Triple-negative Breast Cancer Lines Research Articles

Multicore iron oxide nanoparticles for magnetic hyperthermia and combination therapy against cancer cells

HypothesisMulticore flower-like iron oxide nanoparticles (IONPs) are among the best candidates for magnetic hyperthermia applications against cancers. However, they are rarely investigated in physiological environments and their efficacy against cancer cells has been even less studied. The combination of magnetic hyperthermia, using multicore IONPs, with selected bioactive molecules should lead to an enhanced activity against cancer cells. ExperimentsMulticore IONPs were synthesized by a seeded-growth thermal decomposition approach. Then, the cytotoxicity, cell uptake, and efficacy of the magnetic hyperthermia approach were studied with six cancer cell lines: PANC1 (pancreatic carcinoma), Mel202 (uveal melanoma), MCF7 (breast adenocarcinoma), MB231 (triple-negative breast cancer line), A549 (lung cancer), and HCT116 (colon cancer). Finally, IONPs were modified with a chemotherapeutic drug (SN38) and tumor suppressor microRNAs (miR-34a, miR-182, let-7b, and miR-137), to study their activity against cancer cells with and without combination with magnetic hyperthermia. FindingsTwo types of multicore IONPs with very good heating abilities under magnetic stimulation have been prepared. Their concentration-dependent cytotoxicity and internalization have been established, showing a strong dependence on the cell line and the nanoparticle type. Magnetic hyperthermia causes significant cell death that is dramatically enhanced in combination with the bioactive molecules.

Thermosensitive polymer prodrug nanoparticles prepared by an all-aqueous nanoprecipitation process and application to combination therapy

Despite their great versatility and ease of functionalization, most polymer-based nanocarriers intended for use in drug delivery often face serious limitations that can prevent their clinical translation, such as uncontrolled drug release and off-target toxicity, which mainly originate from the burst release phenomenon. In addition, residual solvents from the formulation process can induce toxicity, alter the physico-chemical and biological properties and can strongly impair further pharmaceutical development. To address these issues, we report polymer prodrug nanoparticles, which are prepared without organic solvents via an all-aqueous formulation process, and provide sustained drug release. This was achieved by the “drug-initiated” synthesis of well-defined copolymer prodrugs exhibiting a lower critical solution temperature (LCST) and based on the anticancer drug gemcitabine (Gem). After screening for different structural parameters, prodrugs based on amphiphilic diblock copolymers were formulated into stable nanoparticles by all-aqueous nanoprecipitation, with rather narrow particle size distribution and average diameters in the 50–80 nm range. They exhibited sustained Gem release in human serum and acetate buffer, rapid cellular uptake and significant cytotoxicity on A549 and Mia PaCa-2 cancer cells. We also demonstrated the versatility of this approach by formulating Gem-based polymer prodrug nanoparticles loaded with doxorubicin (Dox) for combination therapy. The dual-drug nanoparticles exhibited sustained release of Gem in human serum and acidic release of Dox under accelerated pathophysiological conditions. Importantly, they also induced a synergistic effect on triple-negative breast cancer line MDA-MB-231, which is a relevant cell line to this combination.

Abstract 431: Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent triple-negative breast cancer

Abstract Background & Hypothesis: Treatments for triple-negative breast cancer (TNBC) are limited to chemotherapy, PARP inhibitors and immunotherapies. Tumor metabolism seeks to identify metabolic pathways preferentially utilized by cancer. Malic enzyme 2 (ME2) has a role in the malate-aspartate shuttle and pyruvate and NADPH production, contributing to the redox balance and metabolite synthesis. We demonstrated that ME2 knockdown inhibits growth of TNBC cells with upregulated serine biosynthesis pathway. We hypothesized that ME2 knock-down (kd) would be growth inhibitory in vivo. Methods: We used a non-tumorigenic control (MCF10a) and TNBC lines (BT20, MDA-MB-468, HCC1806, HCC70, Hs578T, SUM149). Nude mice received mammary fat pad xenografts of MDA-MB-468, to assess the impact of ME2-shRNA on growth. ME2 relevant metabolite profiling was achieved through the Promega Metabolite-Glo assay, measuring NAD, NADH, NADP, NADPH, Malate, and Glutamate. Cellular proliferation was quantified via the BioTek BioSpa 8 system and Promega CellTiter Glo. Comprehensive profiling of metabolic genes was achieved with the Nanostring nCounter across all cell lines. The potential therapeutic efficacy of NPD-389, MDSA, disodium embonate and doxorubicin was evaluated, alone or in combination, through targeted drug assays. Metabolic flux analyses, specifically mitochondrial and glycolytic function, were conducted using the Seahorse XF96, contrasting ME2-shRNA modified and control cells. Results: ME2 kd resulted in reduced proliferation in several TNBC cell lines to varying degrees, in contrast to control MCF10a, and to Hs578T. In response to the loss of ME2, several cell lines upregulated serine biosynthesis genes, including PSAT1. MDA-MB-468 ME2 kd cells developed significantly smaller tumors in a nude mouse xenograft model. Increased malate levels, but reduced NADH and NADPH, were detected in TNBC cells with inhibited growth post-ME2 kd, but not in MCF10a or Hs578T cells. Among the tested ME2 inhibitors, NPD-389 had superior efficacy vs. MDSA and disodium embonate. Metabolic flux analysis showed compromised mitochondrial and glycolytic processes in the TNBC cell lines exhibiting growth inhibition upon ME2 kd. Conclusions: ME2 knockdown selectively impairs cell proliferation and tumor growth in TNBC cell lines, validating its potential as a therapeutic target. The differential metabolic response, including altered malate and NADH/NADPH levels, confirms ME2's importance in TNBC metabolism. NPD-389 effectively reduced growth in ME2-sensitive lines, meriting further investigation. These findings support targeting ME2 in as a novel treatment strategy. Citation Format: Mark D. Slayton, Zackariah A. Farah, Jin Heon Jeon, Abhinav Achreja, Ben Krinkel, Brisilda Nilaj, Yi-Hsien Eu, Justin Sung, Alyssa Rosenfeld, Alisa Lui, Mason Collard, Liwei Bao, Xu Cheng, Celina Kleer, Kerry Loomes, Deepak Nagrath, Sofia D. Merajver. Malic enzyme 2 inhibition reveals metabolic vulnerability in serine-dependent triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 431.

