Human Triple-negative Breast Cancer Research Articles

The fabrication method impacts biological properties of microfibrous three-dimensional in vitro cell culture scaffolds and their applications in drug screening

Two-dimensional (2D) cell culture models cannot accurately emulate the three-dimensional (3D) in vivo microenvironment; hence, the shift from compound screening in traditional 2D well plates to 3D cell culture models is forthcoming. New scaffold designs must comply with the 3Rs principle to reduce the use of animal-derived products, such as natural proteins. In line with this motivation, we engineered and compared various fibrous scaffolds featuring diverse fibre networks (coarse and fine fibres) and fibre surface properties (porous vs. smooth fibres), and tested them in in vitro cell culture. We fabricated coarse solid fibre scaffolds via a melt-based electrohydrodynamic process, which resulted in fibres with a diameter of 10.6 ± 1.9 µm and a porosity of 87.1 ± 0.9%. Using solution-based cryo-electrohydrodynamic process, we fabricated fine porous fibre and fine solid fibre scaffolds that exhibited fibres of 2.01 ± 0.5 µm and 1.72 ± 0.6 µm in diameter, with porosities of 96.5 ± 0.3% and 94.4 ± 0.5%, respectively. The scaffolds underwent only chemical modification via an alkaline treatment to enhance their hydrophilicity. We used mouse fibroblast L929 cells and human triple-negative breast cancer MDA-MB-231 cells as cell models, and used the MTT assay to measure their viability. After seven days of cell culture, all scaffolds were thoroughly penetrated. However, the coarse scaffolds created by the melt-based electrohydrodynamic process and the porous fibre scaffolds produced by the solution-based cryo-electrohydrodynamic process outperformed the simpler morphology of smooth fine fibres. Furthermore, the 50% half-maximum effective concentration (EC50) of doxorubicin tested in human glioblastoma (U87-MG) and human triple-negative breast cancer (MDA-MB-231) cell lines in 3D cell culture models was approximately twice as high as that achieved in conventional 2D cell cultures. Therefore, we infer that poly (ɛ-caprolactone) (PCL) microfibre scaffolds, devoid of animal-derived product surface treatment, may provide a favourable 3D milieu for various cell types for in vitro studies.

Abstract 1570: Lipid metabolism alterations promote TNBC metastasis and immunotherapy resistance via PD-L1 internalization and signaling

Abstract Programmed death-ligand 1, PD-L1 (CD274), is an immune checkpoint protein upregulated by cancer cells to facilitate immune evasion. PD-L1 also performs oncogenic functions within cancer cells, but the underlying mechanisms of PD-L1 internalization and signaling remain largely unknown. Here, we report a new mechanism whereby altered ceramide metabolism via ceramide synthase 4 (CerS4) drives PD-L1 internalization to potentiate a TGFβ/Shh/β-catenin signaling axis to drive triple-negative breast cancer (TNBC) migration and metastasis. In response to altered ceramide metabolism, we observed that PD-L1 is internalized via clathrin-mediated endocytosis and depends on S278/S279 in its cytoplasmic domain. Once internalized, PD-L1 directly interacts with Caprin-1, an RNA-binding protein previously unassociated with PD-L1 signaling. Through our analysis of clinical specimens, we established a positive association between PD-L1/Caprin-1 interaction and metastatic progression in human TNBC. In terms of mechanism, we determined that the PD-L1/Caprin-1 complex plays a pivotal role in mediating CerS4-dependent cell migration. This complex selectively binds and stabilizes mRNA, resulting in heightened TGFβ/Shh/β-catenin signaling. Conversely, CerS4 restoration in metastatic TNBC cells impaired cellular migration and the interaction between PD-L1 and Caprin-1. Genetic depletion of CerS4 enhanced lung metastasis derived from 4T1 orthotopic allografts and transgenic MMTV-PyMT breast tumors concomitant with PD-L1/Caprin-1 interaction and TGFβ/Shh/β-catenin signaling. Bioinformatics analysis of clinical datasets was used to validate the impact of the CerS4/PD-L1/Caprin-1 axis on metastatic progression in human TNBC. In alignment with enhanced tumor progression, CerS4 depletion in tumors promoted regulatory T-cell infiltration, suggesting an immunosuppressive tumor microenvironment (TME). Treatment with sonidegib, an FDA-approved Hedgehog (Hh) inhibitor, effectively prevented PD-L1 internalization and its interaction with Caprin-1. We hypothesized that sonidegib could synergize with immune checkpoint blockade to sensitize TNBC to immunotherapy, which typically has low response rates. Sonidegib, in combination with αPD-L1 therapy, was significantly more effective in controlling TNBC primary and metastatic tumor growth, especially when Cers4 was downregulated, compared to αPD-L1 monotherapy. Our data also suggested that this potent anti-tumor effect was due to immunotherapy sensitization after combined flow cytometry, multiplexed immunofluorescence, and bulk RNA-sequencing analyses. Altogether, these findings demonstrate a mechanism to define intracellular PD-L1 signaling in response to altered ceramide metabolism, providing a new therapeutic strategy to target TNBC metastasis and improve response to immunotherapy. Citation Format: Wyatt O. Wofford, Jisun Kim, Dosung Kim, Alhaji H. Janneh, Han Gyul Lee, F. Cansu Atilgan, Natalia Oleinik, Elizabeth Hill, Ozge Saatci, Salih Gencer, Paramita Chakraborty, Shikhar Mehrotra, Ozgur Sahin, Besim Ogretmen. Lipid metabolism alterations promote TNBC metastasis and immunotherapy resistance via PD-L1 internalization and signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1570.

