Targeting ferroptosis with flavonoids for cancer therapy: Mechanisms and opportunities.

Ferroptosis, an iron-dependent form of regulated cell death marked by lipid peroxidation, has emerged as a promising therapeutic target in breast cancer, particularly in aggressive subtypes such as triple-negative breast cancer (TNBC). This systematic review explores the molecular mechanisms underlying ferroptosis sensitivity and resistance, focusing on the interplay between iron metabolism, antioxidant defenses, and tumor microenvironmental factors. Literature retrieved from PubMed and Scopus up to May was analyzed in accordance with PRISMA guidelines, including mechanistic studies, preclinical experiments, and ongoing clinical trials. Findings reveal that breast cancer cells evade ferroptosis through enhanced glutathione synthesis, upregulation of GPX4 and system Xc- and adaptive metabolic reprogramming; yet these same mechanisms create exploitable vulnerabilities, including dependence on cystine, polyunsaturated lipids, and dysregulated iron handling. Therapeutic strategies that target key ferroptosis regulators, such as GPX4, ACSL4, and SLC7A11, or that harness agents like statins, sulfasalazine, and nanoparticle-based iron complexes demonstrate strong potential to overcome chemoresistance and selectively eliminate therapy-resistant cancer cell populations. Taken together, the evidence highlights ferroptosis as a critical Achilles’ heel of breast cancer biology and supports further clinical translation of ferroptosis-inducing therapies to improve outcomes in otherwise refractory breast cancer subtypes.

The major challenges in clinical oncology are the selective delivery of large amounts of therapeutic agents into tumor cells, accurate evaluation of the drug delivery, and timely assessment of the therapeutic response. Theranostic nanoparticles with the abilities to target tumors, carry therapeutic agents, and produce contrasts or signals for tumor imaging offer an exciting means to address these challenges and have a great promise for effective cancer treatment. We have developed a multifunctional theranostic magnetic iron oxide nanoparticle (IONP) platform that utilizes receptor-targeted IONPs to carry single or multiple therapeutic agents for drug delivery and optical and magnetic resonance imaging (MRI) for monitoring the delivery and response. To overcome physical and intrinsic barriers that reduce efficiency of drug delivery and confer drug resistance in human cancers, our theranostic IONPs are targeted to urokinase plasminogen activator receptor (uPAR), thereby taking advantage of high levels of uPAR expression in tumor cells, angiogenic endothelial cells, and active tumor stromal cells. These IONPs allow the drug to overcome the physical barrier in stroma-rich tumors, such as pancreatic cancer and triple-negative breast cancer (TNBC), by serving as carrier vehicles for passage through the tumor endothelial cell layer and stromal fibroblasts, thereby increasing the efficiency of delivery into tumors but not into normal tissues. Moreover, these uPAR-targeted IONPs can destroy tumor blood vessels, producing an antiangiogenesis effect that enhances treatment efficacy. To make the IONPs suitable for repeat administrations, a recombinant amino terminal fragment (ATF) of the receptor binding domain of uPA, a high affinity natural ligand for uPAR, was produced in a bacterial expression system to minimize the immune response. ATF peptides were conjugated to amphiphilic polymer-coated IONPs to produce the receptor-targeted MRI contrast nanoparticles. The ATF can also be labeled with a new near-infrared dye (NIR-830) developed by our group prior conjugation to generate uPAR-targeted IONPs with dual optical and MR imaging modalities. Based on the surface functionalization of the IONPs and chemical properties of drug molecules, we developed approaches for encapsulating hydrophobic drugs or conjugating hydrophilic drugs to the IONPs, resulting in theranostic IONPs which carry chemotherapy drugs, such as doxorubicin (ATF-IONP-Dox) and gemcitabine (ATF-IONP-Gem). After delivery into tumor cells, the drug molecules can be released efficiently from the nanoparticles using pH-sensitive and/or lysosomal enzyme-sensitive drug release mechanisms. Targeted delivery, intracellular drug release, and the cytotoxic effect have been demonstrated in breast and pancreatic cancer cell lines as well as in an endothelial cell line. The efficacy of ATF-theranostic IONPs and the ability of MRI to monitor drug delivery and response were examined in orthotopic animal tumor models, including a triple-negative mouse mammary tumor model, a basal type human breast ductal carcinoma in situ xenograft model, and a human pancreatic cancer xenograft model. We found that systemic administration of ATF-IONP-Dox significantly inhibited the growth of orthotopic breast and pancreatic tumors in these animal models. Additionally, preoperative treatment of primary tumors with ATF-IONP-Dox significantly decreased the growth of primary tumors and further inhibited local recurrence and lung metastasis in 4T1 mouse mammary tumor model. Using our established MRI methods, the efficiency of intratumoral drug delivery and changes in tumor sizes and tissue contrasts can be detected by T1- and T2-weighted MRI using a clinical field strength MRI scanner in the tumor models. Histological analysis showed that the uPAR-targeted theranostic IONPs are selectively accumulated in primary breast tumor, and primary and peritoneal metastatic pancreatic tumor lesions. Theranostic IONP-mediated doxorubicin delivery reduced systemic toxicity since pathological changes in the liver and heart tissues and serological abnormalities were detected in mice treated with free doxorubicin at a dose of 10 mg/Kg of body weight but not in mice after treatment with an equivalent dose of ATF-IONP-Dox. The effect of ATF-IONP-Gem on the growth of orthotopic human pancreatic cancer was also examined in an orthotopic human pancreatic cancer xenograft model. ATF-IONP-Gem showed significant tumor growth inhibition in the tumor-bearing mice that received systemic delivery of ATF-IONP-Gem containing 2 mg/kg of body weight of gemcitabine. However, there was no significant tumor growth inhibition in the mice that received an equivalent dose of free gemcitabine or nontargeted IONP-Gem. The presence of IONP-drug in the tumor lesion can be detected as bright signals using an ultra-short TE MRI scan method. At present, we are developing uPAR-targeted IONPs carrying multiple therapeutic agents in a single IONP to further enhance the efficacy of the treatment. Our theranostic IONPs have the potential to significantly impact cancer treatment in neoadjuvant therapy of TNBC or locally advanced pancreatic cancer. Preoperative neoadjuvant chemotherapy is usually administered to patients with TNBC to reduce the size of the primary tumor and to treat locally advanced tumors and micrometastatic lesions in order to lower the incidence of local and distant recurrence. The unique differential response to chemotherapy within the TNBC patient population makes it crucial to assess early tumor responses to the therapy and to ensure the most effective chemotherapies while avoiding unnecessary toxicity. About 40% of pancreatic cancer patients are diagnosed with unresectable locally advanced disease at presentation because their tumors have directly invaded into adjacent normal structures and blood vessels. It is feasible to treat these patients with the targeted theranostic IONPs to reduce the burden of the primary tumor and peritoneal metastatic lesions so that the patients can be candidates for potentially curative surgical resection. Additionally, NIR optical signal produced from the dual imaging theranostic IONPs allows optical image-guided surgery to detect and completely remove residual tumors that are resistant to drug treatment. In summary, uPAR-targeted theranostic IONPs developed by our research team have great potentials for the development of an integrated treatment-imaging protocol for pancreatic cancer and TNBC. Such a protocol can significantly improve the survival rate of cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY20-02. doi:10.1158/1538-7445.AM2011-SY20-02