Identification of lineage-specific epigenetic regulators FOXA1 and GRHL2 through chromatin accessibility profiling in breast cancer cell lines.

Breast cancer is a heterogeneous disease, and breast cancer cell lines are invaluable for studying this heterogeneity. However, the epigenetic diversity across these cell lines remains poorly understood. In this study, we performed genome-wide chromatin accessibility analysis on 23 breast cancer cell lines, including 2 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative (ER+/HER2-), 3 ER+/HER2+, 3 HER2+, and 15 triple-negative breast cancer (TNBC) lines. These cell lines were classified into three groups based on their chromatin accessibility: the receptor-positive group (Group-P), TNBC basal group (Group-B), and TNBC mesenchymal group (Group-M). Motif enrichment analysis revealed that only Group-P exhibited coenrichment of forkhead box A1 (FOXA1) and grainyhead-like 2 (GRHL2) motifs, whereas Group-B was characterized by the presence of the GRHL2 motif without FOXA1. Notably, Group-M did not show enrichment of either FOXA1 or GRHL2 motifs. Furthermore, gene ontology analysis suggested that group-specific accessible regions were associated with their unique lineage characteristics. To investigate the epigenetic landscape regulatory roles of FOXA1 and GRHL2, we performed knockdown experiments targeting FOXA1 and GRHL2, followed by assay for transposase-accessible chromatin sequencing analysis. The findings revealed that FOXA1 maintains Group-P-specific regions while suppressing Group-B-specific regions in Group-P cells. In contrast, GRHL2 preserves commonly accessible regions shared between Group-P and Group-B in Group-B cells, suggesting that FOXA1 and GRHL2 play a pivotal role in preserving distinct chromatin accessibility patterns for each group. Specifically, FOXA1 distinguishes between receptor-positive and TNBC cell lines, whereas GRHL2 distinguishes between basal-like and mesenchymal subtypes in TNBC lines.

A Comparative and Critical Analysis for In Vitro Cytotoxic Evaluation of Magneto-Crystalline Zinc Ferrite Nanoparticles Using MTT, Crystal Violet, LDH, and Apoptosis Assay.

Zinc ferrite nanoparticles (ZFO NPs) are a promising magneto-crystalline platform for nanomedicine-based cancer theranostics. ZFO NPs synthesized using co-precipitation method are characterized using different techniques. UV-visible spectroscopy exhibits absorption peaks specific for ZFO. Raman spectroscopy identifies Raman active, infrared active, and silent vibrational modes while Fourier transforms infrared spectroscopic (FTIR) spectra display IR active modes that confirm the presence of ZFO. X-ray diffraction pattern (XRD) exhibits the crystalline planes of single-phase ZFO with a face-centered cubic structure that coincides with the selected area electron diffraction pattern (SAED). The average particle size according to high-resolution transmission electron microscopy (HR-TEM) is 5.6 nm. X-ray photoelectron spectroscopy (XPS) signals confirm the chemical states of Fe, Zn, and O. A superconducting quantum interference device (SQUID) displays the magnetic response of ZFO NPs, showing a magnetic moment of 45.5 emu/gm at 70 kOe. These ZFO NPs were then employed for comparative cytotoxicity evaluation using MTT, crystal violet, and LDH assays on breast adenocarcinoma epithelial cell (MCF-7), triple-negative breast cancer lines (MDA-MB 231), and human embryonic kidney cell lines (HEK-293). Flow cytometric analysis of all the three cell lines were performed in various concentrations of ZFO NPs for automated cell counting and sorting based on live cells, cells entering in early or late apoptotic phase, as well as in the necrotic phase. This analysis confirmed that ZFO NPs are more cytotoxic towards triple-negative breast cancer cells (MDA-MB-231) as compared to breast adenocarcinoma cells (MCF-7) and normal cell lines (HEK-293), thus corroborating that ZFO can be exploited for cancer therapeutics.

A global sensitivity analysis of a mechanistic model of neoadjuvant chemotherapy for triple negative breast cancer constrained by in vitro and in vivo imaging data

