Human Breast Cancer Cell Lines Research Articles

Dehydrodieugenol isolated from Ocotea cymbarum induces cell death in human breast cancer cell lines by dysregulation of intracellular copper concentration

In this work, two neolignans – dehydrodieugenol (1) and dehydrodieugenol B (2) – were isolated from leaves of Ocotea cymbarum (H. B. K.) Ness. (Lauraceae). When tested against two human breast cancer cell lines (MCF7 and MDA-MB-231), compound 1 was inactive (IC50 > 500 μM) whereas compound 2 displayed IC50 values of 169 and 174 μM, respectively. To evaluate, for the first time in the literature, the synergic cytotoxic effects of compounds 1 and 2 with ion Cu2+, both cell lines were incubated with equimolar solutions of these neolignans and Cu(ClO4)2·6H2O. Obtained results revealed no differences in cytotoxicity upon the co-administration of compound 2 and Cu2+. However, the combination of compound 1 and Cu2+ increases the cytotoxicity against MCF7 and MDA-MB-231 cells, with IC50 values of 165 and 204 μM, respectively. The activity of compound 1 and Cu2+ in MCF7 spheroids regarding the causes/effects considering the tumoral microenvironment were accessed using fluorescence staining and imaging by fluorescence microscopy. This analysis enabled the observation of a higher red filter fluorescence intensity in the quiescence zone and the necrotic core, indicating a greater presence of dead cells, suggesting that the combination permeates the spheroid. Finally, using ICP-MS analysis, the intracellular copper disbalance caused by mixing compound 1 and Cu2+ was determined quantitatively. The findings showcased a 50-fold surge in the concentration of Cu2+ compared with untreated cells (p > 0.0001) – 18.7 ng of Cu2+/mg of proteins and 0.37 ng of Cu2+/mg of protein, respectively. Conversely, the concentration of Cu2+ in cells treated with compound 1 was similar to values of the negative control group (0.29 ng of Cu2+/mg of protein). This alteration allowed us to infer that compound 1 combined with Cu2+ induces cell death through copper homeostasis dysregulation.

Phthalazine sulfonamide derivatives as carbonic anhydrase inhibitors. Synthesis, biological and in silico evaluation.

Carbonic Anhydrases (CAs) are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide involved in several biological processes. They show a wide diversity in tissue distribution and their subcellular localization. Twenty-two novel phthalazine derivatives were designed, synthesized, and evaluated against four human isoforms: hCA I, hCA II, hCA IX, and hCA XII. Compounds appeared to be very active mostly against hCA IX (7) and hCA I (6) isoforms being more potent than reference drug acetazolamide (AAZ). Some compounds appeared to be very selective with a selectivity index up to 13.8. Furthermore, docking was performed for some of these compounds on all isoforms to understand the possible interactions with the active site. Additionally, the most active compounds against hCA IX were subjected to cell viability assay. The anticancer activity of the compounds (3a-d, 5d, 5i, and 5m) was investigated using two human breast cancer cell lines, i.e. MCF-7 and MDA-MB-231 cells, and the normal counterpart, namely MCF10-A cells.

Enhancing Curcumin’s therapeutic potential in cancer treatment through ultrasound mediated liposomal delivery

Improving the efficacy of chemotherapy remains a key challenge in cancer treatment, considering the low bioavailability, high cytotoxicity, and undesirable side effects of some clinical drugs. Targeted delivery and sustained release of therapeutic drugs to cancer cells can reduce the whole-body cytotoxicity of the agent and deliver a safe localized treatment to the patient. There is growing interest in herbal drugs, such as curcumin, which is highly noted as a promising anti-tumor drug, considering its wide range of bioactivities and therapeutic properties against various tumors. Conversely, the clinical efficacy of curcumin is limited because of poor oral bioavailability, low water solubility, instability in gastrointestinal fluids, and unsuitable pH stability. Drug-delivery colloid vehicles like liposomes and nanoparticles combined with microbubbles and ultrasound-mediated sustained release are currently being explored as effective delivery modes in such cases. This study aimed to synthesize and study the properties of curcumin liposomes (CLs) and optimize the high-frequency ultrasound release and uptake by a human breast cancer cell line (HCC 1954) through in vitro studies of culture viability and cytotoxicity. CLs were effectively prepared with particles sized at 81 ± 2 nm, demonstrating stability and controlled release of curcumin under ultrasound exposure. In vitro studies using HCC1954 cells, the combination of CLs, ultrasound, and Definity microbubbles significantly improved curcumin’s anti-tumor effects, particularly under specific conditions: 15 s of continuous ultrasound at 0.12 W/cm2 power density with 0.6 × 107 microbubbles/mL. Furthermore, the study delved into curcumin liposomes’ cytotoxic effects using an Annexin V/PI-based apoptosis assay. The treatment with CLs, particularly in conjunction with ultrasound and microbubbles, amplified cell apoptosis, mainly in the late apoptosis stage, which was attributed to heightened cellular uptake within cancer cells.