Introduction: The triple negative breast cancer (TNBC) phenotype, which lacks the presence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (ErbB-2 or Her-2), is highly aggressive and resistant to chemotherapy. In fact, some of the patients under hormone deprivation and/or Herceptin therapy acquire resistance. Therefore, there is an urgent need to identify new diagnostic as well as therapeutic markers for treatment of such patients. Herceptin, an immunotherapy directed against the Her-2 receptor, inhibits cancer growth and progression in Her-2 positive breast cancer by blocking the downstream survival pathways. In these cells however, the expression of epidermal growth factor receptor (EGFR) is significantly low. Interestingly, EGFR expression is significantly increased in triple negative and Her-2 resistant breast cancer thereby contributing to cancer growth and progression. Present studies were designed to investigate whether or not Annexin A2 (AnxA2), a calcium dependent phospholipid binding protein, regulated EGFR downstream signaling pathway and if the functions of EGFR can be inhibited by the AnxA2 antibody in TNBC and Herceptin resistant cancer cell lines. Methods: AnxA2 function at cell surface of the triple negative breast cancer cell line MDA-MB-231 and Her-2 resistant breast cancer cell line JIMT-1 was blocked incubating with AnxA2 antibody (2μg/ml) after 12h of serum starvation. The cells were treated with/without EGF (50ng/ml) for 20 min after 2h of antibody treatment. The cell lysate was analyzed for pEGFR and EGFR mediated downstream signaling by Western blotting. Cell migration was analyzed by scratch wound healing assay. Results: The results of the present study indicate that AnxA2 interacts with EGFR at the cell surface and plays an important role in the regulation of EGFR mediated downstream signaling. Treatment of MDA-MB-231 and JIMT-1 cells with AnxA2 antibody causes significant decrease in EGF-induced phosphorylation of EGFR at Y845 and Y1068 sites. Our results also demonstrate that blocking cell surface AnxA2 functions causes the downregulation of proteins such as pAKT, and pERK1/2 which are regulated by EGFR resulting in lower cell survival, proliferation, and migration. Conclusions: Our data indicate that association of AnxA2 with EGFR in the membrane domain plays a positive regulatory role in keeping EGFR signaling events in an activated state in both triple negative and Her-2 resistant breast cancer cells, thus making AnxA2 an important therapeutic target. Citation Format: Pankaj Chaudhary, Jamboor K. Vishwanatha. Inhibition of triple-negative and Her-2 resistant breast cancer proliferation and migration by Annexin A2 antibodies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A052.

