Negative Breast Cancer Cells Research Articles

Extracellular vesicles derived from SARS-CoV-2 M-protein-induced triple negative breast cancer cells promoted the ability of tissue stem cells supporting cancer progression.

SARS-CoV-2 infection increases the risk of worse outcomes in cancer patients, including those with breast cancer. Our previous study reported that the SARS-CoV-2 membrane protein (M-protein) promotes the malignant transformation of triple-negative breast cancer cells (triple-negative BCC). In the present study, the effects of M-protein on the ability of extracellular vesicles (EV) derived from triple-negative BCC to regulate the functions of tissue stem cells facilitating the tumor microenvironment were examined. Our results showed that EV derived from M-protein-induced triple-negative BCC (MpEV) significantly induced the paracrine effects of adipose tissue-derived mesenchymal stem cells (ATMSC) on non-aggressive BCC, promoting the migration, stemness phenotypes, and in vivo metastasis of BCC, which is related to PGE2/IL1 signaling pathways, in comparison to EV derived from normal triple-negative BCC (nEV). In addition to ATMSC, the effects of MpEV on endothelial progenitor cells (EPC), another type of tissue stem cells, were examined. Our data suggested that EPC uptaking MpEV acquired a tumor endothelial cell-like phenotype, with increasing angiogenesis and the ability to support the aggressiveness and metastasis of non-aggressive BCC. Taken together, our findings suggest the role of SARS-CoV-2 M-protein in altering the cellular communication between cancer cells and other non-cancer cells inside the tumor microenvironment via EV. Specifically, M-proteins induced the ability of EV derived from triple-negative BCC to promote the functions of non-cancer cells, such as tissue stem cells, in tumorigenesis.

Vanadocene-functionalized mesoporous silica nanoparticles: platforms for the development of theranostic materials against breast cancer

Nanoscale materials have demonstrated a very high potential in anticancer therapy by properly adjusting their functionalization and physicochemical properties. Herein, we report the synthesis of some novel vanadocene-loaded silica-based nanomaterials incorporating four different S-containing amino acids (penicillamine, methionine, captopril, and cysteine) and different fluorophores (rhodamine B, coumarin 343 or Alexa Fluor™ 647), which have been characterized by diverse solid-state spectroscopic techniques viz; FTIR, diffuse reflectance spectroscopies, 13C and 51V solid-state NMR spectroscopy, thermogravimetry and TEM. The analysis of the biological activity of the novel vanadocene-based nanostructured silicas showed that the materials containing cysteine and captopril aminoacids demonstrated high cytotoxicity and selectivity against triple negative breast cancer cells, making them very promising antineoplastic drug candidates. According to the biological results it seems that vanadium activity is connected to its incorporation through the amino acid, resulting in synergy that increases the cytotoxic activity against cancer cells of the studied materials presumably by increasing cell internalization. The results presented herein hold significant potential for future developments in mesoporous silica-supported metallodrugs, which exhibit strong cytotoxicity while maintaining low metal loading. They also show potential for theranostic applications highlighted by the analysis of the optical properties of the studied systems after incorporating rhodamine B, coumarin 343 (possible) in vitro anticancer analysis, or Alexa Fluor™ 647 (in vivo studies of cancer models).

MRI-Guided Tumor Therapy Based on Synergy of Ferroptosis, Immunosuppression Reversal and Disulfidptosis.

Triple negative breast cancer (TNBC) cells have a high demand for oxygen and glucose to fuel their growth and spread, shaping the tumor microenvironment (TME) that can lead to a weakened immune system by hypoxia and increased risk of metastasis. To disrupt this vicious circle and improve cancer therapeutic efficacy, a strategy is proposed with the synergy of ferroptosis, immunosuppression reversal and disulfidptosis. An intelligent nanomedicine GOx-IA@HMON@IO is successfully developed to realize this strategy. The Fe release behaviors indicate the glutathione (GSH)-responsive degradation of HMON. The results of titanium sulfate assay, electron spin resonance (ESR) spectra, 5,5'-Dithiobis-(2-nitrobenzoicacid (DTNB) assay and T1-weighted magnetic resonance imaging (MRI) demonstrate the mechanism of the intelligent iron atom (IA)-based cascade reactions for GOx-IA@HMON@IO, generating robust reactive oxygen species (ROS). The results on cells and mice reinforce the synergistic mechanisms of ferroptosis, immunosuppression reversal and disulfidptosis triggered by the GOx-IA@HMON@IO with the following steps: 1) GSH peroxidase 4 (GPX4) depletion by disulfidptosis; 2) IA-based cascade reactions; 3) tumor hypoxia reversal; 4) immunosuppression reversal; 5) GPX4 depletion by immunotherapy. Based on the synergistic mechanisms of ferroptosis, immunosuppression reversal and disulfidptosis, the intelligent nanomedicine GOx-IA@HMON@IO can be used for MRI-guided tumor therapy with excellent biocompatibility and safety.