Molecular interactions between metformin and D-limonene inhibit proliferation and promote apoptosis in breast and liver cancer cells

BackgroundCancer is a fatal disease that severely affects humans. Designing new anticancer strategies and understanding the mechanism of action of anticancer agents is imperative.Hypothesis/PurposeIn this study, we evaluated the utility of metformin and D-limonene, alone or in combination, as potential anticancer therapeutics using the human liver and breast cancer cell lines HepG2 and MCF-7.Study designAn integrated systems pharmacology approach is presented for illustrating the molecular interactions between metformin and D-limonene.MethodsWe applied a systems-based analysis to introduce a drug–target–pathway network that clarifies different mechanisms of treatment. The combination treatment of metformin and D-limonene induced apoptosis in both cell lines compared with single drug treatments, as indicated by flow cytometric and gene expression analysis.ResultsThe mRNA expression of Bax and P53 genes were significantly upregulated while Bcl-2, iNOS, and Cox-2 were significantly downregulated in all treatment groups compared with normal cells. The percentages of late apoptotic HepG2 and MCF-7 cells were higher in all treatment groups, particularly in the combination treatment group. Calculations for the combination index (CI) revealed a synergistic effect between both drugs for HepG2 cells (CI = 0.14) and MCF-7 cells (CI = 0.22).ConclusionOur data show that metformin, D-limonene, and their combinations exerted significant antitumor effects on the cancer cell lines by inducing apoptosis and modulating the expression of apoptotic genes.

Discovery of Alkyl Triphenylphosphonium Pinostrobin Derivatives as Potent Anti-Breast Cancer Agents.

Pinostrobin demonstrated anticancer properties, but its hydrophobic feature led to a reduction in bioavailability. The mitochondria-targeted approach successfully synthesized eight new alkyl triphenylphosphonium pinostrobin derivatives (1-8) with good yield in this study. Seven compounds (1-3, 5-8) showed greater cytotoxic potency against the human MCF-7 breast cancer cell line than pinostrobin. Molecular docking studies were performed with two important targets in hormone-dependent anticancer strategies, estrogen receptor α (ERα) ligand binding domains, 3ERT (antagonist recognition and antiproliferative function), and 1GWR (agonist recognition and pro-proliferative function). In addition, the MD simulation study of the two most potent compounds (2 and 3) complexed with both ERα forms suggested that compounds 2 and 3 could serve as favourable antagonists. Furthermore, the in silico ADMET prediction indicated that compounds 2 and 3 could be potential drug candidates.

Abstract PO3-24-02: Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers

Abstract Introduction: Current FDA-approved immunotherapeutics for triple-negative breast cancer are only available for a subset of patients, highlighting the need to develop improved immune-targeting strategies. VISTA (V-domain Ig suppressor of T cell activation) is a single-pass transmembrane immune checkpoint receptor that is an emerging clinical target for cancer immunotherapy. It is expressed widely on lymphoid and myeloid cells in healthy individuals, is commonly expressed on cells of the tumor microenvironment and can be abnormally upregulated on tumor cells in a wide variety of tumors. Although VISTA confers quiescence to naïve T cells, molecular mechanisms responsible for this effect remain unclear. Also, the effects of VISTA expression on tumor cells remains poorly defined. Methods: Multi-omic profiling was performed in human breast cancer cells lines to determine immune regulatory genes controlled by growth factor stimulation. Human triple-negative breast cancer tumor specimens (N=248) were examined by immunohistochemistry for expression of VISTA, PDL1, PD1 and FoxP3. VISTA expression was engineered into human and mouse triple-negative breast cancer cell lines and growth metrics were characterized in vitro and in immunocompetent (syngeneic) and immunodeficient (xenograft) mouse tumor models. Membrane receptor trafficking and localization of EGFR was performed in human TNBC cells lines expressing VISTA and VISTA mutants. Proximity biotinylation identified protein partners that interacted with VISTA’s intracellular domain. Deletion mutagenesis was performed to identify motifs required for biochemical interactions between VISTA and clathrin adaptor proteins. Clathrin adapter localization was examined in WT and VISTA knock-out cell lines. Cytokine response, clathrin adaptor localization and tumor growth kinetics were measured in TNBC cell lines expressing VISTA or VISTA mutants. Results: A class of human triple-negative breast cancers were identified with high VISTA expression, low tumor infiltrating lymphocytes and low proliferative index (8-20% of all TNBCs). Tumor models demonstrated that enforced expression of VISTA in tumor cells caused growth suppression, even in the absence of an immune system. This effect was mapped to the cytoplasmic domain of VISTA, which lacks defined signaling domains. Through biochemical approaches, the clathrin-adaptor molecules NUMB and GULP1 were found in complex with a VISTA intracellular NPGF motif. This motif sequestered NUMB at early endosomes, resulting in diminished NUMB recycling for EGFR endocytosis, thereby causing growth suppression. VISTA impaired NUMB recycling by blocking Rab11 effector complex formation. VISTA NPGF mutations did not disrupt cytokine secretion in mixed lymphocyte assays. VISTA-targeted antibodies did not disrupt NUMB sequestration. VISTA+ tumors were selectively sensitive to VISTA-blocking antibodies. Conclusions: VISTA+ TNBC have distinct features including slower proliferative index and increased sensitivity to VISTA-blocking antibodies. This work identifies mechanisms by which VISTA enforces a quiescent cellular state by sequestering clathrin adapter proteins away from sites of endocytosis in cancer cells. This is the first description of intracellular molecular mechanisms controlled by VISTA’s intracellular tail. It may inform features of the VISTA-dependent immune checkpoint that could be exploited to improve anti-tumor immunity. Citation Format: Yan Zhao, Fatima Charles, Andrew Lipchik, Yan Peng, James Ford, Melinda Telli, Song Zhang, Allison Kurian, Michael Snyder, Joshua Gruber. Molecular characterization and therapeutic approach for VISTA+ triple-negative breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-02.