AbstractNeoadjuvant chemotherapy (NAC) is a standard-of-care treatment for locally advanced triple negative breast cancer (TNBC) before surgery. The early assessment of TNBC response to NAC would enable an oncologist to adapt the therapeutic plan of a non-responding patient, thereby improving treatment outcomes while preventing unnecessary toxicities. To this end, a promising approach consists of obtaining in silico personalized forecasts of tumor response to NAC via computer simulation of mechanistic models constrained with patient-specific magnetic resonance imaging (MRI) data acquired early during NAC. Here, we present a new mechanistic model of TNBC growth and response to NAC, including an explicit description of drug pharmacodynamics and pharmacokinetics. As longitudinal in vivo MRI data for model calibration is limited, we perform a sensitivity analysis to identify the model mechanisms driving the response to two NAC drug combinations: doxorubicin with cyclophosphamide, and paclitaxel with carboplatin. The model parameter space is constructed by combining patient-specific MRI-based in silico parameter estimates and in vitro measurements of pharmacodynamic parameters obtained using time-resolved microscopy assays of several TNBC lines. The sensitivity analysis is run in two MRI-based scenarios corresponding to a well-perfused and a poorly perfused tumor. Out of the 15 parameters considered herein, only the baseline tumor cell net proliferation rate along with the maximum concentrations and effects of doxorubicin, carboplatin, and paclitaxel exhibit a relevant impact on model forecasts (total effect index, $$S_{T}$$ S T 0.1). These results dramatically limit the number of parameters that require in vivo MRI-constrained calibration, thereby facilitating the clinical application of our model.

Effect of microRNA-21 blockade in human triple negative breast cancer allografts on progression and immune checkpoint proteins.

e13097 Background: Triple negative breast cancer (TNBC) is an orphan disease that attacks > 46,000 US women every year. TNBC recurs after standard-of-care surgery, chemotherapy and radiation, killing half its victims within 4 years. Poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, immune checkpoint inhibitors, and antibody-drug conjugates extend the survival of a fraction of TNBC patients for a few months. Thus, TNBC shows a critical need for molecularly-targeted therapy. Most TNBC cells show high microRNA 21 (miR-21), identical in humans and mice, which decreases tumor suppressor proteins that keep cell growth in check. Hypothesis: Short anti-miR-21 RNA analogs conjugated to a receptor ligand will direct TNBC cell uptake and slow the growth of TNBC orthotopic allografts with immune activation and minimal toxicity. Methods: We designed an anti-miR-21 RNA analog using aminomethyl bridged nucleic acid (BNA), exhibiting low toxicity, and serum stability. We lipofected a concentration ramp of the anti-miR-21 RNA analog into human TNBC cell lines (MDA-MB-231, MDA-MB-157, MDA-MB-436, MDA-MB-468, BT-20, BT-549, HCC1806, HCC1937, HCC1806) representing a spectrum of TNBC subtypes, then measured proliferation and biochemical markers. We also conjugated a peptide analog for endocytosis by the insulin-like growth factor 1 receptor (IGF1R), overexpressed in most TNBC lesions. The peptide ligand for IGF1R provides a unique strategy for delivering the anti-miR-21 RNA analog preferentially into TNBC cells. We administered 5 mg/kg of the anti-miR-21 RNA-peptide analog in sterile saline intraperitoneally twice a week for 14 days into immunocompetent female Balb/c mice bearing syngeneic EMT6 TNBC tumors in their mammary fat pads. Results: The anti-miR-21 RNA analog slowed proliferation, increased apoptosis, elevated tumor suppressor proteins, and suppressed immune checkpoint gene expression in multiple human TNBC lines. IC50 for inhibiting cell proliferation correlated with miR-21 copies/cell in 7 TNBC cell lines, but not in the non-tumorigenic breast epithelial cell line MCF-10A. The anti-miR-21 RNA-peptide analog stopped orthotopic TNBC allograft growth in syngeneic immunocompetent female Balb/c mice during 14 days of therapy, with apparent safety. Conclusions: This strategy optimizes cancer cell-specific delivery to block proliferation and immune checkpoints. The results support further preclinical proof-of-concept studies to enable an eIND submission for the anti-miR-21 RNA-peptide analog.

Robust eIF4B levels undermine invasive growth and immune evasion mechanisms in murine triple negative breast cancer models.

Abstract Dysregulated protein synthesis is seen in many aggressive cancers, including metastatic breast cancer. However, the specific contributions of certain translation initiation factors to in vivo disease remain undefined. This is particularly true of eIF4B, an RNA-binding protein and cofactor of the RNA helicase eIF4A and associated eIF4F cap-binding complex. While eIF4A, eIF4G, and eIF4E are known to contribute to the progression of many cancer types, the role played by eIF4B in breast cancer remains relatively unclear. We therefore explored how naturally divergent and experimentally modulated eIF4B levels impact tumor growth and progression in murine triple negative breast cancer (TNBC) models. Surprisingly, we found that higher eIF4B levels in mouse and human breast cancers were associated with less aggressive phenotypes. shRNA-mediated eIF4B knockdown in TNBC lines failed to markedly alter proliferation and global translation in cells grown in vitro and only modestly hindered primary mammary tumor growth in mice. However, eIF4B knockdown significantly enhanced invasive growth in vitro and exacerbated both tumor burden and mortality relative to nontargeting shRNA controls in a model of metastatic disease. Deposited patient data reinforced a link between high eIF4B levels and better outcomes. Interestingly, in vitro and in vivo studies associated low eIF4B expression with more formidable immune evasion. These findings suggest that eIF4B levels impact metastatic disease in part through immunomodulatory mechanisms beyond the factor’s traditional role supporting eIF4/F directed translation. Future studies should explore the impact of these findings for personalizing treatment decisions and developing future therapies.