Abstract 4109: Discovery of a novel cancer-specific antigen for therapeutic targeting using the Oncotope platform

Abstract Identification of druggable cancer-specific surface antigens is hindered by the complexity of cancer's molecular diversity and the risk of normal cell cross-reactivity. We developed the Oncotope Platform that rapidly identifies multiple cancer-specific cell surface targets not expressed on normal cells (JCO 2022). In this study, we generated polyclonal antibodies to human Triple Negative Breast Cancer (TNBC; Hs578T, ATCC). Following this, we used the Oncotope Platform to filter the anti-TNBC antibody-enriched serum, to yield cancer specific polyclonal antibodies. Using a Protein Microarray, we assessed the binding affinity of both the Total Anti-TNBC Serum (before filtration) and the Oncotope Filtered Anti-TNBC Serum towards approximately 25,000 human proteins. Our results identified antigens that exhibited significantly higher affinity binding to the Oncotope Filtered Anti-TNBC Serum than the Total Anti-TNBC Serum. One of these antigens ZIP7, a zinc transporter situated in the ER and Golgi, has been shown to activate cell proliferation related signaling pathways in TNBC. Our findings revealed the aberrant cell surface expression of ZIP7 on TNBC cells and its potential as a therapeutic target. Flow cytometry analysis using rabbit polyclonal antibodies against ZIP7 revealed substantial binding to TNBC cells, while no significant binding was observed with normal breast epithelial (NBE) cells from the same patient (Hs578BsT, ATCC). Additionally, cytotoxicity assays, employing the same antibodies and a secondary anti-rabbit ADC, showed the preferential killing of TNBC cells over NBE cells (Table 1). In summary, our study marks a breakthrough in identifying cancer-specific antigens with therapeutic potential. The Oncotope Platform, in combination with protein microarrays, enables rapid identification of unique cancer-specific antigens absent in normal cells. Specifically targeting ZIP7 on TNBC cell surfaces highlights its promise for precision cancer therapy. Table 1. Cell Line Incubated with: %Binding Flow Cytometry (1:400 Ab dilution) %Cytotoxicity (4ug/ml Ab conc.) TNBC (Hs578T) Total Anti-TNBC Serum 96.65 94.5 +/- 2.5 Oncotope Filtered Anti-TNBC Serum 97.46 79.2 +/- 2.1 ZIP7 Polyclonal antibody ((Proteintech 11200-1-AP) 46.2 73.2 +/- 0.5 Normal Breast Epithelial cells (Hs578Bst) Total Anti-TNBC Serum 92.95 92.3 +/- 3.0 Oncotope Filtered Anti-TNBC Serum 20.39 17.9 +/- 4.5 ZIP7 Polyclonal antibody ((Proteintech 11200-1-AP) 6.37 14.1 +/- 1.6 Citation Format: John Sanil Manavalan, Danielle Mor, Joseph Davis, Sharanjot Saini, Ipsita Pal, David Feith, Yuichi Hikichi, Elliot Davis. Discovery of a novel cancer-specific antigen for therapeutic targeting using the Oncotope platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4109.

Abstract 6718: Evaluating toxicity and efficacy of bispecific EGFRXCD3 T cell engager in solid tumor-bearing humanized animal model

Abstract Background: Cancer immunotherapy has currently become a highly active research area of oncology and a common treatment strategy for several cancer types. One prominent reason is that the utilized animal models could not accurately predict the of toxic response of human immune system to anti-cancer drug. For improving the safety of immune drug development, it is urgent to establish a predictive nonclinical animal platform that can accurately predict human immune response to the drugs and detect potential unwanted toxicity to human patient will be critical. We have demonstrated a disseminated tumor-bearing humanized mouse model that can evaluate the toxicity and efficacy of bispecific T cell engager (BiTE). In this study, we co-engrafted disseminated and solid tumor cells to the same mice and compared with mice engrafted with disseminated tumor or solid tumor. The purpose is to develop a novel tumor-bearing mouse model that can simultaneously evaluate the long-term efficacy against solid tumors in addition to cytokine level assessment. Method: Epidermal growth factor receptor (EGFR) expressing human triple negative breast cancer MDA-MB-231/Luciferase-2A-GFP (MDA) cell line was utilized to establish solid tumors in mice. The cells were implanted into PBMC humanized NSG-MHC class I/II double knock out (DKO) mice via orthotopic injection into mammary fat pad (MFP). Disseminated MDA cells were injected intravenously (IV) and tumor cells normally colonize in mouse lungs. Following EGFRxCD3 BiTE dosing initiation, tumor burden in MFP and lungs were evaluated via in vivo Xenogen imaging system, and solid tumor volume in MFP was measured 2 to 3 times a week by a digital caliper. To evaluate the toxicity in response to EGFRxCD3 BiTE treatment, human inflammatory cytokine levels was analyzed by BD cytometric bead. Clinical observations and body weight change, and human immune cell activation were evaluated. Result: EGFRxCD3 BiTE treatments significantly inhibited MFP solid tumor growth regardless the presence of IV implanted with MDA cells. EGFRxCD3 BiTE treatment induced elevated human inflammatory cytokines from humanized mice implanted with combined MFP and IV tumors. By comparison, the serum cytokine levels were low in mice implanted with MDA cells via MFP alone, suggesting that disseminated tumor cells are required for cytokine production induced by EGFRxCD3 BiTE. Conclusion: Without disseminated tumor, MFP solid tumor-bearing mice can be served as a model for drug efficacy but did not induce inflammatory cytokines by EGFRxCD3 BiTE treatment. Combination of solid and disseminated tumor models in the same mice provide a new approach for simultaneously evaluating the toxic cytokine release and efficacy of immunotherapy drugs. Citation Format: Guoxiang Yang, Li-Chin Yao, James G. Keck, Mingshan Cheng. Evaluating toxicity and efficacy of bispecific EGFRXCD3 T cell engager in solid tumor-bearing humanized animal model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6718.