Abstract 1947 Exploring the Interplay of Social- Economic, and Racial Disparities Stress and Their Impact on Breast Cancer, with a Focus on Triple Negative Breast Cancer and Hormonal Imbalance-Induced Early Immune Cell Aging

Abstract 1947 Exploring the Interplay of Social- Economic, and Racial Disparities Stress and Their Impact on Breast Cancer, with a Focus on Triple Negative Breast Cancer and Hormonal Imbalance-Induced Early Immune Cell Aging

Curcumin Inhibits the PPARδ-p-Akt-GLUT1 Pathway and Ameliorates the Antiproliferative Effects of Doxorubicin in MDA-MB-231 Cells.

The aim of the present study was to examine whether GLUT1 was involved in the antiproliferative activity of curcumin and doxorubicin by understanding mechanistically how curcumin regulated GLUT1. Expression level of GLUT1 in MCF-7 and MDA-MB-231 cells were quantitated using quantitative real-time PCR and western blot. GLUT1 activity was inhibited in MDA-MB-231 cells with the pharmacological inhibitor WZB117 to assess the anti-proliferative effects of doxorubicin using MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). To examine cell proliferation, trypan blue assay was used in cells transfected with GLUT1 siRNA or plasmid overexpressing GLUT1 with doxorubicin and/or commercially available curcumin. The role of PPARδ and Akt on the regulation of GLUT1 by curcumin was examined by overexpressing these proteins and western blot was employed to examine their protein expression. The data revealed that there was a 1.5 fold increase in GLUT1 mRNA and protein levels in MDA-MB-231 compared to MCF-7. By inhibiting GLUT1 in triple negative breast cancer cell line, MDA-MB-231 with either the pharmacological inhibitor WZB117 or with GLUT1 siRNA, we observed the enhanced antiproliferative effects of doxorubicin. Additional observations indicated these effects can be reversed by the overexpression of GLUT1. Treatment of MDA-MB-231 with curcumin also revealed downregulation of GLUT1, with further growth suppressive effects when combined with doxorubicin. Overexpression of GLUT1 blocked the growth suppressive role of curcumin and doxorubicin (p< 0.05). Mechanistically, we also observed that the regulation of GLUT1 by curcumin was mediated by the Peroxisome proliferator-activated receptor (PPAR) δ/Akt pathway. Our study demonstrates that regulation of GLUT1 by curcumin via the PPARδ/Akt signaling improves the efficacy of doxorubicin by promoting its growth inhibitory effects in MDA-MB-231 cells.

Combination therapy application of Abemaciclib with Doxorubicin in triple negative breast cancer cell line MDA-MB-231.

Due to lack of clinical biomarkers, Triple Negative Breast Cancer (TNBC) is more likely to have spread to other tissues at time of diagnosis and therapy planning generally involves use of cytotoxic chemotherapy agents, such as Doxorubicin. We aimed to investigate possible advantages of using combination strategy using Doxorubicin alongside Abemaciclib. After determining the IC50 values for Doxorubicin (DOX) and Abemaciclib (ABE); CompuSyn and ComBenefit software were used to reveal the effect resulting from the combination of two drugs. Following the determined effect, cell death was revealed by fluorescence microscopy and a colony forming assay was performed to see the potential of even a single cancer cell with adhesive character to survive over time and form a clone of itself. Detection of changing antioxidant activity following DOX, ABE and DOX+ABE combination therapy in MDAMB231 cells was determined by measuring MDA, SOD and GSH activities. The expression of Cleaved Caspase 3, PARP, Cleaved PARP, Cdk2 and Bax, which changed as a result of DOX, ABE and DOX+ABE application, was shown by Western Blotting.Cyclin-dependent kinase inhibitors appear as promising agents in therapy planning for breast cancer due to their prominent role in cell cycle regulation, where the number of studies interrogating its efficiency in the treatment of cancer such as TNBC is limited. For this reason, in this study, we aimed to determine the impact of the combined use of the CDK4/6 inhibitors ABE and DOX on the cytotoxicity, apoptotic homeostasis, alterations in antioxidative mechanisms, and the molecular pathways that they utilize. Our results showed that when used in combination, Doxorubicin and Abemaciclib showed a synergistic effect on TNBC cell line MDA-MB-231.