Abstract 3921: Malic enzyme 2 identified as metabolic target in triple-negative breast cancers with increased serine biosynthesis

Abstract Background & Hypothesis: Triple-negative breast cancer (TNBC) is a difficult to treat and deadly form of breast cancer. Pyruvate is an essential intermediate component of cellular metabolism which cancerous cells, including breast cancer cells, rely upon. Importantly, pyruvate and phosphoenolpyruvate are important nodes in the glycolytic pathway that support serine synthesis, lactate production and the TCA cycle. As a result of this, inhibition of the metabolic enzymes which regulate pyruvate production is currently used for cancer therapy. Apart from the glycolytic pathway, malic enzyme 2 (ME2) contributes to pyruvate and NADPH production through the oxidation of malate. Therefore, ME2 is a critical pathway supporting multiple metabolic needs of TNBC. Here, we investigate TNBC cell lines and assess their reliance on ME2 to drive accelerated growth, and thus potentially making ME2 a therapeutic target in TNBC. Methods: We investigated four TNBC lines (HCC1806, Hs578T, BT20, MDAMB468) with distinct serine synthesis activity assessed via western blotting of serine metabolism enzymes. To knockdown ME2 in TNBC cell lines, we used siRNA transient transfection and shRNA-encoding lentiviral vectors. The growth rates of TNBC cells were determined by cell counting in the BioTek BioSpa 8. Lastly, we used immunohistology to analyze the expression of ME2 and serine metabolic genes in a TNBC tumor microarray. Results: Modulation of the serine synthesis pathway revealed an unexpected synergy between serine metabolism and ME2. Knockdown of ME2 reduced the expression of serine metabolism genes but did not affect the expression of ME1 or ME3. The proliferative rate of cells with elevated serine synthesis, BT20 and MDAMB468 - was significantly reduced under ME2 knockdown, while Hs578T and HCC1806 experienced a modest reduction. Immunohistologic staining of TNBC tumors corroborated the link between ME2 and serine metabolism. Conclusions: We observed that TNBC cell lines with elevated, but not reduced, expression of serine synthesis genes were growth-inhibited in the absence of ME2. Our results indicate that ME2 is required for optimal growth in some forms of TNBC and may serve as a therapeutic target for the treatment of cancers with elevated serine biosynthesis. Ongoing studies are aimed at discerning additional biomarkers of sensitivity to ME2 inhibition in TNBC and other aggressive cancers. Citation Format: Mark D. Slayton, Jin Heon Jeon, Abhinav Achreja, Zackariah A. Farah, Brisilda Nilaj, Yi-Hsien Eu, Alisa Lui, Mason Collard, Liwei Bao, Celina Kleer, Deepak Nagrath, Sofia D. Merajver. Malic enzyme 2 identified as metabolic target in triple-negative breast cancers with increased serine biosynthesis. [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 3921.

Abstract P1-13-12: Targeting Dynamin-related protein 1 for the management of taxane-resistant triple-negative breast cancer

Abstract The development of drug resistance is a primary cause of chemotherapy failure in the treatment of triple-negative breast cancer (TNBC). Some cancer cells are resistant to drugs with unrelated structures and mechanisms of action, a phenomenon known as multidrug resistance (MDR). Although taxanes such as docetaxel and paclitaxel are effective against non-metastatic and metastatic TNBC and other types of breast cancer, they eventually become ineffective due to development of drug resistance. Mitochondrial dynamics has gained significant attention as a means to treat MDR and non-MDR cancers. Mitochondrial dynamics determine the number, shape, and location of mitochondria within cells, which are fundamental to the health of the cell. Among its key components is the mitochondrial fission-promoting dynamin-related protein 1 (Drp1). There is mounting evidence indicating that both Drp1 and its phosphorylated form promote cancer survival, resistance to apoptosis, and chemoresistance. We observed increased phosphorylation of Drp1 at Ser616 as well as phosphorylation of its upstream kinase ERK1/2 in TNBC lines SUM159PT and its paclitaxel-resistant derivative that we developed called SUM159PT/PAC200 that displays more than an 80-fold increase in resistance to paclitaxel. Using CRISPR-Cas9 technology, we knocked out the gene encoding Drp1 in both lines. When Drp1 is genetically ablated, paclitaxel resistance in SUM159PT/PAC200 was significantly reversed. Furthermore, proliferation, migration, and invasion capabilities were decreased in both parental and paclitaxel-resistant Drp1 knockout lines. In SUM159PT/PAC200 Drp1-KO cells, a mesenchymal to epithelial transition (MET) was observed as indicated by downregulation of mesenchymal markers such as N-cadherin and -catenin, and upregulation of the epithelial marker ZO-1. Interestingly, Drp1 knockout halted the ability of SUM159PT/PAC200 to form colonies in soft agar. SUM159PT/PAC200 cells strongly express the MDR pump P-glycoprotein (ABCB1), to which paclitaxel is a known substrate, and genetically targeting Drp1 reduced the expression to almost undetectable levels. Taken together, these findings suggest that Drp1 is both a resistance factor and a key protein in breast cancer proliferation and dissemination, making it a promising actionable molecular target in the treatment of TNBC. Further studies are in progress to understand the role of Drp1 in the development and maintenance of MDR phenotype, proliferation, and invasion. Citation Format: David Terrero, Amit Tiwari, Dayanidhi Raman. Targeting Dynamin-related protein 1 for the management of taxane-resistant triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-12.

The role of activated androgen receptor in cofilin phospho-regulation depends on the molecular subtype of TNBC cell line and actin assembly dynamics.