Abstract 6630: Non-cell-autonomous cancer progression from chromosomal instability

Abstract Chromosomal instability (CIN) is a driver of cancer metastasis, yet the extent to which this effect depends on the immune system remains unknown. Using ContactTracing—a newly developed, validated and benchmarked tool to infer the nature and conditional dependence of cell-cell interactions from single-cell transcriptomic data—we show that CIN-induced chronic activation of the cGAS-STING pathway promotes downstream signal re-wiring in cancer cells, leading to a pro-metastatic tumor microenvironment. This re-wiring is manifested by type I interferon tachyphylaxis selectively downstream of STING and a corresponding increase in cancer cell-derived endoplasmic reticulum (ER) stress response. Reversal of CIN, depletion of cancer cell STING or inhibition of ER stress response signaling abrogates CIN-dependent effects on the tumor microenvironment and suppresses metastasis in immune competent, but not severely immune compromised, settings. Treatment with STING inhibitors reduces CIN-driven metastasis in melanoma, breast and colorectal cancers in a manner dependent on tumor cell-intrinsic STING. Finally, we show that CIN and pervasive cGAS activation in micronuclei are associated with ER stress signaling, immune suppression and metastasis in human triple-negative breast cancer, highlighting a viable strategy to identify and therapeutically intervene in tumors spurred by CIN-induced inflammation. Citation Format: Jun Li, Melissa Hubisz, Ethan Earlie, Mercedes Duran, Christy Hong, Austin Varela, Emanuele Lettera, Matthew Deyell, Bernardo Tavora, Jonathan Havel, Phyu Su, Amit Dipak Amin, Karolina Budre, Erina Kamiya, Julie-Ann Cavallo, Christopher Garris, Simon Powell, Jorge Reis-Filho, Hannah Wen, Sarah Bettigole, Atif Khan, Benjamin Izar, Eileen Parkes, Ashley Laughney, Samuel Bakhoum. Non-cell-autonomous cancer progression from chromosomal instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6630.

Abstract 5423: Selective inhibition of glucose transporter 3 (GLUT3) suppresses triple-negative breast cancer cell migration by modulating actin cytoskeleton and cell mechanics

Abstract Studies have reported clear clinical observations indicating that obesity and diabetes, which often accompany hyperglycemia, are linked to a poor prognosis across various types of cancer. Specifically, multiple clinical studies involving breast cancer patients have demonstrated that hyperglycemia (>7 mM) is associated with an unfavorable prognosis. Despite the evident correlation between elevated glucose levels and cancer metastasis, the causality remains unclear, and the underlying molecular mechanisms are largely unknown. In both normal and diseased physiology, a broad range of tissue glucose levels has been documented, ranging from normal blood circulation (5.5 mM) to normal tissues (1 - 3 mM) and to tumor tissues (<0.5 mM). Consequently, during the early stages of metastasis, cancer cells undergo a significant increase in extracellular glucose levels. The capacity of cancer cells to deform and exert contractile force, collectively known as cell mechanics, plays a crucial role in various stages of metastasis. In our previous work, we demonstrated that elevated extracellular glucose levels boost glycolysis and activate the RhoA signaling pathway. This leads to heightened cell stiffness and increased contractility in human triple-negative breast cancer (TNBC) cells. Furthermore, the modified cell mechanics are associated with enhanced cell migration and invasion. In this study, we present findings indicating that the inhibition of glucose transporter 3 (GLUT3) significantly diminishes the motility of human TNBC cells, including MDA-MB-231 and its highly metastatic variant, MDA-MB-231/HM cells. Mechanistic investigations reveal that GLUT3 inhibition, facilitated by a well-characterized potent GLUT3-selective inhibitor (G3iA), results in reduced glucose uptake and glycolysis. The diminished glycolysis, induced by GLUT3 inhibition, activates 5' AMP-activated protein kinase (AMPK) and enhances the phosphorylation of Yes-associated protein (YAP). Moreover, GLUT3 inhibition leads to a decrease in non-muscle myosin II activity and induces alterations in F-actin rearrangement by influencing actin-binding proteins such as VASP and cofilin. These changes are associated with a reduction in cell migration. In summary, our results suggest that inhibiting GLUT3 modifies the mechanics of TNBC cells, leading to a decrease in cell motility. Given that GLUT3 is notably elevated in TNBC and is linked to metastatic progression and a poor prognosis in breast cancer patients, our findings underscore the need for further in vivo studies to validate G3iA as a potential anti-metastatic drug in hyperglycemic TNBC animal models. Citation Format: Tae-Hyung Kim, Aadil Qadir Bhat, Cristina V. Iancu, Jun-Yong Choe. Selective inhibition of glucose transporter 3 (GLUT3) suppresses triple-negative breast cancer cell migration by modulating actin cytoskeleton and cell mechanics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5423.

Abstract 1495: Lipocalin-2 promotes breast cancer metastasis via tumor cell intrinsic and extrinsic mechanisms

