Negative Breast Cancer Cells Research Articles

The interplay between IGF-1R signaling and Hippo-YAP in breast cancer stem cells

BackgroundBoth IGF-1R/PI3K/AKT/mTOR and Hippo pathways are crucial for breast cancer stem cells (BCSCs). However, their interplay remains unclear.MethodsFour triple negative breast cancer cell lines derived from CSC of two patient-derived xenografts (PDXs), AS-B145, AS-B145-1R, AS-B244, and AS-B244-1R, were used to elucidate the role of YAP in BCSCs. YAP silenced BCSCs were analyzed by cell proliferation, aldehyde dehydrogenase (ALDH) activity, mammosphere formation, and tumorigenesis. The effects of modulating IGF-1R and IGF-1 on YAP expression and localization were evaluated. The clinical correlation of YAP and IGF-1R signaling with the overall survival (OS) of 7830 breast cancer patients was analyzed by KM plotter.ResultsKnockdown of YAP abates the viability and stemness of BCSCs in vitro and tumorigenicity in vivo. Depletion of IGF-1R by shRNA or specific inhibitor decreases YAP expression. In contrast, IGF-1 addition upregulates YAP and enhances its nuclear localization. YAP overexpression increased the mRNA level of IGF-1, but not IGF-1R. Data mining of clinical breast cancer specimens revealed that basal-like breast cancer patients with higher level of IGF-1 and YAP exhibit significantly shorter OS.ConclusionsYAP contributes to stemness features of breast cancer in vitro and in vivo. The expression and localization of YAP was regulated by IGF-1R and YAP expression in turns upregulates IGF-1, but not IGF-1R. Clinically, higher level of YAP and IGF-1 significantly correlated with shorter OS in basal-like breast cancer. Taken together, these findings suggest the clinical relevance of interplay between YAP and IGF-1/IGF-1R pathway in sustaining the properties of BCSCs.DsuDGZeKddKHpMfgDukzC_Video

IKKα inhibition re-sensitizes acquired adriamycin-resistant triple negative breast cancer cells to chemotherapy-induced apoptosis

IKKα has been shown to be responsible of multiple pro-tumorigenic functions and therapy resistance independent of canonical NF-κB, but its role in acquired chemotherapy resistance in breast cancer remains unclarified. In this study, we obtained pre-treatment biopsy and post-treatment mastectomy specimens from a retrospective cohort of triple-negative breast cancer (TNBC) patients treated with neoadjuvant chemotherapy(NAC) (n = 43). Immunohistochemical methods were used to detect the expression of IKKα before and after NAC, and the relationship between IKKα and the pathologic response to NAC was examined. In addition, we developed a new ADR-resistant MDA-MB-231 cell line(MDA-MB-231/ADR) and analyzed these cells for changes in IKKα expression, the role and mechanisms of the increased IKKα in promoting drug resistance were determined in vitro and in vivo. We demonstrated that the expression of IKKα in residual TNBC tissues after chemotherapy was significantly higher than that before chemotherapy, and was positively correlated with lower pathological reaction. IKKα expression was significantly higher in ADR-resistant TNBC cells than in ADR-sensitive cells, IKKα knockdown results in apoptotic cell death of chemoresistant cells upon drug treatment. Moreover, IKKα knockdown promotes chemotherapeutic drug-induced tumor cell death in an transplanted tumor mouse model. Functionally, we demonstrated that IKKα knockdown significantly upregulated the expression of cleaved caspase 3 and Bax and inhibited the expression of Bcl-2 upon ADR treatment. Our findings highlighted that IKKα exerts an important and previously unknown role in promoting chemoresistance in TNBC, combining IKKα inhibition with chemotherapy may be an effective strategy to improve treatment outcome in chemoresistant TNBC patients.

Synergistic effects of combined cisplatin and Clinacanthus nutans extract on triple negative breast cancer cells

ObjectiveTriple negative breast cancer (TNBC) is the most invasive breast cancer subtype enriched with cancer stem cells. TNBCs do not express estrogen, progesterone, or human epidermal growth factor receptor 2 (HER2) receptors, making them difficult to be targeted by existing chemotherapy treatments. In this study, we attempted to identify the effects of combined cisplatin and Clinacanthus nutans treatment on MDA-MD-231 and MDA-MB-468 breast cancer cells, which represent TNBC subtypes. MethodsThe phytochemical fingerprint of C. nutans ethanolic leaf extract was evaluated by LC–MS/MS analysis. We investigated the effects of cisplatin (0–15.23 μg/mL), C. nutans (0–50 μg/mL), and a combination of cisplatin (3.05 μg/mL) and C. nutans (0–50 μg/mL), on cell viability, proliferation, apoptosis, invasion, mRNA expression in cancer stem cells (CD49f, KLF4), and differentiation markers (TUBA1A, KRT18) in TNBC cells. In addition, we also studied the interaction between cisplatin and C. nutans. ResultsDerivatives of fatty acids, carboxylic acid ester, and glycosides, were identified as the major bioactive compounds with potential anticancer properties in C. nutans leaf extract. Reductions in cell viability (0–78%) and proliferation (2–77%), as well as a synergistic anticancer effect, were identified in TNBC cells when treated with a combination of cisplatin and C. nutans. Furthermore, apoptotic induction via increased caspase-3/7 activity (MDA-MB-231: 2.73-fold; MDA-MB-468: 3.53-fold), and a reduction in cell invasion capacity to 36%, were detected in TNBC cells when compared to single cisplatin and C. nutans treatments. At the mRNA level, cisplatin and C. nutans differentially regulated specific genes that are responsible for proliferation and differentiation. ConclusionOur findings demonstrate that the combination of cisplatin and C. nutans represents a potential treatment for TNBC.

