Non-malignant Breast Epithelial Cells Research Articles

Novel dual inhibitor targeting CDC25 and HDAC for treating triple-negative breast cancer

Triple-negative breast cancer (TNBC) presents a significant challenge for treatment due to its aggressive nature and the lack of effective therapies. This study developed dual inhibitors against cell division cycle 25 (CDC25) and histone deacetylases (HDACs) for TNBC treatment. CDC25 phosphatases are crucial for activating cyclin-dependent kinases (CDKs), the master regulators of cell cycle progression. HDACs regulate various biological processes by deacetylating histone and non-histone proteins, affecting gene expression, chromatin structure, cell differentiation, and proliferation. Dysregulations of HDAC and CDC25 are associated with several human malignancies. We generated a group of dual inhibitors for CDC25 and HDAC by combining the molecular structures of CDC25 (quinoline-5,8-dione) and HDAC (hydroxamic acid or benzamide) pharmacophores. The newly developed compounds were evaluated against various solid-tumor, leukemia, and non-malignant breast epithelial cells. Among the synthesized compounds, 18A emerged as a potent inhibitor, demonstrating significant cytotoxicity against TNBC cells, superior to its effects on other cancer types while sparing non-malignant cells. 18A possessed similar HDAC inhibitory activity as MS-275 and potently suppressed CDC25 activity in vitro and the CDK1 dephosphorylation in cells. Additionally, 18A hindered the progression of S and G2/M phases, triggered DNA damage, and induced apoptosis. These findings underscore the potential of 18A as a targeted therapy for TNBC and warrants further preclinical development.Graphical

Structural Design, Anticancer Evaluation, and Molecular Docking of Newly Synthesized Ni(II) Complexes with ONS-Donor Dithiocarbazate Ligands.

The current article reports the investigation of three new Ni(II) complexes with ONS-donor dithiocarbazate ligands: [Ni(L1)PPh3] (1), [Ni(L2)PPh3] (2), and [Ni(L2)Py] (3). Single-crystal X-ray analyses revealed mononuclear complexes with a distorted square planar geometry and the metal centers coordinated with a doubly deprotonated dithiocarbazate ligand and coligand pyridine or triphenylphosphine. The non-covalent interactions were investigated by the Hirshfeld surface and the results revealed that the strongest interactions were π⋅⋅⋅π stacking interactions and non-classical hydrogen bonds C-H···H and C-H···N. Physicochemical and spectroscopic methods indicate the same structures in the solid state and solution. The toxicity effects of the free ligands and Ni(II) complexes were tested on the human breast cancer cell line MCF-7 and non-malignant breast epithelial cell line MCF-10A. The half-maximal inhibitory concentration (IC50) values, indicating that the compounds were potent in inhibiting cell growth, were obtained for both cell lines at three distinct time points. While inhibitory effects were evident in both malignant and non-malignant cells, all three complexes demonstrated lower IC50 values for malignant breast cell lines than their non-malignant counterparts, suggesting a stronger impact on cancerous cell lines. Furthermore, molecular docking studies were performed showing the complex (2) as a promising candidate for further therapeutic exploration.

Abstract 5680: Circular MALAT1 - the new kid on the block in breast-to-brain metastatic cancer