Abstract PO3-05-12: Randomized Phase 2 of Bria-IMT, an Allogenic Human Cell Line with Antigen Presenting Activity, in Heavily Pretreated Metastatic Breast Cancer

Abstract Introduction Despite progress in MBC, too many continue to succumb due to treatment resistance or intolerance. SV-BR-1-GM is an allogenic human breast cancer cell line with antigen presenting cell activity designed to overcome the immune-suppressive tumor microenvironment while being acceptable to extensively pretreated contemporary MBC patients. We report interim exploratory analysis of the ongoing RCT of the Bria-IMT regimens. Methods An interim exploratory analysis using preliminary partially available evaluable data of the ongoing prospective, randomized phase 2 (NCT03328026; 2018-present) of Bria-IMT (irradiated SV-BR-1-GM) with a PD-1check point inhibitor (CPI) every 3 weeks (25 patients dosed to date). Both regimens include cyclophosphamide 300 mg/m2 48-72 hours prior to intradermal Bria-IMT (~20 x 106 cells) followed by interferon-alpha at the inoculation sites 2 days later. The control arm used the original sequence with simultaneous start of Bria-IMT and CPI in cycle 1. The experimental arm started CPI in cycle 2 after the 2nd inoculation. Candida skin test was performed before cycle 1 to determine anergy status while delayed-type hypersensitivity skin test (DTH), defined as >5mm erythema & induration, was done at every cycle by intradermal injection of a small test dose of Bria-IMT prior to full dose inoculation. Results Median age: 58 (range 37-81); median prior therapies: 8 lines (range 4-18); 89% were grade 2/3 (n=18); 68% hormone receptor positive; 61% HER2 negative, 33% HER2 low, and 6% HER2 high; 33% triple negative. Overall, 53% of subjects had a clinical benefit as best response by BICR, PI or target lesion measurement. The two treatment arms were similar with a trend showing longer median durations on therapy (mDOT) of 3.7 mo in the experimental delayed CPI arm compared with mDOT = 2.9 for original sequence control arm. 47% of patients were anergic to candida. Among all anergic subjects, the mDOT was 3.5 compared to 2.5 mo for non-anergic patients. However, among anergic subjects, the alternative treatment arm had a lower risk of treatment discontinuation (improved HR) with mDOT of 3.7 compared to the 3.3 mo mDOT of the original treatment. 50% of anergic subjects eventually developed DTH to SV-BR-1-GM. mDOT was 4.0 mo for DTH+ pts vs 3.0 mo for those who did not mount DTH. Median OS was not yet reached; mean OS was higher in non-anergic vs anergic pts. Among those who developed an inoculation site reaction to Bria-IMT, OS was greater than for those who did not. Time on the Bria-IMT regimen exceeded the time on penultimate treatment in 22% of subjects while 33% of subjects with prior CPI use exceeded last therapy. AEs were mild with 28% of subjects (n=7) having > grade 3 events. The most common grade 3+ AE was lipase increase (n=3). One subject with encouraging changes in peripheral blood tumor markers and CTCs completed a PET study using Zr-89 crefmirlimab berdoxam, a zirconium-89 labelled truncated antibody specific to human CD8α (CD8 Immuno-PET) and demonstrated increased uptake in some metastatic lesions and draining lymph nodes. Updated results will be provided at the meeting. Conclusion Sequencing of CPI with Bria-IMT is associated with differential clinical outcome trends but not statistically significant in this interim preliminary exploratory analysis regardless of prior CPI use. Bria-IMT is immunogenic with clinical benefit demonstrated for both treatment schedules among patients with inducible DTH reactions. Patients who develop an immune response to Bria-IMT had an increase in OS compared to those who did not. The nodal localization of CD8+ cells on PET may indicate a systemic activation of CD8 positive lymphoid cells. It also provides support that the Bria-IMT + CPI combination immune-based therapy can result in an increase of CD8+ tumor infiltrating lymphocytes in breast cancer metastatic sites. These preliminary results will be evaluated in the ongoing randomized phase 3 pivotal registrational trial. Citation Format: Carmen Calfa, Chaitali Nangia, Minal Barve, John Knecht, Jarrod Holmes, Kendrith Roland, Ralph Boccia, Frances Valdes-Albini, Zach Gostout, Mingjin Chang, William Williams, Charles Wiseman, Bonnie Guerin, Shaker Dakhil, Giuseppe Del Priore, Saranya Chumsri. Randomized Phase 2 of Bria-IMT, an Allogenic Human Cell Line with Antigen Presenting Activity, in Heavily Pretreated Metastatic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-05-12.

Abstract PO1-08-09: Drynaria fortunie, a nutritional herb for prevention in triple negative breast cancer

Abstract Study Rationale: The triple negative breast cancer (TNBC) lacks the expression of hormone and growth factor receptors and responds only to anthracycline/taxol-based conventional chemotherapy. Major therapeutic limitations include systemic toxicity and acquired resistance to chemo-therapeutics. Relatively non-toxic nutritional herbs from traditional Chinese medicine (TCM) may represent testable alternatives against TNBC. These herbs effectively target multiple signaling pathways (Yang et al: J. Ethno-pharmacology 264: 113249, 2021). Growth inhibitory efficacy of nutritional herbs in a cellular model for TNBC is associated with downregulation of RB, RAS, PI3K and AKT signaling pathways (Telang et al: Pharmaceuticals 14: 1318, 2021). Drynaria fortunie (DF) is a nutritional herb that is used in many TCM herbal formulations. The present study is designed to examine the effects of DF in a cellular model for TNBC and to identify mechanistic leads for its efficacy. Experimental Model, herbal extract and biomarkers: Estrogen receptor negative, progesterone receptor negative and human epidermal growth factor receptor-2 negative MDA-MB-231 human breast cancer cell line represented a cellular model for TNBC. Non-fractionated aqueous extract from the bark of DF represented the test agent. Cell cycle progression, RB signaling and caspase 3/7 activity represented quantitative end point biomarkers. Study Outcome: Treatment with DF at cytostatic concentrations induced S phase arrest and inhibited RB signaling via downregulated expression of cyclin E, CDK2, pRB and E2F1. DF treatment also induced caspase 3/7 activity, a marker for cellular apoptosis. Collectively, these data provide potential mechanistic leads for anti-proliferative and pro-apoptotic effects of DF in the present model. Conclusion: Present data validate a mechanism-driven experimental approach to prioritize efficacious nutritional herbs as testable alternatives for prevention/therapy of TNBC. Citation Format: Nitin TELANG, Hareesh Nair, George Wong. Drynaria fortunie, a nutritional herb for prevention in triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-08-09.

