TNBC Cell Lines Research Articles

Abstract LB261: Establishment and characterization of a paclitaxel-resistant human triple-negative breast cancer cell line

Abstract Triple-negative breast cancer (TNBC) is a lethal female cancer, of which paclitaxel resistance is a major factor limiting treatment outcomes, and continued unmet needs for more effective therapeutic strategies are arduous. The aim of this study is to establish a paclitaxel-resistant TNBC cell line and to characterize the underlying mechanism for potential strategies to overcome the resistance. The human TNBC cell line MDA-MB-231 was exposed to stepwise escalating paclitaxel concentration from 0.5 to 50 nM and a resistant cell line T50R was selected. The IC50 of paclitaxel was 2 and >100 nM in MDA-MB-231 and T50R cells respectively. However, T50R cells required 50 nM Taxol for proliferation. When cultured in the absence of paclitaxel, T50R cells arrested at mitotic stage with high levels of spindle abnormalities. Since induction of microtubule (MT) stabilization plays critical roles in paclitaxel’s potency, we thus measure cell MT polymerization and kinetochore-MT stability. The results showed that, compared to parental MDA-MB-231 cells, the interphase MTs were less polymerized and the kinetochore-MTs were less stable in T50R cells, indicating that the MT dynamic instability was significantly increased in T50R cells. Thus, the increased dynamic unstable MTs in T50R cells might contribute to drug requirement for cell growth and counteract with paclitaxel effects, and consequently lead to drug-resistant phenotype. Identification and interference of endogenous regulators involved in the increased MT instability in T50R cells might provide leads to overcome paclitaxel resistance. Citation Format: Ling-Huei Yih, Hsiao-Hui Kuo, Chieh-Ting Fang. Establishment and characterization of a paclitaxel-resistant human triple-negative breast cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB261.

Abstract LB167: Acetalax (Oxyphenisatin acetate, NSC 59687) and bisacodyl (dulcolax) cause oncosis in triple negative breast cancer by poisoning the ion exchange membrane protein TRPM4

Abstract Triple negative breast cancer (TNBC) is generally unresponsive to current therapies, and the development of new anticancer agents is needed. Oxyphenisatin acetate, called Acetalax, which was used as a laxative in the 1950’s, has recently been reported to have anticancer activity in several murine cancer models. In this study we demonstrate that Acetalax and its structural analog laxative analog bisacodyl (Dulcolax) exhibit potent antiproliferative activity in TNBC cell lines. We show that both cause oncosis, a non-apoptotic cell death characterized by cellular and nuclear swelling and cell membrane blebbing that leads to mitochondrial dysfunction and ATP depletion. Acetalax was further shown to enhance immune and inflammatory responses and inhibit protein synthesis. Mechanistically, we provide evidence that TRPM4 (Transient Receptor Potential Melastatin member 4) is poisoned by Acetalax and bisacodyl. By blocking TRPM4, a monovalent cation channel, Acetalax and Bisacodyl cause oncosis, and TNBC cells without endogenous TRPM4 expression as well as TRPM 4 knockout TNBC cells were found to be Acetalax- and Bisacodyl resistant. TRPM4 was also lost in cells with acquired Acetalax resistance. Moreover TRPM4 is rapidly degraded the ubiquitin-proteasome system upon acute exposure to Acetalax and bisacodyl. Together, these results demonstrate that TRPM4 is a previously unknown target of Acetalax and Bisacodyl and that TRPM4 expression in cancer cells is a predictor of Acetalax and Bisacodyl efficacy, and could be used for the clinical development of these drugs as anticancer agents. Citation Format: Makito Mizunuma, Christophe Redon, Liton Saha, Naoko Takebe, Yves G. Pommier. Acetalax (Oxyphenisatin acetate, NSC 59687) and bisacodyl (dulcolax) cause oncosis in triple negative breast cancer by poisoning the ion exchange membrane protein TRPM4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB167.

Abstract LB061: Aurora kinases inhibition enhances neoadjuvant chemotherapy response in triple negative breast cancer

Abstract Triple-Negative Breast Cancer (TNBC) is a type of breast cancer without expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2). TNBC accounts for around 15-20% of all breast cancers, and it remains the most challenging subtype to treat because of its aggressive phenotype and limited treatment strategies. Since TNBC does not respond to endocrine and HER2-targeted therapy, chemotherapy and anti-PD-L1 immunotherapy remain the major treatment strategies so far. However, not all TNBC patients respond to chemotherapy, suggesting that TNBC is a complicated disease. Identifying biomarkers to further predict and subdivide TNBC patients with distinct chemotherapy responses and outcomes is very important. To profile the protein expression and phosphorylation alterations, comprehensive proteogenomics was deployed to probe the molecular basis for differential response to neoadjuvant chemotherapy for TNBC. From a Reverse Phase Protein Array (RPPA) on TNBC cell lines MDA-MB-468 cells and MDA-MB-231 cells treated with Docetaxel, Paclitaxel, Epirubicin, and Doxorubicin, we found only a short list of protein changes after chemotherapy. Among them, the most significant changes in protein are phosphor-AURKA/B/C, AURKA, AURKB, and PLK1, which fall into the mitotic kinase group and are confirmed by western blot analysis. In addition, no significant change in the mRNA level was found after chemotherapy treatment, suggesting a post-transcription level molecular mechanism regulates the upregulation of AURKA and AURKB protein. In fact, the protein level of AURKB decreased more significantly with Cycloheximide (CHX) treatment alone compared with the co-treatment of CHX and Paclitaxel, indicating Paclitaxel extended the lifetime of Aurora Kinases protein and increased the protein stability. Moreover, AURKB or AURKA overexpression in TNBC cells renders them resistant to Paclitaxel treatment and attenuates the apoptosis effect triggered by chemotherapy treatment, suggesting Aurora Kinases could mediate the chemo-resistance in TNBC. To overcome the resistance to chemotherapy, we treated resistant TNBC cells with the combination of Aurora kinase inhibitors and anthracycline/taxane chemotherapy and found that Aurora kinase inhibition significantly enhanced the chemotherapy effect. In summary, we found that Aurora Kinases upregulation by treatment with Taxanes-type and Anthracyclines-type chemotherapy could render chemotherapy resistance to TNBC cells. Citation Format: Jia Xu, Chang Liu, Bohan Ning, Ali Cem Kucukdagli, Yunjia Chen. Aurora kinases inhibition enhances neoadjuvant chemotherapy response in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB061.