Abstract Introduction: Breast-to-brain metastatic (BTB met) cancer constitutes a great challenge for modern medicine and science. As the research on protein-coding genes brought only incremental progress in developing anticancer therapy, much attention is devoted to understanding the role of non-coding RNAs, including a relatively recently discovered group - of circular RNAs. This project aimed to determine the relationship between BTB met cancer cells and cells derived from a deadly brain tumor - glioblastoma stem-like cells (GSCs), considering the circular RNA signatures and selecting a reliable candidate for mechanistic study - a circular form of non-coding oncogene MALAT1 (circMALAT1). Methods: The circular RNA expression profile in patient-derived BTB met cancer cells (n=4), GSCs (n=8), and neural progenitor cells (NPC) as a non-malignant control (n=3) was evaluated by the microarray approach. Expression of circMALAT1 was also validated in non-malignant breast epithelial cells by digital PCR and data mining in the GEO database. CircMALAT1 knockdown by antisense oligonucleotides was followed by functional analysis. Results: 12,660 circular RNAs were detected using the Arraystar platform, and upon applying the cutoff of FC ≥2 and p-value <0.05, 547 circular RNAs were selected as significantly deregulated in all cancer groups compared to NPC. CircMALAT1 was elevated in BTB met compared to NPC and normal breast epithelial cells. Knockdown of circMALAT1 affected cancer cell viability and proliferation while promoting p21 expression, thus decelerating the cell cycle manifested by a reduction in 3D spheroids volume. Conclusions: The analysis of the circular RNA landscape showed for the first time some striking similarities between BTB mets and GSCs despite different tissue-of-origin. Of note, the circRNAome of BTB mets clustered especially closely with mesenchymal GSC - the most aggressive and therapy-resistant subtype of GSCs. These results also demonstrated the role of circMALAT1 in shaping the oncogenic traits of these cells. Therefore, circular RNAs can serve as valuable biomarkers and promising novel mechanistic candidates whose modulation may become a paradigm-shifting therapeutic approach. Citation Format: Adrian Szczepaniak, Agnieszka Bronisz, Jakub Godlewski. Circular MALAT1 - the new kid on the block in breast-to-brain metastatic 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 5680.

Primary breast tumor induced extracellular matrix remodeling in premetastatic lungs

The premetastatic niche hypothesis proposes an active priming of the metastatic site by factors secreted from the primary tumor prior to the arrival of the first cancer cells. We investigated several extracellular matrix (ECM) structural proteins, ECM degrading enzymes, and ECM processing proteins involved in the ECM remodeling of the premetastatic niche. Our in vitro model consisted of lung fibroblasts, which were exposed to factors secreted by nonmalignant breast epithelial cells, nonmetastatic breast cancer cells, or metastatic breast cancer cells. We assessed ECM remodeling in vivo in premetastatic lungs of female mice growing orthotopic primary breast tumor xenografts, as compared to lungs of control mice without tumors. Premetastatic lungs contained significantly upregulated Collagen (Col) Col4A5, matrix metalloproteinases (MMPs) MMP9 and MMP14, and decreased levels of MMP13 and lysyl oxidase (LOX) as compared to control lungs. These in vivo findings were consistent with several of our in vitro cell culture findings, which showed elevated Col14A1, Col4A5, glypican-1 (GPC1) and decreased Col5A1 and Col15A1 for ECM structural proteins, increased MMP2, MMP3, and MMP14 for ECM degrading enzymes, and decreased LOX, LOXL2, and prolyl 4-hydroxylase alpha-1 (P4HA1) for ECM processing proteins in lung fibroblasts conditioned with metastatic breast cancer cell media as compared to control. Taken together, our data show that premetastatic priming of lungs by primary breast tumors resulted in significant ECM remodeling which could facilitate metastasis by increasing interstitial fibrillar collagens and ECM stiffness (Col14A1), disruptions of basement membranes (Col4A5), and formation of leaky blood vessels (MMP2, MMP3, MMP9, and MMP14) to promote metastasis.

Organic Electronic Platform for Real-Time Phenotypic Screening of Extracellular-Vesicle-Driven Breast Cancer Metastasis.

