Treating Triple-negative Breast Cancer Cells Research Articles

Nanoprodrug ratiometrically integrating autophagy inhibitor and genotoxic agent for treatment of triple-negative breast cancer

Effective combination therapies are urgently needed to treat triple-negative breast cancer (TNBC), which is insensitive to the existing treatment regimens. However, the synergistic potency of traditional small-molecule combinations is limited in TNBC mainly due to mismatched molar ratios, inconsistent pharmacokinetics, and intratumoral accumulation of individual drugs. Here, we find that the autophagy inhibitor hydroxychloroquine (HCQ) and the topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin (SN38) exhibit synergistic effects when the molar ratio reaches 5:1. We further develop a glutathione-responsive self-assembled combination nanoparticle (Combo NP) to integrate individual HCQ and SN38 polymeric prodrugs at the optimized ratio. In TNBC cells treated with Combo NP, HCQ-mediated autophagy blockage significantly enhances the DNA damage and apoptotic effect of SN38, manifesting synergistically cytotoxic effects of Combo NP. In vivo evaluations show that Combo NP maintains the molar ratio of HCQ to SN38 within the synergistic range in mouse blood circulation and intratumoral tissues. More importantly, Combo NP elicits superior therapeutic benefit in metastatic TNBC models, compared to free drug combination as well as single drug nanoparticles. Taken together, our engineered nanosystem highlights a nanoprodrug-based chemosensitizing approach for improving the therapeutic response to TNBC, addressing the major challenges of the current combination therapy.

LIFR inhibition enhances the therapeutic efficacy of HDAC inhibitors in triple negative breast cancer

Histone deacetylase inhibitors (HDACi) are identified as novel therapeutic agents, however, recent clinical studies suggested that they are marginally effective in treating triple negative breast cancer (TNBC). Here, we show that first-in-class Leukemia Inhibitory Factor Receptor (LIFRα) inhibitor EC359 could enhance the therapeutic efficacy of HDACi against TNBC. We observed that both targeted knockdown of LIFR with CRISPR or treatment with EC359 enhanced the potency of four different HDACi in reducing cell viability, cell survival, and enhanced apoptosis compared to monotherapy in TNBC cells. RNA-seq studies demonstrated oncogenic/survival signaling pathways activated by HDACi were attenuated by the EC359 + HDACi therapy. Importantly, combination therapy potently inhibited the growth of TNBC patient derived explants, cell derived xenografts and patient-derived xenografts in vivo. Collectively, our results suggest that targeted inhibition of LIFR can enhance the therapeutic efficacy of HDACi in TNBC.

CHFR regulates chemoresistance in triple-negative breast cancer through destabilizing ZEB1

Failures to treat triple-negative breast cancer (TNBC) are mainly due to chemoresistance or radioresistance. We and others previously discovered that zinc finger E-box-binding homeobox 1 (ZEB1) is a massive driver causing these resistance. However, how to dynamically modulate the intrinsic expression of ZEB1 during cell cycle progression is elusive. Here integrated affinity purification combined with mass spectrometry and TCGA analysis identify a cell cycle-related E3 ubiquitin ligase, checkpoint with forkhead and ring finger domains (CHFR), as a key negative regulator of ZEB1 in TNBC. Functional studies reveal that CHFR associates with and decreases ZEB1 expression in a ubiquitinating-dependent manner and that CHFR represses fatty acid synthase (FASN) expression through ZEB1, leading to significant cell death of TNBC under chemotherapy. Intriguingly, a small-molecule inhibitor of HDAC under clinical trial, Trichostatin A (TSA), increases the expression of CHFR independent of histone acetylation, thereby destabilizes ZEB1 and sensitizes the resistant TNBC cells to conventional chemotherapy. In patients with basal-like breast cancers, CHFR levels significantly correlates with survival. These findings suggest the therapeutic potential for targeting CHFR-ZEB1 signaling in resistant malignant breast cancers.

Efficacy of Targeted ECO/miR-200c Nanoparticles for Modulating Tumor Microenvironment and Treating Triple Negative Breast Cancer as Non-invasively Monitored by MR Molecular Imaging.

To investigate the effectiveness of targeted ECO/miR-200c in modulating tumor microenvironment and treating triple negative breast cancer (TNBC) using non-invasive magnetic resonance molecular imaging (MRMI) of extradomain B fibronectin (EDB-FN) with a targeted MRI contrast agent. MDA-MB-231 and Hs578T TNBC cells were transfected with RGD-PEG-ECO/miR-200c. Invasive and migratory potential was evaluated using transwell, scratch wound, and spheroid formation assays. Athymic nude mice bearing orthotopic MDA-MB-231 and Hs578T xenografts were treated with weekly i.v. injection of RGD-PEG-ECO/miR-200c nanoparticles at 1.0 mg/kg/week RNA for 6weeks. MRMI of EDB-FN was performed using a targeted contrast agent MT218 [ZD2-N3-Gd(DO3A)] on a 3T MRS 3000 scanner. T1-weighted images were acquired following intravenous injection of MT218 at dose of 0.1mmol/kg using a fast spin echo axial sequence with respiratory gating. Systemic administration of RGD-PEG-ECO/miR-200c nanoparticles in mice bearing orthotopic TNBC xenografts significantly suppressed tumor progression without toxic side-effects. MRMI with MT218 revealed that the treatment significantly suppressed tumor proliferation as compared to the control. MRMI also showed that the miR-200c treatment altered tumor microenvironment by reducing EDB-FN expression, as evidenced by decreased contrast enhancement in both MDA-MB-231 and Hs578T tumors. The reduction of EDB-FN was confirmed by immunohistochemistry. Targeted delivery of miR-200c with RGD-PEG-ECO/miR-200c nanoparticles effectively modulates tumor microenvironment and suppresses TNBC proliferation in animal models. MRMI of tumor EDB-FN expression is effective to non-invasively monitor tumor response and therapeutic efficacy of RGD-PEG-ECO/miR-200c nanoparticles in TNBC.