Abstract LB178: HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action

Abstract Triple negative breast cancer (TNBC) accounts for 15-20% of all breast carcinomas and has poor prognosis. Chemotherapy is the mainstay treatment for TNBC patients, yet with limited clinical benefit. Androgen receptor (AR) is expressed in approximately 50% of TNBC tumors and thus the anti-androgen therapy could be a promising solution for the treatment of AR+ TNBC. Nevertheless, AR inhibitors including enzalutamide and bicalutamide or the androgen biosynthesis inhibitor abiraterone have failed to demonstrate satisfying anti-tumor activity in clinical trials. We have discovered HP518, an AR-targeting PROTAC (proteolysis targeting chimera) that induces potent degradation of both the wild-type AR and AR mutants. HP518 is currently under the clinical development for the treatment of mCRPC. Here we report the preclinical data on HP518 for the treatment of AR+ TNBC. HP518 robustly degrades AR in multiple TNBC cell lines with a half-maximal degradation concentration (DC50) < 1 nM. HP518 strongly inhibits AR+ TNBC cell growth, whereas the AR inhibitor enzalutamide does not show significant anti-proliferative effect on these cells in the presence or absence of androgen. Consistent with the in vitro results, HP518 significantly and dose-dependently inhibits tumor growth in MDA-MB-453 cell line derived tumor xenograft (CDX) and patient-derived xenograft (PDX) mouse models. PI3KCA activating mutations are frequently expressed in AR+ TNBC. HP518 in combination with alpelisib, a PI3Kα inhibitor shows a synergistic anti-tumor activity. Mechanistically, HP518, but not AR inhibitor enzalutamide downregulates the mRNA expression levels of a set of DNA replication-related genes in AR+ TNBC cells, indicating a novel and ligand-independent role for AR in regulating growth of AR+ TNBC cells. Therefore, HP518, an AR-targeting PROTAC degrader holds great potential to fulfill the unmet clinical needs for the AR+ TNBC therapy. Selected pre-clinical data will be presented. HP518 structure will not be disclosed. Citation Format: Jing Li, Hongwei Yuan, Kefan Chen, Yongxu Huo, Guoqing Liu, Wu Du, Xinghai Li. HP518, an oral AR-targeting PROTAC for the treatment of AR positive triple negative breast cancer with a novel mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB178.

Oncogenic miR-106b-5p promotes cisplatin resistance in triple-negative breast cancer by targeting GDF11.

Cytoplatin (CDDP) is a standard treatment for triple-negative breast cancer (TNB), but patient resistance to CDDP limits its efficacy. A growing study confirms that microRNAs (miRNAs) are significantly important in breast cancer, especially TNBC. This research was carried out to examine the function of miR-106b-5p in CDDP resistance of TNBC as well as the downstream mechanism. The miR-106b-5p and growth-differentiation factor 11 (GDF11) expressions in the tissues from TNBC patients and CDDP-treated TNBC cell lines were measured by RT-qPCR. Thereafter, cell proliferation and migration in the presence of CDDP treatment were evaluated via CCK-8 and Transwell assays in the TNBC cells. A xenograft mice model was also established to verify the miR-106b-5p silencing effect on the growth of CDDP resistance TNBC cells in vivo. Luciferase reporter experiments were performed to predict the relationship between miR-106b-5p and GDF11 expression. The results showed that miR-106b-5p was upregulated in the TNBC tumor cells and TNBC cells treated with CDDP and knockdown of this caused inhibition of the TNBC cell lines' proliferation, migration and suppressed the growth of the TNBC xenografted tumors, in the presence of CDDP treatment. In addition, it was observed that miR-106b-5p can bind to GDF11; as a result in the TNBC tissues and CDDP-treated TNBC cell lines the down-regulation of GDF11 was observed. Moreover, GDF11 silencing promoted CDDP-treated TNBC cell lines' proliferation and migration and reversed the interference effect of miR-106b-5p. MiR-106b-5p was upregulated in TNBC and this upregulation may promote CDDP resistance of the TNBC cells by targeting GDF11 and inhibiting its expression.

Abstract 4492: Deferiprone optimization for the treatment of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is the leading cause of new cancer cases and the second leading cause of cancer deaths in American women disproportionately affecting Black and Hispanic women under 40. Currently, there is no targeted therapy for TNBC and treatment options to manage this lethal disease include surgery, adjuvant chemotherapy and radiotherapy. Among the chemotherapy agents, combination of the PARP inhibitors with DNA-damaging results in a more promising anti-tumor effect. A better understanding of TNBC etiology has aided in unraveling the roles of two additional cellular targets - androgen receptor (AR) and histone lysine demethylase (KDM) - in the viability and survival of TNBC. Interestingly, AR overexpression is the most important driver of prostate cancer (PCa). A class of current medications used to treat PCa act as androgen receptor (AR) antagonists (or antiandrogens). Antiandrogens are under investigation in the clinic as potential therapies for luminal AR (LAR) subtypes of TNBC, a particularly worrisome subtype that frequently metastasizes to regional lymph nodes and bones. Although LAR TNBC constitutes 15-20% of TNBCs, it is characterized by minimal response to chemotherapy and aggressiveness. It is therefore plausible to repurpose and optimize antiandrogens, in the context of designed multiple ligands; to obtain a TNBC-targeting therapy effective against several subtypes of this heterogenous disease, including the basal subtype that is responsible for ~80% of TNBC incidence. The proposed therapeutics are dual-acting antiandrogen-KDM inhibitors. The antiandrogen moiety of these agents will enable TNBC cell-targeting and translocation of the drug into the nucleus where inhibition of KDM will prevent transcription of KDMs that promote tumor growth. The three pharmacophores of these agents are (i) an AR binding group, (ii) a linker, and (iii) an iron-binding group. Two functional classes of these compounds, alkyl- and aryl-derivatives, vary in the design of the AR binding moiety. To optimize these agents, the methylene linker length is varied in each class for in vitro determination of the optimal length for increased drug efficacy. The compounds are tested on TNBC cell lines (MDA-MB-231, low AR+; and MDA-MB-453, high AR+), and ER+ BCa (MCF-7) while PCa cell lines (LNCaP PCa, AR+; and DU 145, PCa, AR-) serve as controls for AR dependency of the compounds. Longer linker length compounds exhibited low micromolar cytotoxicity and selectivity for AR+ TNBC and PCa cell lines. Further analysis of lead compounds in TNBC cells aided in understanding the effects on targets, transcriptomic effects via RNA sequencing analysis and the mechanism of action. Thus far, the designed dual-acting therapeutics have potential to revolutionize the design of anticancer drugs and improve treatment outcome for TNBC patients. Citation Format: Alexis Johnston, Adegboyega K. Oyelere. Deferiprone optimization for the treatment of triple-negative 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 4492.

