Lupu, Ruth

HER2 transactivation by the HER3 ligand heregulin (HRG) promotes an endocrine-resistant phenotype in the estrogen receptor-positive (ER+) luminal-B subtype of breast cancer. The underlying biological mechanisms that link them are, however, incompletely understood. Here, we evaluated the putative role of the lipogenic enzyme fatty acid synthase (FASN) as a major cause of HRG-driven endocrine resistance in ER+/HER2-negative breast cancer cells. MCF-7 cells engineered to stably overexpress HRG (MCF-7/HRG), an in vitro model of tamoxifen/fulvestrant-resistant luminal B-like breast cancer, showed a pronounced up-regulation of FASN gene/FASN protein expression. Autocrine HRG up-regulated FASN expression via HER2 transactivation and downstream activation of PI-3K/AKT and MAPK-ERK1/2 signaling pathways. The HRG-driven FASN-overexpressing phenotype was fully prevented in MCF-7 cells expressing a structural deletion mutant of HRG that is sequestered in a cellular compartment and lacks the ability to promote endocrine-resistance in an autocrine manner. Pharmacological inhibition of FASN activity blocked the estradiol-independent and tamoxifen/fulvestrant-refractory ability of MCF-7/HRG cells to anchorage-independently grow in soft-agar. In vivo treatment with a FASN inhibitor restored the anti-tumor activity of tamoxifen and fulvestrant against fast-growing, hormone-resistant MCF-7/HRG xenograft tumors in mice. Overall, these findings implicate FASN as a key enabler for endocrine resistance in HRG+/HER2- breast cancer and highlight the therapeutic potential of FASN inhibitors for the treatment of endocrine therapy-resistant luminal-B breast cancer.

One of the greatest challenges in the cancer immunotherapy field is the need to biologically rationalize and broaden the clinical utility of immune checkpoint inhibitors (ICIs). The balance between metabolism and immune response has critical implications for overcoming the major weaknesses of ICIs, including their lack of universality and durability. The last decade has seen tremendous advances in understanding how the immune system’s ability to kill tumor cells requires the conspicuous metabolic specialization of T-cells. We have learned that cancer cell-associated metabolic activities trigger shifts in the abundance of some metabolites with immunosuppressory roles in the tumor microenvironment. Yet very little is known about the tumor cell-intrinsic metabolic traits that control the immune checkpoint contexture in cancer cells. Likewise, we lack a comprehensive understanding of how systemic metabolic perturbations in response to dietary interventions can reprogram the immune checkpoint landscape of tumor cells. We here review state-of-the-art molecular- and functional-level interrogation approaches to uncover how cell-autonomous metabolic traits and diet-mediated changes in nutrient availability and utilization might delineate new cancer cell-intrinsic metabolic dependencies of tumor immunogenicity. We propose that clinical monitoring and in-depth molecular evaluation of the cancer cell-intrinsic metabolic traits involved in primary, adaptive, and acquired resistance to cancer immunotherapy can provide the basis for improvements in therapeutic responses to ICIs. Overall, these approaches might guide the use of metabolic therapeutics and dietary approaches as novel strategies to broaden the spectrum of cancer patients and indications that can be effectively treated with ICI-based cancer immunotherapy.

Abstract Fatty acid synthase (FASN) is a novel therapeutic target for cancer as it has increased expression in many different types of cancers. There has been significant understanding that cancer cells can hijack and modify tightly regulated metabolic pathway for its own survival. FASN is the enzyme that synthesizes palmitate from malonyl-CoA for energy storage, structure and biomolecules. FASN expression is low to undetectable in normal cells with the exception of liver and adipose tissue. In this study, we investigated the role of FASN in breast cancer cells. We observed that in highly expressing FASN breast cancer cells, TVB-3166, FASN inhibitor, results in a decrease in cellular proliferation and an increase in cellular death. FASN inhibition increased reactive oxygen species (ROS) and altered NADPH/NADP+ ratio. Increase in ROS and NADPH have been linked to the intrinsic apoptosis pathway. Additionally, there is an increase in expression of the BH3-only proteins Bim, Puma and Noxa, leading to the permeabilization of the mitochondria and the release of cytochrome c. Furthermore, after prolong exposure to TVB-3166 a number of cells enter into a senescent state. This was observed the in detection of β-galactosidase and confirmed by the expression of senescent markers including ARF, INK4A, and CIP. Additionally, when the cells were treated with a CDK4/6 inhibitor in combination with the FASN inhibitor TVB-3166, an increase in stress induced premature senescence was observed. Our data demonstrates that inhibition of FASN induces intrinsic apoptosis while it stimulates cellular senescence to the apoptotic resistant cells. Therefore, the ability for TVB-3166 to both eradicate and overturn cell growth makes it potential therapeutic modality. Citation Format: Travis Van der Steen, George Kemble, Ruth Lupu. Targeting fatty acid synthase induces apoptosis and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 708.