Abstract While metastasis is the primary factor predicting poor outcomes in solid tumors, survival is lowest in patients initially diagnosed with a localized malignancy that then progresses to metastatic disease. Notably, nearly 25% of early-stage breast cancers (BCs) metastasize during treatment or following an initial positive response to therapeutic intervention. To define the molecular and cellular factors that contribute to metastatic progression of BC, we previously developed the Py230-C57Bl/6 syngeneic experimental metastasis model for interrogating mechanisms by which soluble factors condition premetastatic lung tissue to potentiate BC cell seeding/expansion. Using this model and the TCGA, we identified lipocalin-2 (Lcn2) as a molecular candidate that is upregulated in lung-tropic Py230 cells and other human triple-negative breast cancer (TNBC) cells, and that predicts poor outcomes in human BCs. While Lcn2 has been previously reported to mediate BC progression in genetically engineered mouse models, its mechanism(s) of action remain undefined. Here, we demonstrate that Py230 cell-secreted Lcn2 is necessary for optimal reprogramming of the premetastatic lung during metastatic progression. In agreement with these data, we defined a cell intrinsic role for Lcn2 in BC cell organoid assembly/invasion and expression of proinflammatory factors. To define BC cell extrinsic roles for Lcn2 in remodeling the premetastatic lung, we collected global transcriptome and single-cell spatial proteome data for lung tissue from C57Bl/6 mice systemically educated with mock media or media conditioned with Py8119 (Lcn2low) or Py230 (Lcn2hi) BC cells. Mice receiving Py230 conditioned media treatments were further divided between two groups – one receiving an isotype control IgG and the other receiving an Lcn2 immunoneutralizing antibody. Global transcriptome data was used to determine novel gene set enrichment signatures and clinically relevant interactomes – notable candidates predicted to cooperate with Lcn2 in decreasing patient survival were affiliated with cilia biogenesis and function. Single-cell spatial proteome data were analyzed for 28 different cell populations within the premetastatic lung. Interestingly, Py230 cell-derived Lcn2 did not affect the percentage of ciliated epithelial cells indicating that the global transcriptome data referenced above represent a real increase in expression of cilia-related genes and not an increase in cilia-positive cell types. Notably, we identified that Lcn2 increased the percentage of CD45neg/Despos mechanomodulatory cells and proliferating CD45pos/CD3pos/CD4neg/CD8pos cytotoxic T cells in the premetastatic lung. Taken together, these data define new targets around which new therapeutic strategies can be developed to improve BC patient outcomes. Citation Format: Joshua Gamez, Francesca Sanchez, Gabriela Ortiz-Soto, Jane Cox, Amir Gerami, Jonathan A. Kelber. Lipocalin-2 promotes breast cancer metastasis via tumor cell intrinsic and extrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1495.

Abstract 482: Advancing treatment for metastatic triple negative breast cancer: Liposomal oncolytic adenovirus as a novel neoadjuvant therapy

Abstract Introduction: Adenovirus type-5 (Ad) is considered a prominent viral vector in cancer therapy due to its modifiability for the selective lysis of cancer cells. However, challenges are posed in Ad delivery to cancers lacking coxsackievirus and adenovirus receptors (CARs). In response to this limitation, Ad-encapsulated DOTAP-folate liposomes were developed, which improved Ad delivery and heightened therapeutic efficacy through membrane fusion and folate receptor-mediated endocytosis. Methods: The 4T1 mammary carcinoma model, a CAR-negative triple-negative breast cancer (TNBC) cell line (ER-, PR-, HER2-), resistant to adenoviral infection, was utilized for studying the comparative in vitro transduction of green fluorescent protein (GFP)-expressing Ad (Ad-GFP). The study assessed Ad-GFP with and without DOTAP-folate liposomal encapsulation at a multiplicity of infection (MOI) of 50. Additionally, an oncolytic adenovirus, Ad-hTERT, with a human telomerase promoter, underwent liposomal encapsulation. CAR-negative 4T1 GFP-expressing cells and CAR-positive MDA-MB-231-GFP human breast adenocarcinoma TNBC cells were transduced in vitro at MOI 100 with Ad-hTERT, both with and without DOTAP-folate liposomal encapsulation. Results: CAR-negative 4T1 cells exhibited superior Ad-GFP transduction when encapsulated in DOTAP-folate liposomes, compared to the unencapsulated form (p < 0.001). For oncolytic adenovirus Ad-hTERT, encapsulation resulted in 100% cell lysis within 24 hours in 4T1-GFP cells, contrasting with 13% with unencapsulated Ad-hTERT (P < 0.0001). In CAR-positive MDA-MB-231-GFP TNBC cells, encapsulated Ad-hTERT showed enhanced efficacy, resulting in 100% cell lysis within 24 hours compared to 75% with unencapsulated Ad-hTERT (P < 0.001). Discussion: DOTAP-folate liposomal encapsulation enhances the efficacy of oncolytic Ad-hTERT in both CAR-negative and CAR-positive TNBC cells, establishing it as an optimal therapy for tumors with CAR heterogeneity. Ongoing in vivo studies with Balb/C immune-competent mice and the 4T1 tumor model will assess the effectiveness of encapsulation through intratumoral administration. This approach may extend the recurrence-free period post-surgical tumor removal. Citation Format: Jaimin R. Shah, Tao Dong, Abraham Phung, Sarah L. Blair, Omonigho Aisagbonhi, William C. Trogler, Andrew C. Kummel. Advancing treatment for metastatic triple negative breast cancer: Liposomal oncolytic adenovirus as a novel neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 482.

Abstract 5858: Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer

Abstract Antibody drug conjugates (ADCs) are revolutionizing precision therapeutics in oncology. Sacituzumab Govitecan (SG), combining a Topoisomerase I (TOP1) inhibitor payload (SN38) and the tumor-selective antigen TROP2-targeting antibody hRS7, has demonstrated efficacy in challenging cancers including triple negative breast cancer (TNBC). Despite its promising clinical efficacy, the intricacies of sensitivity and resistance mechanisms to SG remain elusive. To systematically interrogate ADC sensitivity mechanisms, we employed genome-wide CRISPR-Cas9 knockout screening to discover novel regulators influencing SG sensitivity in human TNBC cells. Interactome analysis of top SG sensitizing hits showed clustered pathways of interest, including DNA repair/replication, mTORC1/metabolism, ubiquitin/proteolysis and endosome trafficking/sorting. A top druggable hit sensitizing to SG was PARP1, encoding the poly (ADP-ribose) polymerase 1 that is a key enzyme required for single-strand break repair pathways and for DNA replication fork stability. Furthermore, the synergistic lethality between SG and PARP1 was confirmed in experiments combining SG with PARP inhibitor treatment of TNBC cell lines. To selectively identify SG/ADC-specific genes and pathways, we performed a secondary round of CRISPR knockout and activation screens using a custom library comprising top hits discovered in the primary screen, treating cells in parallel with either SG or cytotoxic payload SN38 alone. Pathway analysis of genes selectively modulating sensitivity to SG but not SN38 revealed novel mediators involved in TROP2 turnover and recycling. We then performed validation and mechanistic studies to elucidate how specific genetic permutations regulate antibody delivery and sensitivity to SG. In summary, employing a systematic CRISPR screening method, we uncovered numerous novel pathways associated with both resistance and sensitization, playing a role in ADC delivery and target protein turnover. Our current focus involves prioritizing druggable genes and pathways, laying the foundation for proof-of-concept studies that will combine ADCs with specific targeted therapeutics, aiming to enhance tumor-killing efficacy. Citation Format: Bogang Wu, Sheng Sun, Nayana Thimmiah, Aiko Nagayama, James T. Coates, Ting Liu, Win Thant, Elena Bitman, David Li, John Doench, Aditya Bardia, Leif W. Ellisen. Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5858.