C/EBPβ-LIP mediated activation of the malate-aspartate shuttle sensitizes cells to glycolysis inhibition

ObjectiveCancer cells use glycolysis for generation of metabolic intermediates and ATP needed for cell growth and proliferation. The transcription factor C/EBPβ-LIP stimulates glycolysis and mitochondrial respiration in cancer cells. We initially observed that high expression of C/EBPβ-LIP makes cells vulnerable to treatment with the glycolysis inhibitor 2-deoxyglucose. The aim of the study was to uncover the involved mechanisms of C/EBPβ-LIP induced sensitivity to glycolysis inhibition. MethodsWe used genetically engineered cell lines to examine the effect of C/EBPβ-LIP and -LAP protein isoforms on glycolysis and NADH/NAD+ metabolism in mouse embryonic fibroblasts (MEFs), and triple negative breast cancer (TNBC) cells that endogenously express high levels of C/EBPβ-LIP. Analyses included assays of cell proliferation, cell survival and metabolic flux (OCR and ECAR by Seahorse XF96). Small molecule inhibitors were used to identify underlying metabolic pathways that mediate sensitivity to glycolysis inhibition induced by C/EBPβ-LIP. ResultsThe transcription factor C/EBPβ-LIP stimulates both glycolysis and the malate-aspartate shuttle (MAS) and increases the sensitivity to glycolysis inhibition (2-deoxyglucose) in fibroblasts and breast cancer cells. Inhibition of glycolysis with ongoing C/EBPβ-LIP-induced MAS activity results in NADH depletion and apoptosis that can be rescued by inhibiting either the MAS or other NAD+-regenerating processes. ConclusionThis study indicates that a low NADH/NAD+ ratio is an essential mediator of 2-deoxyglucose toxicity in cells with high cytoplasmic NAD+-regeneration capacity and that simultaneous inhibition of glycolysis and lowering of the NADH/NAD+ ratio may be considered to treat cancer.

Meroterpenoids from Ganoderma petchii inhibiting migration of triple negative breast cancer cells

Ganoderpetchoids A−E and (−)-dayaolingzhiol H, six undescribed meroterpenoids, were isolated from Ganoderma petchii. Their structures including the relative configurations were identified by means of spectroscopic methods and 13C NMR calculations. Chiral separation of the new racemics was performed to afford their respective enantiomers. The absolute configurations of the new isolates were clarified by computational approaches, CD comparisons and X-ray diffraction analysis. Biological studies toward triple negative breast cancer indicated that (+)-6 and (−)-6 significantly inhibit the migration of MDA-MB-231 cell line.

Vitamin K2 enhances the tumor suppressive effects of 1,25(OH)2D3 in triple negative breast cancer cells

K vitamins are well known as essential cofactors for hepatic γ-carboxylation of coagulation factors, but their potential role in chronic diseases including cancer is understudied. K2, the most abundant form of vitamin K in tissues, exerts anti-cancer effects via diverse mechanisms which are not completely understood. Our studies were prompted by previous work demonstrating that the K2 precursor menadione synergized with 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to inhibit growth of MCF7 luminal breast cancer cells. Here we assessed whether K2 modified the anti-cancer effects of 1,25(OH)2D3 in triple negative breast cancer (TNBC) cell models. We examined the independent and combined effects of these vitamins on morphology, cell viability, mammosphere formation, cell cycle, apoptosis and protein expression in three TNBC cell models (MDA-MB-453, SUM159PT, Hs578T). We found that all three TNBC cell lines expressed low levels of the vitamin D receptor (VDR) and were modestly growth inhibited by 1,25(OH)2D3 in association with cell cycle arrest in G0/G1. Induction of differentiated morphology by 1,25(OH)2D3 was observed in two of the cell lines (MDA-MB-453, Hs578T). Treatment with K2 alone reduced viability of MDA-MB-453 and SUM159PT cells but not Hs578T cells. Co-treatment with 1,25(OH)2D3 and K2 significantly reduced viable cell number relative to either treatment alone in Hs578T and SUM159PT cells. The combination treatment induced G0/G1 arrest in MDA-MB-453 cells, Hs578T and SUM159PT cells. Combination treatment altered mammosphere size and morphology in a cell specific manner. Of particular interest, treatment with K2 increased VDR expression in SUM159PT cells suggesting that the synergistic effects in these cells may be secondary to increased sensitivity to 1,25(OH)2D3. The phenotypic effects of K2 in TNBC cells did not correlate with γ-carboxylation suggesting non-canonical actions. In summary, 1,25(OH)2D3 and K2 exert tumor suppressive effects in TNBC cells, inducing cell cycle arrest leading to differentiation and/or apoptosis depending on the specific cell line. Further mechanistic studies to clarify common and unique targets of these two fat soluble vitamins in TNBC are warranted.