Abstract PO5-26-09: Digital image analysis and a novel set of cell line samples as aids in the development of a quantitative external quality assessment programme for Ki-67

Abstract Background and Aims Ki-67 is a well-established biomarker of proliferation in breast cancer (BC). However, its value in treatment decision making is hampered by a lack of analytical reproducibility. Regular participation in external quality assessment (EQA) substantially improves inter-laboratory concordance. Pre-requisites in establishing a fit-for-purpose EQA for Ki-67 are a well-validated testing substrate and a reproducible method of assessing Ki-67 scores in participants’ submitted materials. We report here on the results of work using cell line controls analysed by digital image analysis (DIA) as a first step towards providing such an EQA. Materials and Methods A formalin-fixed paraffin embedded (FFPE) cell line microarray (CLMA) was designed and produced in conjunction with Array Sciences LLC (Sausalito, USA). It was comprised of cores taken from a pure population of Sf9 caterpillar cells, which have been shown to be completely unreactive with most commercial antibodies to human Ki-67, together with cores of Sf9 cells mixed with four different human BC cell lines. These were BT-20 (85% BC cells), ZR-75-1 (75%), BT-474 (65%) and BT-483 (55%). Sections from the CLMA were mounted onto glass microscope slides together with sections from a FFPE tonsil sample and two BC samples; one BC showed high (~30%, BC-high) and the second, low proliferation (~5%, BC-low). In both BC samples proliferating cancer cells were distributed homogeneously throughout the the block. Unstained sections were distributed to laboratories participating in the Scheme’s Ki-67 BC programme; after routine IHC-staining for Ki-67 returned slides were centrally visually assessed for stain quality and subjected to DIA using an in-house application developed on Visiopharm software (Visiopharm A/S, Hoersholm, Denmark). Results Slides were returned by 37 laboratories. Analysis of Ki-67 scores obtained on the Sf9 core identified two distinct groups. The first (n = 27) were negative or showed low Ki-67 scores (mean = 1.1%, 95% CIs: 0.2-1.9%), the second (n = 10) displayed a step-change in scores (mean = 49.9%, 95% CIs: 33.2-66.7%); the means of the two groups were significantly different (P< 0.0001). When Ki-67 scores for each of the tissue samples were dichotomized into similar groups, the means of those groups differed significantly for the two BC samples (P< 0.001), but not for tonsil. Quality scores generated by visual assessment did not differ significantly between the two groups. However, when slides bearing Sf9 cores demonstrating aberrant Ki-67 scores were visually examined nuclear staining was clearly visible, and non-specific nuclear staining could also be identified in the matched BC tissue samples, but not in the tonsil sections. Correlation of Ki-67 scores between the four BC cell line cores and each of the BC tissue samples was examined using Pearson’s correlation statistics. The r statistic range was 0.57-0.71 in comparisons between BC cell line cores and the BC-high sample, and 0.53-0.70 for those with BC-low; in each case BT-483 showed the highest correlation score and BT-20 the lowest. A similar analysis was undertaken between the tonsil and the two BC tissues. The r statistic for correlation between tonsil and BC-high scores was 0.72; it was 0.64 for those between tonsil and BC-low. Conclusions By using a pure population of Sf9 cells we have developed a sensitive indicator of non-specific nuclear staining in IHC preparations stained for Ki-67 which identifies the presence of the artefact quantifiably. Cores made from Sf9/BC cell line mixtures (especially BT-483) produce Ki-67 scores which correlate with those obtained in breast cancer samples at a similar level to those achieved between tonsil and BC samples; this is true for both high and the low proliferation ranges. Cell line mixtures can be adjusted to show Ki-67 scores in the clinically relevant ranges, and they do not show the inherent biological variations seen in tissue controls such as tonsil. Citation Format: Andrew Dodson, Fitim Berisha, Dawn Wilkingson, Lila Zabaglo, Suzanne Parry. Digital image analysis and a novel set of cell line samples as aids in the development of a quantitative external quality assessment programme for Ki-67 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-26-09.