Co-loading of an anthracycline analogue Pirarubicin and Salinomycin in a 1:3 ratio into Quatramerbiodegradable polymeric nanoparticles synergistically inhibit growth of triple-negative breast cancer.

e15047 Background: Triple negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers and differ from other types of invasive breast cancers in that they grow and spread faster. TNBCs have limited treatment options and a worse prognosis. Therapy with anthracyclines considered to be one of the most effective agents in the treatment. Unfortunately, resistance to anthracycline therapy is very common due to drug efflux mediated by overexpression of ABC transporter. Pirarubicin (PIRA), an analogue of doxorubicin (DOX), is approved in Japan, Korea and China and is shown to be less cardiotoxic than DOX. Recent studies suggest that cancer stem cells (CSCs) play an important role in tumorigenesis and biology of TNBC. Targeting CSCs may be a promising, novel strategy for the treatment of this aggressive disease. Recent studies have shown that salinomycin (SAL) preferentially targets the viability of CSCs. Methods: SAL and PIRA were co-encapsulated in polylactic acid (PLA)-based block copolymeric nanoparticles (NPs) to efficiently co-deliver these agents to treat TNBC cells. Results: Generated SAL-PIRA co-encapsulated dual drug-loaded NPs showed an average diameter of 110 ± 7 nm, zeta potential of -12.5 mV and PDI of less than 0.25. Both of these anti-cancer agents showed slow and sustained release profile in non-physiological buffer (PBS, pH 7.4) from these dual drug-encapsulated NPs. Additionally, multiple ratios (PIRA:SAL = 3:1, 1:1, 1:3) were encapsulated to generate diverse dual drug-loaded NPs. The results demonstrate that, in contrast to 1:1 and 3:1, treatment of TNBC cells with 1:3 ratio of PIRA:SAL dual drug-loaded NPs, was associated with significant inhibition of growth in vitro in multiple TNBC cell lines. Interestingly, PIRA:SAL (1:3) was synergistic as compared to either SAL- or PIRA single drug-loaded NPs. The IC50 of PIRA and SAL in single drug-encapsulated NPs is 150 nM and 700 nM respectively in MDA-MB-468. Importantly, the IC50 of PIRA in dual drug-encapsulated NPs dropped down to 30 nM (5-fold). Similar results were obtained in SUM-149 TNBC cell line. Studies are underway to evaluate in vivo biological activity of PIRA:SAL (1:3) on tumor growth in a TNBC xenograft mice model. Conclusions: These results demonstrate that a novel dual drug-loaded NP formulation of PIRA and SAL in a unique ratio of 1:3 represents an approach for successful targeting of CSCs and bulk tumor cells in TNBC and potentially other cancer types.

Unravelling the human triple negative breast cancer suppressive activity of biocompatible zinc oxide nanostructures influenced by Vateria indica (L.) fruit phytochemicals

The present study delineates the biosynthesis of ZnOVI nanostructures by using aqueous fruit extract of V. indica. The study has disclosed the role of V. indica fruit extract as both reducing and capping agents, ushering the formation of ZnOVI nanostructures with distinct morphologies. The formation of ZnOVI nanostructures was corroborated by FT-IR and UV–visible spectroscopy which was further substantiated by the elemental composition study through EDS spectroscopy. The nanostructures were also investigated by Rietveld refinement of PXRD data, FE-SEM, and BET analysis. The morphology, size, and surface area were found to be precursor stoichiometry dependent. The in-vitro cytotoxicity study of ZnOVI nanostructures carried out on MDA-MB468 human triple-negative breast cancer (TNBC) cells has revealed their potential cytotoxicity (91.18 ± 1.98). MTT assay performed on the NIH3T3 mouse fibroblast cells has unfolded the non-toxic nature of ZnOVI nanostructures. Additionally, the results of the AO-EB dual staining assay indicated early apoptosis in TNBC cells by displaying greenish yellow-fluorescence in the nuclei. Reactive oxygen species (ROS) measurement study has confirmed the elevated intracellular levels of ROS, supporting the oxidative-stress induced cytotoxicity in ZnOVI nanostructures treated TNBC cells. Furthermore, the haemocompatibility of ZnOVI nanostructures was evaluated using human erythrocytes. Thus, the obtained results have shown greater potential in the anticancer activity of bio-fabricated ZnOVI nanostructures.