Tumor-derived extracellular vesicles (TEVs) induce the epithelial-to-mesenchymal transition (EMT) in nonmalignant cells to promote invasion and cancer metastasis, representing a novel therapeutic target in a field severely lacking in efficacious antimetastasis treatments. However, scalable technologies that allow continuous, multiparametric monitoring for identifying metastasis inhibitors are absent. Here, the development of a functional phenotypic screening platform based on organic electrochemical transistors (OECTs) for real-time, noninvasive monitoring of TEV-induced EMT and screening of antimetastatic drugs is reported. TEVs derived from the triple-negative breast cancer cell line MDA-MB-231 induce EMT in nonmalignant breast epithelial cells (MCF10A) over a nine-day period, recapitulating a model of invasive ductal carcinoma metastasis. Immunoblot analysis and immunofluorescence imaging confirm the EMT status of TEV-treated cells, while dual optical and electrical readouts of cell phenotype are obtained using OECTs. Further, heparin, a competitive inhibitor of cell surface receptors, is identified as an effective blocker of TEV-induced EMT. Together, these results demonstrate the utility of the platform for TEV-targeted drug discovery, allowing for facile modeling of the transient drug response using electrical measurements, and provide proof of concept that inhibitors of TEV function have potential as antimetastatic drug candidates.

Discovery of Ruthenium(II) Metallocompound and Olaparib Synergy for Cancer Combination Therapy.

Synergistic drug combinations can extend the use of poly(ADP-ribose) polymerase inhibitors (PARPi) such as Olaparib to BRCA-proficient tumors and overcome acquired or de novo drug resistance. To identify new synergistic combinations for PARPi, we screened a "micro-library" comprising a mix of commercially available drugs and DNA-binding ruthenium(II) polypyridyl complexes (RPCs) for Olaparib synergy in BRCA-proficient triple-negative breast cancer cells. This identified three hits: the natural product Curcumin and two ruthenium(II)-rhenium(I) polypyridyl metallomacrocycles. All combinations identified were effective in BRCA-proficient breast cancer cells, including an Olaparib-resistant cell line, and spheroid models. Mechanistic studies indicated that synergy was achieved via DNA-damage enhancement and resultant apoptosis. Combinations showed low cytotoxicity toward non-malignant breast epithelial cells and low acute and developmental toxicity in zebrafish embryos. This work identifies RPC metallomacrocycles as a novel class of agents for cancer combination therapy and provides a proof of concept for the inclusion of metallocompounds within drug synergy screens.

Highly modular hepatitis B virus-like nanocarriers for therapeutic protein encapsulation and targeted delivery to triple negative breast cancer cells.

Protein therapeutics offer enormous clinical impact in treating a variety of diseases by offering high selectivity with limited off-target effects. However, delivery challenges severely hinder functional proteins from reaching their target cells and necessitate frequent administration. To address these problems, nanocarrier encapsulation can provide protease protection and enhanced targeted transportation of functional proteins to their intended disease site. Inspired by their viral analogues, virus-like particles (VLPs) are non-infectious viral capsids that have potential for drug delivery applications because of their shared structural characteristics, such as high loading capacity, particle stability, and structural uniformity. Here, we describe a modular hepatitis B virus (HBV) VLP delivery platform offering tunable modifications of both the exterior and interior viral capsid surfaces via SpyCatcher-SpyTag bioconjugation and a multi-expression system, respectively. This new platform facilitates modification with epidermal growth factor receptor (EGFR)-targeting proteins and encapsulation with both model green fluorescent protein (GFP) and prodrug-converting yeast cytosine deaminase (yCD) enzyme. The resultant targeted VLPs demonstrated enhanced uptake and toxicity in EGFR-overexpressing triple negative breast cancer (TNBC) cells in contrast to non-malignant breast epithelial cells.

Effectiveness of hypofractionated and normofractionated radiotherapy in a triple‐negative breast cancer model

Breast cancer (BC) is one of the most diagnosed malignant carcinomas in women with a triple-negative breast cancer (TNBC) phenotype being correlated with poorer prognosis. Fractionated radiotherapy (RT) is a central component of breast cancer management, especially after breast conserving surgery and is increasingly important for TNBC subtype prognosis. In recent years, moderately hypofractionated radiation schedules are established as a standard of care, but many professionals remain skeptical and are concerned about their efficiency and side effects. In the present study, two different triple-negative breast cancer cell lines, a non-malignant breast epithelial cell line and fibroblasts, were irradiated daily under normofractionated and hypofractionated schedules to evaluate the impact of different irradiation regimens on radiation-induced cell-biological effects. During the series of radiotherapy, proliferation, growth rate, double-strand DNA break-repair (DDR), cellular senescence, and cell survival were measured. Investigated normal and cancer cells differed in their responses and receptivity to different irradiation regimens, indicating cell line/cell type specificity of the effect. At the end of both therapy concepts, normal and malignant cells reach almost the same endpoint of cell count and proliferation inhibition, confirming the clinical observations in the follow-up at the cellular level. These result in cell lines closely replicating the irradiation schedules in clinical practice and, to some extent, contributing to the understanding of growth rate or remission of tumors and the development of fibrosis.