Rationale and Background: Immunotherapy of cancer has gained much attention in the past decade with the development of immune checkpoint inhibitors and chimeric antigen receptor (CAR) technology that can activate and redirect patient T cells to kill tumors that over-express a specific antigen. CARs are fusion receptors that are comprised of an antibody-derived single-chain variable fragment (scFv) coupled via hinge and transmembrane elements to a T cell signaling and co-stimulatory domain. This technology is in its early stages of development and has not been fully exploited for the treatment of metastatic epithelial cancers. We focus our studies on Pancreatic Ductal Adenocarcinomas (PDAC) and Triple Negative Breast Cancer (TNBC). However, if successful, the studies will be applicable to other epithelial tumors. For CAR-T cells to work, and to avoid off target toxicities, both the target antigen and the antibody recognizing the target have to be highly specific. The challenge is that there are few such antigen-antibody combinations for solid tumors. We have recently developed a novel patented antibody (designated TAB 004) that specifically recognizes ONLY the tumor-associated form of MUC1 (tMUC1) but not the normal form of MUC1 (nMUC1) in several subtypes of breast cancers including TNBC and in PDAC. We show compelling data that TAB004 recognizes tMUC1 in >90% of human TNBCs and 85% of PDAC but spares all normal epithelial tissues. The antigenic isoform that TAB004 recognizes is completely hidden in normal epithelia making it extremely safe for development of CAR-T cells. Hypothesis: TNBC and PDAC can be specifically targeted with the tMUC1-CAR-engineered T cells, whilst sparing normal organs. Methodology and Results: We have engineered several TAB-specific CAR constructs using the scFv fragment of TAB 004. Six constructs are developed, 3 for human T cells and 3 for mouse T cells: 1. TAB- CD28-CD3zeta (2nd generation CAR), 2. TAB-CD28-41BB-CD3zeta (3rd generation CAR), and 3. TAB-CD28-OX40-CD3zeta (3rd generation CAR). Data shows that we can successfully engineer human T cells to express the TAB-CAR on their surface and that these engineered T cells can bind specifically to tumor cells expressing the unique tMUC1 epitope, become activated, and effectively kill the tumor cells. We show that these engineered T cells only minimally bind and kill normal epithelial cell lines. However, some of the cell lines are more resistant than others. We are therefore conducting combination therapy with various drugs that are known to enhance immune based therapies including checkpoint inhibitors, COX-2 inhibitors, cyclophosphamide and others. We may also have data to show that the engineered TAB-CAR T cells kill tMUC1-expressing TNBC and PDAC cells in vivo. We will show data that TAB-CAR-T cell kill human PDAC and TNBC cells in vitro and propose to conduct the same in vivo xenograft model of human PDAC and metastatic TNBC. In the following months, we will test if TAB-CAR-T cell retards tumor growth in an orthotopic syngeneic mouse model of PDAC and metastatic BC in human MUC1.Tg immune competent mice. We further propose to demonstrate that the TAB-CAR-T cell can mediate apoptosis in the immune competent KCM mice (KC X human MUC1.Tg mice) that develop spontaneous PDAC and the MMT mice (PyVMT X human MUC1.Tg mice) that develop spontaneous mammary gland tumors. Both models mimic the human disease progression and express human MUC1 in a tissue specific manner. This is important since all normal epithelia in these mice express the nMUC1 except the tumors that expresses the target, tMUC1. Impact: If successful, this project will have a major impact and accelerate progress toward a clinical trial for PDAC and metastatic TNBC. This will be the first attempt to test the efficacy of a CAR-T cell in an immune competent, human MUC1.Tg mouse model that develops spontaneous tumors within the appropriate stromal and hormonal microenvironment. Citation Format: Pinku Mukherjee, Ru Zhou, Mahboubeh Yazdanifar, Das Roy Lopamudra. Development and future of CAR T cell therapy for pancreatic ductal adenocarcinoma and triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A37.

CDK4/6 inhibition in combination with endocrine therapy is the standard of care for estrogen receptor (ER+) breast cancer, and although cytostasis is frequently observed, new treatment strategies that enhance efficacy are required. Here, we perform two independent genome-wide CRISPR screens to identify genetic determinants of CDK4/6 and endocrine therapy sensitivity. Genes involved in oxidative stress and ferroptosis modulate sensitivity, with GPX4 as the top sensitiser in both screens. Depletion or inhibition of GPX4 increases sensitivity to palbociclib and giredestrant, and their combination, in ER+ breast cancer models, with GPX4 null xenografts being highly sensitive to palbociclib. GPX4 perturbation additionally sensitises triple negative breast cancer (TNBC) models to palbociclib. Palbociclib and giredestrant induced oxidative stress and disordered lipid metabolism, leading to a ferroptosis-sensitive state. Lipid peroxidation is promoted by a peroxisome AGPAT3-dependent pathway in ER+ breast cancer models, rather than the classical ACSL4 pathway. Our data demonstrate that CDK4/6 and ER inhibition creates vulnerability to ferroptosis induction, that could be exploited through combination with GPX4 inhibitors, to enhance sensitivity to the current therapies in breast cancer.