Abstract 6800: The tumor progression mechanism of increasing CXCL16-CXCR6 pathway within the tumor microenvironment in TNBC

Abstract Background: Triple negative breast cancer (TNBC) is characterized rapid progression, higher relapse, and poor prognosis, so the establishment of an effective therapeutic target is required. CXCL16, a chemokine abundantly present in the TNBC tumor microenvironment (TME), is secreted by tumor-associated macrophages (TAM) and is known to promote tumor growth and metastasis. However, the exact mechanism through which CXCL16 facilitates tumor growth and metastasis remains unclear. This study aims to study the role of CXCL16 in the tumor progression and confirm the anti-tumor effects of CXCL16 neutralization in TNBC model. Method: MDA-MB-231 human TNBC cell lines were utilized for in vitro validation, and a syngeneic model involving 4T1 mouse TNBC cell lines and Balb/c mice was used for in vivo studies. Results: Stimulation of TNBC cells with recombinant human CXCL16 protein (rhCXCL16) activates the tumor growth signaling pathways, including pAKT and STAT3, as well as EMT markers. These results shows that CXCL16 can promote tumor growth and metastasis. Treatment with CXCL16 neutralizing antibodies inhibited the promotion of cancer growth and metastasis by rhCXCL16. Additionally, combination treatment of CXCL16 antibodies with PD-1 antibodies in the 4T1 syngeneic model increased the proportion of CD8+ T cells and CD80+ M1 like macrophages. These results shows that neutralization of CXCL16 can regulate polarization of macrophage and proportion of T cells. These regulation and changes of immune cells allow neutralization of CXCL16 control the TME to resist tumor cells, suggesting that optimal condition can be constructed to maximize the effectiveness of anti-cancer drugs. Conclusion: This study confirms that the CXCL16-CXCR6 pathway within the TME triggers tumor growth and metastasis, while neutralization of CXCL16 can inhibit tumor progression. This effect is attributed to the increase in CD8+ T cell and M1 macrophage proportions within the TME. In conclusion, these research findings propose the potential of CXCL16 as a novel biomarker and therapeutic target for TNBC. Citation Format: Yun sun Lee, Cham Han Lee, Seong Yun Ha, Sang Hyeob Lee, Jin Yi Yang, Young Sang Kim, Sung Moo Kim, Seong Keun Kim. The tumor progression mechanism of increasing CXCL16-CXCR6 pathway within the tumor microenvironment in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6800.

Abstract 5645: HuR regulates aerobic glycolysis in triple negative breast cancer (TNBC)

Abstract Introduction: The RNA-binding protein Human Antigen R (HuR/ELAVL1) is ubiquitously expressed and exerts post-transcriptional regulation on mRNAs characterized by U/AU-rich sequences in their 3' UTR. Elevated cytoplasmic expression of HuR has been associated with various malignancies, influencing key cellular processes such as proliferation, apoptosis, angiogenesis, and metastasis. The involvement of HuR influences prognostic outcomes and hence is a potential biomarker and therapeutic target in cancer research. The present study investigates the oncogenic role of HuR in TNBCs, an aggressive subtype of breast cancer. Methods: Immunohistochemistry was performed on TNBC patient tumor tissue (n=40) to asses the expressions of HuR and MMP9. Human TNBC cell lines MDA-MB-231 and MDA-MB-468 were used for in vitro studies after confirming cytoplasmic HuR accumulation in these cells via Immunofluorescence. The RNA binding activity of HuR was disrupted using CMLD2, a specific inhibitor of HuR, and subsequent physiological changes were assessed through functional assays. An exploratory investigation of HuR-moderated metabolic changes was performed employing NANOSTRING nCounter XT gene expression assay using metabolic pathways panel. Investigation and/or validation of molecular changes were done via immunoblotting. Results: Meta-analysis using Liu-2014 TNBC dataset correlated high HuR protein expression with reduced patient survival (p=0.028). HuR (77%) and MMP9(55%) were found to be overexpressed in TNBC tissues. Inhibiting HuR with CMLD2 resulted in the downregulation of the protein and its downstream targets CDK2, MMP9, and β catenin. HuR inhibition compromised proliferation, migration, Matrigel invasion and EMT (downregulated E-Cadherin, N-Cadherin, and Vimentin) in TNBC cell lines. Further, HuR inhibition resulted in decreased clonogenicity in 2D and 3D TNBC cultures. Transcriptomics studies showed that HuR inhibition in MDA-MB-231 altered the profile of glycolytic genes in addition to other pathways such as cell cycle and DNA damage repair. In accordance, the expression of glycolytic proteins was reduced upon CMLD2 treatment. Similar results were obtained on HuR knockdown by siRNA suggesting that HuR-mediated effects on TNBC progression might be in part through its modulation of aerobic glycolysis. Conclusion: Overall, our results demonstrate HuR as a positive regulator of aerobic glycolysis in TNBCs and the ability of CMLD2 to inhibit HuR provides a rationale to consider it as a potential anticancer agent for TNBCs. Citation Format: Arundhathi Dev J R, Ajay Gogia, Sandeep R. Mathur, S.V.S. Deo, Chandra Prakash Prasad. HuR regulates aerobic glycolysis in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5645.