Abstract 502: ISM3091, a novel selective USP1 inhibitor as a targeted anticancer therapy

Abstract Ubiquitin-specific protease 1 (USP1), a well-characterized member of the deubiquitinating enzymes family, cleaves ubiquitin from various target proteins. USP-1 expression is dysregulated in many cancers where it acts as an oncogenic driver with roles in various DNA damage repair processes including translesion synthesis and the Fanconi anemia pathway. Previous data show that USP1 plays a critical role in protecting the replication fork in BRCA1-deficient cells and that its loss results in reduced cell survival and replication fork degradation, suggesting that USP1 inhibitors may be particularly useful in BRCA-deficient tumors. Though PARP inhibitors (PARPi) have shown clinical benefit in BRCA-mutated (BRCAm) populations, some patients either do not respond to therapy or develop resistance. USP1 inhibitors may have the potential to address this unmet clinical need. We evaluated ISM3091, a novel and selective USP1 inhibitor, against tumor cell lines and in vivo models with BRCAm and other homologous recombination repair deficiency (HRD), as well as in homologous recombination (HR)-proficient models.ISM3091 demonstrated potent anti-proliferation activity (IC50, 20 nM) in the BRCA1m triple negative breast cancer (TNBC) cell line, MDA-MB-436, with a ~1500-fold selectivity for BRCAm vs BRCA WT. Synthetic lethality between USP1 and HRD was due to persistent monoubiquitinated PCNA. In vitro data revealed that the combination of ISM3091 and olaparib, a PARPi, had synergistic activity in cell lines with HRD. Multiple in vivo CDX and PDX models confirmed the dose-dependent, single-agent inhibitory activity of ISM3091, with Tumor Growth Inhibition (TGI) of 66% at 30 mg/kg BID (TNBC) and 60% at 50 mg/kg BID (ovarian). The combination of ISM3091 and olaparib yielded a more robust and durable anti-tumor response, even at low doses of ISM3091, with TGI of 91% (TNBC; ISM3091, 3 mg/kg BID + olaparib, 50 mg/kg QD) and 110% (ovarian; ISM3091, 50 mg/kg BID + olaparib, 50 mg/kg QD). Notably, ISM3091 displayed strong single-agent activity (TGI 72% at 50 mg/kg BID) in an ovarian PDX model with acquired resistance to olaparib (BRCA WT), indicating that it has the potential to treat tumors with HRD beyond BRCAm, and also to overcome PARPi resistance. In vivo evaluation against the HR-proficient lung adenocarcinoma cell line, NCIH1792, showed potent efficacy (TGI 86% at 30 mg/kg BID) suggesting a broader ISM3091 potential. ISM3091also displayed very favorable ADME properties and PK profiles, and GLP toxicology studies indicated that it was well tolerated without significant gastrointestinal toxicity or hematological toxicity. These data support the future clinical development of ISM3091 as a potential best-in-class USP1 inhibitor not only for PARPi-resistant/responsive HRD-mutant cancers, both, as a single agent as well as in combination with PARPi, but also for subsets of HR-proficient cancers. Citation Format: Yangguang Li, Jianping Wu, Jinxin Liu, Luoheng Qin, Xin Cai, Junwen Qiao, Ling Wang, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM3091, a novel selective USP1 inhibitor as a targeted anticancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 502.

Abstract 1433: Targeting the mitotic kinase NEK2 potentiates CDK4/6 inhibitor efficacy in breast cancer

Abstract Mitotic kinases are essential for cell cycle progression, and several mitotic kinase inhibitors have been developed to target the uncontrolled proliferation of cancer cells. However, despite their efficacy in preclinical models, mitotic kinase inhibitors are generally considered poor therapeutic targets due to their off target effects in other rapidly proliferating tissues and subsequent toxicity. However, preliminary data in our lab shows that relative to non-transformed cells, triple negative breast cancer (TNBC) cells are selectively sensitive to inhibition of the mitotic kinase NEK2 compared to other mitotic kinase inhibitors. Therefore, we hypothesize that NEK2 is a targetable vulnerability of TNBC. To begin to understand the role of NEK2 in TNBC, we performed RNA-sequencing following NEK2 silencing and found increased expression of genes important at the G1/S cell cycle transition. This transition is largely controlled by the RB-E2F signaling axis, where CDK4 and CDK6 are key players. CDK4/6 inhibitors have been FDA approved for the treatment of a subset of breast cancers. We investigated the impact of silencing NEK2 and treating with CDK4/6 inhibitors and found that loss of NEK2 sensitizes TNBC cells to CDK4/6 inhibition. Mechanistically, this combination causes a significant increase in genomic instability phenotypes. We tested the combination of NEK2 inhibition and CDK4/6 inhibition in vivo, and found that the combination significantly decreases mouse tumor volume without inducing toxicity. Mechanistically, the combination altered mitotic spindle genes in vivo, indicating an increase in genomic instability. Together, these data suggest that that NEK2 is a viable therapeutic target that could be used in combination with FDA approved CDK4/6 inhibitors for the treatment of aggressive breast cancers such as TNBC. Citation Format: Jessica R. Bobbitt, Leslie Cuellar-Vite, Benjamin Bryson, Kristen Weber-Bonk, Ruth Keri. Targeting the mitotic kinase NEK2 potentiates CDK4/6 inhibitor efficacy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1433.