Ferroptosis, a genetically and biochemically distinct form of programmed cell death, is characterized by iron-dependent accumulation of lipid peroxides. Ferroptosis is induced in cancer cells by inhibition of lipid peroxide quencher GPx4. System xc - imports cystine into cytosol for the biosynthesis of glutathione. Tumor cells that are resistant to chemotherapeutic drugs called ‘drug tolerant’ or ‘Persister’ cells which have distinct vulnerability towards iron mediated cell death or ferroptosis. Unfolded Protein Response (UPR) plays critical role for cancer cells to become drug tolerant. Tweaking the balance of UPR to make drug tolerant cells susceptible to Ferroptotic cell death could be an attractive therapeutic strategy. To decipher the emerging contribution of ER-stress in ferroptosis, we investigated the status of UPR following treatment of potent ferroptosis inducer RSL3 (Ras Selective Lethal) in colon cancer cells. We observed an overall up-regulation of UPR activators (ATF6, IRE1α and PERK) and their downstream effectors along with a marked overexpression of cystine-glutamate transporter (System xc -). To further delineate the contribution of particular UPR arm in modulating System xc - expression and subsequent ferroptosis, we made stable knock down cells of each UPR arm and discovered that PERK is selective and critical in inducing ferroptosis in colon cancer cells. Loss of PERK function not only promotes ferroptosis via increasing lipid peroxidation but also limits in vivo tumor growth in colon xenograft model. Further, we find that low PERK expression is associated with higher patient survival as per TCGA COLON CANCER (COAD) database. Overall, our experimental data indicate that PERK is a negative regulator of ferroptosis and genetic silencing of PERK sensitizes colon cancer cells to selective Ferroptotic cell death. Therefore, small molecule PERK inhibitors hold huge promise as novel therapeutics that can sensitize apoptosis resistant cancer cells towards Ferroptotic cell death. Citation Format: Krishan K. Saini, Priyank Chaturvedi, Ayushi Verma, Mushtaq A. Nengroo, Abhipsa Sinha, Akhilesh Singh, Sanjeev Meena, Muqtada A. Khan, Manish P. Singh, Dipak Datta. PERK arm of UPR selectively regulates ferroptosis in colon cancer cells by modulating the expression of system xc - (SLC7A11) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6249.

Osteosarcoma is a malignant bone sarcoma prevalent in children, adolescents, and young adults, with limited treatment options. Targeting ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, presents a potential therapeutic strategy for cancers. However, the molecular mechanisms that regulate iron metabolism in ferroptosis remain unclear. In this study, we explored cell defense mechanisms against ferroptosis mediated by intracellular iron regulation and immune signaling pathway in osteosarcoma cells. First, we found that xCT and GPX4 protein were detected with variable expressions across five osteosarcoma cell lines. Erastin, an xCT inhibitor, and RSL3, a GPX4 inhibitor, induced ferroptotic cell death in these cells. Specifically, higher inhibitory dose of RSL3 induced a significant reduction in GPX4 levels in U2OS and MG-63 cells in a dose- and time-dependent manner. In contrast, xCT expression increased in these cells, suggesting an adaptive mechanism to counteract oxidative stress following GPX4 depletion. RSL3 treatment elevated lipid peroxidation levels in U2OS and MG-63 cells, which were restored by Ferostatin-1. Our RNA-seq analysis identified 18 differentially expressed genes in response to RSL3 treatment in U2OS cells, with the highest expression in AKR1C1 gene, which plays a significant role in oxidative stress. We confirmed that GPX4 inhibition upregulated AKR1C1 gene expression as well as other oxidative stress markers, including NRF2, HMOX1, and NQO1.Our data also showed that osteosarcoma cells modulated intracellular iron homeostasis in response to GPX4-mediated ferroptotic stress. GPX4 inhibition significantly enhanced FPN and FTH1 expression, which are essential for iron efflux and storage, in U2OS cells, while MG-63 cells only increased FTH1 with minimal change in FPN. Furthermore, macrophage stimulating protein receptor (MSPR) protein expression was significantly increased by GPX4 inhibitor using Proteome Profiler analysis. We observed that combination treatment with dual MSPR and MET inhibitor, BMS-777607, showed synergistic effects with GPX4 inhibition. Altogether, our findings demonstrate that ferroptotic cell death is inducible in osteosarcoma cells, and the potential resistance mechanism to ferroptosis is mediated by oxidative stress mechanism and intracellular iron regulation. We also identified novel target, MSPR, regulated by GPX4 inhibition, which potentially contribute to the activation of immunosuppressive signals from osteosarcoma. In conclusion, this study underscores that targeting the ferroptotic pathway holds a great promise for therapeutic potential in osteosarcoma by regulating both intracellular pathway and the immunosuppressive tumor microenvironment. Citation Format: Md Abdullah, Donghee Lee, Rong Li, Eslenur Nipa, Jong Hyuk Kim. GPX4-mediated Pathway Regulates Iron Efflux to Prevent Ferroptotic Cell Death and Promote Immunosuppressive Signals in Osteosarcoma Cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1637.

4, 9-dihydroxy-α-lapachone as a potent antiproliferation agent for triple-negative breast cancer via ferroptosis.