Triple negative breast cancer (TNBC) is highly metastatic and of poor prognosis. Metastasis involves coordinated actin filament dynamics mediated by cofilin and associated proteins. Activated androgen receptor (AR) is believed to contribute to TNBC tumorigenesis. Our current work studied roles of activated AR and cofilin phospho-regulation during migration of three AR+ TNBC cell lines to determine if altered cofilin regulation can explain their migratory differences. Untreated or AR agonist-treated BT549, MDA-MB-453, and SUM159PT cells were compared to cells silenced for cofilin (KD) or AR expression/function (bicalutamide). Cofilin-1 was found to be the only ADF/cofilin isoform expressed in each TNBC line. Despite a significant increase in cofilin kinase caused by androgens, the ratio of cofilin:p-cofilin (1:1) did not change in SUM159PT cells. BT549 and MDA-MB-453 cells contain high p-cofilin levels which underwent androgen-induced dephosphorylation through increased cofilin phosphatase expression, but surprisingly maintain a leading-edge with high p-cofilin/total cofilin not found in SUM159PT cells. Androgens enhanced cell polarization in all lines, stimulated wound healing and transwell migration rates and increased N/E-cadherin mRNA ratios while reducing cell adhesion in BT549 and MDA-MB-453 cells. Cofilin KD negated androgen effects in MDA-MB-453 except for cell adhesion, while in BT549 cells it abrogated androgen-reduced cell adhesion. In SUM159PT cells, cofilin KD with and without androgens had similar effects in almost all processes studied. AR dependency of the processes were confirmed. In conclusion, cofilin regulation downstream of active AR is dependent on which actin-mediated process is being examined in addition to being cell line-specific. Although MDA-MB-453 cells demonstrated some control of cofilin through an AR-dependent mechanism, other AR-dependent pathways need to be further studied. Non-cofilin-dependent mechanisms that modulate migration of SUM159PT cells need to be investigated. Categorizing TNBC behavior as AR responsive and/or cofilin dependent can inform on decisions for therapeutic treatment.

Overexpression of miR-181a regulates the Warburg effect in triple-negative breast cancer

Objective Triple-negative breast cancer (TNBC) is highly aggressive and leads to a poor prognosis. microRNA-181a (miR-181a) exhibits strong antineoplastic effects in many types of cancer. In this study, we examine the responses of human miR-181a-transfected TNBC cells and explore the mechanisms underlying the observed effects. Methods A series of cellular assays were conducted using cells from the MDA-MB-231 TNBC line to assess the impact of miR-181a overexpression. The extracellular acidification rate, lactate production and glucose uptake were evaluated as a measure of aerobic glycolysis (i.e. the Warburg effect). The expressions of glycolysis-related gene were analyzed. Results Viability, migration and survival of miR-181a-transfected MDA-MB-231 cells were all significantly reduced. miR-181a inhibited glycolysis in TNBC cells by reducing the rates of glucose uptake and lactate production and a substantial downregulation of factors known to contribute to the Warburg effect, including the serine/threonine kinase, AKT3, hypoxia-inducible factor-1α (HIF-1α) and progesterone receptor membrane component 1 (PGRMC1). Conclusion Our results demonstrate that miR-181a may regulate glycolysis in MDA-MB-231 TNBC cells, potentially via interference with components of the AKT3–HIF-1α and PGRMC1 pathways. These results suggest that miR-181a might be developed as a therapeutic agent for use in antineoplastic regimens directed at TNBC and PGRMC1-overexpressing breast cancers.

Chondroitin Sulfate Targeting Nanodrug Achieves Near-Infrared Fluorescence-Guided Chemotherapy Against Triple-Negative Breast Primary and Lung Metastatic Cancer.

Lack of highly expressed tumor target and ligands limits application of nano-medicine against triple-negative breast cancer (TNBC). Previous study reported that placenta-derived oncofetal chondroitin sulfate glycosaminoglycan chain (CSA) expressed on 90% of stage I-III invasive ductal breast carcinomas. Our study found the CSA anchor protein VAR2CSA derived small peptide plCSA had strong binding activity with TNBC cell lines and tumor tissue. Here, we combined the AIEgens TBZ-DPNA and therapy drug paclitaxel (PTX) to fabricate near-infrared fluorescence-guided nanodrug (plCSA-NP) to investigate its targeting and anti-tumor effect on TNBC. We synthesized and purified TBZ-DPNA with one step, measured optical properties and photoluminescence (PL) spectra. We prepared nanodrug plCSA-NP by encapsulating TBZ-DPNA and PTX and conjugating them with peptide plCSA. We evaluated plCSA-NP targeting activity by examining AIEdots fluorescence signal on TNBC cell lines and subcutaneous and lung metastatic mouse model. We assessed PTX delivery effect by cytotoxicity assay on TNBC line and tumor growth of subcutaneous and lung metastatic mouse models. PL spectra and TEM imaging results showed plCSA-NP had maximum emission feature at 718 nm and nearly monodispersed nanosphere with an average diameter of 70 nm. In vitro studies showed plCSA-NPs had high affinity and cytotoxicity with TNBC cell lines. In vivo subcutaneous and lung metastasis mouse studies showed plCSA-NPs accumulated on TNBC tumor tissue, and significantly prevented TNBC subcutaneous and lung metastasis tumor growth. In conclusion, we provide solid evidence for chondroitin sulfate targeting peptide plCSA guided nanodrug, exhibit good targeting efficiency and therapeutic effect against TNBC primary and lung metastatic tumor growth.