Effective Anticancer Potential of a New Sulfonamide as a Carbonic Anhydrase IX Inhibitor Against Aggressive Tumors.

This study examines efficiency of a newly synthesized sulfonamide derivative 2-bromo-N-(4-sulfamoylphenyl)propanamide (MMH-1) on the inhibition of Carbonic Anhydrase IX (CA IX), which is overexpressed in many solid tumors including breast cancer. The inhibitory potential of MMH-1 compound against its four major isoforms, including cytosolic isoforms hCA I and II, as well as tumor-associated membrane-bound isoforms hCA IX and XII, was evaluated. To this context, the cytotoxic effect of MMH-1 on cancer and normal cells was tested and found to selectively affect MDA-MB-231 cells. MMH-1 reduced cell proliferation by holding cells in the G0/G1 phase (72 %) and slowed the cells' wound healing capacity. MMH-1 inhibited CA IX under both hypoxic and normoxic conditions and altered the morphology of triple negative breast cancer cells. In MDA-MB-231 cells, inhibition of CA IX was accompanied by a decrease in extracellular pH acidity (7.2), disruption of mitochondrial membrane integrity (80 %), an increase in reactive oxygen levels (25 %), and the triggering of apoptosis (40 %). In addition, the caspase cascade (CASP-3, -8, -9) was activated in MDA-MB-231 cells, triggering both the extrinsic and intrinsic apoptotic pathways. The expression of pro-apoptotic regulatory proteins (Bad, Bax, Bid, Bim, Cyt-c, Fas, FasL, TNF-a, TNF-R1, HTRA, SMAC, Casp-3, -8, P21, P27, and P53) was increased, while the expression of anti-apoptotic proteins, apoptosis inhibitor proteins (IAPs), and heat shock proteins (HSPs) (Bcl-2, Bcl-w, cIAP-2, HSP27, HSP60, HSP70, Survivin, Livin, and XIAP) was decreased. These results propose that the MMH-1 compound could triggers apoptosis in MDA-MB-231 cells via the pH/MMP/ROS pathway through the inhibition of CA IX. This compound is thought to have high potential and promising anticancer properties in the treatment of aggressive tumors.

G-4 inhibits triple negative breast cancer by inducing cell apoptosis and promoting LCN2-dependent ferroptosis

Compound G-4 is a derivate of cyclin-dependent kinase inhibitor Rocovitine and showed strong sensitivity to triple negative breast cancer (TNBC) cells. In this study, the antitumor activity, mechanism and possible targets of G-4 in TNBC were investigated. Flow cytometry and immunoblotting showed that G-4 not only arrested the S phase of the cell cycle, but also induced apoptosis in TNBC cells via the mitochondrial pathway through inhibiting epidermal growth factor receptor (EGFR), AKT and MAPK pathways. In addition, G-4 induced the iron-mutagenesis process in TNBC cells and down-regulated differentially expressed gene lipid carrier protein 2 (LCN2) by RNA-seq. Moreover, G-4 elevated levels of cytosolic reactive oxygen species (ROS), lipid ROS, Fe and malondialdehyde (MDA), but decreased levels of superoxide dismutase (SOD) and glutathione (GSH), consistent with the effects of iron-mutagenic agonists Erastin and RSL3, which were inhibited by the iron inhibitor ferrostatin-1 (Fer-1). Furthermore, a LCN2 knockdown cell model was established by siRNA transfection, the IC50 of G-4 was increased nearly 100-fold, accompanied by a trend of no ferroptosis characteristic index. The results indicated that G-4 suppressed the malignant phenotype of TNBC, induced apoptosis by inhibiting EGFR pathway and promoted LCN2-dependent ferroptosis.