Triple-negative breast cancer (TNBC) is a form of breast cancer that does not express estrogen receptor (ER), progesterone receptors (PR) or human epidermal growth factor receptor 2 (HER2). It accounts for 15-20% of all breast carcinomas and preferentially affects women who are African American, younger than age 40, or have a BRCA1 mutation. The TNBC subtype is highly aggressive and heterogeneous and has a poor prognosis relative to other breast cancer subtypes due to a lack of highly recurrent and actionable biomarkers that can be used to direct therapy. Although standard chemotherapies show a high initial response rate in TNBC, tumor relapses and metastasis often occur rapidly. Moreover, for patients who have recurrent disease or progressive disease during chemotherapy, treatment options are limited. Therefore, there is an unmet need to identify effective therapies targeting TNBC. By analyzing public human datasets, we found that CCDC88A (also known as Girdin or GIV), a putative oncogene, is highly expressed in TNBC compared to other breast cancer subtypes. CCDC88A has been found to be involved in key cancer development events such as tumor angiogenesis, invasion, wound healing, and metastasis. Interestingly, we found that, although the high expression of CCDC88A conferred the ability of lung metastasis, it increased the susceptibility of TNBC to ferroptosis, a type of cell death induced by iron-dependent lipid peroxidation. Mechanistically, CCDC88A sensitized TNBC cells to ferroptosis by limiting intracellular glutathione (GSH) levels. shRNA-mediated CCDC88A knockdown in TNBC cells significantly increased GSH levels and in turn blunted cellular sensitivity to ferroptosis. On the other hand, inhibition of GSH synthesis with a glutamate-cysteine ligase (GCL) inhibitor, BSO, restored the ferroptosis sensitivity in CCDC88A knocked-down TNBC cells. Thus, our findings uncover CCDC88A as a novel ferroptosis regulator and also suggest that ferroptosis is a therapeutically tractable target for metastatic TNBC that overexpresses CCDC88A. Citation Format: Wei-Ling Tu, Mu-En Wang, Ji-Hoon Kim, Alyssa Bawcom, Yi Lu, Ming Chen. Targeting ferroptosis in CCDC88A-overexpressing metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1635.

Introduction: We evaluated the potential therapeutic benefit of conventional chemotherapy combined with hetIL-15 immunotherapy in two mouse models of pancreatic and breast cancer. IL-15 is an important cytokine that stimulates the proliferation and cytotoxic functions of CD8+ T cells and NK cells. We have produced the native heterodimeric form of IL-15 (hetIL-15), which has advanced in clinical trials due to its anticancer activities. Study design and methods: We assessed the efficacy of hetIL-15 immunotherapy in combination with the chemotherapeutic agents gemcitabine or doxorubicin on the growth of primary pancreatic and breast tumors, and on the metastatic disease. We used the genetically engineered mouse model (GEMM) KPC of pancreatic cancer (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) and the orthotopic 4T1 triple negative breast cancer (TNBC) model. We analyzed the tumor infiltrating lymphocytes (TILs) by flow cytometry and immunohistochemistry (IHC) and evaluated the extent of necrosis on the primary tumors by H&E and cytokeratin staining. We also evaluated the lung metastatic burden by histology. Results: The combination of hetIL-15 and doxorubicin significantly delayed 4T1 tumor growth. In addition, histological analysis showed an increase in the percentage of the necrotic areas of the primary tumors in the combination group. hetIL-15 treated groups also showed increased necrotic areas of the primary pancreatic tumors, although tumor growth showed no significant change compared to the control group. Flow analysis of TILs showed increased CD8+/CD4+ T cell ratios in tumors of hetIL-15 treated groups in both cancer models, which was confirmed by IHC. Importantly, the evaluation of the metastatic disease showed that hetIL-15 treated animals in both cancer models had reduced metastatic foci and in some cases were completely disease free. Chemotherapy did not significantly reduce the numbers of the total metastatic lesions but reduced their size. Conclusions: Chemotherapy can be combined with hetIL-15 treatment in both breast and pancreatic mouse models. Despite small beneficial results in the size of primary tumor, histological evaluation revealed increased tumor necrosis and potential synergy of chemotherapy and immunotherapy. The observed decreased metastatic burden in the lungs in both cancer models indicates that hetIL-15 strongly reduces metastases in several cancer types and suggests that hetIL-15 is a promising therapeutic agent against metastatic disease. Citation Format: Dimitris Stellas, Vasiliki Stravokefalou, Sevasti Karaliota, Bethany Nagy, Serguei Koslov, Konstantinos Dimas, Barbara K. Felber, George N. Pavlakis. Combination of hetIL-15 with chemotherapy in triple negative breast and pancreatic cancer mouse models increases tumor necrosis and alleviates metastatic disease [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 6651.