Secreted Factors from Adipose Tissue Reprogram Tumor Lipid Metabolism and Induce Motility by Modulating PPARα/ANGPTL4 and FAK

Recent studies indicate that adipose tissue in obesity promotes breast cancer progression by secreting protumorigenic chemokines, growth factors, and fatty acids. However, the detailed mechanisms by which hypertrophic adipose tissue influences breast cancer cells are still not well understood. Here we show that co-culture with adipose tissue from high-fat diet induced obese C57BL/6 mice alters transcriptome profiles in triple-negative breast cancer (TNBC) cells, leading to upregulation of genes involved in inflammation and lipid metabolism, such as IL1B, PLIN2, and ANGPTL4. Similar results were obtained by treating TNBC cells with adipose tissue conditioned media (ACM) generated from fat tissue of obese female patients. Many of the upregulated genes were activated by PPAR nuclear receptors, as shown by pathway analyses and gene expression experiments using PPAR agonists and antagonists. Metabolic analysis revealed that TNBC cells cultivated with ACM had significantly higher levels of β-oxidation. Furthermore, ACM-treated TNBC cells displayed a pronounced aggressive cell phenotype, with enhanced wound healing, proliferation, and invasion capabilities. ACM-induced invasion was dependent on the PPAR-target ANGPTL4 and activated FAK signaling, as shown by ANGPTL4 depletion and FAK inhibition. Together, our data suggest that factors released by adipose tissue change PPAR-regulated gene expression and lipid metabolism and induce a more aggressive TNBC cell phenotype. These effects are, at least in parts, mediated by fatty acids provided by the adipose tissue. IMPLICATIONS: Adipose tissue provides factors for increased progression of TNBC cells, identifying PPAR- and FAK-signaling as potential novel targets for treatment of TNBC, especially in obese women.

Nanoparticle-delivered miriplatin ultrasmall dots suppress triple negative breast cancer lung metastasis by targeting circulating tumor cells

No effective therapy is yet available to treat triple negative breast cancer (TNBC), which has poor prognosis due to frequent metastasis. Cancer stem cells (CSCs) or CSC-like cells play crucial roles in cancer metastasis and are exceptionally tolerant with genetic lesions. The extent of DNA damages has an important impact on the fate of CSCs. Despite the importance of platinum [Pt(II)] agents in cancer therapy, accumulating reports showed the treatment failure of conventional Pt(II) drugs, which is likely due to their inadequate DNA damage effects. Miriplatin is a clinically approved drug only being locally-used for treating liver cancer. In this study, we developed a novel ultrasmall Pt(II) dot (uPtD) from miriplatin and encapsulated it into our recently-reported integrin α5(ITGA5) active targeting nanoparticles (uPtDs NPs) and tested their therapeutic efficacy against TNBC metastasis. It was found that uPtDs NPs displayed a superior DNA damage capability via enhanced-interactions with DNA and a significantly stronger effect in reducing CSC-like property of TNBC cells, compared to conventional cisplatin and miriplatin. Mechanistically, the severe DNA damages induced by uPtDs NPs activated the CHK1/2-CDC25A-cyclin A/E pathway to induce cell cycle arrest. Moreover, uPtDs NPs could target the in vivo circulating tumor cells (CTCs) to suppress TNBC lung metastasis. Given the desired-safety profile of miriplatin, the uPtDs represent a promising therapeutic agent of the metal-based nanomedicines to reduce cancer metastasis. SignificanceThe miriplatin ultrasmall dots developed from clinically-prescribed miriplatin may serve as a potent systemically-administered agent to target CTCs and reduce cancer metastasis.

Abstract 5213: Drug repurposing to treat triple negative breast cancer (TNBC) based on a novel ER beta-p53-p73 signaling axis

Abstract That endocrine therapy or HER2 targeted therapies are not effective against TNBC makes it the most difficult breast cancer subtype to treat. Although these tumors initially respond to chemotherapy, there is high risk of relapse. Therefore, there is urgent need to develop new therapeutic strategies against these aggressive cancers. Reports including the Cancer Genome Atlas (TCGA) show that about 80% of TNBC express mutant p53 and it is a major driver of these cancers. Furthermore, 60-80% of TNBC express estrogen receptor-beta (ERβ). We have shown that tumor suppressor protein p53 status (wild type versus mutant) is a determinant of pro-versus anti-tumorigenic roles of ERβin breast cancer (JNCI, 2019, 111:1202-1215). These observations, along with our novel discovery that Tamoxifen (Tam) increases interaction between ERβ and mutant p53 leading to decreased binding of mutant p53 to tumor suppressor p73 resulting in reactivation of p73 in TNBC cells, led us to hypothesize that Tam can be repurposed to treat TNBC stratified based on combined expression of mutant p53 and ERβ. Experimental procedures to test this hypothesis include use of cell culture, TNBC cell line-derived xenograft (CDX), and patient tumor-derived organoid (PDO) models, proximity ligation assay (PLA), co-immunoprecipitation (Co-IP), quantitative chromatin immunoprecipitation (qChIP), quantitative real time PCR (qPCR), RNAi-mediated knockdown, gene knockout with CRISPR, RNA-seq, and bioinformatics data analysis. Our data show that Tam when combined with the widely used chemotherapeutic agent doxorubicin (Doxo) reduces several fold the IC50 of Doxo in TNBC cells. Importantly, Tam was unable to decrease the IC50 of Doxo in isogenic TNBC cells where mutant p53 was knocked out demonstrating that the effect of Tam mediated by ERβ and p73 is directed against the oncogenic gain-of-functions of mutant p53. Consistent with this, the combination treatment elicited robust antitumor effect on CDX tumors in vivo. The drug combination also increases apoptosis as a consequence of restored p73 activity. Conversely, PHTPP (an ERβ-specific antagonist) disrupts the ERβ-mutant p53 interaction enabling mutant p53 to bind and inactivate p73. Our finding that Tam in combination with Doxo activates p73 leading to robust apoptotic response and inhibition of tumor growth suggest that Tam, a drug that is well tolerated with relatively less side effects, can be repurposed in combination with Doxo to treat TNBCs expressing mutant p53 and ERβ and this approach can reach the clinic relatively fast as both the drugs are FDA-approved and have been in the clinic for several years to treat other forms of cancer. Furthermore, based on our data that Tam decreases the IC50 of Doxo considerably, there is potential for using Doxo at a dose much lower than what is currently used in the management of TNBC, thereby reducing major side effects of Doxo. Citation Format: Chetan C. Oturkar, Christina Adams, Utpal K. Mukhopadhyay, Alexander Caradori, Manasori Oshi, Yoshihisa Tokumaru, Chad J. Creighton, Jun H. Park, Nishant Gandhi, Kazuaki Takabe, Kristopher M. Attwood, Benny A. Kaipparettu, Gokul M. Das. Drug repurposing to treat triple negative breast cancer (TNBC) based on a novel ER beta-p53-p73 signaling axis [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 5213.