Increased matrix stiffness enhances pro-tumorigenic traits in a physiologically relevant breast tissue- monocyte 3D model

High mammographic density, associated with increased tissue stiffness, is a strong risk factor for breast cancer per se. In postmenopausal women there is no differences in the occurrence of ductal carcinoma in situ (DCIS) depending on breast density. Preliminary data suggest that dense breast tissue is associated with a pro-inflammatory microenvironment including infiltrating monocytes. However, the underlying mechanism(s) remains largely unknown. A major roadblock to understanding this risk factor is the lack of relevant in vitro models. A biologically relevant 3D model with tunable stiffness was developed by cross-linking hyaluronic acid. Breast cancer cells were cultured with and without freshly isolated human monocytes. In a unique clinical setting, extracellular proteins were sampled using microdialysis in situ from women with various breast densities. We show that tissue stiffness resembling high mammographic density increases the attachment of monocytes to the cancer cells, increase the expression of adhesion molecules and epithelia-mesenchymal-transition proteins in estrogen receptor (ER) positive breast cancer. Increased tissue stiffness results in increased secretion of similar pro-tumorigenic proteins as those found in human dense breast tissue including inflammatory cytokines, proteases, and growth factors. ER negative breast cancer cells were mostly unaffected suggesting that diverse cancer cell phenotypes may respond differently to tissue stiffness. We introduce a biological relevant model with tunable stiffness that resembles the densities found in normal breast tissue in women. The model will be key for further mechanistic studies. Additionally, our data revealed several pro-tumorigenic pathways that may be exploited for prevention and therapy against breast cancer. Statement of significanceWomen with mammographic high-density breasts have a 4–6-fold higher risk of breast cancer than low-density breasts. Biological mechanisms behind this increase are not fully understood and no preventive therapeutics are available. One major reason being a lack of suitable experimental models. Having such models available would greatly enhance the discovery of relevant targets for breast cancer prevention. We present a biologically relevant 3D-model for studies of human dense breasts, providing a platform for investigating both biophysical and biochemical properties that may affect cancer progression. This model will have a major scientific impact on studies for identification of novel targets for breast cancer prevention.

Role of β3 subunit of the GABA type A receptor in triple negative breast cancer proliferation, migration, and cell cycle progression

ABSTRACT Triple negative breast cancer (TNBC) is known for its heterogeneous nature and aggressive onset. The unresponsiveness to hormone therapies and immunotherapy and the toxicity of chemotherapeutics account for the limited treatment options for TNBC. Ion channels have emerged as possible therapeutic candidates for cancer therapy, but little is known about how ligand gated ion channels, specifically, GABA type A ligand-gated ion channel receptors (GABAAR), affect cancer pathogenesis. Our results show that the GABAA β3 subunit is expressed at higher levels in TNBC cell lines than non-tumorigenic cells, therefore contributing to the idea that limiting the GABAAR via knockdown of the GABAA β3 subunit is a potential strategy for decreasing the proliferation and migration of TNBC cells. We employed pharmacological and genetic approaches to investigate the role of the GABAA β3 subunit in TNBC proliferation, migration, and cell cycle progression. The results suggest that pharmacological antagonism or genetic knockdown of GABAA β3 subunit decreases TNBC proliferation and migration. In addition, GABAA β3 subunit knockdown causes cell cycle arrest in TNBC cell lines via decreased cyclin D1 and increased p21 expression. Our findings suggest that membrane bound GABAA receptors containing the β3 subunit can be further developed as a potential novel target for the treatment of TNBC.

Fractionation and phytochemical composition of an ethanolic extract of Ziziphus nummularia leaves: antioxidant and anticancerous properties in human triple negative breast cancer cells.

Natural products have long been utilized in traditional medicine as remedies to improve health and treat illnesses, and have had a key role in modern drug discovery. Recently, there has been a revived interest in the search for bioactives from natural sources as alternative or complementary modalities to synthetic medicines; especially for cancer treatment, which incidence and mortality rates are on the rise worldwide. Ziziphus nummularia has been widely used in traditional medicine for the treatment of various diseases. Its traditional uses and numerous ethnopharmacological properties may be attributed to its richness in bioactive metabolites. However, its phytochemical composition or chemopreventive effects against the aggressive triple-negative breast cancer (TNBC) are still poorly explored. Here, phytochemical composition of an ethanolic extract of Z. nummularia leaves (ZNE) and its chromatographically isolated fractions was identified both qualitatively by spectrophotometric assays and analytically by HPLC-PDA-MS/MS. The anti-proliferative effects of ZNE were tested in several cancer cell lines, but we focused on its anti-TNBC effects since they were not explored yet. The anti-cancerous potential of ZNE and its fractions was tested in vitro in MDA-MB-231, a TNBC cell line. Results showed that ZNE and its Fraction 6 (F6) reduced the viability of MDA-MB-231 cells. F6 decreased MDA-MB-231 viability more than crude ZNE or its other fractions. ZNE and F6 are rich in phytochemicals and HPLC-PDA-MS/MS analysis identified several metabolites that were previously reported to have anti-cancerous effects. Both ZNE and F6 showed potent antioxidant capacity in the DPPH assay, but promoted reactive oxygen species (ROS) production in MDA-MB-231 cells; an effect which was blunted by the antioxidant N-acetyl cysteine (NAC). NAC also blunted ZNE- and F6-induced reduction in TNBC cell viability. We also demonstrated that ZNE and F6 induced an arrest of the cell cycle, and triggered apoptosis- and autophagy-mediated cell death. ZNE and F6 inhibited metastasis-related cellular processes by modifying cell migration, invasion, and adhesion. Taken together, our findings reveal that Z. nummularia is rich in phytochemicals that can attenuate the malignant phenotype of TNBC and may offer innovative avenues for the discovery of new drug leads for treatment of TNBC and other cancers.