Cytotoxic Activity of Carrageenan on Malignant MCF-7 Breast Cancer and the non-Malignant SVCT Breast Epithelial Cell Lines

Objective: Recent studies have shown that sulfated polysaccharides obtained from marine algae have many biological and physiological activities. Kappa-carrageenan, a sulfated polysaccharide obtained from red marine algae, is known to have anti-proliferative effects in various cancer cell lines. In this study, the cytotoxic effect of kappa-carrageenan on two different cell lines, namely the malignant MCF-7 breast cancer cell line and the non-malignant SVCT breast epithelial cell lines, was investigated in an in vitro model. Materials & Methods: Three different concentrations of carrageenan were prepared for each cell in its own medium, with an initial dose of 1000 µg/ml (Dilution I: 1000 µg/ml; Dilution II: 250 µg/ml; Dilution III: 62.5 µg/ml). Cells were incubated with carrageenan at the doses set for each experimental group. Cells incubated in a carrageenan-free medium comprised the control group. Results: To measure cell viability, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) analysis was performed. Cell morphology was investigated by the acridine orange (AO)/propidium iodide (PI) fluorescent staining method. The present results indicated that carrageenan caused cytotoxic effects on both malignant and non-malignant cell lines. Conclusion: Considering that the different phenotypic features of the subtypes of the MCF-7 cell line may affect cell viability and cell proliferation, cell selection should be performed very carefully in cytotoxicity studies. We suggest that using the MCF-7 cell line for cytotoxicity experiments needs to contemplate this important phenomenon for further experimental setups.

ErbB2/Her2-dependent downregulation of a cell death-promoting protein BLNK in breast cancer cells is required for 3D breast tumor growth

A significant proportion of breast cancers are driven by ErbB2/Her2 oncoprotein that they overexpress. These malignancies are typically treated with various ErbB2-targeted drugs, but many such cancers develop resistance to these agents and become incurable. Conceivably, treatment of ErbB2-positive cancers could be facilitated by use of agents blocking oncogenic signaling mechanisms downstream of ErbB2. However, current understanding of these mechanisms is limited. The ability of solid tumor cells to resist anoikis, cell death triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. In an effort to understand the mechanisms of ErbB2-driven breast cancer cell anoikis resistance we found that detachment of non-malignant breast epithelial cells from the ECM upregulates a cell death-promoting tumor suppressor adapter protein BLNK and that ErbB2 blocks this upregulation by reducing tumor cell levels of transcription factor IRF6. We further observed that trastuzumab, a therapeutic anti-ErbB2 antibody, upregulates BLNK in human trastuzumab-sensitive but not trastuzumab-resistant ErbB2-positive breast cancer cells. Moreover, we established that BLNK promotes anoikis by activating p38 MAP kinase and that ErbB2-dependent BLNK downregulation blocks breast cancer cell anoikis. In search for pharmacological approaches allowing to upregulate BLNK in tumor cells we found that clinically approved proteasome inhibitor bortezomib upregulates IRF6 and BLNK in human breast cancer cells and inhibits their 3D growth in a BLNK-dependent manner. In addition, we found that BLNK upregulation in human ErbB2-positive breast cancer cells blocks their ability to form tumors in mice. Furthermore, we used publicly available data on mRNA levels in multiple breast cancers to demonstrate that increased BLNK mRNA levels correlate with increased relapse-free survival in a cohort of approximately 400 patients with ErbB2-positive breast cancer. In summary, we discovered a novel mechanism of ErbB2-driven 3D breast tumor growth mediated by ErbB2-dependent BLNK downregulation.