Abstract 332: Simultaneous inhibition of EZH2 and activation of dopamine D1 in an isotropic triple-negative breast cancer microgel model

Abstract Triple negative breast cancer (TNBC) cells overexpress enhancer of zeste homolog 2 (EZH2) the catalytic subunit of polycomb repressive complex 2 (PRC2). EZH2 is a potential driver of TNBC metastasis, and its high expression strongly associates with the TNBC phenotype as compared with other molecular subtypes of breast cancer. Furthermore, several studies have also shown the involvement of the neurotransmitter, dopamine, in proliferation, apoptosis, tumor angiogenesis, and drug resistance among different cancers, including the breast. Dopamine D1 receptor (D1R) activation in TNBC cell line induces apoptosis, autophagy, inhibit the invasion, and regress mammary tumors. The oncogenic nature of EZH2 in driving aggressiveness in TNBC cells led us to simultaneously target D1R and EZH2 to completely ablate tumor growth and metastasis. Conventional in vitro models utilize monolayers to investigate the effect of drugs on cancer cells but do not accurately recapitulate the in vivo microenvironment as the cells are subjected to homogeneous growth conditions. Microgels are miniature compartmentalized hydrogels that yield isotropic cell culture conditions unbiased by gravitational or hydrodynamic forces. As such, microgels lend suitable microenvironments conducive to the growth of tumor spheroids, which impose radial-dependent growth conditions as observed in vivo. While non-transformed epithelial cells with reconstituted basement membrane form hollow, growth-arrested, polarized three-dimensional (3D) structures, tumorigenic cells form large, solid, proliferating and invasive structures characteristic of in vivo tumor, which are recapitulated by tumor spheroids. As such, tumor spheroids become an ideal model to study the sensitivity of drugs combination, which led us to investigate the efficacy of EZH2 inhibitor and D1R agonist on TNBC tumor spheroids. Tumor spheroids were formed in monodisperse calcium-alginate microgels generated via droplet-based microfluidics, which mimic the viscoelastic properties of decellularized ECM and induce aggregation by removing integrin-binding sites. Tumor spheroids formed after 2 days were subjected to single and combination therapies of GSK126, an EZH2 inhibitor, and A77636, a dopamine agonist over the span of 4 days. Single doses demonstrated a dose-dependent decrease in tumor spheroid area. Furthermore, the combination therapy demonstrated a regression in growth, synergistic decrease in area (CI < 0.6), and the induction of necrosis. Furthermore, knockout of EZH2 in the TNBC cells resulted in inability for tumor spheroid formation and a sensitivity to A77636. Our findings imply that EZH2 is critical for spheroid formation and growth in TNBC and suggest that targeting the EZH2 alongside D1R presents a novel strategy for enhancing EZH2 inhibition (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: Xilal Y. Rima, Gautham Sarathy, Lara Rizzotto, Anagh Kulkarni, Dario Palmieri, Meisam Naeimi Kararoudi, Eduardo Reátegui, Bhuvaneswari Ramaswamy, Eswar Shankar. Simultaneous inhibition of EZH2 and activation of dopamine D1 in an isotropic triple-negative breast cancer microgel model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 332.

Abstract 4965: Targeting the mitotic kinesin, KIF18A, in chromosomally unstable cancers

Abstract Background: Chromosome instability (CIN), characterized by frequent and ongoing loss or gain of chromosome number, is commonly observed in tumor cells. Although long recognized as a vulnerability of cancer cells, potential CIN-selective therapeutic targets have only recently been discovered. Genetic studies from multiple groups have identified the mitotic kinesin, KIF18A, as selectively essential for the proliferation of CIN and aneuploid cells. By targeting KIF18A genetically and with novel small molecule inhibitors here we present data supporting KIF18A as therapeutic target in CIN tumors. Methods and Materials: To explore the therapeutic potential of KIF18A, we evaluated the effects of KIF18A genetic depletion and small molecule inhibition in both CIN-positive and CIN-negative cell lines. Biochemical, cell proliferation and phenotypic assays were used to characterize the potency and cellular activity of reference and novel KIF18A small molecule inhibitors. Anti-tumor activity of KIF18A inhibitors was assessed in CIN-positive cell line xenograft models. Results: In triple negative breast and colorectal CIN positive cancer cell lines siRNA mediated KIF18A knockdown and small molecule inhibition of the KIF18A ATPase activity both lead to a reduction in proliferation associated with an increase in mitotic index and multi-polar spindles. Consistent with KIF18A knockdown, KIF18A inhibition results in increased spindle length and chromosome alignment defects. Importantly, these effects are not observed with KIF18A knockdown or KIF18A inhibition in non-transformed, near diploid cells. In vivo, treatment of CIN-positive xenograft tumors with potent, novel KIF18A inhibitors results in robust anti-tumor activity with minimal impact on body weight. Conclusions: Collectively our data support KIF18A as a therapeutic target and provide rationale for the continued development of potent selective small molecule KIF18A inhibitors for the treatment of CIN positive cancers. Citation Format: James D. Joseph, Katherine Schutt, Kira Fisher, Katelyn Queen, Olivia Budington, Weifeng Mao, Wei Liu, Zhengqing Zhu, Xiangping Zhang, Yisong Xiao, Kunmin Lai, Xiaohui Gu, Jason Stumpff, Fred Aswad. Targeting the mitotic kinesin, KIF18A, in chromosomally unstable cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4965.