Synthesis of Self Permeable Antisense PMO Using C5-Guanidino-Functionalized Pyrimidines at the 5'-End Enables Sox2 Downregulation in Triple Negative Breast Cancer Cells.

Delivery of macromolecular drugs inside cells has been a huge challenge in the field of oligonucleotide therapeutics for the past few decades. Earliest natural inspirations included the arginine rich stretch of cell permeable HIV-TAT peptide, which led to the design of several molecular transporters with varying numbers of rigid or flexible guanidinium units with different tethering groups. These transporters have been shown to efficiently deliver phosphorodiamidate morpholino oligonucleotides, which have a neutral backbone and cannot form lipoplexes. In this report, PMO based delivery agents having 3 or 4 guanidinium groups at the C5 position of the nucleobases of cytosine and uracil have been explored, which can be assimilated within the desired stretch of the antisense oligonucleotide. Guanidinium units have been connected by varying the flexibility with either a saturated (propyl) or an unsaturated (propargyl) spacer, which showed different serum dependency along with varied cytoplasmic distribution. The effect of cholesterol conjugation in the delivery agent as well as at the 5'-end of full length PMO in cellular delivery has also been studied. Finally, the efficacy of the delivery has been studied by the PMO mediated downregulation of the stemness marker Sox2 in the triple-negative breast cancer cell line MDA-MB 231. These results have validated the use of this class of delivery agents, which permit at a stretch PMO synthesis where the modified bases can also participate in Watson-Crick-Franklin base pairing for enhanced mRNA binding and protein downregulation and could solve the delivery problem of PMO.

Abstract B022: Targeting epithelial-mesenchymal plasticity and CD73 to enhance responses of breast cancers to immune checkpoint blockade therapies

Abstract Immune checkpoint blockade (ICB) therapy has generated dramatic responses in certain types of human tumors however, the response of breast cancers has been largely limited. Epithelial-mesenchymal plasticity (EMP) enables carcinoma cells to metastasize, gain tumor-initiating ability and mount resistance to chemotherapies. In addition, we have demonstrated that this plasticity program also contributes to the establishment of an immunosuppressive tumor microenvironment and confers resistance to ICB therapy (Dongre and Weinberg 2019). By establishing novel, preclinical models of more-epithelial or more-mesenchymal breast tumors, we observed that epithelial tumors recruit CD8+ T-cells to their microenvironment and are sensitive to anti-CTLA4 ICB. In contrast, mesenchymal tumors recruit Tregs and M2-like macrophages and are resistant to the same treatment (Dongre et al., 2017). Strikingly, abrogation of CD73 from more-mesenchymal cells completely sensitizes otherwise refractory tumors to anti-CTLA4 ICB (Dongre et al., 2021). These findings demonstrated for the first time that more-mesenchymal breast cancer cells can be completely sensitized to anti-tumor immunity by interrupting certain cell-intrinsic signaling channels. However, the underlying mechanisms of such sensitization remain elusive. Additionally, the regulatory networks that control the expression of CD73 itself in more-mesenchymal but not epithelial breast cancer cells are not well defined. To determine the subset of T-cells that is functionally important in sensitizing mesenchymal tumors lacking CD73, we depleted either conventional CD4+ T-cells or CD8+ cytotoxic T-cells in combinatoin with ICB. While responding mice recruited both T-cells to their tumors, only CD4+ T-cells (and not CD8+ T-cells) were functionally important in driving sensitization. Along these lines, more-mesenchymal cancer cells lacking CD73 failed to respond to anti-CTLA4 ICB when propagated in genetically engineered mice lacking CD4+ T-cells relative to wild type controls. Importantly, depletion of CD4+ T-cells also prevented the recruitment of peripheral CD8+ T-cells to responding tumors, thereby driving resistance to ICB. In addition, we observed that human triple negative breast cancer cell lines expressed the highest amount of CD73 relative to hormone receptor-positive cell lines. Most importantly, activating EMP in human breast cancer cells by doxycycline-controlled expression of various transcription factors, induced the expression of CD73. Taken together, our work demonstrates that (i) the phenotypic plasticity of breast cancer cells can predict responses to ICB (ii) targeting CD73 can completely sensitize more-mesenchymal tumors to anti-CTLA4 ICB in a CD4+ T-cell dependent fashion and; (iii) altering the cellular plasticity of cancer cells can regulate CD73 expression. Given the highly refractory nature of more-mesenchymal cancer cells, our findings hold tremendous potential in aiding translational efforts to target cancer plasticity to enhance responses of breast cancers to ICB therapy. Citation Format: Anushka Dongre. Targeting epithelial-mesenchymal plasticity and CD73 to enhance responses of breast cancers to immune checkpoint blockade therapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B022.