Abstract Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States and continues to be hindered with limited treatment options. One of the current therapeutic regimens for pancreatic cancer often include the nucleoside analog gemcitabine (Gem), while it has been used for many years, the overall survival rate lingers around 6%. New therapies are needed to treat this disease. Fatty acid synthase (FASN) has received increased attention as a potential target for cancer therapy in the last decade. FASN is the key enzyme in the de novo synthesis of palmitate from malonyl-CoA. Palmitate is the precursor other long-chain fatty acids (FA). However, the precise mechanism by which pharmacological interference with endogenous FA biogenesis might facilitate apoptosis in tumor cells remains unresolved. We herein explored the molecular relationship between FASN activity, oxidative stress/redox balance, and the intrinsic apoptotic pathway in FASN overexpressing-dependent pancreatic cancer cells. About 68% of Pancreatic Ductal Carcinomas (PDA) expresses high levels of Fatty acid synthase (FASN), and its expression is correlated with disease free survival. A newly developed FASN inhibitor with excellent pharmaceutical properties was used for our studies TVB-3166 (in vitro) and TVB-3664 (in vivo) and ten different PDX derived cells were used for the studies. Pharmacological blockade of FASN activity significantly increased the NADPH/NADP+ ratio, promoted the production of reactive oxygen species (ROS). Accordingly, the inhibition of endogenous FA synthesis promoted mitochondrial membrane permeabilization, cytochrome c release, and apoptotic cell death. Each step of the FASN inhibition/BH3-only protein axis could be reversed with a ROS scavenger or the FASN end-product palmitate, thus confirming that the delineated pro-apoptotic cascade likely reflects on-target effects of FASN inhibition. Most importantly, the in vivo studies using PaCa-PDX reveal a significant decrease in tumor growth and furthermore a synergistic effect of between TVB-3664 and Gem. In conclusion, our findings uncover a novel FASN-dependent mitochondrial priming that links de novo FA biosynthesis with the intrinsic apoptotic threshold in cancer cells. The discovery that FASN-inhibited cancer cells exist in an apoptosis-prone state highly sensitive to BH3 mimetics warrants clinical exploration in FASN-overexpressing pancreatic cancer patients. Citation Format: Travis Vander Steen, Li Ding, Yu Zhang, George Kemble, Daniel Billadeau, Ruth Lupu. Fatty acid synthase: A new therapeutic target for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3723.

Abstract Fatty Acid Synthase (FASN), the key multienzyme in de novo lipogenesis, is upregulated and activated in numerous carcinomas, including breast cancer. Fatty acids play a fundamental role in cellular structure, energy production and storage and are intermediates in the biosynthesis of hormones and other biologic molecules. Therefore, FASN is a novel therapeutic target for treatment in cancer cells that have upregulated FASN protein. BT474 breast cancer cells, which overexpress FASN, were treated with a pharmacologic FASN inhibitor and resulted in apoptosis in a dose-dependent manner. Inhibition of FASN simultaneously upregulates the pro-apoptotic BCL2 family members Bim, Puma, and Noxa; additionally, FASN inhibition alters the NDAPH/NADP+ balance and induces reactive oxygen species (ROS) production. Most significantly, FASN inhibition induces sensitization of breast cancer cells to the BH3 mimetic navitoclax, a potential opportunity to use BH3 mimetics for the treatment of breast carcinomas. These results support further preclinical and potentially clinical development of a dual FASN and Bcl-2 treatment for breast cancer tumors that express FASN. In addition to apoptosis, there is an increase in cellular senescence in breast cancer cells overexpressing FASN when treated with FASN inhibitors. The increase in ROS, a known effect of FASN inhibition, is known to induce cellular stress, resulting in both apoptosis and senescence. Increased senescence was initially observed by x-gal staining of BT474 cells treated with FASN inhibitors via microscopy and FLOW. We also observed an increased expression of several pro-senescent genes both at the protein and mRNA levels, including p16/INK4a, p14ARF/p19ARF, p15, and p21. A hallmark of age-induced cellular senescence is the shortening of telomeres. However, telomere shortening was not observed, suggesting that senescence was a result of FASN inhibition and activation of senescence-induced genes. Therefore, inhibition of FASN appears to be an ideal target for therapeutic treatment of breast cancers that overexpress FASN through both apoptosis and senescence. Citation Format: Travis Vander Steen, Ruth Lupu. Inhibition of FASN induces apoptosis and senescence in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 318.

Evidence is provided that the fibroproliferative actions of TGF‐β are dependent on a metabolic adaptation that sustains pathologic growth. Specifically, profibrotic TGF‐β signaling is shown to require fatty acid synthase (FASN), an essential anabolic enzyme responsible for the de novo synthesis of fatty acids. With the use of pharmacologic and genetic approaches, we show that TGF‐β‐stimulated FASN expression is independent of Smad2/ 3 and is mediated via mammalian target of rapamycin complex 1. In the absence of FASN activity or protein, TGF‐β ‐driven fibrogenic processes are reduced with no apparent toxicity. Furthermore, as increased FASN expression was also observed to correlate with the degree of lung fibrosis in bleomycin‐treated mice, inhibition of FASN was examined in a murine‐treatment model of pulmonary fibrosis. Remarkably, inhibition of FASN not only decreased expression of profibrotic targets, but lung function was also stabilized/improved, as assessed by peripheral blood oxygenation.—Jung, M.‐Y., Kang, J.‐H., Hernandez, D. M., Yin, X., Andrianifahanana, M., Wang, Y., Gonzalez‐Guerrico, A., Limper, A. H., Lupu, R., Leof, E. B. Fatty acid synthase is required for profibrotic TGF‐β signaling. FASEB J. 32, 3803–3815 (2018). www.fasebj.org