Antidiabetic, Antiglycation, and Antioxidant Activities of Ethanolic Seed Extract of Passiflora edulis and Piceatannol In Vitro.

The objective of this work was to investigate the antidiabetic, antiglycation, and antioxidant potentials of ethanolic extract of seeds of Brazilian Passiflora edulis fruits (PESE), a major by-product of the juice industry, and piceatannol (PIC), one of the main phytochemicals of PESE. PESE, PIC, and acarbose (ACB) exhibited IC50 for alpha-amylase, 32.1 ± 2.7, 85.4 ± 0.7, and 0.4 ± 0.1 µg/mL, respectively, and IC50 for alpha-glucosidase, 76.2 ± 1.9, 20.4 ± 7.6, and 252 ± 4.5 µg/mL, respectively. The IC50 of PESE, PIC, and sitagliptin (STG) for dipeptidyl-peptidase-4 (DPP-4) was 71.1 ± 2.6, 1137 ± 120, and 0.005 ± 0.001 µg/mL, respectively. PESE and PIC inhibited the formation of advanced glycation end-products (AGE) with IC50 of 366 ± 1.9 and 360 ± 9.1 µg/mL for the initial stage and 51.5 ± 1.4 and 67.4 ± 4.6 µg/mL for the intermediate stage of glycation, respectively. Additionally, PESE and PIC inhibited the formation of β-amyloid fibrils in vitro up to 100%. IC50 values for 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) scavenging activity of PESE and PIC were 20.4 ± 2.1, and 6.3 ± 1.3 µg/mL, respectively. IC50 values for scavenging hypochlorous acid (HOCl) were similar in PESE, PIC, and quercetin (QCT) with values of 1.7 ± 0.3, 1.2 ± 0.5, and 1.9 ± 0.3 µg/mL, respectively. PESE had no cytotoxicity to the human normal bronchial epithelial (BEAS-2B), and alpha mouse liver (AML-12) cells up to 100 and 50 µg/mL, respectively. However, 10 µg/mL of the extract was cytotoxic to non-malignant breast epithelial cells (MCF-10A). PESE and PIC were found to be capable of protecting cultured human cells from the oxidative stress caused by the carcinogen NNKOAc at 100 µM. The in vitro evidence of the inhibition of alpha-amylase, alpha-glucosidase, and DPP-4 enzymes as well as antioxidant and antiglycation activities, warrants further investigation of the antidiabetic potential of P. edulis seeds and PIC.

Abstract 5653: Increased expression of brain-specific serine/threonine-protein kinase BRSK2 contributes to breast cancer survival by enhancing autophagy under nutrient stress