Abstract 3801: Oxidative stress induces glutamine-dependent GD2+ triple negative breast cancer stem cells

Abstract Breast cancer stem cells (BCSCs) have been shown to be associated with tumor recurrence, metastasis and drug resistance. We reported that the ganglioside GD2 selectively identifies BCSCs and its over-expression in Triple-Negative Breast Cancer (TNBC). However, the factors regulating GD2+ BCSCs in a growing tumor are not known. Here we hypothesize that nutrient deprivation-induced oxidative stress upregulates GD2+ BCSCs in TNBC. TNBC cell lines including SUM159 and MDA-MB-231 were cultured under different nutrient-deprived conditions and analyzed for GD2 expression by flow cytometry. We have identified a steady increase of GD2+ cells from 5%±2% on day 3 to 20%±5% on day 6 in cells cultured in nutrient deprived condition. We further performed the in vivo by injecting GFP+ MDA-MB-231 cells into NSG mice and measured GD2+ expression in the growing tumor once a week. Interestingly, we also noticed a positive correlation between the percentage of GD2+ cells and tumor volume in vivo with r=0.9829 and p<0.0004 suggesting nutrient deprived conditions in the growing tumors results in higher GD2 expression. Next, the cells were cultured in medium with or without glucose or media containing 2-deoxy glucose (2DG, glycolysis inhibitor). We found that the percentage of GD2+ cells increased 2-fold in the absence of glucose or in the presence of 2DG (10mM) compared to cells cultured with glucose (6g/L) or without 2DG, suggesting that glucose deprivation enhances GD2 biosynthesis in TNBC cells. Global metabolomics profiling using liquid chromatography-mass spectrometry followed by KEGG pathway analysis identified signatures such as glutathione mediated detoxification and glutathione biosynthesis to be most highly upregulated in GD2+ compared to GD2- cells. As glutamine is a key precursor for glutathione biosynthesis, we cultured SUM159 and MDA-MB-231 cells in media with or without glutamine and found that the percentage of GD2+ cells positively correlates with glutamine concentration in the medium. Next, to target glutamine metabolism, we treated TNBC cells with glutaminase inhibitor, CB-839 or glutamine transporter inhibitor, V9302. This resulted in a 70-80% reduction of GD2 expression in a dose dependent manner. CB-839 and V9302 also inhibited mammosphere formation by 40-50% and increased reactive oxygen species (ROS) in a dose-dependent manner in TNBC cells. In conclusion, oxidative stress results in GD2+ BCSC phenotype in TNBC cells. Inhibition of glutamine uptake or its utilization by V9302 or CB-839 inhibits GD2+ BCSC phenotype and alters redox homeostasis in BCSCs by inducing ROS levels. Targeting glutamine transporter or glutaminase could complement conventional chemotherapy by targeting BCSCs in TNBC. Citation Format: Appalaraju Jaggupilli, Stanely J. Ly, Roshan Borkar, Khoa Nguyen, Bin Yuan, Nagireddy Putluri, Michael Andreeff, V. Lokesh Battula. Oxidative stress induces glutamine-dependent GD2+ triple negative breast cancer stem cells [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 3801.

