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Supplementary figure 2. Aspirin treatment results in activation of AMPK and decrease in mTORC1 signaling.

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiologic studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA. Here, we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on COX-2 and NF-κB. We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mTORC1 inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, whereas combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports the evaluation of aspirin and PI3K pathway inhibitors as a combination therapy for targeting breast cancer. Cancer Res; 77(3); 790–801. ©2016 AACR.

Supplementary figure 1. Expression of mutant PIK3CA increases COX-2 expression and IKKbeta/NF-KB signaling.

SummaryFerroptosis is a form of regulated cell death with roles in degenerative diseases and cancer. Ferroptosis is driven by excessive iron-dependent peroxidation of membrane phospholipids, especially those containing the polyunsaturated fatty acid arachidonic acid. Here, we reveal that an understudied Golgi membrane scaffold protein, MMD, promotes susceptibility to ferroptosis in ovarian and renal carcinoma cells. Upregulation of MMD correlates with sensitization to ferroptosis upon monocyte-to-macrophage differentiation. Mechanistically, MMD interacts with ACSL4 and MBOAT7, two enzymes that catalyze consecutive reactions in the biosynthesis of phosphatidylinositol (PI) containing arachidonic acid. MMD increases cellular levels of arachidonoyl-phospholipids and heightens susceptibility to ferroptosis in an ACSL4- and MBOAT7-dependent manner. We propose that MMD potentiates the synthesis of arachidonoyl-PI by bridging ACSL4 with MBOAT7. This molecular mechanism not only clarifies the biochemical underpinnings of ferroptosis susceptibility, with potential therapeutic implications, but also contributes to our understanding of the regulation of cellular lipid metabolism.Graphical Abstract

Ferroptosis—an iron-dependent, non-apoptotic cell death process—is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2–5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR–Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome–ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis. The cellular organelles peroxisomes contribute to the sensitivity of cells to ferroptosis by synthesizing polyunsaturated ether phospholipids, and changes in the abundances of these lipids are associated with altered sensitivity to ferroptosis during cell-state transitions.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers

// Whitney S. Henry 1,* , David G. Hendrickson 2,3,* , Francisco Beca 1 , Benjamin Glass 1 , Marianne Lindahl-Allen 4 , Lizhi He 4 , Zhe Ji 4 , Kevin Struhl 4 , Andrew H. Beck 1 , John L. Rinn 2,3 and Alex Toker 1 1 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 2 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA 3 Broad Institute of MIT and Harvard, Cambridge, MA, USA 4 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA * These authors have contributed equally to this work Correspondence to: Alex Toker, email: // John L. Rinn, email: // Keywords : lncRNA, Src, PI3K, breast cancer, LINC00520 Received : March 29, 2016 Accepted : September 02, 2016 Published : September 10, 2016 Abstract Long non-coding RNAs (lncRNAs) have been implicated in normal cellular homeostasis as well as pathophysiological conditions, including cancer. Here we performed global gene expression profiling of mammary epithelial cells transformed by oncogenic v-Src , and identified a large subset of uncharacterized lncRNAs potentially involved in breast cancer development. Specifically, our analysis revealed a novel lncRNA, LINC00520 that is upregulated upon ectopic expression of oncogenic v-Src , in a manner that is dependent on the transcription factor STAT3. Similarly, LINC00520 is also increased in mammary epithelial cells transformed by oncogenic PI3K and its expression is decreased upon knockdown of mutant PIK3CA. Additional expression profiling highlight that LINC00520 is elevated in a subset of human breast carcinomas, with preferential enrichment in the basal-like molecular subtype. ShRNA-mediated depletion of LINC00520 results in decreased cell migration and loss of invasive structures in 3D. RNA sequencing analysis uncovers several genes that are differentially expressed upon ectopic expression of LINC00520, a significant subset of which are also induced in v-Src -transformed MCF10A cells. Together, these findings characterize LINC00520 as a lncRNA that is regulated by oncogenic Src, PIK3CA and STAT3, and which may contribute to the molecular etiology of breast cancer.

75% of women suffering from ovarian cancer are diagnosed at late stage of the disease often associated with cancer cell infiltration into the peritoneal cavity. Standard therapy is tumor resection and subsequent platin-based chemotherapy. Relapse is frequently observed caused by persisting cancer cells. Persister cells comprise a therapy-tolerant subpopulation of cancer cells and repeated therapies presumably select for increased tolerance. The present project aims to find molecular markers for the identification and targeted eradication of persister ovarian cancer cells. Persister cells of the cisplatin-sensitive high-grade serous ovarian cancer cell line, OVCAR-3, were selected after cisplatin incubation with clinically relevant doses. Individual clones were isolated from control or cisplatin challenged OVCAR-3 cells. A subpopulation of the resulting clonal persister cells showed a distinct morphological phenotype characterized by increased migration and high viability in short- and long-term survival after cisplatin treatment. A significant upregulation (p Additionally, the ovarian cancer OVCAR-8 luciferase reporter cell line was transduced with the “Watermelon” (WM) library [1]. The WM library is a complex barcode library that enables simultaneous tracing of cell lineage and the cellular transcriptional and proliferative states. To study cancer relapse in a CDX model, WM-labelled OVCAR-8-Luc cells were injected into mice and pulse-treated with carboplatin. Single cell RNA sequencing of 900 cells isolated from solid metastases, ascites and treatment-naïve mice revealed an increase in multiple ER stress markers including ATF3, ATF4, JUN and XBP1 in the most abundant solid relapse lineages. Furthermore, analysis of clinical data from GEO, EGA and TCGA showed that low ATF3 expression is associated with increased 5-year-survival of patients with high-grade serous ovarian cancer (Logrank p=6.3e-6). We found that increased ER stress correlates with survival of cancer persister cells and incidence of metastases. This phenomenon was observed in cell lines, a CDX model and clinical data from human ovarian cancer samples. We propose ER stress response, exemplified by ATF3, as a molecular marker to identify and therapeutic targeting of ovarian cancer persister cells. [1] Metabolic switching underlies the ability of cancer persister cells to cycle under drug treatment, Oren Y et al., unpublished Citation Format: Kathrin Böpple, Meng Dong, Markus Kleih, Andrea Gaißler, Yaara Oren, Whitney S. Henry, Moshe Oren, Yael Aylon, Bernd Winkler, Aviv Regev, Robert A. Weinberg, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Ovarian cancer persister cells are characterized by enhanced ER stress gene expression correlating with poor survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4320.