Abstract 689: BUB1 interferes with the repair of radiation-induced DNA damage to radiosensitize triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is associated with locoregional recurrence and low survival rates. Novel molecular targets are expected to improve the efficacy of radiation therapy (RT) in TNBC. BUB1, a mitotic checkpoint Serine/Threonine-protein kinase is known to impact chromosomal segregation during mitosis. However, its precise role in DNA damage repair in response to RT remains largely unknown. We demonstrated that BUB1 is overexpressed in BC (vs. normal breast) and considerably raised in TNBC with poor survival outcomes using differential gene expression analysis. In multiple TNBC cell lines, cell proliferation assays revealed that pharmacological (BAY1816032) or genomic (CRISPR) ablation of BUB1 was cytotoxic. Using clonogenic survival assays, we observed that BUB1 ablation radiosensitized TNBC cell lines (SUM159, MDA-MB-231, MDA-MB-468, BT-549) while its ablation did not radiosensitized breast epithelial cells (MCF10A). Furthermore, by depleting endogenous BUB1 (siRNAs and CRISPR) and reintroducing wild-type (WT) or kinase-dead (KD) BUB1, we demonstrated that inhibition of BUB1 kinase function is responsible for the radiosensitization phenotype. BUB1 ablation increased tumor doubling time in SUM159 xenografts when combined with radiation. Ki67 levels were also reduced in these tumors, confirming the efficacy of the combined treatment. BUB1 ablation led to persistent radiation-induced DNA DSB (gH2AX foci) indicating decreased DNA-DSB repair. BLRR assays confirmed that BUB1 impacts the non-homologous end joining (NHEJ) pathway. Biochemical analyses showed that BUB1 ablation stabilized total- and phospho- DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1i and DNAPKi prevented stabilization. To evaluate the recruitment of DDR proteins to the chromatin following RT, soluble nuclear and chromatin-enriched fractionations were performed which illustrated an increase in recruitment of phospho- and total-DNAPK, KAP1, and ATM to chromatin indicating that BUB1 is important in the activation and recruitment of critical NHEJ proteins to DSBs. In addition, BUB1 staining of TNBC TMAs demonstrated significant correlation between BUB1 protein expression with tumor grade and stage in TNBC patients. Our findings demonstrate that ablation of BUB1 sensitizes TNBC cell lines and xenograft to RT and BUB1 mediated radiosensitization may occur through NHEJ. Our results suggest that BUB1 may serve as a novel molecular target for radiosensitization in women with TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Pin Li, Stephen L. Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. BUB1 interferes with the repair of radiation-induced DNA damage to radiosensitize triple-negative 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 689.

Abstract 7205: PLK1 inhibition overcomes PARP inhibitor resistance in triple-negative breast cancer

Abstract Patients with aggressive breast cancer (BC) subtypes usually do not have favorable prognosis despite improvements in treatment modalities. These cancers remain a major cause of morbidity and mortality in women globally. This has fostered a major effort to discover actionable molecular targets to treat these patients. Polo-like Kinase (PLK1) is one of these molecular targets that are under comprehensive investigation for the treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not been explored. Therefore, we examined the expression of PLK1 protein in a large cohort of more than 1500 Middle Eastern ethnicity BC by immunohistochemistry. Correlation with clinico-pathological parameters and the prognosis was performed. PLK1 overexpression was observed in 27.4% of all BC and was significantly associated with aggressive clinico-pathological markers. The antitumor effect of PLK1 and PARP inhibitors either alone or in combination was tested in two BRCA mutated, and one BRCA proficient TNBC cell lines. We showed that combined inhibition significantly reduced cell survival and induced apoptosis in TNBC cell lines. Furthermore, our data suggest that PLK1 inhibition overcomes PARP inhibitor resistance in TNBC cell lines. These findings would pave the way to explore the future use of PLK1 inhibitors in the clinical setting of PARPi-resistant patients. Citation Format: Abdul K. Siraj, Pratheesh Kumar Poyil, Divya Padmaja, Sandeep Kumar Parvathareddy, Khadija Alobaisi, Rafia Begum, Osama Almalik, Fouad Al-Dayel, Khawla S. Al-Kuraya. PLK1 inhibition overcomes PARP inhibitor resistance in triple-negative 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 7205.