Abstract 3803: Synergistic inhibition of thioridazine combined with carboplatin on triple negative breast cancer by targeting cancer stem cells

Abstract In the past decades, chemotherapy has shown encouraging but limited efficacy treating triple negative breast cancer (TNBC). Targeting cancer stem cells (CSCs) has been recognized as a promising method to inhibit tumor growth and dissemination. This study aimed to determine whether an anti-psychotic drug thioridazine (THZ) could sensitize metastatic TNBC cells to anti-cancer drug carboplatin (CBP). To this end, metastatic TNBC (4T1)-bearing animals were treated with THZ, CBP and their combination respectively. MTT, flow cytometry, electron microscope and western blot were performed to evaluate the treated 4T1 cells. Tumor size and lung metastasis were monitored. The percentage of residual 4T1 cancer stem cells and the tumorigenecity of residual tumor cells from each treated group were compared. We found that both mono- and combination treatments suppressed 4T1 cells and induced apoptosis in a dose-dependent manner. These effects involved mitochondrial dysfunction and mitochondria-mediated apoptosis. The structure of mitochondrial membrane was impaired as observed by electron microscope, and the mitochondrial membrane stability was interrupted as evident by down-regulated Bcl-2 & Bcl-xL expression, and up-regulated BAX expression. Both extrinsic and intrinsic apoptosis were demonstrated by enrichment of activated caspase 3, 8, 9. Tumor growth and lung metastasis in 4T1-bearing animals were markedly inhibited by CBP combined with THZ. We also found suppression of PI3k/AKT/mTOR pathway and activation of ER stress by CSP + THZ both in vitro and in vivo. Moreover, angiogenesis was inhibited, demonstrating an important mechanism for tumor metastasis inhibition. More importantly, THZ demonstrated the capability of destroying CSCs and this effect was amplified when combined with CBP. The tumorigenecity of CBP + THZ combination-treated residual tumor cells was significantly impaired. These results show that THZ could inhibit 4T1 tumor cell proliferation and induce apoptosis and significantly destroy the stability of mitochondrial membrane, causing extrinsic and intrinsic apoptosis when combined with CBP. A signature of dysangiogenesis indicated that angiogenesis might be involved in this process. These findings suggest that the anti-psychotic drug THZ is a promising chemosensitizer in TNBC tumor and TNBC patients may benefit from THZ + CBP administration treatment by targeting cancer stem cells. Keywords: Triple-negative breast cancer (TNBC); cancer stem cells (CSCs); thioridazine (THZ); carboplatin (CBP). Citation Format: Yi Wang, Leiming Xia, Li Gong, Yang Xia, Liu Liu, Yuanyuan Liu, Wicha Max, Alfred E. Chang, Qiao Li, Yangyi Bao. Synergistic inhibition of thioridazine combined with carboplatin on triple negative breast cancer by targeting cancer stem cells [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 3803.

Abstract 126: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in PTEN-null TNBC model

Abstract Background: The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating triple-negative breast cancer (TNBC); but not all patients respond to this combination. The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors. In this study, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in PTEN-null TNBC cells in vitro and tumors in vivo to try to identify the combination with the most clinical potential. Methods: The PTEN-null TNBC cell line, MDA-MB-468, was used in this study. For in vitro studies, proliferation, cellular apoptosis, and 3D on-top clonogenic growth were measured, tube formation assays and western blot analysis were performed. For in vivo studies, a xenograft model was used to examine treatment responses, and immunohistochemical analysis was performed. Results: Interestingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation of MDA-MB-468 cells. Importantly, addition of dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further increased the activation of caspase 3 and PARP, thereby enhancing the antitumor efficacy of veliparib plus carboplatin. In an MDA-MB-468 xenograft model, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination. Conclusion: Here, we provide novel evidence that veliparib combined with carboplatin drives Src activation in the preclinical model tested. Moreover, we provide provocative in vitro and in vivo data highlighting the potential for increasing therapeutic efficacy by combining dasatinib with veliparib and carboplatin in PTEN-null TNBC. Citation Format: Yuliang Sun, Xiaoqian Lin, Jennifer Aske, Casey Williams, Mark Abramovitz, Brian Leyland-Jones. Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth in PTEN-null TNBC model [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 126.

Targeting epidermal growth factor-overexpressing triple-negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor.

ObjectivesTraditional cancer therapy and regular immunotherapy are ineffective for treating triple‐negative breast cancer (TNBC) patients. Recently, chimeric antigen receptor‐engineered natural killer cells (CAR NK) have been applied to target several hormone receptors on different cancer cells to improve the efficacy of immunotherapy. Furthermore, epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. Here, we demonstrated that EGFR‐specific CAR NK cells (EGFR‐CAR NK cells) could be potentially used to treat patients with TNBC exhibiting enhanced EGFR expression.Materials and methodsWe investigated the cytotoxic effects of EGFR‐CAR NK cells against TNBC cells in vitro and in vivo. The two types of EGFR‐CAR NK cells were generated by transducing lentiviral vectors containing DNA sequences encoding the single‐chain variable fragment (scFv) regions of the two anti‐EGFR antibodies. The cytotoxic and anti‐tumor effects of the two cell types were examined by performing cytokine release and cytotoxicity assays in vitro, and tumor growth assays in breast cancer cell line‐derived xenograft (CLDX) and patient‐derived xenograft (PDX) mouse models.ResultsBoth EGFR‐CAR NK cell types were activated by TNBC cells exhibiting upregulated EGFR expression and specifically triggered the lysis of the TNBC cells in vitro. Furthermore, the two EGFR‐CAR NK cell types inhibited CLDX and PDX tumors in mice.ConclusionsThis study suggested that treatment with EGFR‐CAR NK cells could be a promising strategy for TNBC patients.