Loss of E-cadherin, a core member of the epithelial adherens junction (AJ), leads to the induction of the epithelial-mesenchymal transition (EMT), a phenomenon where epithelial cells de-differentiate towards a more motile and invasive mesenchymal cell type. Because of its stabilizing function in the AJ, p120-catenin has caught attention in the context of tumor development and progression. We previously reported that expression of protein kinase C alpha (PKCα) imparts a hormone-independent, and tamoxifen-resistant phenotype in T47D:A18/PKCα breast cancer cells. Our preliminary data strongly indicate a positive relationship between PKCα and FOXC2, a forkhead transcription factor shown to be a central mediator of the EMT program and tumor metastasis. Data mining from Oncomine database revealed a strong correlation between PRKCA and FOXC2 in basal A triple negative breast cancer (TNBC) cell lines. In this study, two representative cell lines, HCC1937 and HCC1143, were chosen to investigate the relationship between PKCα, FOXC2, and p120-catenin, and how their interplay alters migration and invasion of TNBC and endocrine resistant breast cancer. Cell lines (T47D:A18/PKCα, HCC1143, HCC1937) were maintained as previously described and according to ATCC guidelines. Expression of PKCα and FOXC2 were manipulated using siRNA targeting PKCα and FOXC2 respectively. Transcripts level of FOXC2 (FOXC2) and CTNND1(p120-catenin) were measured using quantitative PCR. PKCα, FOXC2, and p120-catenin protein expression was measured by Western blot using PKCα (GE-Healthcare, 1:200), FOXC2 (Abcam, 1:500), and p120-catenin (Cell Signaling, 1:500) antibodies respectively. All three cell lines were found to co-express high level of PKCα and FOXC2. Knockdown of PKCα resulted in a down-regulation of FOXC2 transcripts and protein. However, knockdown of FOXC2 did not alter expression of PKCα, indicating PKCα is an upstream regulator of FOXC2, and not vice versa. Downregulation of either PKCα or FOXC2 led to a rescue of p120-catenin, and not E-cadherin, transcripts. Re-expression of p120-catenin transcripts also resulted in re-expression of the protein. Knockdown of FOXC2 in T47D:A18/ PKCα cells led to a significant reduction in migratory and invasive potential of these cells, to the same extent as knockdown of PKCα For the first time, we are reporting PKCα as a novel regulator of FOXC2 in TNBC and endocrine-resistant breast cancer. We also identified p120-catenin as a potential target of PKCα and FOXC2. Down-regulation of p120-catenin by either PKCα or FOXC2 may be a critical event in the dissolution of the AJ, which facilitates migration of breast cancer cells. Citation Format: Thao ND Pham, Bethany Perez-White, Debra A. Tonetti. Protein kinase C alpha (PKCα) is a novel regulator of FOXC2 and p120-catenin in triple negative and endocrine resistant 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 4095. doi:10.1158/1538-7445.AM2015-4095

Background: Tumor infiltrating lymphocytes (TILs) are strong prognostic biomarkers for early TNBC. We evaluated the role of CD8, FOXP3 and PD-L1 expression in refining prognostic models for non-metastatic TNBC in a large cohort of patients treated with standard therapy. Methods: Consecutive patients diagnosed with stage I-III TNBC (ER/PgR <10%, HER2 0/1+ or ISH non amplified) between May 2012 and December 2015 were included. All patients received treatment with surgery, chemotherapy (neoadjuvant or adjuvant) and radiotherapy (when indicated). For each case, three FFPE tumor slides were stained for CD8 (Clone C8/144B, Dako Cytomation), FOXP3 (Clone 236A/E7, dilution 1:200, Abcam) and PD-L1 (IHC 73-10 Research Use Only assay developed by Agilent Technologies). One slide was stained for cytokeratins with MNF116. Digital slides were evaluated by a Visiopharm® software application, following alignment of the CD8, FOXP3 and PD-L1 slides with the MNF116 slide. The density of CD8 and FOXP3 expression was calculated as the number of cells/mm2 of stroma area. For PD-L1, the % of positive stromal cells over the total of stromal cells was analyzed. Disease-free survival (DFS) was calculated from diagnosis to relapse or death. The Harrell’s c-index was used to determine the cut-offs for CD8, FOXP3 and PD-L1 to be used in survival analyses. Results: 265 TNBC pts were evaluated. Median TILs was 10% (Q1-Q3 3%-25%), median CD8 was 249 (Q1-Q3 109-568), median FOXP3 was 57 (Q1-Q3 21-134), median PD-L1 was 5.2% (Q1-Q3 0.2%-25.4%). TILs, CD8, FOXP3 and PD-L1 were positively correlated with each other (p<0.001): CD8 showed strong correlation with TILs (Spearman’s coefficient 0.753), FOXP3 and PD-L1 showed moderate correlation with TILs (Spearman’s coefficient 0.535 and 0.587). Higher TILs, CD8, FOXP3 and PD-L1 were associated with age ≤50yrs (p=0.002, p=0.004, p=0.065, p=0.011), Grade 3 (p=0.001, p=0.006, p=0.006, p=0.003) and Ki67 ≥30% (p=0.056, p=0.024, p=0.004, p=0.005). There was no association between immune markers and stage. Among classic clinicopathologic factors, TILs (10% increments) and stage at diagnosis were independent prognostic parameters in multivariate analysis (HR 0.81, 95% CI 0.69-0.94 p=0.005 for TILs, HR 2.01 95% ci 1.01-4.23 P=0.047 for stage II vs stage I and HR 5.31 95% CI 2.54-11.11 p<0.001 for stage III vs stage I). In univariate analysis, high CD8 (≥443), high FOXP3 (≥57) and high PD-L1 (>20%) were all significantly associated with improved DFS (HR 0.36 95%CI 0.18-0.72, p=0.004 for CD8; HR 0.48 95%CI 0.28-0.80, p=0.005 for FOXP3; HR 0.52 95%CI 0.28-0.97, p=0.039 for PD-L1). FOXP3 and PD-L1 provided significant additional prognostic information beyond a model containing TILs and stage: likelihood ratio χ2 5.12, p=0.024 for FOXP3; likelihood ratio χ2 5.52, p=0.019 for PD-L1. CD8 did not add relevant prognostic information beyond TILs and stage (likelihood ratio χ2 2.76, p=0.097). Including both FOXP3 and PD-L1 did not add further prognostic information to models already containing TILs, stage and either FOXP3 or PD-L1. Conclusions: FOXP3 and PD-L1 expression evaluated with a software-assisted method were prognostic for stage I-III TNBC pts treated with standard therapy and may contribute to refine the prognostic stratification beyond stage and TILs. This study was supported by a grant from Merck KGaA. Citation Format: Maria Vittoria Dieci, Vassilena Tsvetkova, Gaia Griguolo, Federica Miglietta, Deborah Bacchin, Giulia Tasca, Carlo Alberto Giorgi, Enrico Cumerlato, Enrico Orvieto, Valentina Guarneri, Pierfranco Conte. Integrating CD8, FOXP3 and PD-L1 expression in prognostic models for triple negative breast cancer (TNBC): An analysis of 265 patients treated with standard therapy for stage I-III disease [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 P5-06-14.