Abstract 3646: New functional role of tamoxifen in breast cancer immunomodulation: role of RACK7 in activation of type I interferon signaling and CEACAM1/TIM-3-dependent immunosuppression

Abstract Despite advancements of immunotherapy against various cancers, breast cancer still retains poor response to immune checkpoint blockade (ICB) therapy. Therefore, the identification of promising target or new strategy to enhance ICB therapy in breast cancer is crucial. Here, we uncover that the DNA-damaging potential of tamoxifen (TAM) can shape the unfavorable but ready-to-fire tumor immune microenvironment in breast cancer. We discover that a long-term TAM administration unexpectedly induces JAK/STAT signaling and Type I interferon (IFN)-stimulated gene expression to promote T-cell infiltration. Furthermore, TAM also induces the expression of CEACAM1, which acts as an alternative T-cell inhibitory ligand via binding to TIM-3, a “checkpoint” receptor expressed on CD4+ and CD8+ T cells. The chromatin ‘‘reader’’ RACK7/ZMYND8, which functions as a transcriptional repressor of IFN-stimulated gene (ISG) and a critical factor in DNA repair, is found to be downregulated upon tamoxifen exposure and involved in the tamoxifen-mediated cellular modulation. We demonstrate that TAM in conjunction with RACK7-knockdown (KD) triggers robust upregulation of ISGs and CEACAM1 in both estrogen receptor-positive (ER+) and triple negative breast cancer (TNBC) cells. This immunomodulatory effect lead by loss of RACK7 is specific to TAM treatment, and is not observed when combined with other endocrine therapeutics. TAM combined with RACK7-KD promotes mitochondrial DNA damage, which leads to accumulation of cytosolic DNA and subsequent activation of the cGAS/STING pathway. The murine breast orthotopic models with TS/A, EO771 and 4T1 cells further demonstrate that TAM-mediated immunomodulatory in conjunction with RACK7-KD evokes cytokine/chemokine secretion and further induces T-cell infiltration into tumor microenvironment. However, the tumor killing effect is limited due to promotion of T-cell exhaustion from CEACAM1-TIM-3 interaction between tumor and T cells. Thus, our study indicates that targeting CEACAM1-TIM-3 interaction is crucial for TAM-mediated tumor immune response. This brings promising therapeutic approach with TAM by combining RACK7-KD with blockage to CEACAM1-TIM-3 interaction in breast cancer. The resistance of tamoxifen treatment may be overcome, and RACK7 may serve as both a therapeutic target and a biomarker to enable ICB therapy. Citation Format: Marvin A. Aberin, Yu-Ling (Pony) Lee, Xin-Guo Hsu, Chen-Yang Shen, Yao-Ming Chang, Kun-Yuan Lin, Chandan Guha, Shu-Ping Wang. New functional role of tamoxifen in breast cancer immunomodulation: role of RACK7 in activation of type I interferon signaling and CEACAM1/TIM-3-dependent immunosuppression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3646.

Abstract 3831: The molecular composition of a water-soluble extract from the leaves of the indigenous Southern African plant Tulbaghia violacea that displays anti-cancer activity against a triple negative breast cancer cell line

Abstract Approximately 20% of all breast cancer cases are Triple negative breast cancer (TNBC), whichis more difficult to treat and has a higher mortality rate than other types of breast cancer. Thereis an active practice of traditional medicine in in South Africa, in the form of infusions madeusing plant material. Previously we demonstrated that water soluble extracts from the leaves ofone of these plants, African garlic (Tulbaghia violacea), is able to induce apoptosis in TNBCcell lines, while also inhibiting the ability of these cells to migrate or penetrate a basementmembrane. In an attempt to isolate the molecular compounds that are present in this extract,water soluble leaf extracts were subjected to Fourier transform infrared spectroscopy (FITR),Gas chromatography–mass spectrometry (GC-MS) and Nuclear magnetic resonance (NMR)spectroscopy. Identified compounds of interest were then used in computational dockingstudies with the anti-apoptotic protein Cox2. The FITR analysis indicated that the extractconsisted of multiple compounds, belonging to a variety of classes. GC-NS and NMRspectroscopy identified 36 and 61 separate compounds respectively. Many of these compoundswere similar in structure to compounds with known anti-cancer properties. Many compoundswith established medical uses were also identified in the extract. Five compounds identified inthe extract, d-Glycero-d-galacto-heptose, H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, and Benzaldehyde and 4-(1-methylethyl)- demonstrated high binding affinity towardsCOX2. With d-Glycero-d-galacto-heptose having a higher docking score than manycommercially available COX2 inhibitors. Based on our findings, the compounds within theextract could be used as the basis for the development of safe and effective drugs for thetreatment of TNBC and other cancers. In order to fulfil this potential, future work shouldexamine the molecular pathways affected by the treatment of TNBC cells with this extract orindividual compounds. Citation Format: Mohammed Alaouna, Clement Penny, Rodney Ulll, Zodwa Dlamini. The molecular composition of a water-soluble extract from the leaves of the indigenous Southern African plant Tulbaghia violacea that displays anti-cancer activity against a triple negative breast cancer cell line. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3831.

