized that by inhibiting FA biosynthesis, HER2 inhibitors make HER2+ BC cells dependent on extracellular FA uptake, which could contribute to resistance to anti-HER2 treatments. Methods: In tumor biopsy specimens from HER2+ BC patients treated with preoperative T-containing chemotherapy (ChT), we performed quantitative real time PCR (qRT-PCR) to measure expression levels of the lipoprotein lipase (LPL) enzyme and the FA transporter CD36, two key players in the uptake of extracellular FAs. The same analyses were also performed in surgical tumor specimens from patients failing to achieve pathologic complete response (pCR), and paired Wilcoxon test was used to compare CD36 expression levels of pre- versus post-treatment tumors. In MDA-MB-361, HCC1954 and BT474 HER2+ BC cell lines, we investigated the association between baseline CD36 expression and cells’ sensitivity to L, or their ability to uptake the docosahexanoic acid (DHA) from culture media. Finally, we assessed the antiproliferative effect of combining L with the CD36 inhibitor Sulfo N-Succinimidoyl Oleate (SSO). Results: 82 patients with stage II-III HER2+ BC were treated with preoperative ChT plus T at Fondazione IRCCS Istituto Nazionale dei Tumori between January 2013 and December 2017. As expected, achieving pCR was associated with better relapse-free survival (RFS) and overall survival (OS). Baseline LPL and CD36 expression did not correlate with pCR probability (p=0.15 and 0.1, respectively), nor with RFS (p=0.28 and 0.7, respectively) or OS (p=0.42 and 0.35, respectively). On the other hand, in 20 patients failing to achieve pCR, we found significantly increased LPL (p=0.00096) and a trend towards increased CD36 (p=0.12) expression in post-treatment surgical specimens. In a parallel cohort of 17 stage II-III HER2+ BC patients enrolled in a prospective study, LPL and CD36 expression was significantly increased 21 days after the first administration of single-agent T (p=0.0002 and p&lt;0.0001, respectively). In keeping with our working hypothesis, cell susceptibility to L was inversely associated with CD36 expression or DHA uptake, with L-sensitive BT474 cells displaying the lowest CD36 expression and DHA uptake from culture media. Consistent with analyses on tumor specimens from patients, treatment with L increased CD36 gene expression and protein levels in L-resistant HCC1954 cells, but not in L-sensitive BT474 ones. Finally, SSO showed additive antiproliferative effects in combination with L in HCC1954, but not in BT474 cells. Conclusions: This is the first study to show that pharmacological inhibition of HER2 induces short- and long-term upregulation of LPL/CD36 expression in HER2+ BC cell lines and tumor specimens, and that CD36 inhibition sensitizes intrinsically resistant HER2+ BC cells to L. Prospective clinical studies are required to validate the role of enhanced FA uptake as a potential mechanism of resistance to anti-HER2 treatments in HER2+ BC.
Citation Format: Claudio Vernieri, Lorenzo Castagnoli, Francesca Ligorio, Antonino Belfiore, Elena Fasano, Paola A. Corsetto, Simona Faraci, Marta Brambilla, Francesca Corti, Tiziana Triulzi, Daniele Generali, Andrea Vingiani, Angela M. Rizzo, Giulia V. Bianchi, Giuseppe Capri, Elda Tagliabue, Filippo de Braud, Giancarlo Pruneri, Serenella M. Pupa. Fatty acid uptake as a potentially new resistance mechanism to anti-HER2 treatments in HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-01.