Abstract 4149: Real-time intravital trafficking of tumor infiltrating lymphocytes in breast cancer models

Abstract Cancer immunotherapy utilizing the immunoreactive efficiency of immune cells via adoptive cell therapy (ACT) has been a novel method of cancer treatment. In tumor microenvironment, immune cell reactivity of tumor-infiltrating lymphocytes (TILs) can facilitate cancer cell attack, which finally derives efficient suppression of tumor growth. However, the conventional evaluation for cancer immunotherapy has been generally performed by ex vivo methods, providing restricted information on lymphocyte motility and immune cell dynamics within the tumor microenvironment. In vivo data with the dynamic behavior of TILs in breast cancer can provide new insight to investigate novel targets of immune checkpoint inhibitors. In this research, longitudinal intravital imaging of motility trafficking of TILs in breast cancer animal models has been performed by utilizing intravital microscopy to unveil the immunoreactive dynamics of T cell activity in early stage. In vivo analysis of TIL motility within different breast cell lines, including 4T1 (mouse breast cancer cell), MDA-MB-231 (human triple-negative breast cancer cell), and MCF7 (human breast cancer cell), respectively, has been investigated for immune responses to cancer. We’ve monitored spatiotemporal dynamics of TIL over 11 days in breast cancer xenograft mouse model generated by tumor implantation into the dorsal skinfold imaging chamber and adoptive T cell transfer by blood circulation. In the results, the adoptive immunoreactivity of TIL has been differentiated by the timepoint of adoptive T cell transfer, distance proximity of T cells in tumors, and breast cancer types. Furthermore, anti-cancer effect coordinated with dendritic cells behaviors showed induced immune responses with accumulated dendritic cells with increased TIL count over time in breast cancer model treated with various cancer therapy such as immunotherapy, radiation therapy and anti-cancer drug (anti-PD-L1), which implied a relationship between programmed death-ligand 1 expression levels and TIL/dendritic cell motility. The result demonstrates that intravital TIL motility trafficking analysis in cancer model can be an invaluable assessment method to explain the detailed cellular/molecular mechanism of immune response to cancer involved with T lymphocytes, dendritic cells, and immune checkpoint inhibitors to accelerate the efficiency of cancer immunotherapy. Citation Format: Soyeon Ahn, Kubra Akyildiz, Hyunseok Kim, Pilhan Kim. Real-time intravital trafficking of tumor infiltrating lymphocytes in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4149.

Abstract 6108: Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor (TF) activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus (sn) ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=33), Kenyan (n=25), and European American (EA, n=24) women in relation to genetic ancestry, risk factor exposures, clinical characteristics, and 5-year survival. To achieve this, we successfully isolated intact, high-quality single nuclei from archival frozen breast tumor tissue through an optimized combination of enzymatic digestion and automated tissue homogenization. We performed snMultiome sequencing of 82 tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Harmony), our dataset includes a total of 296,557 nuclei. Cancerous (163,419 nuclei) and non-cancerous (133,138 nuclei) cells were distinguished based on DNA copy number (CopyKat). Within the microenvironment, 11 major immune, epithelial, and stromal cell types were successfully annotated, exhibiting distinct patterns by population group (e.g. AA tumors showed markedly increased abundance of myeloid and T-cells, while Kenyan tumors showed increased abundance of pericytes and fibroblasts). A large number of enriched TFs within each cell type varied significantly by population group, suggesting distinct chromatin accessibility patterns related to genetic ancestry. Within cancerous cells, striking intra- and inter-tumoral heterogeneity was observed by genetic ancestry even within molecular subtype groups. Current efforts focus on in-depth molecular characterization of ancestry- and risk factor-related differences in the tumor epithelium and microenvironment and distinct signatures present in lethal disease. This project holds the potential to yield crucial insights into how ancestry or other factors may influence the etiology of different breast cancer subtypes, as well as produce clinically actionable biomarkers and therapeutic targets to enhance precision medicine within patient populations at high risk for aggressive disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany Jenkins-Lord, Tiffany H. Dorsey, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6108.

Abstract 3716: Enhancing anti-tumor responses in triple-negative breast cancer by combining SIRPα inhibition with immunogenic chemotherapy or antibody-drug conjugates

Abstract Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer defined by the absence of hormone receptors and HER2 receptor on tumor cells. Because of a lack of targetable receptors and poor response to PD1/PDL1 immunotherapy, TNBC has limited systemic treatment options. In previous work using a genomic screen (Shuptrine et al., 2017), we identified CD47 as one of the leading factors that contributes to TNBC immune evasion. The CD47-SIRPα axis is an innate immune checkpoint that primarily regulates myeloid cells. In the context of cancer, tumor cells upregulate CD47 to engage SIRPα inhibitory signaling to block phagocytosis. While CD47 or SIRPα blockade alone show limited antitumor effect, therapies that combine blockade of this axis with other treatment agents, such as tumor-antigen specific antibodies, show superior tumor inhibition. Thus, we hypothesize that the addition of SIRPα inhibition to existing TNBC treatments (chemotherapy and Trop2-antibody drug conjugates (ADC)) will improve their treatment efficacy. Methods: To ascertain the impact of SIRPα inhibition combined with chemotherapy and Trop2-ADC, we utilized and validated a novel SIRPα KO mouse, along with SIRPα mAbs, in our previously utilized models of Triple-Negative Breast Cancer (TNBC) (Crosby et al., 2018). Using both in vitro and in vivo tumor models, we tested the therapeutic impact of SIRPα inhibition/blockade combined with different immunogenic chemotherapies (doxorubicin/cyclophosphamide) and Sacituzumab govitecan (SG) in TNBC. Results: After validating the loss of SIRPα in KO mice, we found that E0771 TNBC cells had comparable growth rates in WT and SIRPα KO mice. Similarly, we did not observe different anti-tumor responses to Doxorubicin between WT and SIRPα-KO mice. However, we observed more profound suppression of E0771 tumors after cyclophosphamide treatment in SIRPα KO mice than in WT mice. To evaluate the efficacy of SG in inducing antibody-dependent cellular phagocytosis (ADCP), we co-cultured SIRPα KO and WT mouse macrophages with a syngeneic mouse TNBC model (MM3MG-Trop2-WT). We found that SG promotes ADCP in WT macrophages, and SIRPα loss further increased this ADCP, suggesting the potential importance of antibody-mediated phagocytosis as a mechanism of action for SG. In vivo, we implanted human TNBC cells (MDA-MB-468) in mice and treated them with SG with and without SIRPα antibody blockade. These studies demonstrated an anti-tumor effect from SG that was potentially enhanced by SIRPα blockade, suggesting the clinical potential of this combination in TNBC. Conclusion: Our study demonstrates that the addition of SIRPα inhibition to chemotherapy or SG may promote tumor phagocytosis and stimulate stronger antitumor immunity in TNBC. These results support further investigation of SIRPα-targeting combination therapies for TNBC. Citation Format: Xingru Ma, Li-Chung Tsao, Tao Wang, Cong-Xiao Liu, Melissa Gajda, Xiao Yang, Gangjun Lei, Junping Wei, Zachary Hartman. Enhancing anti-tumor responses in triple-negative breast cancer by combining SIRPα inhibition with immunogenic chemotherapy or antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3716.