Abstract A092: DNA repair proficiency predicts disparities in triple negative breast cancer outcomes

Abstract Breast cancer remains as one of the most lethal type of gynecological cancers in women. Among various subtypes, triple negative breast cancer (TNBC) is the most aggressive and difficult to treat subtype due to limited therapeutic options. African American women have higher death rate from breast cancer than women of other racial and ethnic groups. Additionally, the incidence of germline BRCA1 mutations is much lower among African American women diagnosed with TNBC, than the women of the other races or ethnicities. Mechanistically, increased DNA repair and cell cycle checkpoint activation remain as the foremost reasons behind TNBC tumor resistance to chemotherapy and recurrence. We used a CHK1 inhibitor prexasertib to inhibit cell cycle checkpoint activation in TNBC cells. Interestingly, we found that prexasertib treatment promotes both proteasome-mediated degradation of BRCA1 and RAD51 proteins and affects their transcript levels. Analysis of DNA repair efficiency in prexasertib treated TNBC cells revealed reduction in homologous recombination efficiency compared to control cells. Based on these results, we hypothesized that prexasertib treatment induced homologous recombination deficiency (HRD) should cause synergistic lethality when combined with PARP inhibitors (PARPi) in TNBC cells. As predicted, combined treatment of TNBC cells with prexasertib and PARPi olaparib resulted in increased DNA strand breaks, gH2AX foci and nuclear disintegration, an indicator of mitotic catastrophe. Similarly, these drug combinations also caused synergistic lethality in multiple TNBC cell lines when compared to single drug-treatments, as indicated by combination index (CI) values. Since RAD51 is the downstream effector repair protein in FA-BRCA pathway-mediated HR, we evaluated TCGA data for RAD51 gene expression using UALCAN portal. Computational analysis revealed that RAD51 overexpression in breast tumors compared to normal breast tissues, in particular, TNBC subtype showed highest RAD51 expression compared to other subtypes of breast cancer. Overall, these data show RAD51 as a poor prognostic marker for breast cancer patients. Interestingly, there was a discrepancy in RAD51 expression levels in distinct racial groups, where African American and Asian breast cancer patients showed high RAD51 expression compared to Caucasian breast cancer patients. Consistent with these observations, African American and Asian TNBC patients show decreased survival probability. Based on these data, RAD51 could be a biomarker for aggressive TNBC and racial disparity in breast cancer therapeutic outcomes. As positive correlation exists between RAD51 and CHEK1 expression in breast cancer, in-vitro preclinical data presented here provides additional mechanistic insights for further evaluation of the rational combination of prexasertib and olaparib for improved prognosis and to reduce racial disparity in TNBC. Citation Format: Chinnadurai Mani, Jonnalagadda Shirisha, Awasthi Sanjay, Manne Upender, Palle Komaraiah. DNA repair proficiency predicts disparities in triple negative breast cancer outcomes [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr A092.

Exploring the Role of Mitochondrial ClpP Activators on Immune Signaling in Breast Cancer

Approximately 20% of breast cancer cases diagnosed yearly are of the triple‐negative breast cancer (TNBC) type. Current treatments for TNBC patients are limited to surgical intervention and traditional chemotherapies. ONC201 is a recently discovered orally‐active imipridone molecule in phase I/II clinical trials for a large variety of aggressive cancers including glioblastoma, breast, endometrial, and others. We have been studying highly potent analogs of ONC201, known as the TR compounds (TRs), for their efficacy in TNBC. To better understand the mechanism of action, we coupled a TR compound to agarose beads and applied proteomics to identify potential targets. Our studies identified the mitochondrial caseinolytic peptidase (ClpP), as a major ONC201 and TRs binding protein. We further demonstrated that these compounds activated the proteolytic activity of ClpP and that this mitochondrial signal was required for subsequent activation of the integrated stress response as determined by increased CHOP and ATF4 proteins. In addition to activation of the integrated stress response, other recent studies have suggested that the antitumor effects of ONC201 may in part be dependent on increased immune cell recruitment and cancer growth inhibition through this mechanism. Thus, one of our major objectives is to understand how activation of ClpP by ONC201 or TRs impacts cellular signaling events that lead to enhanced immune cell recruitment. We hypothesize that activation of ClpP promotes activation of key immune signaling proteins, like NF‐kB, which subsequently leads to an increase in proteins required for immune cell recruitment. Accordingly, our preliminary data shows activation or up‐regulation of proteins connected to innate immune signaling including MAVS, NF‐kB, and TBK1 in response to ONC201 or TRs treatment. Additionally, our qPCR data shows that treatment of TNBC cells with ONC201 or TRs leads to increased mRNA levels of specific Interleukins (IL‐12, IL‐10). Collectively, these results suggest that activation of ClpP by ONC201 or TRs increases innate immune signaling and expression of cytokines, which are crucial events for successful recruitment of immune cells. Future experiments will evaluate if activation of NF‐κB by ONC201 or TRs treatment is ClpP dependent using a ClpP knockout in TNBC cell lines. Lastly, we will also measure changes in levels of known NF‐κB regulated cytokines (TNFα, IL‐6, IL‐18 and IL‐1β) after treating TNBC cells with ONC201 or TRs using flow cytometry. Overall, this work provides valuable new insight into potential mechanisms by which ClpP activators mediate their anti‐cancer effects through immune signaling and recruitment.Support or Funding InformationL.J.A.‐C was supported by the NIH 5 T32 GM007040

Potent Suppression of Proliferation of Breast Carcinoma Cells by a Novel Anthranilic Acid Derivative