Abstract 6969: Determining the role of obesity in breast cancer progression

Abstract Breast cancer, especially triple negative breast cancer, impacts a large proportion of the female population. Approximately ten to fifteen percent of all breast cancer diagnoses will be triple negative. Triple negative breast cancer lacks receptors that are used as therapeutic targets for other forms of breast cancer and has the poorest prognosis. Notably, the age of onset has also decreased, which correlates with the rise in prevalence of obesity. In addition, past studies revealed that maintaining a high BMI from childhood correlates with higher cancer mortality in adulthood. However, the mechanisms by which obesity contributes to cancer progression are not clear. Adipose tissue from different locations (visceral vs. subcutaneous, abdominal vs. femoral) and from patients with obesity from childhood rather than as adults could have different molecular characteristics. Further, depending on the genetic background, different profiles may be more susceptible to environmental cues that could drive their progression. To address this, we are using conditioned media collected from tissues that reflect these differences and determining how they affect TNBC cell lines with distinct genetic profiles. Preliminary data revealed that adipose tissue from both the abdominal and femoral regions of high BMI individuals caused an increase in the proliferation rate of TNBC cell lines. To determine if the adipocytes vs. other cell types in the adipose tissue drive this growth, we are co-culturing mature adipocytes from these tissues with the TNBC cell lines. Recent studies by other groups showed that adipocyte-derived extracellular vesicles could drive the expression of EMT genes in cancer cells. We will purify EVs from the adipocytes and characterize differences in their molecular signatures for different patients to determine which factors cause the change in TNBC progression and correlate this with different fat depots and/or age of onset. We will also measure the effect of adipose tissue and/or adipocytes on other cancer hallmarks including metastasis (migration and invasion) and changes in metabolism. These studies should reveal how adipocytes confer changes in TNBC progression, and how to consider obesity in different treatment regimes. Citation Format: Alexandra Perlman, Alisa Piekny, Elena Kuzmin, Sylvia Santosa. Determining the role of obesity in breast cancer progression [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 6969.

Abstract 167: Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy

Abstract Introduction: Statins, known as inhibitors of HMG-CoA, not only reduce cholesterol but also exhibit pleiotropic effects which include anti-tumor activities by targeting tumor microenvironments. They inhibit the synthesis of FPP and GGPP, which is important for prenylation of signaling proteins. Although PD-1/PD-L1 inhibitors has been approved for treatment for TNBC patients with PD-L1 positive, the response remains unsatisfactory. Also, it has been reported that statins affect PD-L1 expression in other tumors, but it is still uncertain in TNBC. Therefore, we aimed to identify the effect of statins on the expression of PD-L1 in TNBC and to verify the efficacy of combination therapy with statins and immune checkpoint inhibitors. Methods: Human and mouse TNBC cell lines, mouse macrophage cell line (RAW 264.7), and clinically approved simvastatin were used. Flow cytometry, western blot, proliferation assay, qRT-PCR, immune phenotyping and immunohistochemistry were conducted. A co-culture of macrophages and cancer cells was performed. An orthotopic syngeneic mouse model by injection of EMT6 cells into mammary gland fat pad were produced. Mice were treated with simvastatin(20mg/kg) by oral gavage daily and anti-PD-1(200μg) by intraperitoneal injection two times per week. Results: Among thirteen human TNBC cell lines, we confirmed that TNBC cell lines showed various PD-L1 expression, and especially MDA-MB-231 and HCC38 highly expressed endogenous/constitutive PD-L1. Statins reduced PD-L1 expression and exerted anti-proliferative effects in MDA-MB-231, HCC38, and EMT6 in a dose- and time-dependent manner. Phosphorylation of AKT and STAT3 was reduced when simvastatin was treated. Mice with combination therapy of anti-PD-1 and simvastatin had less effective inhibitory effect on tumor growth than mice with anti-PD-1 monotherapy, which showed the highest tumor suppression effect. In immune phenotyping, tumor-infiltrated T cells did not have significant differences among groups. However, M1:M2 macrophage ratio was highest in anti-PD-1 group but decreased in combination therapy group with simvastatin. To determine the effect of simvastatin on macrophages, RAW264.7 cells were stimulated with MDA-MB-231 conditioned medium, revealing an upregulation of PD-1 expression. Conclusions: Our findings show that simvastatin has an anti-tumor effect, which reduces Pd-L1 expression and kills breast cancer cells, reducing phosphorylation of STAT3 and AKT. However, when combined with PD-1 checkpoint inhibitor, it reduces the anti-tumor effect by increasing PD-1 expression in macrophages and promoting M1 to M2 polarization. Further study is needed to demonstrate the mechanism by which simvastatin regulate polarization of macrophages and to confirm the polarization of macrophages in samples of TNBC patients who had been taking simvastatin. Citation Format: Sangeun Lee, Ju Hee Kim, A Young Park, Jihong Baik, Ji-Jung Jung, Han-Byoel Lee, Wonshik Han. Modulation of PD-1/PD-L1 by simvastatin in TNBC and macrophages and its impact on the efficacy of immunotherapy [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 167.

Abstract 4131: Combination therapeutic strategy to fight against triple-negative breast cancer

Abstract Triple-negative breast cancers (TNBCs) lack estrogen-and progesteron receptors and human epidermal growth factor receptor-2 (HER2) which limits the therapeutic approaches. The high metastasizing capability of TNBCs is in correlation with the poor oxygenation of the cancerous breast tissue. This leads to the activation of the hypoxia-inducible-factor 1 (HIF-1), which induces multiple target genes that are involved in tumor progression and metastasis formation. This makes HIF-1 an attractive drug target, however, HIF-1 inhibitors failed if applied in monotherapy. Therefore, our aim is to investigate the combined therapeutic effect of HIF-1 inhibitor acriflavine and paclitaxel, a commonly used taxane in TNBC treatment. The synergy assessment of the two drugs was carried out by utilizing Sulforhodamine B assay on TNBC cell lines. The in vivo toxicity and efficacy of the drug combination was tested on NOD SCID mice with orthotopically inoculated MDA-MB-231 cell line. Quantitative and qualitative analysis of multiple genes involved in tumor progression was performed by mRNA and protein expression level assessment with qPCR, western blot and immunohistochemistry methods. HIF-1 stimulation was achieved by either incubating the cells at 1% oxygen level or through CoCl2 treatment. The in vitro mRNA expression studies demonstrated that acriflavin possesses a hypoxic-effect-mitigating property on the expression of metastasis-involved- and HIF-1 target genes. Acriflavine alleviated the epithelial-mesenchymal transition of TNBC cells by increasing the expression of the epithelial marker, E-cadherin. At protein level, we detected decrease in HIF-1alpha and Phospho-Protein kinase B (Akt) and increase in p53 tumor suppressor gene expression in response to acriflavine treatment. We further established MDA-MB-231 xenograft model in mice for investigating in vivo toxicity and efficacy. The drug combination efficiently reduced the tumor size and the macrometastasis formation compared to the control groups. The protein analysis of frozen tumor tissue samples indicated that the combination therapy decreased the HIF-1alpha expression and the amount of Phospho-Akt and Phospho-Mitogen-activated protein kinases (pMAPK/pERK) in CoCl2treated mice. Additionally, expression of epithelial markers increased and that of mesenchymal markers decreased. The combination therapy led to a decrease in the platelet-derived growth factor receptor beta (PDGFRB) as well. To conclude, we identified a novel drug combination which can efficiently combat TNBC by inhibiting HIF-1 signaling, mitigating cell migration and metastasis formation and suppressing the activation of the Phosphoinositide 3-kinase (PI3K) and MAPK pathways. This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Citation Format: Laura Svajda, Ivan Ranđelović, Sára Eszter Surguta, Marcell Baranyi, István Kenessey, Mihály Cserepes, József Tóvári. Combination therapeutic strategy to fight against triple-negative 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 4131.