Hexachlorobenzene as a differential modulator of the conventional and metronomic chemotherapy response in triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is usually treated with high doses of paclitaxel, whose effectiveness may be modulated by the action of environmental contaminants such as hexachlorobenzene. High doses of paclitaxel cause adverse effects such as low cellular selectivity and the generation of resistance to treatment due to an increase in the expression of multidrug resistance proteins (MRPs). These effects can be reduced using a metronomic administration scheme with low doses. This study aimed to investigate whether hexachlorobenzene modulates the response of cells to conventional chemotherapy with paclitaxel or metronomic chemotherapy with paclitaxel plus carbachol, as well as to study the participation of the MRP ATP-binding cassette transporter G2 (ABCG2) in human TNBC MDA-MB231 cells. Cells were treated with hexachlorobenzene alone or in combination with conventional or metronomic chemotherapies. The effects of treatments on cell viability were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the nuclear factor kappa B pathway participation was evaluated using a selective inhibitor. ABCG2 expression and its modulation were determined by western blot. Results confirmed that paclitaxel reduces MDA-MB231 cell viability in a concentration-dependent manner. Results also showed that both conventional and metronomic chemotherapies reduced cell viability with similar efficacy. Although hexachlorobenzene did not modify cell viability per se, it did reverse the effect induced by the conventional chemotherapy, without affecting the efficacy of the metronomic chemotherapy. Additionally, a differential modulation of ABCG2 expression was determined, mediated by the nuclear factor kappa B pathway, which was directly related to the modulation of cell sensitivity to another cycle of paclitaxel treatment. The findings indicate that, in human TNBC MDA-MB231 cells, in the presence of hexachlorobenzene, the metronomic combination of paclitaxel plus carbachol is more effective in affecting the tumor biology than the conventional therapeutic administration scheme of paclitaxel.

Unveiling the relationship between WWOX and BRCA1 in mammary tumorigenicity and in DNA repair pathway selection

Breast cancer is the leading cause of cancer-related deaths in women worldwide, with the basal-like or triple-negative breast cancer (TNBC) subtype being particularly aggressive and challenging to treat. Understanding the molecular mechanisms driving the development and progression of TNBC is essential. We previously showed that WW domain-containing oxidoreductase (WWOX) is commonly inactivated in TNBC and is implicated in the DNA damage response (DDR) through ATM and ATR activation. In this study, we investigated the interplay between WWOX and BRCA1, both frequently inactivated in TNBC, on mammary tumor development and on DNA double-strand break (DSB) repair choice. We generated and characterized a transgenic mouse model (K14-Cre;Brca1fl/fl;Wwoxfl/fl) and observed that mice lacking both WWOX and BRCA1 developed basal-like mammary tumors and exhibited a decrease in 53BP1 foci and an increase in RAD51 foci, suggesting impaired DSB repair. We examined human TNBC cell lines harboring wild-type and mutant BRCA1 and found that WWOX expression promoted NHEJ repair in cells with wild-type BRCA1. Our findings suggest that WWOX and BRCA1 play an important role in DSB repair pathway choice in mammary epithelial cells, underscoring their functional interaction and significance in breast carcinogenesis.

Siglec15/TGF‐β bispecific antibody mediates synergistic anti‐tumor response against 4T1 triple negative breast cancer in mice

AbstractAn ideal tumor‐specific immunomodulatory therapy should both preferentially target the tumor, while simultaneously reduce the immunosuppressive environment within the tumor. This guiding principle led us to explore engineering Siglec‐15 (S15) targeted bispecific antibody (bsAb) to enhance therapy against triple negative breast cancer (TNBC). S15 appears to be exclusively expressed on macrophages and diverse tumor cells, including human and mouse 4T1 TNBC. TGF‐β is a growth hormone frequently associated with increased tumor invasiveness, including in TNBC. Here, to overcome the immune‐suppressive environment within TNBC tumors to enable more effective cancer therapy, we engineered a bispecific antibody (bsAb) targeting both Siglec15 and TGF‐β. In mice engrafted with orthotopic 4T1 tumors, S15/TGF‐β bsAb treatment was highly effective in suppressing tumor growth, not only compared to control monoclonal antibody (mAb) but also markedly more effective than mAbs against S15 alone, against TGF‐β alone, as well as a cocktail of both anti‐S15 and anti‐TGF‐β mAbs. We did not detect liver and lung metastasis in mice treated with S15/TGF‐β bsAb, unlike all other treatment groups at the end of the study. The enhanced anti‐tumor response observed with S15/TGF‐β bsAb correlated with a less immunosuppressive environment in the tumor. These results underscore S15‐targeted bsAb as a promising therapeutic strategy for TNBC, and possibly other S15 positive solid tumors.