Background/objective: Chemotherapy remains the mainstay therapy for the most intrusive-type of breast carcinoma, triple-negative breast cancer (TNBC) that has a higher tendency for visceral metastases, relapses, and poor prognoses. It is well recognized that the reactivation of a ribonucleoprotein enzyme, telomerase is among the key determinants of breast carcinogenesis, cellular immortalization and metastatic progression. Therefore, our objectives were to assess (i) short- and (ii) long-term effects of a novel anthranilic acid (GV6) developed at our institute and compare it to a known analogue, BIBR1532 on TNBC (MDA-MB 231) and non-TNBC (MCF-7) cells. Methods: Seeded TNBC and non-TNBC flasks were supplemented with 25 µM of either BIBR 1532 or GV6 or solvent (DMSO) alone for 14 (Short-term) or 27 (Long-term) days. Trypan-blue dye exclusion test was utilized to determine viable cells, senescence-associated β-galactosidase activity was employed to detect senescent cells and qPCR was used to quantitate transcript abundance. Results: Cell viability assay revealed that short- and long-term growth inhibitions of TNBC and non-TNBC cells were comparable between BIBR1532 and GV6. Cytochemical detection of βGalactosidase demonstrated that GV6 was equally effective in inducing replicative senescence in treated TNBC and non-TNBC cells. Short- and long-term regimen of BIBR1532 and GV6 showed similar drug-induced downregulation of hTERT in TNBC and non-TNBC cells. Conclusion: Results indicate that GV6 is an equally potent inhibitor of hTERT that induces growth impedance and triggers senescence in TNBC, as well as non-TNBC cells and merits further studies for improved treatment options.

Abstract P3-11-19: Preclinical model of TNBC: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth

Abstract Background: The combination of a poly ADP ribose polymerase (PARP) inhibitor with a DNA-damaging agent has shown promise in treating triple-negative breast cancer (TNBC); however, not all patients respond to this combination. The Src protein kinase modulates multiple cancer cell properties and plays a key role in tumorigenic processes. However, Src inhibitors as single agents have shown limited effects in solid tumors. In this study, we examined the antitumor effects of the Src inhibitor dasatinib, the PARP inhibitor veliparib, and the DNA-damaging agent carboplatin in TNBC cells in vitro and tumors in vivo to try to identify the combination with the most clinical potential. Methods: The BRCA-mutated and PTEN null TNBC cell line, SUM149PT, was used in this study. For in vitro studies, proliferation, cellular apoptosis, and 3D on-top clonogenic growth were measured, tube formation assays and western blot analysis were performed. For in vivo studies, a xenograft model was used to examine treatment responses, and immunohistochemical analysis was performed. Results: Surprisingly, treatment with the combination of veliparib plus carboplatin led to an increase in Src phosphorylation. Importantly, addition of dasatinib attenuated Src overexpression induced by veliparib plus carboplatin and further inhibited the downstream signaling of Src, thereby enhancing the antitumor efficacy of veliparib plus carboplatin. In an SUM149PT xenograft model, the triple combination of dasatinib with veliparib plus carboplatin showed greater tumor growth inhibitory effects compared with single agents or double combinations. No systemic toxicity was observed in mice treated with the triple combination. Conclusion: Here, we provide novel evidence that veliparib combined with carboplatin drives Src activation in the preclinical model tested. Moreover, we provide provocative in vitro and in vivo data highlighting the potential for increasing therapeutic efficacy by combining dasatinib with veliparib and carboplatin in BRCA-mutated and PTEN null TNBC. Citation Format: yuliang Sun, Xiaoqian Lin, Jennifer Aske, Casey Williams, Mark Abramovitz, Brian Leyland-Jones. Preclinical model of TNBC: Dasatinib attenuates Src pathway signaling and combined with veliparib and carboplatin potently inhibits tumor growth [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-19.

Abstract P1-05-13: Dissecting the interplay of the long non-coding RNA NORAD and PARP inhibitors in triple negative breast cancer