Abstract A031: The role of nuclear epidermal growth factor receptor in cell migration and wound healing

Abstract In breast cancer cells, epidermal growth factor receptor (EGFR) can retrotranslocate to the nucleus where it acts as a transcription cofactor and activates pro-oncogenic pathways such as stemness and migration. This retrotranslocation can occur through interaction with the BAR domain-containing protein Sorting Nexin 1 (SNX1). Upon retrotranslocation, EGFR is removed from the membrane via the Sec61 translocon, where the now soluble EGFR can interact with heat shock proteins and nuclear importins via its conserved nuclear localization sequence (NLS). Presence of the NLS in all wildtype EGFR indicates nuclear EGFR (nEGFR) also plays a role in non-cancerous tissue. EGFR is a known contributor to wound-healing, migration, and mammary gland development but the role of nEGFR in these processes has yet to be elucidated. Our lab has created cSNX1.3, a peptide-based therapeutic that recapitulates the BAR domain of Sorting Nexin 1. CSNX1.3 competitively binds to EGFR to prevent nuclear trafficking, and this treatment is effective at killing cancer cells that express EGFR, while leaving non-cancerous cells alive. In addition, cSNX1.3 is a potent inhibitor of EGF-dependent cell migration in Triple Negative Breast Cancer cell lines. Based on this data, we hypothesize that, in response to a wound, nEGFR activates cell migration pathways to promote wound healing. To better understand the role of nEGFR in wound healing and migration of non-cancer cell types, we have utilized CRISPR/Cas9 to insert mScarlet or mVenus, improved fluorescent proteins, onto the C-terminus of endogenous EGFR in immortalized MCF10A cells. Using a live cell imaging platform, we discovered that cells on the leading edge of a wound have increased levels of nEGFR. Furthermore, treatment with cSNX1.3 inhibits the migration of MCF10As and reduces levels of nEGFR. This data implies a role for nEGFR in the migration of MCF10As during wound healing. Future studies will delve deeper into the triggers of EGFR retrotranslocation to the nucleus and investigate the specific proteins nEGFR interacts with to affect cell migration. By elucidating the intrinsic role of nuclear EGFR in non-cancerous tissue types, we can better understand how cancer takes advantage of these molecular pathways and possibly its role in the cell migration essential to metastatic progression. Citation Format: Danielle M DiFranco, Joyce Schroeder. The role of nuclear epidermal growth factor receptor in cell migration and wound healing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A031.

Influence of copper(I) nicotinate complex on the Notch1 signaling pathway in triple negative breast cancer cell lines