Abstract 5728: YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors

Abstract Metastatic breast cancer (BC) is the 2nd leading cause of death in women in the US, annually accounting for more than 43,000 deaths and 281,000 new cases of invasive BC. Amongst genetically distinct BC subtypes, those classified as being “triple-negative” (TNBC) are especially devastating due to their highly metastatic behavior, their propensity to recur rapidly, and their low response to standard-of-care therapies. In fact, the acquisition of chemoresistant phenotypes represents the main cause of disease recurrence, metastasis, and death in TNBC patients. Currently, the molecular mechanisms that regulate TNBC progression and metastasis remain unknown, as does the way these metastatic tumors acquire resistance to standard-of-care therapies. We recently established YB1 as a novel driver of these deadly TNBC activities, doing so by stimulating the cancer stem cell phenotype, and by disrupting normal cell cycle progression, therefore promoting therapy resistance and metastasis of TNBC tumors. YB1 is a multifunctional protein that acts as a transcription factor of cancer stem cell genes Nanog, Oct and Sox. Moreover, aberrant activation of YB1 contributes to the metastatic progression of several cancers, including TNBC. Our investigations revealed a major role of YB1 in the regulation of several hallmarks of cancer that drive TNBC tumors progression and metastasis, both in vitro and in preclinical mouse models of TNBC tumors. In extending these discoveries, we now show that aberrant YB1 expression activates oncogenic signaling leading to the dysregulation of cell cycle progression through the regulation of the Cyclin D/CDK4/6 complex signaling and the RB pathway. We also report the identification of a novel small molecule inhibitor, SU056, that specifically targets YB1 and inhibits its oncogenic activity in TNBC cell lines. We further show that the SU056-mediated inhibition of YB1, either as monotherapy or in combination with the CDK4/6 inhibitors, has a significant impact on inhibiting TNBC tumor progression and metastasis. In fact, the SU056-CDK4/6 inhibitors combination showed a synergistic effect when compared to monotherapy, at the same time reducing the toxicity associated with CDK4/6 inhibitors. Mechanistically, our studies revealed that genetic or pharmacologic targeting of YB1 inhibits TNBC tumor progression and metastasis through the regulation cyclin D-CDK4/6-RB pathway and blockade of cell cycle progression. Together, our data support the notion that targeting YB1 represents a potential therapeutic option for the treatment of TNBC, which could be enhanced when combined with standard of care treatment modalities. Citation Format: Wei Wang, Lamyae El Khalki, Neelum Yousaf Zai, Justin Szpendyk, Akram Alkrekshi, Bin Su, Khalid Sossey-Alaoui. YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors [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 5728.

Abstract 530: The effect of cardamonin on PD-L1 expression in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) accounts for about 10% of all breast cancers and does not express estrogen, progesterone, or HER2 receptors. It has an aggressive nature, it is highly metastatic, and because of the lack of therapies, it continues to be a challenge. These cancer cells employ mechanisms to escape the immune system that induce the upregulation of PD-L1 expression, a ligand encoded by the CD274 gene. Because cancer cells tend to produce higher levels of PD-L1 compared to normal cells, therapies that inhibit PD-L1 may be helpful. Evidence also shows that high levels of oxidative stress and inflammation may participate in both the initiation and progression of cancer. Cardamonin, an aromatic enone flavonoid, has displayed an array of pharmacological activities, including modulation of different signaling molecules involved in the development and progression of cancer. This work investigated the cardamonin’s ability to modulate PD-L1 expression and NF-KB in genetically distinct MDA-MB-231 (Caucasian) and MDA-MB-468 (African American) TNBC cell lines. The methodology included cytotoxic assays, ELISA, Real Time-Polymerase Chain Reaction (RT-PCR) assays, and Wes analysis. The results showed that cardamonin treatment caused a dose-dependent decrease in cell viability in both cell lines, ranging from 3.12 µM to 200 µM. The RT-PCR and ELISA data showed that even though IFN-γ stimulated MDA-MB-231 cells showed a higher expression of PD-L1, cardamonin reduced mRNA and protein expression of PD-L1 in both cell lines. As a possible molecular mechanism for PD-L1 inhibition, cardamonin modulated MUC-1, STAT3, and JAK, which are genes that activate the PD1/PD-L1 mechanism. Moreover, data also show that cardamonin modulated NF-KB1 and NF-KB2 genes involved in activating NF-KB signaling. NF-KB is a transcription factor of inflammation and immunity, emerging as a key positive regulator of PD-L1 expression in cancer. Therefore, cardamonin may have the potential utilization as a checkpoint inhibitor that acts by blocking PD-L1 protein, allowing the immune system to recognize and attack the TNBC cancer cells, providing a novel cancer therapy. (Supported by a grant from NIH-NIMHD U54 MD 007582) Citation Format: Patricia Mendonca, Bhonessa Kirpal, Sukhmandeep Kaur, Karam F. A. Soliman. The effect of cardamonin on PD-L1 expression in triple-negative breast cancer cells [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 530.