240 (PB020) - Antitumor activity of Antibody-Drug Conjugates targeting cancer-expressed EGFR in Preclinical Models

Background: The epidermal growth factor receptor (EGFR) plays a key role in the growth and survival of many human tumors of epithelial origin. The monoclonal antibody, 40H3, targeting overexpressed EGFR and the truncated form EGFR variant III (EGFRvIII) on tumor cells, was conjugated to small molecules with potent cytotoxic activity, generating five antibody-drug conjugates (ADCs). Their lethality for tumor cells was evaluated in vitro and in vivo. Materials and methods: ADC construction: Purified 40H3 antibody was conjugated to five different payloads: two tubulin inhibitors, monomethyl auristatin E (MMAE) and DM1, two topoisomerase inhibitors, SN38 and deruxtecan (DXd1) and a PBD dimer (SG-3199). Binding assay: The binding affinity constants of the various 40H3-based ADCs were determined against the His-tagged EGFR peptide loop (aa 287– 302) immobilized on Ni-NTA biosensors with the Octet Red96 analyzer. Bystander assay: Cocultured F98npEGFRvIII and F98 cells were treated with media, free payload (SG-3199), 40H3 antibody, 40H3-tesirine or IgG-tesirine at the indicated concentrations. After 48 hours, the cells were labeled with cetuximab-PE and SYTOXTM Red viability dye and then analyzed for bystander killing by flow cytometry. In vivo studies: MDA-MB-468 or BT-20 tumor xenografts were treated with unmodified 40H3 antibody or 40H3-tesirine. Tumor volumes and mouse weights were measured at least three times weekly. Results: The five ADCs retained antigen binding activity of the unmodified 40H3 antibody. They showed a potent cytotoxicity on a panel of EGFR-expressing cells including three triple negative breast cancer lines. Cell killing was correlated to the number of binding sites for the 40H3 antibody. The 40H3 conjugate with the PBD dimer (40H3-tesirine) was the most active killing agent and it also exhibited bystander killing of cells not expressing human EGFR. Moreover, in vivo, on two different models of tumor xenografts, treatment with 40H3-tesirine achieved complete remissions. Conclusions: The 40H3 antibody is a valid delivery agent for toxic payloads. Among the five ADCs, 40H3-tesirine showed the highest cytotoxic effect toward EGFR-expressing tumor cells in vitro and in vivo. No conflict of interest.

Lack of Oestrogen Receptor Expression in Breast Cancer Cells Does Not Correlate with Kisspeptin Signalling and Migration.

Kisspeptin is an anti-metastatic mediator in many cancer types, acting through its receptor, KISS1R. However, controversy remains regarding its role in breast cancer since both pro- and anti-metastatic roles have been ascribed to it. In KISS1R overexpressing triple-negative breast cancer (TNBC) cells, stimulation has been associated with increased invasion and MMP-9 expression, leading to the suggestion that hormone receptor status determines the metastatic effects of kisspeptin. To assess the veracity of this claim, we compared endogenous KISS1R signalling and physiological output in the hormone receptor-negative MDA-MB-231 and BT-20 cell lines after KP-10 (shortest active kisspeptin peptide) stimulation. MDA-MB-231 cells are metastatic when implanted in mice while BT-20 are not and remain epithelial-like. We show that both cell lines express KISS1R mRNA and respond to KP-10 by elevating calcium mobilisation. However, KP-10 stimulation induced migration of MDA-MB-231, but not BT-20 cells, in a calcium-dependent manner. Moreover, only BT-20 cells responded to KP-10 by increasing ERK phosphorylation in a β-arrestin-dependent manner. Interestingly, both cell lines displayed different complements of β-arrestin 1 and 2 expression. Overall, our data shows that, in TNBC, it is not universally true that kisspeptin/KISS1R stimulate migration or pro-metastatic behaviour, as divergent responses were observed in the two TNBC lines tested. Whether this divergence is related to the observed differences in β-arrestin complements warrants further investigation and may enable further stratification of the ability of kisspeptin to influence breast tumour behaviour.

Cytotoxic and antiparasitic activities of diphosphine-metal complexes of group 10 containing acylthiourea as ligands

In this work, group 10 transition metal complexes bearing dppe [1,2-bis(diphenylphosphino)ethane] and acylthiourea ligands were evaluated for their cytotoxic and antiparasitic activities. Six new complexes with a general formula [M(Ln)(dppe)]BF4 [where M = NiII, PdII or PtII; Ln = N, N′-dimethyl-N-benzoyl thiourea (L1) or N, N′-dimethyl-N-tiofenyl thiourea (L2) were synthesized and characterized by infrared, NMR (31P{1H}, 1H and 13C{1H}) spectroscopies, elemental analysis and molar conductivity. The structures of the complexes were confirmed by X-ray diffraction technique. The biological activity of the complexes was evaluated on breast cancer cells (MDA-MB-231 and MCF-7) and causative agents of chagas disease and leishmaniasis. The complexes presented higher cytotoxicity for breast cancer cell lines compared to non-tumor cells. Nickel complexes stood out when evaluated against the triple-negative breast cancer line (MDA-MB-231), presenting considerably lower IC50 values (about 10 to 22×), when compared to palladium and platinum complexes, and the cisplatin drug. When evaluated on the triple-negative line (MDA-MB-231), the complexes [Ni(L2)(dppe)]BF4(2), [Pd(L2)(dppe)]BF4(4) and [Pt(L2)(dppe)]BF4(6) were able to induce cell morphological changes, influence on the cell colony formation and the size of the cells. The complexes inhibit cell migration and cause changes to the cell cytoskeleton and nuclear arrangement. In the same cell line, the compounds caused cell arrest in the Sub-G1 phase of the cell cycle. The compounds were also tested against the Trypanosom Cruzi (T. cruzi) and Leishmania sp. parasites, which cause Chagas and leishmaniasis disease, respectively. The compounds showed good anti-parasitic activity, mainly for T. cruzi, with lower IC50 values, when compared to the commercial drug, benznidazole. The compounds interact with CT-DNA, indicating that interaction occurs by the minor groove of the biomolecule.