Abstract PO1-16-04: Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression

Abstract Purpose: Low molecular weight isoforms of cyclin E (LMW-E) have been implicated in various human cancers, including triple negative breast cancer (TNBC), and are associated with a poor prognosis. However, targeted therapies for TNBC based on biomarkers are currently lacking. This study aims to investigate LMW-E as a potential therapeutic target in TNBC and evaluate the efficacy of RP-6306, a selective inhibitor of the Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1), in LMW-E-positive breast tumors. Experimental Design: Immunohistochemical (IHC) analysis was performed on pre-treatment tumor specimens from TNBC patients (n=36) to assess the correlation between LMW-E expression, CDK1 phosphorylation at Threonine 14 (pT14), and pathologic complete response to neoadjuvant chemotherapy. LMW-E inducible human mammary epithelial cells (hMEC) and breast cancer cell lines were used to investigate the regulatory effect of LMW-E on PKMYT1, the kinase responsible for CDK1 phosphorylation at T14, and the response to RP-6306, a first in-class and selective inhibitor of PKMYT1. Patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models of TNBC expressing human LMW-E (hLMW-E) were also utilized to assess LMW-E as a biomarker for predicting response to RP-6306. Results. Analysis of TNBC tumor biopsies revealed a significant positive correlation between LMW-E expression and CDK1 pT14, and both biomarkers were associated with a lack of pathological complete response to neoadjuvant chemotherapy. In vitro results using LMW-E inducible hMECs and breast cancer cell lines demonstrated that LMW-E up-regulates PKMYT1 and CDK1 pT14, acting as a PKMYT1 binding protein and enhancing PKMYT1 protein stability. High LMW-E protein levels predicted a favorable response to RP-6306, resulting in the accumulation of sub-G1 and polyploid cells, decreased tolerance to replication stress, increased DNA damage, chromosomal breakage, and apoptosis. In vivo treatment of TNBC PDX models and hLMW-E transgenic tumors with RP-6306 resulted in a significant reduction in tumor volume only in mice harboring high LMW-E tumors, while low cyclin E models showed no response. Immunohistochemical analysis confirmed increased γ-H2AX and decreased CDK1-pT14 and Ki67 levels, indicating the efficacy of RP-6306 in both PDX and transgenic models. Conclusion: This study highlights the regulatory axis from LMW-E to PKMYT1 and its predictive value for pathological complete response in TNBC patients receiving neoadjuvant chemotherapy. The selective PKMYT1 kinase inhibitor RP-6306 consistently induced DNA damage and inhibited tumor growth in in vitro and in vivo pre-clinical breast tumor models. Co-expression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306, emphasizing its therapeutic significance. Citation Format: Mi Li, Amriti Lulla, Cansu Karakas, Spiridon Tsavaschidis, Yan Wang, Tuyen Nguyun, Tuyen Bui, Gary Marshall, Kelly Hunt, Khandan Keyomarsi. Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-04.

Abstract PO5-24-01: Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models

Abstract Despite improvement of outcomes for HR+, HER2- metastatic breast cancer by cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors, safety concerns and disease progression raise a critical need to identify novel approaches. Narazaciclib (ON123300), a novel CDK4/6i, designed to enhance efficacy and safety by its multi-targeted kinase inhibitor activity. Narazaciclib is in Ph I trials; NCT04739293 and CXHL1900340; studying different administration regimens and in endometrial cancer in combination with letrozole (NCT05705505). Our study explored the activity of narazaciclib and its metabolite ON1232580 in comparisons to the FDA approved CDK4/6i and identify additional targets engaged by narazaciclib. To identify direct and secondary targets engaged by narazaciclib and palbociclib, proteome wide Cellular Thermal Shift Assay (CETSA) and Integrative Inferred Kinase Activity (INKA) analysis were performed. Both CETSA and INKA analysis identified additional targets engaged by narazaciclib compared to palbociclib in both MDA-MB-231 lysates and intact cells such as BUB1, CHEK1, AURKA, GSK3α and GSK3β. In TNBC patients with BUB1 overexpression, bioinformatics analysis indicates a low survival correlation. Molecular docking data showed a higher affinity of narazaciclib with BUB1 compared to palbociclib and abemaciclib. The differential activity of narazaciclib was explored by using cohorts of human and mouse breast cancer cell lines and cell based assays. Comparison of narazaciclib’s in vitro IC50 profile to other CDK4/6i was studied against a panel of 370 kinases (HotSpot) and Kd values were determined by KINOMEscan. Intracellular IC50 kinase values were determined by NanoBret technology. Narazaciclib and abemaciclib were found to be the most promiscuous in vitro kinase inhibitors and ribociclib the most specific. Abemaciclib and narazaciclib had similar profiles against the CDK family members. Kd values of CDK4/cyclinD1 binding show similar trends; abemaciclib (0.08 nM), narazaciclib (0.18 nM), palbociclib (0.75 nM) and ribociclib (1.3 nM). Although narazaciclib displayed nM IC50 values in the in vitro assays against many CDKs, narazaciclib was very specific in cellular kinase assays with highest activity against CDK4/6, CSF1R and NUAK1. A stronger inhibition of cell proliferation, as well as induction of apoptosis and senescence were detected in narazaciclib and its metabolite ON1232580 treated PYMT cells, a murine mammary carcinoma model which recapitulates luminal BC subtype, compared to the other CDK4/6i. Both narazaciclib and ON1232580 enhanced CCL5 and CXCL10 mRNA levels, while a higher reduction of PD-L1 and pRb protein levels and a promoting effect on the H2D1 and B2M mRNA levels was shown in narazaciclib and its metabolite treated PYMT cells. Narazaciclib showed significant synergy with anti-PD1 in the EMT-6 syngeneic breast cancer model. A differential effect of narazaciclib on the expression levels of 62 cytokines was detected compared to abemaciclib and palbociclib. Lastly, inhibition of autophagy sensitizes cancer cells to both ON123300 and ON1232580, and induces irreversible cell proliferation inhibition, providing a novel therapeutic approach. Our findings identify important differences generated from assay models studying kinase inhibition, binding and pathway engagement. Understanding the role of the differential targets engaged by narazaciclib, the potential enhanced antitumor immunity and the sensitization by autophagy inhibitors to cell death, will guide future clinical studies. Citation Format: Petros Kechagioglou, Debomita Chakraborty, Camille Dupont, Hajime Yurugi, Ute Distler, Stefan Tenzer, Alexey Chernobrovkin, Kristina Riegel, Julianne Mooz, Mahil Lamber, Volker Dötsch, Stephen Cosenza, Steven Fruchtman, Krishnaraj Rajalingam. Narazaciclib’s differential targets and kinase inhibitory activity contribute to the enhanced inhibition of tumor growth in preclinical models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-01.