Triple negative breast cancer (TNBC) is a subtype of breast cancer which is characterized by its aggressiveness, poor and short overall survival. In this concept, there is a growing demand for metal-based compounds in TNBC therapy as copper complex that have a less toxic effect on normal cells and could stimulate apoptotic cell death. Additionally, Notch1 signaling pathway has received great attention as one of the most important potential targets for developing a novel therapeutic strategy. The present study is an attempt to assess the promising chemotherapeutic activities of copper(I) nicotinate (CNC) through its impact on the expression of downstream genes of Notch1 signaling pathway and the cell fate of TNBC. The co-treatment of TNBC cells with doxorubicin (Doxo) and CNC was also investigated. To approach the objective of the present study, TNBC cell lines; HCC1806 and MDAMB231, were utilized. MTT assay was used to determine the IC50 values of CNC and Doxo. After treatment, microtubule-associated protein light chain3 (LC3) were determined by flow cytometry. Additionally, qRT-PCR technique was used to detect the changes in genes levels that are involved Notch1 signaling pathway. Moreover, autophagosomes were monitored and imaged by Transmission electron microscopy. Treatment of TNBC cells with CNC modulated Notch1 signaling pathway in different manners with respect to the type of cells and the applied dose of CNC. The observed effects of CNC may reflect the possible anti-cancer activities of CNC in both types of TNBC. However, cell type and CNC dose should be considered.

IMN041 is an immunotherapeutic and an effective treatment in mouse xenograft models of pancreatic cancer, renal cancer and triple negative breast cancer

BackgroundiMN013 (5-aza-2',2'-difluorodeoxycytidine), a DNA methyl transferase inhibitor and ribonucleotide reductase inhibitor, and its prodrug iMN041 (3',5'-di-trimethylsilyl-2',2'-difluro-5- azadeoxycytidine), have been shown to be active in mouse xenograft models of hematogenous and solid tumors. In a xenograft of non-small cell lung cancer (NCI-H460), iMN041 treated mice demonstrated a marked inflammatory response upon analysis of tumor histology, which was hypothesized to be mediated by upregulation of natural killer (NK) cells. This study aimed to characterize the antitumor immune responses generated by iMN041 and test the efficacy iMN041 in solid tumors with poor prognosis.MethodsIn the Renca syngeneic mouse model, tumors were harvested following two doses of iMN041 or vehicle control, and analyzed by fluorescent-activated cell sorting for an antitumor immune response. iMN041 was also tested for tumor growth inhibition and animal survival for up to 42 days in xenograft models of pancreatic, renal, and triple negative breast cancer.ResultsTumors from mice implanted with the Renca cell line and treated with iMN041 demonstrated an increase in granzyme B in NK (p = 0.024) and NKT cells (p = 0.004), an increase in the ratios of CD8-T to regulatory T cells (Treg) (p = 0.0026) and CD4-T to Treg cells (p = 0.022) and a decrease in myeloid-derived suppressor cells (p = 0.040), compared to vehicle controls. A significant decrease in MAGE-A positive tumor cells in treated mice, concordant with a proportional decrease in all live tumor cells, suggests that these cells are one of the main targets of the activated immune system. Xenograft models of the triple negative breast cancer cell line DU4475, renal cancer cell lines 786-O and Caki-1, and pancreatic cancer cell lines CFPAC-1 and SW1990, demonstrated significantly lower tumor volumes, and, where there were a sufficient number of events, significantly improved survival in treated mice compared to vehicle control mice.ConclusionsIn mouse cancer models, iMN041 is an effective treatment for solid tumors mediated in part through a unique antitumor immune response.

Tumor therapy by targeting extracellular hydroxyapatite using novel drugs: A paradigm shift.

It has been shown that tumor microenvironment (TME) hydroxyapatite (HAP) is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME-HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue. We provide strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME-HAP) using a novel therapeutic can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects. We developed an injectable cation exchange nano particulate sulfonated polystyrene solution (NSPS) that we engineered to dissolve TME-HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism. This was evaluated in cell culture using 4T1, MDA-MB-231 triple negative breast cancer cells, MCF10 normal breast cells, and H292 lung cancer cells, and in vivo using orthotopic mouse models of cancer that contained detectable microenvironment HAP including breast (MMTV-Neu, 4T1, and MDA-MB-231), prostate (PC3) and colon (HCA7) cancer using 18 F-NaF for HAP and 18 F-FDG for glucose metabolism with PET imaging. On the other hand, H292 lung tumor cells that lacked detectable microenvironment HAP and MCF10a normal breast cells that do not produce HAP served as negative controls. Tumor microenvironment pH levels following injection of NSPS were evaluated via Chemical Exchange Saturation (CEST) MRI and via ex vivo methods. Within 24 h of adding the small concentration of 1X of NSPS (~7 μM), we observed significant tumor cell death (~ 10%, p < 0.05) in 4T1 and MDA-MB-231 cell cultures that contain HAP but ⟨2% in H292 and MCF10a cells that lack detectable HAP and in controls. Using CEST MRI, we found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration. in the tumors that contained TME-HAP in mouse models, MMTV-Neu, 4T1, and MDA-MB-231, PC3, and HCA7, there was a significant reduction (p<0.05) in 18 F-Na Fuptake post NSPS treatment as expected; 18 F- uptake in the tumor = 3.8 ± 0.5 %ID/g (percent of the injected dose per gram) at baseline compared to 1.8 ±0.5 %ID/g following one-time treatment with 100 mg/kg NSPS. Of similar importance, is that 18 F-FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 h of treatment with one-time NSPS which persisted for at least one week. Additionally, tumor growth was significantly slower (p < 0.05) in the mice treated with one-time NSPS. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone. Dissolution of TME-HAP using our novel NSPS has the potential to provide a new treatment paradigm to enhance the management of cancer patients with poor prognosis.