Abstract Aberrant expression of certain cancer-promoting genes is the associative factor of metastasis and cancer-related death. Autophagy is a highly conserved catabolic process that can be regulated by starvation or stress. Among several cellular stresses, hypoxia and nutrient stress can strongly activate autophagy-related genes. Autophagy plays dual roles in cancer; it can be pro-death or pro-survival. The ability of the cancer cells to survive long-term under stress is still a vast question to cancer researchers. The nutrient status of is sensed by LKB1 downstream kinases AMP-activated protein kinase (AMPK). Recent studies revealed that BRSK2 is one of the AMPK related kinases regulated by LKB1 is the activator of WIPI protein function as building blocks for the formation of the autophagosome. Brain-specific kinases 1 and 2 are highly expressed in the mammalian forebrain regulating neuronal polarity and have lower expression in the testis and pancreas. We identified BRSK2 as one of the top genes highly expressed in 10 primary breast cancer tissue with a record of future bone metastasis compared to 10 primary breast tumors with no metastasis record through RNA seq analysis. Our extended analysis using TCGA and METABRIC cohort data unfolded that BRSK2 is highly elevated in breast cancer tumors compared to control breast tissue (p< 0.001), mainly in stage IV patients compared to other stages (p= 0.01). Furthermore, we observed that BRSK2 is differentially expressed in various breast cancer cell lines, in contrast, no expression in nonmalignant breast epithelial cells (MCF10A). We selected MDA-231 and BT474 cell lines having relatively lower BRSK2 expression, and expressed BRSK2 ectopically to understand details about BRSK2-mediated autophagy under nutrient stress. Interestingly, we observed that BRSK2 augmented nutrient starvation mediated autophagic flux in Triple-negative and ER/PR-positive breast cancer cell lines, along with elevated AKT, p-NF-kB, and STAT3 signaling pathways for survival under nutrient stress conditions. We further observed that several inflammatory cytokines CXCL1, CXCL6, TNF-α, and IL1-β, were significantly upregulated with BRSK2 overexpressed cells compared with the control cells. Knockdown of BRSK2 reduces autophagy and enhances apoptosis in breast cancer cells. Altogether, our data revealed that BRSK2 play a role in increasing autophagy to support cell survival under nutrient stress condition through AKT signaling pathways. Blocking of BRSK2 with inhibitor has reduced autophagy and increased apoptosis and cell death. Thus, BRSK2 can be a potential molecule as a therapeutic target for breast cancer metastasis. Citation Format: Nitai Hait, APARNA MAITI, Rongrong Wu, Kazuaki Takabe, Masanori Oshi. Increased expression of brain-specific serine/threonine-protein kinase BRSK2 contributes to breast cancer survival by enhancing autophagy under nutrient stress [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 5653.

Expression analysis and function of mitochondrial genome-encoded microRNAs

MicroRNAs (miRNAs) play a significant role in nuclear and mitochondrial anterograde and retrograde signaling. Most of the miRNAs found inside mitochondria are encoded in the nuclear genome, with a few mitochondrial genome-encoded non-coding RNAs having been reported. In this study, we have identified 13 mitochondrial genome-encoded microRNAs (mitomiRs), which were differentially expressed in breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231), non-malignant breast epithelial cell line (MCF-10A), and normal and breast cancer tissue specimens. We found that mitochondrial DNA (mtDNA) depletion and inhibition of mitochondrial transcription led to reduced expression of mitomiRs in breast cancer cells. MitomiRs physically interacted with Ago2, an RNA-induced silencing complex (RISC) protein, in the cytoplasm and inside mitochondria. MitomiRs regulate the expression of both nuclear and mitochondrial transcripts in breast cancer cells. We showed that mitomiR-5 targets the PPARGC1A gene and regulates mtDNA copy number in breast cancer cells. MitomiRs identified in the present study may be a promising tool for expression and functional analysis in patients with a defective mitochondrial phenotype, including cancer and metabolic syndromes. This article has an associated First Person interview with the first author of the paper.

Abstract P140: Chromomycin A5, a marine-derived antibiotic, targets the oncogenic TBX2 in breast cancer