Purpose: Recent studies evaluating the efficacy of bev in BC have shown conflicting results, particularly in hormone receptor (HR)+ BC. Identification of predictive biomarkers and their relationship to the pharmacodynamic effects of bev would facilitate the identification of BC patients (pts) most likely to benefit from bev. To examine this, we conducted a unique preop phase II trial with a run-in of single agent bev followed by dose-dense AC-T with bev in two cohorts, one with HR+HER2- BC pts, and a smaller triple negative (TN) BC pts. Methods: Pts with HR+, HER2- or TNBC were eligible. Treatment consisted of a single dose of bev 10 mg/kg, followed two wks later by A 60 mg/m2 and C 600 mg/m2 with bev 10 mg/kg q2 wks x 4, followed by T 175 mg/m2 with bev 10 mg/kg q2 wks x 3, followed by T 175 mg/m2 x1. Core biopsies and interstitial fluid pressure (IFP) were assessed pre- and post- bev alone. Pathologic response was confirmed centrally and Miller-Payne (MP) was assessed. Results: The study enrolled 84 pts with HR+ and 20 pts with TN BC. Amongst HR+ pts, 79 had surgical tissue centrally reviewed, and 6 (8%) had a pCR. Amongst TN BC pts, 19 pts had tissue centrally reviewed and 9 (47%) had a pCR. Grade was found to predict MP response in both HR+ and TN pts (p=0.001). Tissue biomarkers and IFP were evaluated as predictors of response to bev. Single-agent bev reduced the mean IFP in the overall cohort and HR+ patients by 20 (p=0.020) and 24.5% (p=0.001), respectively. The IFP decreased > 50% in 24/65 pts and did not change in others. Bev reduced the mean vascular density (MVD) by 33.0% (p<0.05) in TN BC pts, but did not affect the vessel area fraction covered by perivascular cells (PCs). Bev did not modify the fraction of tumor tissue positive for the hypoxia marker CAIX. In TN BC pts, pre- and post-bev, the MVD was inversely correlated with the CAIX fraction (p<0.01). In addition, a drop in MVD associated with increased CAIX+ fraction post-BEV (p=0.05). MP score was more favorable for TN BC pts with lower CAIX+ fraction at baseline (p=0.058) and post-BEV (p<0.05), and higher MVD at baseline (p<0.05) and post-BEV (p<0.05). In contrast, in HR+ BC, BEV reduced MVD non-significantly by 15% (p=0.25) and increased the vessel area fraction covered by PCs (p<0.05). There was no significant correlation between MVD and CAIX+ fraction in HR+ BCs. In contrast to TN BC, in HR+ BCs the fraction of CAIX+ tumor was directly correlated with MP score (p<0.01). Discussion: Collectively, these results indicate that vascular pruning post-BEV may reduce vascular function and increase hypoxia, which is associated with less favorable pathologic response after BEV with chemotherapy in TN BC. In conclusion, our exploratory study suggests that elevated hypoxia and reduced MVD in TN BC reduces the effectiveness of chemotherapy. Citation Format: Yves Boucher, John D. Martin, Sara M. Tolaney, Giogio Seano, Shom Goel, Marek Ancukiewicz, Steven J. Isakoff, Eric P. Winer, Ian E. Krop, Rakesh K. Jain. Tissue biomarkers and interstitial fluid pressure in a phase II study of preoperative (preop) bevacizumab (bev) followed by dose-dense doxorubicin (A)/cyclophosphamide (C)/paclitaxel (T) in combination with bev in HER2-negative operable breast cancer (BC) [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-76. doi:10.1158/1538-7445.AM2013-LB-76