Abstract 599: Combination of CDK4/6 inhibitors with biguanides for treatment of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC) and is associated with early relapse and high frequency of metastasis. This has been attributed in part to the lack of targeted therapies. TNBC occurs more frequently in younger women of African and Hispanic ancestry and with a higher risk of mortality than women of European ancestry. Emerging data shows activation of the AKT/mTOR pathway by insulin may occur in aggressive TNBC and inhibition of mTOR signaling by rapamycin, a potent mTOR inhibitor, induces cell cycle arrest and downregulation of cyclin D1 in TNBC cells. CDK4/6 inhibitors have demonstrated to be an effective therapeutic for estrogen receptor positive BC and although their use in TNBC patients remains uncertain their use in combination with other targeted therapies may be promising. We have developed a set of newly designed biguanides with potent anticancer action and safety doses in vivo that in combination with CDK4/6 inhibitors block TNBC cell growth in vitro and in vivo. Using TNBC cell proliferation in vitro assays, new biguanides show dose-dependent inhibition of cell proliferation (P<0.01). Further, combination of biguanides with ribocliclib, an orally bioavailable CDK4/6 inhibitor, produced additive and synergistic effects in TNBC cell lines in vitro (P<0.01). Biguanides are known to activate de LKB1-AMPK pathway. Western immunoblots showed our biguanide analogues also induce AMPK phosphorylation and significantly reduce phosphorylation of downstream mTOR signaling pathway components including S6 ribosomal protein and 4E-BP1, an effect that is potentiated by combination treatment with ribociblib. Further, cyclin D1 is known to be overexpressed in about 50% of BCs and binding to CDK 4/6 induces phosphorylation of Rb and cell cycle progression. Treatment with biguanides downregulated cyclin D1 expression in TNBC cells and combination treatment of ribociclib with biguanide JD006 was more effective at decreasing Rb phosphorylation than each drug alone. In addition, seahorse analysis evaluated mitochondrial respiratory function. Treatment with biguanides inhibited oxidative phosphorylation in a dose dependent manner, an important metabolic pathway that can drive drug resistance to therapy in cancer cells. In vivo, combination treatment of ribociclib with biguanide JD006 was more effective than each agent alone in stopping human TNBC xenograft progression in nude mouse models (P<0.05). Our preliminary data offer evidence that human TNBC are highly vulnerable to metabolic reprogramming by new biguanides. This work can yield insight on unanticipated mechanisms of biguanide action and rationale for new combination therapies with CDK 4/6 inhibitors as a new option for treatment of TNBC. Funding: CBCRP B27IB3869, JCCC Breast Cancer Award, Hickey Family Foundation, NCI U54 CA143930, Team Research Grant, UCLA TDG. Citation Format: Mario Morales Martinez, Eduardo Mauricio Gonzalez, Gang Deng, Gaoyuan Ma, Hyunsoo Kim, Linsey Stiles, Nalo Hamilton, Michael E. Jung, Richard J. Pietras, Diana C. Marquez-Garban. Combination of CDK4/6 inhibitors with biguanides for treatment of triple negative 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 599.

Abstract 2753: Muc4 is a biomarker of metastasis in TNBC and its downregulation by blocking soluble TNF prevents metastasis in combination with immunotherapy

Abstract Triple negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers and has the worst survival rate. Immunotherapy is a new treatment option but resistance is frequent. We have demonstrated that TNF induces trastuzumab resistance through mucin 4 (MUC4) upregulation and it is an independent biomarker of poor response to therapy in HER2+ breast cancer. MUC4 is a transmembrane glycoprotein involved in metastasis (MTS) dissemination. Here, we evaluated the impact of the axis TNF/MUC4 on the invasive capacity of TNBC cell lines and in a preclinical model of spontaneous MTS. Finally, we assessed the clinical impact of MUC4 expression in TNBC patients. TNF blockade was achieved with etanercept (E), which blocks the soluble (sTNF) and transmembrane isoform of TNF, or with the dominant negative protein INB03 (DN) which neutralizes only sTNF. BT-549 and MDA-MB-231 human TNBC cell lines treated with E or DN exhibit a decrease in MUC4 expression. To assess the impact of TNF blockade on tumor cells and its effect on the tumor microenvironment, we collected conditioned media (CM) from MDA-MB-231 and BT-549 cells treated with E or DN which were used to evaluate the invasive capacity of the TNBC cell lines. The invasion of BT-549 and MDA-MB-231 was impaired with CM-Eta (p≤0.01) and CM-DN (p≤0.01 and p≤0.05, respectively). Female BALB/c mice bearing the TNBC LMM3 tumors were treated with IgG, DN, anti-PD-1 antibodies or DN+anti-PD-1 for two weeks. After the treatments we performed surgery to remove the primary tumor and the animals were sacrificed 2 weeks later. No effect on tumor growth was observed. However, the DN+anti-PD-1 group was the only one with less of 3 MTS per lung (p≤0.05). We obtained tumor extracts and found that the tumors of the animals treated with DN or DN+anti-PD-1 had decreased MUC4 expression measured by Western blot. We determined the presence of TILs by H & E and the expression of MUC4, Ki67, PD-L1 (SP142), androgen receptor (AR) and cytokeratin 5 by immunohistochemistry in a cohort of 56 early TNBC patients. MUC4 expression was inversely correlated with TILs presence (p=0.0003). Since TILs are associated with good prognosis, we evaluated the effect of BT-549-CM and MDA-MB-231-CM on T cell migration. CM-DN from both cell lines increased T cell migration compared to control CM (p≤0.01). MUC4 expression was inversely correlated with Ki67 (≥30%, p=0.036), PD-L1 (p=0.001) and AR (p=0.047) in our cohort. Moreover, MUC4 proved to be an independent predictor of poor overall survival (p=0.02), and is associated with a higher MTS risk (p=0.005). MUC4 is associated with poorly-infiltrated TNBC, and sTNF blockade downregulates its expression decreasing MTS when combined with anti-PD-1. We propose the TNF as a new target for the treatment of TNBC, and MUC4 as a predictive biomarker to guide a combined treatment of TNF blockers with immunotherapy. Citation Format: Florencia Luciana Mauro, Sofia Bruni, Agustina Dupont, Gloria Inurrigarro, Silvina Figurelli, Sabrina Barchuk, Daniel Lopez Della Vecchia, Rosalia Cordo Russo, Ernesto Gil Deza, Maria Florencia Mercogliano, Roxana Schillaci. Muc4 is a biomarker of metastasis in TNBC and its downregulation by blocking soluble TNF prevents metastasis in combination with immunotherapy [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 2753.