Abstract 1702: SunCatcher: Clonal barcoding with qPCR-based detection enables live cell functional analyses and generation of custom cell pools

Abstract Single cell “omics” and lineage tracing analyses are valuable tools for studying heterogeneity and clonal dynamics. However, current clonal analyses do not easily enable live cell retrieval. That is a particular issue when further study of cells that were targeted for elimination or otherwise not selected during an experiment could provide critical information. We report a clonal molecular barcoding method, called SunCatcher, that enables longitudinal tracking and retrieval of live cells. Single cell-derived clonal populations are generated from complex cell population, each is infected with a unique, heritable molecular barcode, and stocks of each individual barcoded clone (BC) are retained. BCs can be combined to create a version of the original cell population or to generate custom BC pools. We developed a highly sensitive, accurate, rapid, and inexpensive qPCR-based method for identifying and quantifying BCs in vitro and in vivo.We applied SunCatcher to various breast cancer cell lines, including murine Met1 triple negative breast cancer line. A heterogeneous pool comprised of 31 Met1 BCs reliably reproduced the proliferation rate, tumor-forming capacity, and cancer progression as the original parental cell line. Individual BCs, however, displayed significant differences in proliferation, tumor formation, and breast cancer cell hallmarks: cytokeratin 8/14, Zeb1 (mesenchymal marker), EpCAM (epithelial marker) PD-L1 (immune checkpoint protein) and MHC-I (antigen presentation). Five BCs consistently dominated the primary tumors at the ethical end point of 21 days: BC2, BC8, BC25, BC53, BC67. Interestingly, not all 5 BCs were the fastest growing clones, suggesting in vitro proliferation does not correlate with clone expansion in vivo. Two clones (BC43, 18) were not detected in the primary tumors at ethical endpoint, yet when injected alone, had tumor-forming capacity after a long latency (~60 days). Therefore, SunCatcher enabled identification of clones that are consequential for disease progression that would otherwise not be identified during pre-clinical experimentation. Using SunCatcher, we successfully detected Met1 spontaneous metastases as early as 3 weeks in the lung, long bone, and mandible. Long bone had the highest incidence and metastatic burden and were dominated by BC53 and 8 in 3/5 mice. Lung had lower metastatic burden and BC53 was the dominant clone in 4/4 mice. BC8 was exclusive to bone, suggesting site-specific metastasis capacity for different tumor clones. SunCatcher is a reliable, inexpensive, and rapid clonal barcoding method that enables deeper understanding of heterogeneity and clonal dynamics. We identified and analyzed BCs that were responsible for cancer progression, and, for the first time, could analyze BCs that were negatively selected during cancer progression. We envision that SunCatcher will be a useful cancer research tool. Citation Format: Qiuchen Guo, Milos Spasic, Adam Maynard, Gregory Goreczny, Amanuel Bizuayehu, Jessica Olive, Sandra McAllister. SunCatcher: Clonal barcoding with qPCR-based detection enables live cell functional analyses and generation of custom cell pools [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1702.

Abstract 2637: Inhibition of novel kinase MAP3K19 reverses EMT and kills triple negative breast cancer cell lines

Abstract The Triple-Negative Breast Cancer (TNBC) subtype is a breast cancer subpopulation whose phenotype lacks estrogen and progesterone growth receptors, and lacks HER2 amplification. This contrasts with the Luminal and HER2+ subpopulations for which clinical precision therapies exist to target cancer proliferation and improve prognoses. Consequently, the discovery of new targets is necessitated by the TNBC subtype, which has the poorest patient outcomes. Our studies have identified MAP3K19 as one such potential target. MAP3K19 an understudied protein kinase with poorly characterized physiological functions outside of its roles in the Mitogen-Activated Protein Kinase (MAPK) signaling cascade. Broadly, the MAPK signaling has been associated with cell proliferation and differentiation in pathways both intracellular and extracellular, which in the context of cancer cell biology warrants investigation into cancer cell behavior as well as the associated tumor microenvironment (TME). The Published Kinase Inhibitor Set (PKIS) is an open-access drug panel designed by GlaxoSmithKline to identify novel kinases. Preliminary PKIS cell-viability screening identified GSK1440913, GW494601, and GSK907232A as compounds with the most adverse effects on cell phenotype and growth in TNBC lines MDA-MB-231 and PDX-derived TU-BcX-49S. Analysis of common targets revealed MAP3K19 a common target, suggesting its importance in cancer cell growth and proliferation. AT-9283, a more specific MAP3K19 inhibitor, was used to support the initial results. Treatment with AT-9283 resulted in a dose-dependent killing effect. Furthermore, MAP3K19 inhibition resulted in decreased EMT gene signatures by qPCR. To summarize, MAP3K19 was identified by the PKIS screen as a major kinase in TNBC. Treatment with AT-9283, a more specific MAP3K19 inhibitor, resulted in cellular death and reversal in EMT gene signature by qPCR. Future directions for this project involve genetic knock-down and overexpression of MAP3K19 to evaluate its roles in biology and its signaling mechanisms. Citation Format: Khoa Nguyen, Thomas Cheng, Madlin Alzoubi, Steven Elliott, Sean Lee, David Drewry, Bridgette Collins-Burow, John Brognard, Matthew Burow. Inhibition of novel kinase MAP3K19 reverses EMT and kills triple negative breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2637.