The cystine transporter (system xC-) is an antiporter of cystine and glutamate. It has relatively low basal expression in most tissues and becomes upregulated in cells under oxidative stress (OS) as one of the genes expressed in response to the antioxidant response element promoter. We have developed 18F-5-fluoroaminosuberic acid (FASu), a PET tracer that targets system xC- The goal of this study was to evaluate 18F-FASu as a specific gauge for system xC- activity in vivo and its potential for breast cancer imaging. Methods:18F-FASu specificity toward system xC- was studied by cell inhibition assay, cellular uptake after OS induction with diethyl maleate, with and without anti-xCT small interfering RNA knockdown, in vitro uptake studies, and in vivo uptake in a system xC--transduced xenograft model. In addition, radiotracer uptake was evaluated in 3 breast cancer models: MDA-MB-231, MCF-7, and ZR-75-1. Results: Reactive oxygen species-inducing diethyl maleate increased glutathione levels and 18F-FASu uptake, whereas gene knockdown with anti-xCT small interfering RNA led to decreased tracer uptake. 18F-FASu uptake was robustly inhibited by system xC- inhibitors or substrates, whereas uptake was significantly higher in transduced cells and tumors expressing xCT than in wild-type HEK293T cells and tumors (P < 0.0001 for cells, P = 0.0086 for tumors). 18F-FASu demonstrated tumor uptake in all 3 breast cancer cell lines studied. Among them, triple-negative breast cancer MDA-MB-231, which has the highest xCT messenger RNA level, had the highest tracer uptake (P = 0.0058 when compared with MCF-7; P < 0.0001 when compared with ZR-75-1). Conclusion:18F-FASu as a system xC- substrate is a specific PET tracer for functional monitoring of system xC- and OS imaging. By enabling noninvasive analysis of xC- responses in vivo, this biomarker may serve as a valuable target for the diagnosis and treatment monitoring of certain breast cancers.

Background: Biallelic mutations in PALB2 (Partner and Localizer of BRCA2) are known to cause Fanconi Anemia Type N. Multiple reports have demonstrated an increased risk for cancer in individuals heterozygous for PALB2 mutations. For example, a recent study by Antoniou et al reported a 33-58% lifetime risk for breast cancer in PALB2 mutation carriers, with 30% of carriers reporting triple negative breast cancer (TNBC). Other studies have suggested associations between PALB2 heterozygosity and pancreatic cancer, ovarian cancer, male breast cancer, and prostate cancer as well. We aimed to better define PALB2 phenotypes by assessing clinical history of TNBC, pancreatic, ovarian, and prostate cancers amongst PALB2 mutation carriers identified via multigene panel testing. Methods: We reviewed clinical histories of 11,007 individuals who underwent PALB2 sequence and deletion/duplication analysis as part of a multigene hereditary cancer panel. Descriptive statistics were utilized for clinical histories of PALB2 carriers, and chi square analysis was used to compare clinical histories of PALB2 mutation carriers to mutation-negative controls. Individuals with mutations in other cancer susceptibility genes were excluded from analysis. Results: A total of 98 PALB2 mutation carriers identified among 9610 individuals were included in our analysis. The majority of mutation carriers were Caucasian (80%) and female (92.8%). All identified mutations were truncating (nonsense, frameshift, or gross deletions). No pathogenic missense mutations were identified in this cohort. 77.6% (n=76) of mutation carriers had breast cancer, diagnosed at a mean age of 48. Hormone receptor status was available for 48 mutation carriers and 2469 controls. 37.5% (18/48) of breast cancers in mutation carriers were reported as triple negative, compared to 17.1% (423/2469) of breast cancers in controls (OR: 2.9 ; p= 0.0002). 7.8% (n= 8) of PALB2 mutation carriers had ovarian cancer. There was no significant difference in the incidence of ovarian cancer between PALB2 mutation carriers and controls (OR: 0.65 ; p= 0.25). Additionally, mutation carriers were significantly less likely to have a family history of ovarian cancer than controls (OR: 0.5; p= 0.02). 5.9% (n=6) of mutation carriers had pancreatic cancer, diagnosed at a mean age of 57.8, compared to 61 for controls. PALB2 mutation carriers were 1.3 times more likely to have personal and/or family history of pancreatic cancer, although this was not statistically significant (p= 0.22). Similarly, PALB2 mutation carriers were 1.5 times more likely to have a family history of prostate cancer, although this was not statistically significant (p= 0.09). Conclusions: Our data supports existing literature associating PALB2 mutations with TNBC. We did not observe significant associations between PALB2 carrier status and a clinical history of pancreatic, prostate, or ovarian cancers. However, this data should be interpreted with caution, as it is possible that unidentified genetic factors contributed to the clinical history of cancer in our mutation-negative controls. Investigation of PALB2-associated cancer risks in an unselected prospective cohort would help to further elucidate the PALB2 phenotype. Citation Format: Emily K Dalton, Rachel McFarland, Holly Laduca, Shuwei Li, Chia-Ling Gau. Teasing out the PALB2 phenotype [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-16.