Abstract 6994: MerTK drives proliferation and metastasis activity in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is characterized by the absence of estrogen, progesterone, and HER2 receptors. It exhibits a higher level of aggressiveness compared to other types of breast cancer, with a greater likelihood of recurrence following standard treatments such as surgery, chemotherapy, and radiation. Despite limited FDA-approved targeted therapies for TNBC, there is an ongoing need for additional molecular targeting agents.Our investigation focused on the role of the receptor tyrosine kinase MerTK and its impact on proliferation and invasion/metastatic potential. Immunohistochemistry analysis revealed that approximately 86% (128/148) expressed varying levels of MerTK in human TNBC samples, whereas approximately 50% (23/47) of TNBC patient-derived xenografts (PDX) expressed MerTK. Additionally, three established TNBC cell lines (BT549, MDAMB231, and MDAMB436) exhibited elevated MerTK expression levels.To further explore the role of MerTK in TNBC, we stably overexpressed MerTK in human TNBC cell lines SUM102 and SUM159, which naturally have low MerTK levels. The results demonstrated that MerTK overexpression led to increases in proliferative potential, robust in vivo tumor growth, heightened migration/invasion potential, and increased lung metastasis. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, including PI3k-Akt, PDGF, and Myc, ultimately driving cell cycle progression, reducing apoptosis, and enhancing cell survival in TNBC.Further investigation using a cytokine array showed increased endoglin production in SUM102-MerTK cells compared to vector control cells. This suggested that MerTK might be creating a conducive environment for increased proliferative and metastatic activity via elevated endoglin expression in TNBC. To ascertain the role of endoglin in increase proliferation and/or invasion potential, we knocked out endoglin in SUM102-MerTK cells. While endoglin knockout SUM102-MerTK cells exhibited similar growth to SUM102-MerTK cells, the number of lung metastases was significantly impacted, indicating that MerTK regulates invasion and metastasis through endoglin in TNBC. Collectively, our data suggests that MerTK regulates a unique proliferative signature allowing for robust tumor growth and increased metastatic potential through endoglin regulation. This suggests that targeting MerTK and endoglin simultaneously may be a viable approach for TNBC patients. Citation Format: Christine Glitchev, Mari Iida, Bridget E. Crossman, Kourtney L. Kostecki, Carlene A. Kranjac, Jillian M. Adams, Peng Liu, Irene Ong, David T. Yang, Irene Kang, Ravi Salgia, Deric L. Wheeler. MerTK drives proliferation and metastasis activity in triple negative 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 6994.

Abstract 461: Dual-kinase targeted miRNA nanotherapy for the treatment of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) represents 20% of breast cancer (BC) patients (51,000 cases/year in the US). It is associated with a highly aggressive course, early metastasis and relapses, and drug-resistant phenotype. About 40% of the patients die within 5 years despite available therapies. If the disease is metastatic, patients survive only 13 months. Since TNBC does not express targetable receptors such as ER, PR, and HER2, and it is a highly heterogeneous cancer with six genetically defined sub-types, currently, there are no common actionable molecular targets and no effective targeted therapies for TNBC. microRNAs (miRNAs) are 18-20 nt long non-coding RNAs and are often dysregulated in cancer cells. Tumor-suppressive miRNAs specifically silence their protooncogenic target genes (mRNAs) by binding specifically to their 3’-UTRs located in their target mRNAs, leading to inhibition of oncogenes that play critical roles in cell proliferation, cell cycle, migration, invasion, angiogenesis, drug resistance, tumor growth, and progression. We have previously demonstrated that Eukaryotic Elongation factor-2 kinase (eEF2K) and AXL are oncogenic kinases, and their higher expression is associated with poor survival in patients with TNBC. However, currently, there is no FDA-approved eEF2K and AXL-targeted therapeutics. To effectively co-target these kinases, we extensively analyzed miRNA databases using miRNA target prediction algorithms and recently, we discovered that miR-329-3p has a specific binding site at both 3’-UTRs of eEF2K and AXL mRNAs. Analyzing the TCGA TNBC database, we found that low expression of miR-329-3p is correlated with shorter overall survival in TNBC patients, and the expression of miR-329-3p is commonly reduced or lost in TNBC patient tumors. Since the successful clinical application of miRNA-based therapeutics requires safe and effective nano-delivery systems, we have developed magnetic iron-oxide-based nanoparticles (MNPs) for the delivery of miR-329-3p. We showed that miR-329-3p significantly suppresses cell proliferation, invasion, and migration by targeting both eEF2K and AXL and their downstream mediators such as SRC/FAK and Cyclin D1 in TNBC cell lines. In vivo systemic treatment with MNPs-miR-329 completely blocks tumor growth in orthotopic MDA-MB-231 and MDA-MB-436 TNBC xenograft models in mice. Analysis of tumors shows remarkable inhibition in eEF2K and AXL expression, and clinically significant downstream targets including SRC, FAK, and Cyclin D1. We also demonstrated that miR-329 nanotherapy induces apoptosis and inhibits intratumoral proliferation in TNBC tumors in mice. Toxicity markers in mouse blood samples show that miR-329 nanotherapy is safe and not toxic to mice. Taken together, our study suggests that miR-329-3p nanotherapy may be used as a novel potential therapeutic approach in TNBC patients. Citation Format: Goknur Kara, Pinar Atalay Dundar, Nermin Kahraman, Emir Baki Denkbas, Bulent Ozpolat. Dual-kinase targeted miRNA nanotherapy for the treatment of triple-negative 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 461.