Abstract Background and Aims: Triple-negative breast cancer (TNBC) is the most aggressive subtype, and is often associated with mutations in the breast cancer susceptibility protein (BRCA) genes, which are known tumor-suppressor genes involved in DNA damage repair. Chromosomal instability (CIN) has been added to the hallmarks of cancer and is characterized by a frequent gain or loss of chromosomes during mitosis (i.e. chromosomal aberrations). BRCA1 and BRCA2 breast tumors develop by specific and distinct evolutionary paths, as their gene profiles and genome aberration spectra differ from each other and from those in sporadic BC. The lncRNA Non-Coding RNA Activated by DNA Damage (NORAD) has been demonstrated to contribute to the progression of several cancer entities by regulating genomic stability. Therefore, we wanted to explore a potential additive anti-cancerous effect by treating TNBC cells with a combination of NORAD silencing and PARP inhibition. Material and Methods: To clarify the potential clinical significance of NORAD in human TNBC, patient data from publicly available databases were analyzed. In order to further characterize NORAD, we performed phenotypic experiments after establishing an siRNA-mediated knock-down approach. Expression levels of NORAD as well as DNA-damage repair proteins were analyzed by qRT-PCR and Western Blot after combined cell treatment with NORAD silencing and PARP inhibition. To further unravel the molecular mechanism behind, we performed immunofluorescent experiments against markers for DNA-double strand breaks and homologous recombination efficiency (i.e. Rad51 and yH2AX) after TNBC treatment with NORAD siRNAs and PARP inhibitors. Results: High NORAD levels were identified to be a negative prognostic factor for disease-free survival of TNBC patients (HR with 95% CI: 2.4 (1.35-4.25); n=161; p=0.002). Furthermore, the NORAD gene is amplified in up to 3% of BC cases and mRNA levels are higher in tumor tissue when compared to healthy tissue. A qPCR based screening of several breast cancer cell lines showed an at least 4-times increased expression of NORAD when compared to normal mammary cells (i.e. MCF 12A). Knock-down of NORAD in TNBC cells resulted in a slightly reduced proliferation as well as to a decreased expression of several DNA repair proteins. Immunofluorescent experiments of TNBC cells treated with the PARP inhibitor olaparib and/or after NORAD silencing revealed increased yH2AX-dependent nuclear foci formation when compared to control conditions, indicating augmented DNA double-strand breaks after treatment. Conclusion: Our first data let us hypothesize that a combination of NORAD knock-down and PARPi treatment increases inhibition of DNA repair mechanisms and leads to a more pronounced “anti-tumorigenic” phenotype in TNBC cells. This approach could lead to the optimization of current treatment concepts. Furthermore, as increased NORAD expression is associated with an unfavorable patient survival, this lncRNA could serve as potential prognostic biomarker. Citation Format: Christiane Klec, Anita Kapeller, Felix Prinz, Christoph Trenk, Herbert Stöger, Martin Pichler. Dissecting the interplay of the long non-coding RNA NORAD and PARP inhibitors in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-13.

An isoflavone derivative potently inhibits the angiogenesis and progression of triple-negative breast cancer by targeting the MTA2/SerRS/VEGFA pathway.

Objective: Angiogenesis plays a vital role in tumor growth and metastasis. Here, we aimed to find novel efficient antiangiogenic molecules targeting vascular endothelial growth factor A (VEGFA ) at the transcriptional level to treat triple-negative breast cancer (TNBC).Methods: We used a cell-based seryl tRNA synthetase (SerRS) promoter-driven dual-luciferase reporter system to screen an in-house library of 384 naturally occurring small molecules and their derivatives to find candidate molecules that could upregulate the expression of SerRS, a potent transcriptional repressor of VEGFA. The levels of SerRS and VEGFA were examined by quantitative RT-PCR (qRT-PCR), western blotting, and/or ELISAs in TNBC cells after candidate molecule administration. Zebrafish, the Matrigel plug angiogenesis assay in mice, the TNBC allograft, and xenograft mouse models were used to evaluate the in vivo anti-angiogenic and anti-cancer activities. Furthermore, the potential direct targets of the candidates were identified by proteomics and biochemical studies.Results: We found the most active compound was 3-(4-methoxyphenyl) quinolin-4(1H)-one (MEQ), an isoflavone derivative. In TNBC cells, MEQ treatment resulted in increased SerRS mRNA (P < 0.001) and protein levels and downregulated VEGFA production. Both the vascular development of zebrafish and Matrigel plug angiogenesis in mice were inhibited by MEQ. MEQ also suppressed the angiogenesis in TNBC allografts and xenografts in mice, resulting in inhibited tumor growth and prolonged overall survival (P < 0.05). Finally, we found that MEQ regulated SerRS transcription by interacting with MTA2 (Metastasis Associated 1 Family Member 2).Conclusions: Our findings suggested that the MTA2/SerRS/VEGFA axis is a drug-treatable anti-angiogenic target, and MEQ is a promising anti-tumor molecule that merits further investigation for clinical applications.

Structural Modifications on CORM-3 Lead to Enhanced Anti-angiogenic Properties Against Triple-negative Breast Cancer Cells.

Carbon monoxide-releasing molecules (CORMs) are a special class of organometallic complexes that have been reported to offer beneficial effects against different conditions including several subtypes of cancer. Especially for the aggressive and poorly treated triplenegative breast cancer (TNBC), early CORMs have been shown to diminish malignant angiogenesis and may be considered as an alternative approach. So, this study aimed at testing novel CORM molecules against angiogenesis in TNBC seeking potent drug candidates for new therapies. Based on previous studies, CORM-3 was chosen as the lead compound and a group of 15 new ruthenium-based CORMs was synthesized and subsequently evaluated in vitro for potential anti-angiogenic properties. A similar anti-angiogenic behaviour to the lead complex was observed and a new CORM, complex 4, emerged as a promising agent from this study. Specifically, this complex offered better inhibition of the activation of VEGFR2 and other downstream proteins of vascular endothelial cells. Complex 4 also retained the ability of the parent molecule to reduce the upregulated VEGF expression from TNBC cells and inhibit endothelial cell migration and new vessel formation. The lack of significant cytotoxicity and the downregulating activity over the cytoprotective enzyme haem oxygenase-1 (HO-1) in cancer cells may also favour CORMs against this poorly treated subtype of breast cancer. Since the anti-angiogenic approach is one of the few available targeted strategies against TNBC, both CORM-3 and the new complex 4 should be considered for further research as combination agents with existing anti-angiogenic drugs for more effective treatment of malignant angiogenesis in TNBC.