Abstract P1-08-20: In silico analysis of a novel mathematical model integrating in vitro and in vivo imaging data reveals driving mechanisms of breast cancer response to NAT for personalized tumor forecasting

Abstract The early assessment of neoadjuvant therapy (NAT) response in triple-negative breast cancer (TNBC) would enable a treating oncologist to adjust a therapeutic plan of a non-responding patient, and thereby enhance outcomes while preventing unnecessary toxicities. To address this challenge, we propose leveraging personalized, in silico forecasts of tumor response to therapeutic regimens via a mechanistic mathematical model calibrated with patient-specific longitudinal magnetic resonance imaging (MRI) data acquired early during NAT. Here, we focus on identifying the driving mechanisms involved in the model formulation through a global sensitivity analysis. Our model describes the dynamics of TNBC cell density as a combination of mobility, which is formulated as a diffusion process constrained by local tumor-induced mechanical stress, and net proliferation, which is represented with a logistic term. To model TNBC response to NAT drug combinations, we adjust the tumor cell proliferation rate with a recent pharmacodynamic model, MuSyC, which also accounts for synergy of potency and efficacy. Tumor cell density is estimated from diffusion-weighted MRI data, while tissue mechanical properties are defined from segmented contrast-enhanced T1-weighted MRI data. To model the heterogeneous intratumoral delivery of drugs, we use perfusion maps estimated from dynamic contrast-enhanced MRI data. NAT drug pharmacokinetics are approximated with a linear model, which reasonably represents their temporal decay during NAT. Sobol’s method is used for the global sensitivity analysis of the NAT response of two different tumors (one well-perfused and one poorly-perfused) on a 3D, tissue-scale domain. This allows us to assess the total effect (ST) of each model parameter on tumor volume and global cellularity. Here, we focus on two standard NAT regimens: doxorubicin plus cyclophosphamide, and paclitaxel plus carboplatin. The parameter space is constructed by integrating three approaches. First, we use prior patient-specific in silico estimates of tumor cell mobility and proliferation. Second, we experimentally constrain the parameters accounting for potential synergistic drug activity by using time-resolved, high-throughput, automated microscopy assays that capture drug-induced changes in proliferation rates of various TNBC lines (HCC1143, SUM149, MDAMB231, and MDAMB468; perfosfamide was used in lieu of the pro-drug cyclophosphamide). Third, we scale the resulting in vitro parameter ranges to be clinically-relevant through an in silico study with our mechanistic model. Our results show that out of the 15 model parameters considered in the sensitivity analysis only a minority exhibited a driving role (ST > 0.1) in representing the dynamics of TNBC response to NAT, namely: the baseline tumor cell proliferation rate along with the effect and ratio of peak concentration to EC50 of doxorubicin, paclitaxel, and carboplatin. The other parameters have a marginal effect and can thus be fixed to any value within the parameter space. We select these constant parameters such that they contribute to simplifying the model formulation, leading to a surrogate reduced model. We further show that the reduced and the original model produce distributions of tumor volume and global cellularity that are in remarkable agreement both qualitatively (Dice similarity coefficient > 0.90 and > 0.94, respectively) and parameter-wise (concordance correlation coefficient > 0.85 and > 0.89, respectively). Thus, we conclude that our reduced model constitutes a feasible surrogate for future clinical calibration-forecasting studies, thereby facilitating personalized NAT response forecasting with patient-specific imaging datasets acquired in vivo. Citation Format: Guillermo Lorenzo, Angela M. Jarrett, Christian T. Meyer, Darren R. Tyson, Vito Quaranta, Thomas E. Yankeelov. In silico analysis of a novel mathematical model integrating in vitro and in vivo imaging data reveals driving mechanisms of breast cancer response to NAT for personalized tumor forecasting [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-20.

DOT1L Is a Novel Cancer Stem Cell Target for Triple-Negative Breast Cancer.

Although chemotherapies kill most cancer cells, stem cell-enriched survivors seed metastasis, particularly in triple-negative breast cancers (TNBC). TNBCs arise from and are enriched for tumor stem cells. Here, we tested if inhibition of DOT1L, an epigenetic regulator of normal tissue stem/progenitor populations, would target TNBC stem cells. Effects of DOT1L inhibition by EPZ-5676 on stem cell properties were tested in three TNBC lines and four patient-derived xenograft (PDX) models and in isolated cancer stem cell (CSC)-enriched ALDH1+ and ALDH1- populations. RNA sequencing compared DOT1L regulated pathways in ALDH1+ and ALDH1- cells. To test if EPZ-5676 decreases CSC in vivo, limiting dilution assays of EPZ-5676/vehicle pretreated ALDH1+ and ALDH1- cells were performed. Tumor latency, growth, and metastasis were evaluated. Antitumor activity was also tested in TNBC PDX and PDX-derived organoids. ALDH1+ TNBC cells exhibit higher DOT1L and H3K79me2 than ALDH1-. DOT1L maintains MYC expression and self-renewal in ALDH1+ cells. Global profiling revealed that DOT1L governs oxidative phosphorylation, cMyc targets, DNA damage response, and WNT activation in ALDH1+ but not in ALDH1- cells. EPZ-5676 reduced tumorspheres and ALDH1+ cells in vitro and decreased tumor-initiating stem cells and metastasis in xenografts generated from ALDH1+ but not ALDH1- populations in vivo. EPZ-5676 significantly reduced growth in vivo of one of two TNBC PDX tested and decreased clonogenic 3D growth of two other PDX-derived organoid cultures. DOT1L emerges as a key CSC regulator in TNBC. Present data support further clinical investigation of DOT1L inhibitors to target stem cell-enriched TNBC.