Abstract PS12-03: p53 loss enables HR+ breast cancer escape from CDK4/6 inhibitor-induced quiescence via CDK2

Abstract Background: Inhibition of CDK4/6 kinases has led to improved clinical outcomes in hormone receptor positive (HR+) breast cancer. While these are highly effective therapies, only a minority of patients experience long-term disease control. We sought to determine the genomic configurations and underlying mechanisms associated with long-term response. Methods: To identify genomic patterns associated with clinical outcomes, we analyzed a cohort of 447 patients with metastatic HR+ breast cancer treated at MSK with first-line CDK4/6 inhibitors (CDK4/6i) for which tumor-normal sequencing and long-term clinical follow up were available. To identify the pattern of genomic features associated with longer, intermediate, and short response, we implemented an elastic net Cox regression on binary pathogenic variant status of each gene as well as select clinical features (prior endocrine therapy, endocrine therapy partner, de novo metastatic status). Our principal aim was variable interpretability over pure predictive accuracy. Human HR+ breast cancer models including human breast cancer organoids and cell lines were utilized for mechanistic studies. For validation in a clinical setting, we analyzed the association between Ki67 score after neoadjuvant ribociclib plus endocrine therapy and pre-treatment gene mutation from the FELINE trial [NCT02712723] using a Fisher exact test. Results: Our model identified a “longer response” group (n = 124, 27.7%) from patients with a median progression free survival (PFS) of 32.5 months, compared with an “intermediate” (n = 224, 50.1%, median PFS = 13.7 months) and “short response” group (n = 99, 22.1%, median PFS = 5.84 months). TP53 and MDM2 pathogenic variant status were the most important variables to stratify between these groups, obtaining variable selection frequencies of 1.0 and 0.93 and mean hazard ratios of 2.02 and 1.38, respectively. To elucidate the mechanisms whereby the p53 pathway supports long term response, we generated isogenic and patient derived models of TP53 loss or MDM2 overexpression. Using immunoblotting and cell cycle assays, we found that drug-treated p53 KO cells and MDM2 overexpressing cells effectively suppressed RB1 phosphorylation and blocked in G1 after 24-48 hours. However, upon drug withdrawal, these cells could reenter the cell cycle and promote long-term tumor outgrowth. These effects we observed both in vitro and in vivo. Measures of long-term CDK4/6i response such as expression of senescence associated secretory phenotype genes was abrogated by TP53 loss. Mechanistically, we found persistent phosphorylation of the p130 RB1-like protein in the p53 KO cells. Phosphorylation of p130 impaired its interaction with E2F4, thereby blocking DREAM complex assembly and promoting cell cycle reentry. Inhibition of phosphorylation of p130 via p21 overexpression or by selective CDK2 inhibitors could restore irreversible cell cycle arrest in p53 KO cells. The combination of CDK2 and CDK4/6 inhibition led to long-term tumor growth suppression in models with mutant TP53. To validate the human relevance of TP53 mediating CDK4/6i response, we analyzed longitudinal samples from the FELINE trial that evaluated efficacy and feasibility of neoadjuvant ribociclib plus endocrine therapy. Of 45 evaluable patients, 13 (28.9%) harbored a pre-treatment TP53 loss of function variant. Of these 13 cases, 7 (53.8%) did not achieve a low ( < 10%) Ki-67 upon surgical resection as compared to TP53 wildtype tumors (n=32), only one (3.2%) of which did not achieve a low Ki-67 [OR 32.1, 95% CI 3.28 – 1660.3, p = 0.00026]. Conclusion: Loss of p53 was strongly associated with lack of long-term response to CDK4/6i in patients. Complete inhibition of both CDK4/6 and CDK2 appears to be necessary in order to convert quiescent HR+ tumors cells into durably inhibited and effectively dormant cancers. Citation Format: Rei Kudo, Anton Safonov, Edaise M. da Silva, Qing Li, Hong Shao, Marie Will, Harikrishna Nakshatri, Jorge Reis-Filho, Shom Goel, Andrew Koff, Britta Weigelt, Qamar Khan, Pedram Razavi, Sarat Chandarlapaty. p53 loss enables HR+ breast cancer escape from CDK4/6 inhibitor-induced quiescence via CDK2 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS12-03.