G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer.

P38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G-Quadruplex (G4) structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G4 motifs upstream of the MAPK12 promoter, forming parallel G4 structures. They exist in an equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin that bind to these G4 motifs and regulate MAPK12 transcriptional homeostasis. To underscore the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analysed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44High/CD24Low population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.

The Soft Nanodots as Fluorescent Probes for Cell Imaging: Analysis of Cell and Spheroid Penetration Behavior of Single Chain Polymer Dots.

This study describes the formation, size control, and penetration behavior of polymer nanodots (Pdots) consisting of single or few chain polythiophene-based conjugated polyelectrolytes (CPEs) via nanophase separation between good solvent and poor solvent of CPE. Though the chain singularity may be associated with dilution nanophase separation suggests that molecules of a good solvent create a thermodynamically driven solvation layer surrounding the CPEs and thereby separating the single chains even in their poor solvents. This statement is therefore corroborated with emission intensity/lifetime, particle size, and scattering intensity of polyelectrolyte in good and poor solvents. Regarding the augmented features, Pdots are implemented into cell imaging studies to understand the nuclear penetration and to differentiate the invasive characteristics of breast cancer cells. The python based red, green, blue (RGB) color analysis depicts that Pdots have more nuclear penetration ability in triple negative breast cancer cells due to the different nuclear morphology in shape and composition and Pdots have penetrated cell membrane as well as extracellular matrix in spheroid models. The current Pdot protocol and its utilization in cancer cell imaging are holding great promise for gene/drug delivery to target cancer cells by explicitly achieving the very first priority of nuclear intake.

Abstract A007: Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair

Abstract Ser/Thr kinase BUB1 is known to regulate chromosomal segregation during mitosis. However, its exact role in DNA damage repair in response to radiotherapy remained elusive. Here, we demonstrate that inhibition of BUB1 kinase activity (BAY1816032) or genomic depletion (CRISPR) lead to radiation sensitization in triple negative breast cancer and lung cancer cell lines and in TNBC tumor xenograft models. Mechanistic studies demonstrated reduced DNA-DSB repair (γ-H2AX foci assay), and effect on the NHEJ pathway by the bioluminescent DNA repair reporter (BLRR). Gene expression analysis show that BUB1 expression correlates tightly with key members of the NHEJ machinery including H2AX, ATM, 53BP1, DNAPKcs, Ku70, Ku80, Lig4 among others. Cycloheximide-chase experiments demonstrate that BUB1 ablation (pharmacological or genomic) stabilizes DNAPKcs after irradiation. The half-lives (t1/2) of total-DNAPK, phospho-DNAPK could not be estimated in BUB1 ablated cells post radiotherapy. Conversely, we observed BUB1 stabilization following radiotherapy alone while pre-treatment with BUB1i or DNAPKi caused robust degradation of BUB1 (significantly shorter t1/2). Nuclear and chromatin fractionation assays revealed an increase in the phospho- and total- DNAPK, ATM, and KAP1 in nuclear fractions and in chromatin enriched fractions in BUB1 inhibited or depleted cells. There was no significant change in the levels of Ku70 and Ku80 in the nuclear fractions or in the chromatin enriched fractions. This data indicates that BUB1 may be involved in the activation and recruitment of key NHEJ proteins (ATM, DNAPKcs) to DSBs. Further experiments are underway to determine how BUB1 stabilizes DNAPKcs and regulate chromatin loading of key NHEJ factors following radiation. Additionally, we demonstrate that BUB1 mediate apoptotic cell death of cancer cells post-radiation. Our findings suggest that BUB1 may serve as a novel molecular target for radiosensitization in various solid cancers including TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Stephen Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A007.