NFAT transcription factors are key regulators of gene expression in immune cells. In addition, NFAT1-induced genes play diverse roles in mediating the progression of various solid tumors. Here we show that NFAT1 induces the expression of the IL8 gene by binding to its promoter and leading to IL8 secretion. Thapsigargin stimulation of breast cancer cells induces IL8 expression in an NFAT-dependent manner. Moreover, we show that NFAT1-mediated IL8 production promotes the migration of primary human neutrophils in vitro and also promotes neutrophil infiltration in tumor xenografts. Furthermore, expression of active NFAT1 effectively suppresses the growth of nascent and established tumors by a non cell-autonomous mechanism. Evaluation of breast tumor tissue reveals that while the levels of NFAT1 are similar in tumor cells and normal breast epithelium, cells in the tumor stroma express higher levels of NFAT1 compared to normal stroma. Elevated levels of NFAT1 also correlate with increased neutrophil infiltrate in breast tumors. These data point to a mechanism by which NFAT1 orchestrates the communication between breast cancer cells and host neutrophils during breast cancer progression.

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiological studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA. Here we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on cyclooxygenase-2 (COX-2) and nuclear factor-kappa B (NF-κB). We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mammalian target of rapamycin complex 1 (mTORC1) inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, while combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports evaluation of aspirin and PI3K pathway inhibitors as combination therapy for targeting breast cancer.

Although aspirin is primarily administered as an antipyretic and analgesic, numerous observational and randomized clinical trials have suggested a potential chemotherapeutic use. Recently, two independent studies demonstrated that aspirin use is associated with superior survival in colorectal cancer patients with mutant-PIK3CA but not among those with the wild-type gene. Despite these seminal observations, the molecular basis for this phenomenon remains poorly understood. Furthermore, whether this observation occurs in breast cancer is unknown. Given that (1) mutation of PIK3CA, the gene encoding the catalytic subunit of the phosphoinositide 3-kinase (PI 3-K) is highly prevalent in breast cancer and that (2) aspirin use is associated with a decrease in breast cancer mortality and distant recurrence, the relationship between PI 3-K pathway activation and the protective benefit of aspirin in breast cancer was investigated. Here, we utilize both PIK3CA mutant breast cancer cell lines and a well-characterized system of immortalized mammary epithelial cells stably expressing either wild-type and mutant PIK3CA E545K and H1047R respectively. Mutant PIK3CA breast cancer cells show a dose-dependent decrease in cell viability with increasing concentration of aspirin/salicylate. In these cells, co-treatment of aspirin with PI 3-K pathway inhibitors BYL719 or BKM120 caused a more significant decrease on cell growth compared to each single agent alone. In 3D culture, MCF10A cells expressing mutant PIK3CA-H1047R showed greater sensitivity to aspirin compared to cells expressing wild-type PIK3CA. Gene expression profiling and quantitative qRT-PCR was employed to identify and validate mutant PIK3CA-regulated genes respectively. Many of these genes were associated with inflammation; among them was the PTGS2 gene which encodes for cyclooxygenase-2 (COX-2). We observed that mutant PIK3CA regulates cyclooxygenase-2 (COX-2) mRNA and protein expression as well as production of prostaglandin E2α (PGE2α). Expression of mutant PIK3CA also increases phosphorylation of IKKβ and IκB; increases mTORC1 signaling and reduces AMPK activity. This is abrogated upon pharmacologic inhibition of mutant PIK3CA with BYL719 and BKM120. Notably, both aspirin and its metabolite salicylate can inhibit these PIK3CA-induced pathways and activate AMPK signaling. An understanding of these key survival and metabolic signaling pathways, which are modulated by aspirin, may help to explain aspirin's beneficial effect in PIK3CA-mutant breast cancers. Given the growing interest in personalized medicine, this pre-clinical study may provide insight for the stratification of patients who are most likely to benefit from adjuvant aspirin therapy. Citation Format: Whitney Henry, Alex Toker. Investigating the chemotherapeutic effects of aspirin in mutant PIK3CA breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2634. doi:10.1158/1538-7445.AM2015-2634