Facile synthesis and surface characterization of platinum and gold bimetallic nanoparticles prepared using hydroalcoholic extract of Bryophyllum Pinnatum: Enhancing efficacy against breast cancer

Primary aim of the proposed study was to explore the green technique for synthesis of bimetallic nanoparticles of platinum and gold metal by employing Bryophyllum pinnatum hydroalcoholic extract (BrPi-P-G-NPs) to explore their potential medicinal properties. Green synthesised bimetallic nanoparticles were characterized by techniques including UV spectroscopy, XRD, FTIR, SEM, TEM, DSC, Particle size and Zeta Potential analysis. Experimental findings confirmed that BrPi-P-G-NPs demonstrate irregular shape, with an average size 34.5 nm. Zeta potential for BrPi and BrPi-P-G-NPs were found to be −8.1 and −24.1 mV, respectively. At a concentration of 75 µg mL−1, the MTT and trypan blue assays of the BrPi-P-G-NPs revealed a cell inhibition rate of 63.54 % and 60.23 %, respectively. In the MCF-7 cell line, the MTT and trypan blue assays recorded IC50 values of 153.54 and 158.04, respectively, for the BrPi-P-G-NPs at the same concentration. Thus, biologically synthesized BrPi-P-G-NPs demonstrated promising anti-cancer properties against human breast cancer cell lines, while showed no signs of toxicity. The enhanced biological activity of BrPi-P-G-NPs can be attributed to their distinctive properties at the nanoscale, as both exhibit lower polydispersity and nanoparticle size, which contribute to increased reactivity and interactions with biological systems. As a result, these BrPi-P-G-NPs nanoparticles synthesized through the biogenic approach using plant extract of Bryophyllum pinnatum have immense potential for a wide range of pharmaceutical applications.

Comprehensive studies on the biological activities of human metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer cell lines, directly or combinedly treated using non-thermal plasma-based approaches

Progressive incidence and a pessimistic survival rate of breast cancer in women worldwide remains one of the most concerning topics. Progressing research indicates a potentially high effectiveness of use cold atmospheric plasma (CAP) systems. The undoubted advantage seems its simplicity in combination with other anti-cancer modalities. Following observed trend of studies, one inventory CAP system was applied to directly treat human breast cancer cell lines and culturing in two different Plasma Activated Media (PAM) for combined utilization. Proposed CAP treatments on MCF-10 A, MCF-7, and MDA-MB-231 cell lines were studied in terms of impact on cell viability by MTT assay. Disturbances in cell motility following direct and combined CAP application were assessed by scratch test. Finally, the induction of apoptosis and necrosis was verified with annexin V and propidium iodide staining. Reactive species generated during CAP treatment were determined based on optical emission spectrometry analysis along with colorimetric methods to qualitatively assess the NO2−, NO3−, H2O2, and total ROS with free radicals concentration. The most effective approach for CAP utilization was combined treatment, leading to significant disruption in cell viability, motility and mostly apoptosis induction in breast cancer cell lines. Determined CAP dose allows for mild outcome, showing insignificant harm for the non-cancerous MCF-10 A cell line, while the highly aggressive MDA-MB-231 cell line shows the highest sensitivity on proposed CAP treatment. Direct CAP treatment seems to drive the cells into the sensitive state in which the effectiveness of PAM is boosted. Observed anti-cancer response of CAP treatment was mostly triggered by RNS (mostly NO2− ions) and ROS along with free radicals (such as H2O2, OH•, O2-•, 1O2, HO2•). The combined application of one CAP source represent a promising alternative in the development of new and effective modalities for breast cancer treatment.

An efficient synthesis and anticancer evaluation of 1,2,3-triazolo-indole-2-carboxylate derivatives

A new series of 1,2,3-Triazole bearing Indole derivatives (6a–j, 8a & 9a) was developed and examined for their preliminary anticancer effects against five human cancer cell lines such as breast cancer cell line (MCF-7), human lung cancer cell line (A549), human colon cancer cell line (Colo-205), human prostate cancer cell line (DU-145) and human breast cancer cell line (MDA MB-231). The well-known chemotherapeutic agent as etoposide used as a positive control. Among synthesized compounds, six compounds (6a, 6b, 6c, 6d, 6f and 8a) were possessed more potent anticancer activities than standard drug.

MEIS2 suppresses breast cancer development by downregulating IL10.

Breast cancer (BC) is the most commonly diagnosed female cancer. Homeobox protein MEIS2, a key transcription factor, is involved in the regulation of many developmental and cellular processes. However, the role of MEIS2 in the development of breast cancer is still unclear. We aimed to examine the role of myeloid ecotropic insertion site (MEIS2) in breast cancer and the association of MEIS2 with breast cancer clinical stages and pathological grades. We revealed the underlying mechanism by which MEIS2 affected breast cancer cell growth and tumor development. Using human BC cell lines, clinical samples and animal xenograft model, we reveal that MEIS2 functions as a tumor suppressor in breast cancer. The expression of MEIS2 is inversely correlated with BC clinical stages and pathological grades. MEIS2 knockdown (MEIS2-KD) promotes while MEIS2 overexpression suppresses breast cancer cell proliferation and tumor development in vitro and in animal xenograft models, respectively. To determine the biological function of MEIS2, we screen the expression of a group of MEIS2 potential targeting genes in stable-established cell lines. Results show that the knockdown of MEIS2 in breast cancer cells up-regulates the IL10 expression, but MEIS2 overexpression opposed the effect on IL10 expression. Furthermore, the suppressive role of MEIS2 in breast cancer cell proliferation is associated with the IL10 expression and myeloid cells infiltration. Our study demonstrates that the tumor suppressor of MEIS2 in breast cancer progression is partially via down regulating the expression of IL10 and promoting myeloid cells infiltration. Targeting MEIS2 would be a potentially therapeutic avenue for BC.

Detection of Anti-cancer Activity of Silver Nanoparticles Synthesized using Aqueous Mushroom Extract of Pleurotus ostreatus on MCF-7 Human Breast Cancer Cell Line

In this research, silver nanoparticles (AgNPs) were manufactured using aqueous extract of mushroom Pleurotus ostreatus. Anticancer potential of AgNPs was investigated versus human breast cancer cell line (MCF-7). Cytotoxic response was assessed by MTT assay. AgNPs showed inhibition effect at the following concentrations 12.5, 25, 50, 100 and 200 µg/ml versus MCF-7 cell line, and all treatments had a positive result. The MCF-7 cells were inhibited up to 85.14 % at the concentration 200 μg/ml of AgNPs which reduced cells viability to 14.86%, while 12.5 μg/ml of AgNPs caused 24.23% cells inhibition with reduction of cells viability to 75.77%.