Abstract 551: Investigation of the antipoliferative effect of the Xylopia Aethiopicaextract in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and has very limited treatment options. Xylopia Aethiopica (XA) is a common spice that is widely used in the regions of central and western Africa. It is known to have various pharmacological properties, one of which is anti-inflammatory activity. The anti-inflammatory effect may portend antiproliferative activity. We hypothesized that the extract of XA will exert antiproliferative activity against TNBC cells. The study aimed to establish the molecular mechanism underlying the antiproliferative activity of XA extract (XAE) against TNBC. The dried fruits of XA were crushed in a mixer and macerated using diethyl ether for 72 h. The macerate was subjected to vacuum and rotary evaporation to remove the organic solvent, and a stock solution was prepared in dimethyl sulfoxide (DMSO). Assessment of the efficacy of XAE was performed using serial dilutions (100 µg/ml, 50 µg/ml, 10 µg/ml, 5 µg/ml, and 1µg/ml) of the XAE stock solution, followed by in vitro testing against two TNBC cell lines (MDA-MB-231, and MDA-MB-468 cells). The cytotoxic effect of the XAE was then measured using the resazurin dye assay. XAE resulted in a concentration-dependent cell death in both cell lines. At the highest concentration (100 µg/ml), XAE induced cytotoxic cell death of 89% and 91% in MDAMB231 and MDAMB468 cells, respectively. This was consistent with IC50 values of 32.95 ± 1.32 µg/ml (MDAMB231) and 20.83 ± 1.05 µg/ml (MDAMB468). At the molecular level, western blot analysis of mitogenic markers associated with MAPK oncogenic signaling pathway demonstrated the inhibition of EGFR and AKT protein levels after a 48-h treatment. Acridine orange-ethidium bromide (AO/EB) staining showed a concentration-dependent induction of apoptosis in both cell lines. Flow cytometric analysis was performed to quantify the apoptotic cells. A significant increase of apoptotic cells was observed in treatment groups as compared to control after 72 h. Apoptotic induction was associated with increased p53 protein levels after 48 h treatment compared to control. The study results underscore the anticancer effects of XAE in TNBC cells mediated through induction of apoptosis. Citation Format: Shailvi Soni, Shail Rakesh Modi, Terrick Andey. Investigation of the antipoliferative effect of the Xylopia Aethiopicaextract in triple-negative breast cancer cells [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 551.

Abstract 2790: Hematopoietic stem cells tropism and conditioned media for targeting the triple negative breast cancer stem cells and associated mechanism: An in vitro study

Abstract Triple-negative breast cancers are the most challenging to treat with targeted therapy because of their non-expression of cell surface receptors, unlike other types of breast cancer. However, conventional therapies such as chemotherapy, radiotherapy, and surgery are standard treatment modalities. Moreover, drug encapsulation strategies to deliver drugs at the targeted site have been sought-after alternatives. Hematopoietic Stem Cells (HSCs) have unique features to migrate towards inflammatory sites and other body organs during any injuries and release various cytokines, which lead to interaction with molecular pathways and results in reduced inflammation and tissue injuries. From this characteristic, we have hypothesized that hematopoietic stem cells can show tumor tropism and modulate the metabolic alteration in cancer and cancer stem cells, mainly focusing on Triple Negative Breast Cancer Stem Cells (TNBC CSCs). In this study, we have sorted both cancer stem cells and hematopoietic stem cells from TNBC and hematological cell lines, respectively. Sorted HSCs were cultured in specific conditions to prepare conditioned media. Then, both cell’ types were used for co-cultured assay and conditioned media cultured with TNBC-CSCs were performed. Metabolomics studies were performed to decipher the molecular interactions and TNBC-CSCs metabolic alterations. The result indicates that the HSCs are showing positive tropism towards TNBC stem cells. Furthermore, HSCs-derived conditioned media (CM) interact with the cell cycle pathway by downregulating the CDK genes and causing DNA breakage in the TNBC-CSCs. Moreover, the Metabolomics studies further confirmed that HSCs CM-derived cytokines interact with various pathways, dysregulating the TNBC-CSCs proliferation and altering the metabolic activities. Citation Format: Sumit Mallick, Sudheer Shenoy P, Bipasha Bose. Hematopoietic stem cells tropism and conditioned media for targeting the triple negative breast cancer stem cells and associated mechanism: An in vitro study [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 2790.

Abstract 533: Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture models

Abstract Triple Negative breast cancer (TNBC) accounts for 10-15% of all breast cancers where patients encounter poor clinical outcomes. Treatment modalities for TNBC are conventional cytotoxic chemotherapy and radiation leads to 35%-40% relapse within 5 years of diagnosis. There is a growing need for more effective targeted therapeutics to inhibit TNBC growth. The role of EZH2 in maintaining tumor growth and metastasis in TNBC is well documented. EZH2 promotes breast tumor-initiating cell expansion (BITC) and cancer progression by impairing the DNA damage repair process favoring activation of oncogenes. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity, resistance to these small molecules limits their use in solid tumors. The neurotransmitter dopamine via its D1 receptor activation in TNBC cell lines induces apoptosis and autophagy, as well as inhibits the invasion and regress in mammary tumors in vivo. Here we hypothesize that the presence of dopamine D1 receptor agonist (A77636) enhances the efficacy of EZH2 inhibitors (GSK126) to inhibit in vitro TNBC tumor growth and metastasis. To test the efficacy of the combination we employed a 3D culture system of MDA-MB-231 cells encapsulated in calcium-alginate microgels seeded from a microfluidic droplet generator. We also employed a 3D organ-on-chip-based microphysiological (MPS) platform (SynTumor) to study the effect of the drug combination on metastasis in vitro. The SynTumor MPS devices replicate the pathophysiological architecture of native vascularized breast tumors. The combination treatment enhanced a significant reduction of tumor spheroid diameter compared to the vehicle and indicated spheroid regression over treatment. Furthermore, the combination therapy induced necrosis and caused complete inhibition of EZH2 expression. The knockdown of EZH2 inhibited tumor spheroid formation and displayed cytoskeleton de-arrangement. In the microfluidic SynTumor model, circulating tumor cell numbers were reduced by half at 96 hrs after EZH2 combination treatment. Our data indicate that the combinatorial effect of DRD1 agonist and EZH2 inhibitor efficiently attenuates the EZH2-mediated in vitro tumor growth and metastasis (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: xilal Rima, Gautam Sarathy, Chunyu Hu, Divya S. Patel, Deborah Ramsey, Lara Rizotto, Dario Palmeri, Gwen Fewell, Bhuvaneswari Ramaswamy, Eduardo Reátegu, Eswar Shankar. Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture 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 533.