Cell morphology best predicts tumorigenicity and metastasis in vivo across multiple TNBC cell lines of different metastatic potential

BackgroundMetastasis is the leading cause of death in breast cancer patients. For metastasis to occur, tumor cells must invade locally, intravasate, and colonize distant tissues and organs, all steps that require tumor cell migration. The majority of studies on invasion and metastasis rely on human breast cancer cell lines. While it is known that these cells have different properties and abilities for growth and metastasis, the in vitro morphological, proliferative, migratory, and invasive behavior of these cell lines and their correlation to in vivo behavior is poorly understood. Thus, we sought to classify each cell line as poorly or highly metastatic by characterizing tumor growth and metastasis in a murine model of six commonly used human triple-negative breast cancer xenografts, as well as determine which in vitro assays commonly used to study cell motility best predict in vivo metastasis.MethodsWe evaluated the liver and lung metastasis of human TNBC cell lines MDA-MB-231, MDA-MB-468, BT549, Hs578T, BT20, and SUM159 in immunocompromised mice. We characterized each cell line's cell morphology, proliferation, and motility in 2D and 3D to determine the variation in these parameters between cell lines.ResultsWe identified MDA-MB-231, MDA-MB-468, and BT549 cells as highly tumorigenic and metastatic, Hs578T as poorly tumorigenic and metastatic, BT20 as intermediate tumorigenic with poor metastasis to the lungs but highly metastatic to the livers, and SUM159 as intermediate tumorigenic but poorly metastatic to the lungs and livers. We showed that metrics that characterize cell morphology are the most predictive of tumor growth and metastatic potential to the lungs and liver. Further, we found that no single in vitro motility assay in 2D or 3D significantly correlated with metastasis in vivo.ConclusionsOur results provide an important resource for the TNBC research community, identifying the metastatic potential of 6 commonly used cell lines. Our findings also support the use of cell morphological analysis to investigate the metastatic potential and emphasize the need for multiple in vitro motility metrics using multiple cell lines to represent the heterogeneity of metastasis in vivo.

Assessment of the Efficacy of the Combination of RNAi of lncRNA DANCR with Chemotherapy to Treat Triple Negative Breast Cancer Using Magnetic Resonance Molecular Imaging.

Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is overexpressed in human triple-negative breast cancer (TNBC) and promotes cell migration and proliferation. TNBC is limited in treatment options relative to hormone-receptor-positive breast cancer and is commonly treated with chemotherapy, which is often compromised by acquired resistance. DANCR has been implicated in the development of chemoresistance across multiple cancer types. Here, we applied magnetic resonance molecular imaging (MRMI) with a targeted contrast agent, MT218, specific to extradomain-B fibronectin (EDB-FN), a marker for epithelial-to-mesenchymal transition, to assess the therapeutic efficacy of the combination of paclitaxel and ZD2-PEG-ECO/siDANCR nanoparticles (ZD2-siDANCR-ELNP) to treat TNBC. The treatment of orthotopic MDA-MB-231 TNBC in mice with paclitaxel significantly suppressed tumor growth but with a significant increase of EDB-FN in the tumor, as revealed by MRMI and immunohistochemistry. Combining ZD2-siDANCR-ELNP with paclitaxel further reduced tumor sizes, along with reduced EDB-FN expression. Interestingly, MT218-MRMI revealed a lower reduction of tumor signal enhancement with the combination treatment than that with the siDANCR treatment alone, which was supported by higher cell density in the tumors treated with the combination therapy, as shown by histochemical analysis. MT218-MRMI clearly revealed the changes of the tumor microenvironment in response to various therapies and is effective to noninvasively assess the response of TNBC tumors to the therapies. Regulating oncogenic lncRNA DANCR is an effective strategy for improving the outcomes of chemotherapy in TNBC.

Epigenetic Effects of Resveratrol on Oncogenic Signaling in Breast Cancer.

The crosstalk between oncogenic signaling pathways plays a crucial role in driving cancer development. We previously demonstrated that dietary polyphenols, specifically resveratrol (RSV) and other stilbenoids, epigenetically target oncogenes for silencing via DNA hypermethylation in breast cancer. In the present study, we identify signal transduction regulators among RSV-hypermethylated targets and investigate the functional role of RSV-mediated DNA hypermethylation in the regulation of Hedgehog and Wnt signaling. Non-invasive ER-positive MCF-7 and highly invasive triple-negative MCF10CA1a human breast cancer cell lines were used as experimental models. Upon 9-day exposure to 15 µM RSV, pyrosequencing and qRT-PCR were performed to assess DNA methylation and expression of GLI2 and WNT4, which are upstream regulators of the Hedgehog and Wnt pathways, respectively. Our results showed that RSV led to a DNA methylation increase within GLI2 and WNT4 enhancers, which was accompanied by decreases in gene expression. Consistently, we observed the downregulation of genes downstream of the Hedgehog and Wnt signaling, including common targets shared by both pathways, CCND1 and CYR61. Further analysis using chromatin immunoprecipitation identified increased H3K27 trimethylation and decreased H3K9 and H3K27 acetylation, along with abolishing OCT1 transcription factor binding. Those changes indicate a transcriptionally silent chromatin state at GLI2 and WNT4 enhancers. The inhibition of the Wnt signal transduction was confirmed using a phospho-antibody array that demonstrated suppression of positive and stimulation of negative Wnt regulators. In conclusion, our results provide scientific evidence for dietary polyphenols as epigenetics-modulating agents that act to re-methylate and silence oncogenes, reducing the oncogenic signal transduction. Targeting such an action could be an effective strategy in breast cancer prevention and/or adjuvant therapy.