Abstract 634: The impact of IL-4 on migration in triple negative breast cancer cell lines

Abstract In the United States, triple-negative breast cancer (TNBC), an aggressive form of breast cancer (BCa), disproportionately affects women of African descent and contributes to racial disparities in BCa mortality. Various risk factors are thought to drive this disparity, including socioeconomic status, tumor characteristics, comorbidities, and quality of treatment. Complex diseases and environmental insults all invoke and/or disrupt host inflammation, with individual immune and variable responses to therapeutic agents being dictated in part by genetic variants. This latter has seldom been studied with regards to precision diagnosis and prognosis of individual patient responses, in the frame of geographical ancestry and TNBC. Interleukin-4 receptors (IL4R) have been shown to be upregulated in many epithelial cancers and could be a promising target for metastatic tumor therapy. Yet, IL4R overexpression and/or IL4R variants have not been evaluated in TNBC cell lines of varying genetic ancestry. In view of this, we investigated the effects of IL-4 treatment in four TNBC cell lines varying in proportions of West African Ancestry (WAA) in each cell line: MDA-MB-231 (0.019), HCC-1143 (0.012), HCC-1806 (0.807), MDA-MB-468 (0.803). The frequency of IL4R variants also differed in each cell line. We used high content live cell imaging to evaluate the effect of IL-4 treatment on cellular migration by measuring wound healing. We show that IL-4 treatment on TNBC cells enhances migration as indicated by increased wound healing when compared to non-treated cells. High glucose, at a concentration of 10 g/L, delays cell migration in IL4-treated TNBC cells. This data suggests that comorbidities, such as high glucose levels (i.e., uncontrolled diabetes) could impact tumor behavior and mortality disparities between populations. Citation Format: Portia Andrews, Kevin S. Kimbro. The impact of IL-4 on migration in triple negative breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 634.

Abstract 2846: The mitochondrial dynamics of breast cancer regulate P-glycoprotein expression

Abstract Multidrug efflux transporter ABCB1 (MDR1) encodes P-glycoprotein (P-gp), a well-known transporter that is believed to contribute to the development of multidrug resistance (MDR). Furthermore, P-gp plays an important role in drug disposition, pharmacokinetics, and drug development. Thus, P-gp transporter expression, function, and modulation of MDR in human cancers have been extensively studied. In spite of significant progress in understanding P-gp, ABCB1 regulation and its impact on clinical trials remain unclear. Through sequential exposure of SUM159PT to paclitaxel, we created a paclitaxel resistant triple negative breast cancer cell line SUM159PT-PAC200. We observed a significant increase in the phosphorylation of the mitochondrial fission protein dynamin related protein kinase 1 (Drp1) at Ser616 and its upstream kinase ERK1/2a of Drp1. Through the use of CRISPR-Cas9 technology, we knocked out the gene encoding Drp1 in PAC200. When Drp1 is genetically abated (PAC200 Drp1-/-), paclitaxel resistance is significantly reversed. It was also observed that clonogenicity under paclitaxel pressure was significantly decreased in both the PAC200/Drp1-/- line and the partial KO Drp1-/+ line, compared to the PAC200 line. Parallelly, a significant increase in the expression of P-gp transporters was also observed in the PAC200 cell line. As a surprise, genetically targeting Drp1 in PAC200 cells (i.e., PAC200 Drp1/KO) reduced P-gp expression to virtually undetectable levels. Until now, there has been no description of how Drp1 and P-gp are related. Furthermore, PAC200 Drp1/KO cells showed reduced proliferation, migration, and invasion potential, while their soft agar clonogenic capacities were almost nonexistent, suggesting that Drp1 plays a key role in breast cancer proliferation and dissemination. It appears that modulating or targeting Drp1 might be a useful strategy for modulating P-gp in the development and maintenance of the cancer's MDR phenotype, proliferation, and invasion. Understanding how mitochondrial dynamics determine the transactivation and coordination of drug-resistant P-gp phenotypes would make Drp1 an attractive molecular target for cancer treatment. Citation Format: David Terrero, Saloni Malla, Dayanidhi Raman, Jayachandra Babu, Amit Tiwari. The mitochondrial dynamics of breast cancer regulate P-glycoprotein expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2846.

Abstract 6251: High expression level of GSK3β in basal triple negative breast cancer: Effects of the HDAC inhibitor, vorinostat

Abstract High expression levels of glycogen synthases kinase 3 beta (GSK3β) have been correlated with poor prognosis in triple negative breast cancer (TNBC) patients. GSK3β is a multifaceted kinase and is a key regulator of a number of cellular processes. It is known to function as a positive regulator of epithelial-mesenchymal transition (EMT). Usually, cells that undergo EMT are known to acquire stem cell properties and these cells have properties that are known to be resistant to chemotherapies. Inhibition of GSK3β has been shown to decrease markers of EMT and reduce cancer stem cells. High levels of GSK3β were found in the basal-2 subtype of triple negative breast cancer cells, HCC1806. Histone deacetylases (HDACs) are expressed at high levels in some cancers, including TNBC. In this study, vorinostat, a histone deacetylase inhibitor, was shown to down regulate GSK3 expression in HCC1806 cells at both the gene and protein level using qPCR and western blots. Furthermore, cancer stem cells (CSC) cell markers CD44/CD24 using flow cytometry and immunostaining were significantly decreased. Previous studies published from our laboratory have shown vorinostat’s ability to decrease TNBC cells invasion compacity, as well as cell growth. This study suggests that targeting GSK3β may be a potential therapeutic target for certain subtypes of TNBC. Triple negative breast cancer remains one of the most aggressive breast cancers, often having a high mortality rate in younger women. Citation Format: Beverly D. Lyn-Cook, Fatemeh Nouri Emamzaden, Beverly Word, George Hammons. High expression level of GSK3β in basal triple negative breast cancer: Effects of the HDAC inhibitor, vorinostat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6251.