Antibacterial, antioxidant, and anticancer effects of green synthesized silver/silver chloride nanoparticles using Spondias pinnata bark extract

In this study, the green synthesis of silver/silver chloride nanoparticles (Ag/AgCl-NPs) using Spondias pinnata bark extract was confirmed by the color changes and a sharp absorbance peak at 415 nm determined by a UV-visible spectrometer. Scanning electron microscopy (SEM), X-ray powder diffraction (XRD), Thermal gravimetric analysis (TGA), and Fourier-transformed infrared spectroscopy (FTIR) were used to characterize the synthesized Ag/AgCl-NPs. The disc diffusion method was applied to screen the antibacterial activity of synthesized Ag/AgCl-NPs against four pathogenic bacteria such as Shigella boydii, Escherichia coli, Shigella dysenteriae, and Staphylococcus aureus. The highest antibacterial activity with the maximum zone of growth inhibition was observed against Shigella boydii. The Minimum inhibitory concentration (MIC) was recorded as 3 µg/ml for S. boydii and S. aureus, 6 µg/ml for E. coli, and 12 µg/ml for S. dysenteriae. Synthesized Ag/AgCl-NPs displayed comparatively higher scavenging activity for ABTS (IC50 = 29.87±1.36 µg/ml) than DPPH (IC50 = 27.66±4.54 µg/ml). Additionally, synthesized Ag/AgCl-NPs inhibited 76.83±0.60 % Ehrlich ascites carcinoma (EAC) cells, 78.43±1.97 % human breast cancer (MCF-7) cell lines, and 68.44±1.95 % human lung cancer (A549) cell lines growth at the concentration of 128 µg/ml. Next, synthesized Ag/AgCl-NPs showed potent toxicity against brine shrimp nauplii with an LC50 value of about 30 µg/ml.

Synthesis, molecular docking studies and biological evaluation of N-(4-oxo-2-(trifluoromethyl)-4H-chromen-7-yl) benzamides as potential antioxidant, and anticancer agents

A series of novel chromone derivatives of (N-(4-oxo-2-(trifluoromethyl)-4H-chromen-6-yl) benzamides) were synthesized by treating 7-amino-2-(trifluoromethyl)-4H-chromen-4-one with K2CO3 and/or NaH, suitable alkyl halides and acetonitrile and/or 1,4-dioxane. The obtained products are in high yields (87 to 96%) with various substituents in short reaction times with no more by-products and confirmed by FT-IR, 1H, and 13C-NMR Spectral data. The in vitro cytotoxic activity was examined against two human cancer cell lines, namely the human lung adenocarcinoma (A-549) and the human breast (MCF-7) cancer cell line. Compound 4h showed promising cytotoxicity against both cell lines with IC50 values of 22.09 and 6.40 ± 0.26 µg/mL respectively, compared to that of the standard drug. We also performed the in vitro antioxidant activity by DPPH radical, hydrogen peroxide, NO scavenging, and total antioxidant capacity (TAC) assay methods, and they showed significant activities. The possible binding interactions of all the synthesized chromone derivatives are also investigated against selective pharmacological targets of human beings, such as HERA protein for cytotoxic activity and Peroxiredoxins (3MNG) for antioxidant activity which showed closer binding free energies than the standard drugs and evidencing the above two types of activities.

Synthesis, physico-chemical properties, and AC induction heating of polycaprolactone-coated bio-compatible Fe3O4 nanoparticles

Ex-situ functionalization of Fe3O4 nanoparticles, synthesized using a coprecipitation method, with bio-compatible polycaprolactone (PCL) polymer is reported in this study. The formation of the crystalline structure of Fe3O4 is confirmed from the X-ray diffraction patterns. The presence of the PCL polymer on the surface of the coated MNPs is confirmed using Fourier transform infrared and X-ray photoelectron spectroscopy techniques and thermogravimetric analysis. Scanning and transmission electron microscopy imaging studies reveal the nearly spherical morphology of the synthesized nanoparticles. Room temperature magnetization measurements show the superparamagnetic nature of the PCL coated nanoparticles with high saturation magnetizations varying from ∼61–66 emu/g. Further, the room temperature coercivity is found to be reduced by ∼78 % for the PCL coated nanoparticles, which is attributed to the lowering of the interparticle interactions upon surface functionalization. Magnetic fluid hyperthermia studies on the PCL coated nanoparticles dispersed in ∼1 wt% agar gel shows significant alternating magnetic field induced heating efficiency, with temperature rise in the vicinity of the hyperthermia limit (∼42 °C). The highest specific absorption rate is found to be ∼85.4 ± 3.9 W/gFe, indicating an intrinsic loss power of ∼0.62 nHm2kg−1, which is significantly higher than the previously reported values for PCL coated Fe3O4 MNPs. The field induced heating efficiency is found to be correlated with the saturation magnetization of the PCL coated nanoparticles. In vitro cytotoxicity studies on human breast cancer (MCF-7) cell lines confirm the superior bio-compatibility of the PCL coated Fe3O4 nanoparticles. Superior field induced heating efficiency, coupled with good bio-compatibility makes the PCL functionalized Fe3O4 nanoparticles a potential candidate for magnetic fluid hyperthermia.