Abstract 2397: An advanced approach technique for tracking point between immune check point inhibitor and cancer cell mechanism in immune response motility by infiltration a breast cancer cell

Abstract In Breast Cancer, Tumor-infiltrating lymphocyte (TIL) motility has been reported that it is reflecting tumor microenvironment response by adoptive cell therapy (ACT). ACT is well-known to be best applied for suppressing cancer cell growth with immune response and it also was well-known the immunotherapy. However, ACT response tracking by general analysis method included immunohistochemistry and flow cytometry was provided limited information for cancer therapy in recent years. Therefore, by assessing relationship immunotherapy marker, PD-L1, known as immune check point inhibitor and TIL using intravital microscopy in breast cancer model, we also imaged whether TIL response was related to immunotherapy following dependent on time point in a various breast cancer cell implanted mouse model such as 4T1 (mouse breast cancer cell), MDA-MB-231 (human triple negative breast cancer cell) and MCF7 (human breast cancer cell). These images result in PD-L1 expression was decreased in cancer cell implanted until 10days, whereas TIL expression increased in a various breast cancer cell when it is measuring displacement length, track mean velocity and confinement ratio called tumor microenvironment. These results indicated that we can imaged immune response related to TIL response in breast cancer cell line by intravital microscopy instead of general microscopy. It means that intravital microscope was an advanced approach technique to understand cancer mechanism accompanied by the immune response and established the method of high-resolution intravital cellular visualization compared to a general histopathological method for evaluating immune response in cancer tissue Citation Format: Hyunseok Kim, Soyeon Ahn, Hyungjin Kwon, Hsuan-Ju Chen, Inwon Park, Pilhan Kim, Akyildiz Kubra. An advanced approach technique for tracking point between immune check point inhibitor and cancer cell mechanism in immune response motility by infiltration a breast cancer cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2397.

Abstract 3789: Circular RNA expression is enriched in breast cancer extracellular vesicles and is associated with chemotherapy resistance

Abstract Up to 90% of failures in chemotherapy treatment are associated with metastatic-related drug resistance. Extracellular vesicles (EVs) are believed to play a role in the spread of chemotherapy resistance through the horizontal transfer of oncoproteins and noncoding RNA species, but the underlying mechanisms for this are not fully understood. Here, we study EV biogenesis, release, and uptake in the context of breast cancer chemoresistance. Specifically, we characterized the bioactive cargoes in EVs secreted by chemoresistant triple negative breast cancer cells and chemoresistant estrogen receptor-positive breast cancer cell lines. Preliminary data show that these EVs contain unusually high levels of circular RNAs, small RNA species formed covalently through backsplicing and are typically noncoding. When chemosensitive cells absorb these circRNA-enriched EVs, they experience a significant increase in resistance phenotypes. Moreover, knockdown of the circRNAs via antisense oligonucleotides results in decreased chemoresistance, suggesting a role of circRNAs in the development of chemotherapy resistance. Previous studies have shown that tumor-derived exosomal circRNAs may confer resistance by sponging microRNAs. We are exploring what the circRNAs do mechanistically to promote tumor survival, and clarifying these mechanisms as a whole will serve to potentially provide therapeutic targets against chemotherapy resistance and cancer recurrence. Citation Format: Harrison Ngue, Jitendra Shrestha, Hyejin Kim, Shobha Vasudevan. Circular RNA expression is enriched in breast cancer extracellular vesicles and is associated with chemotherapy resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3789.

Abstract 2258: Starvation response to chemotherapy induces MHC class 1 upregulation in cancer cells

Abstract In tumor Major Histocompatibility Complex (MHC) class 1 expression pathway, antigen presenting peptides get hijacked and thus cancer cells escape immune recognition and promote immunosuppressive microenvironment. The restoration of altered MHC 1 expression is important for the treatment of cancer. Inhibition of amino acid uptake or tRNA synthase which mediates amino acid incorporation into protein, mimics starvation. The cancer cell responds to starvation by upregulating proteases to mobilize fatty acids and cellular surface molecules to scavenge exogenous molecules. EPRS (Glutamyl prolyl tRNA synthetase) catalyzes the aminoacylation of glutamic acid and proline to cognate tRNAs. Apart from that, the overexpression of EPRS1 gene is related to tumor angiogenesis, but the mechanism is not clear. Research showed that inhibition of EPRS is effective for the inhibition of triple negative breast cancer (TNBC) and colon cancer. Camptothecin (CPT) is a kind of chemotherapy drug that causes DNA damage by binding topoisomerase enzyme and DNA complex and arrest cells cycle in the S and G2 phases resulting in apoptosis. The knockdown (KD) of EPRS in TNBCs and the use of CPT for the treatment of colon cancer are both likely to have an impact on the accumulation of self epitopes, PTMs, and the creation of neoepitopes within tumors as a result. In this study, we examined the effects of EPRS and CPT treatment on the stimulation of MHC 1 in TNBC and colon cancer cells, as well as the diversity of peptide repertoires in EPRS KD breast cancer cells and CPT treated colon cancer cells. We conducted flow cytometer to detect the HLA specific antibodies on CPT treated RKO and SW620 colon cancer as well as EPRS KD MDA MB 231 triple negative breast cancer cells in our laboratory. In addition, we also performed immunopeptidomes on these cell lines to gain a deeper understanding of the biochemistry and immune pathway. Furthermore, we conducted cell cycle assays in both CRISPR and lentiviral mediated EPRS KD MDA MB 231 cells after CPT treatment. The human MHC class 1 expression were higher in both CPT treated RKO and SW620 colon cancer and EPRS KD MDA MB 231 TNBC cells as compared to the control in both our laboratory and Cayman Immunopeptidome analysis tests. In addition, the cell cycle result showed increment in S phase and reduction in G1 phase in some of the CPT treated EPRS KD ShRNA samples as compared to the control. These results showed that CPT as well as EPRS may play some role in the MHC Class 1 presentation in cancer cells. There were no significant changes observed in the total peptide content of TNBC cells after EPRS knockdown and colon cancer cells after CPT treatment. However, the knockdown of EPRS gene and the CPT treatment resulted in a novel peptide repertoire presented by the cells, which could have relevant to antigen presentation. Further studies are needed for detailed understanding of the biochemistry and immune pathway. Citation Format: Reshma Gurung, Alehegne Yirsaw, Mohammad Hassan, Temesgen Samuel, Clayton Yates, Deepa Bedi. Starvation response to chemotherapy induces MHC class 1 upregulation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2258.

Abstract 3820: Changes in the transcriptome of a triple negative breast cancer cell line following treatment with a water-soluble extract from the leaves of the indigenous Southern African plant Tulbaghia violacea

Abstract Triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancer cases. And is defined by the absence of oestrogen, progesterone, and human epidermal growth factor 2 receptors. This makes it difficult to treat and leads to a low five-year survival rate. In South Africa a large percentage of the population still relies on traditional medicine based on the use of medicinal plants. One of these plants, African garlic (Tulbaghia violacea), that has been used to treat symptoms similar to that observed in cancer. However, the usefulness of these treatments has yet to be clinically proven. Previous studies have revealed that the water-soluble extract from the leaves of T. violacea had anticancer activity against a TNBC cell line. In order to identify the signalling pathways involved in this anti-cancer activity whole transcriptome sequencing was performed using RNA extracted from both TNBC and normal breast cells before and after treatment with the water-soluble T. violacea extract. Paired end sequencing was performed at a depth of 10 million bases for 300 cycles. Data analysis was performed using the Galaxy platform/Sequencing results led us to perform an antibody array assay for proteins involved in the receptor tyrosine kinase pathway. In line with the increased levels of apoptosis previously observed following treatment of the TNBC cell line with the extract, there was an increase in the transcription of genes associated with apoptosis. Decreases were observed in the transcription of genes involved in growth receptor signalling, angiogenesis, and cancer related pathways such as the Wnt, Notch and the PI3K pathway. This work highlights the pathways affected by compounds present within a water-soluble extract of the leaves of T, violacea. Combined with the results of our [previous work, this indicates that the compounds isolated from T, violacea could serve as lead compounds for the development of future therapeutic treatments for TNBC. Citation Format: Zodwa Dlamini, Rodney Hull, Mohammed Alouna, Clement Penny. Changes in the transcriptome of a triple negative breast cancer cell line following treatment with a water-soluble extract from the leaves of the indigenous Southern African plant Tulbaghia violacea. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3820.

Abstract 2539: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis

Abstract TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting that there are unknown modulatory mechanisms responsible for its lack of TRAIL activity in vivo. We hypothesized that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu, modulating the therapeutic efficacy. To investigate the hypothesis, we performed RNAseq analysis of MDA-MB231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. TRAIL treatment of the TNBC significantly induced expression of a number of cytokines, such as CXCL1, CXCL2, CXCL3, CXCL8, CXCL11, IL6, which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by Death Receptor 5 and Caspase-8 protein, but not Caspase-8 enzymatic activity. Gene Set Enrichment Analysis of the RNAseq indicated that NFKB pathway-mediated TNF-alpha signaling was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL. However, siRNA knockdown experiments indicated that the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs’ (1, 2, 3, 8) and IL6 significantly increased neutrophil chemotaxis in a NFKB2-dependent manner. Moreover, neutrophils pre-incubated with supernatants from TRAIL-treated TNBC significantly inhibited its cytotoxic effect in TNBCs in a NFKB2-dependent pathway. Further RNAseq and RT-PCR of neutrophils incubated with by either TRAIL or supernatant of MDA-MB231 cells treated with TRAIL revealed significant enrichment of inflammatory pathway-related genes as well as increased expression of immune modulating cytokines. These results suggested that TRAIL exerts a pro-inflammatory role towards immune cells in tumor microenvironment. CODEX (CO-Detection by indEXing) analysis of in vivo TNBC xenografts from mice treated with the TRAIL agonist MEDI3039 confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Using TNBC organoids and humanized mice models, the changes in tumor immune environment caused by TRAIL are currently under investigation. Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production that affects neutrophil functions, leading to modulation of the immune response in TNBC. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa A. Ridnour, David A. Wink, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2539.