Abstract Breast cancer (BC) continues to pose a major health burden. In 2020 alone, over 2 million people were diagnosed with BC globally and of these 684 996 people succumbed to the disease. Cancer cell drug resistance and the notion of transcription factors as undruggable targets has slowed progress for novel BC therapies. The transcription factor, TBX2, is commonly over-expressed in breast cancer with various hormone receptor statuses where it functions as a powerful pro-proliferative factor by directly repressing p21, PTEN, CST-6, and NDGR1 expression. Furthermore, TBX2 mediates Cisplatin drug resistance by inducing an S-phase cell cycle arrest allowing for cisplatin-induced DNA damage repair and breast cancer cell survival. Targeting TBX2 with DNA-damaging chemotherapies therefore emerges as a promising anti-cancer strategy to treat BC. To this end, we performed a reverse affinity screen to fish for marine-derived compounds with DNA-damaging- and TBX2 binding- activity and identified the antibiotic Chromomycin A5 (CMA5) as a promising agent. This project confirms the binding affinity of CMA5 for TBX2 by thermophoresis and fully characterizes the in vitro anticancer activity of CMA5 in TBX2-driven BC cells. Short-term and long-term selective cytotoxicity of CMA5 were investigated in two TBX2-driven BC cell lines (MCF-7 and T47D) and a non-malignant breast epithelial cell line (MCF-12A) using MTT and clonogenic assays. Our results show that CMA5 exhibited potent (IC50 ≤6.5 nM) and selective (SI ~ 3.3 ) cytotoxicity against BC cells. Importantly, CMA5 inhibited TBX2 protein levels and co-treatment of the BC cells with CMA5 and the proteasome 26S inhibitor MG132 rescued TBX2 levels. These results show that CMA5 targets TBX2 for protein degradation. Furthermore, whereas knocking down TBX2 using shRNA blocked the cytotoxicity of CMA5 (IC50 increased by 5-fold), ectopically over-expressing TBX2 increased the cytotoxic activity of CMA5 (IC50 decreased by 1.2-fold). These results suggest that CMA5 cytotoxicity is dependent, in part, on the presence of TBX2. To further explore the significance of TBX2 inhibition by CMA5 we investigated the impact of CMA5 on expression levels of the TBX2 target genes p21, PTEN, CST-6 and NDGR1 by quantitative real-time PCR analyses. The results revealed that all four genes were upregulated in the presence of CMA5 treatment which suggested that CMA5 inhibits BC cell viability and survival by targeting TBX2 and thus releasing its ability to repress these tumor suppressors. To elucidate the mechanism(s) underpinning the cytotoxic activity of CMA5 we investigated its effects on several markers of DNA damage (yH2AX expression) and apoptosis (cleaved caspase 3, 7, 8, 9 and PARP) by western blotting and immunocytochemistry. Our results show that CMA5 induced DNA damage and killed cells, in part, by both the intrinsic and extrinsic apoptotic programmed cell death pathways. Overall, this study identifies CMA5 as a promising anti-breast cancer agent because of its capability to induce DNA damage and to inhibit the oncogenic TBX2. Citation Format: Claire Bellis, Suparna Chakraborty, Mihlali Mlaza, Bianca Del Bianco Sahm, Paula Rezende Teixeira, Leticia Costa-Lotufo, Sharon Prince. Chromomycin A5, a marine-derived antibiotic, targets the oncogenic TBX2 in breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P140.

Inhibition of \u03b1v\u03b23 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles

BackgroundExtracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells.MethodsTo probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments.ResultsWe find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content.ConclusionIn summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs.6Ak9CS6CfK5Ku3JS4e63coVideo Graphical abstract

The use of patient-derived breast tissue explants to study macrophage polarization and the effects of environmental chemical exposure.

Ex vivo mammary explant systems are an excellent model to study interactions between epithelium and stromal cell types because they contain physiologically relevant heterotypic interactions in the background of genetically diverse patients. The intact human mammary tissue, termed patient‐derived explant (PDE), can be used to investigate cellular responses to a wide variety of external stimuli in situ. For this study, we examined the impact of cytokines or environmental chemicals on macrophage phenotypes. We demonstrate that we can polarize macrophages within human breast tissue PDEs toward M1 or M2 through the addition of interferon‐γ (IFNγ) + lipopolysaccharide (LPS) or interleukin (IL)‐4 + IL‐13, respectively. Elevated expression levels of M(IFNγ + LPS) markers (HLADRA and CXCL10) or M(IL‐4 + IL‐13) markers (CD209 and CCL18) were observed in cytokine‐treated tissues. We also examined the impact of the endocrine‐disrupting chemical, benzophenone‐3, on PDEs and measured significant, yet varying effects on macrophage polarization. Furthermore, a subset of the PDEs respond to IL‐4 + IL‐13 through downregulation of E‐cadherin and upregulation of vimentin which is reminiscent of epithelial‐to‐mesenchymal transition (EMT) changes. Finally, we were able to show immortalized nonmalignant breast epithelial cells can exhibit EMT characteristics when exposed to growth factors secreted by M(IL‐4 + IL‐13) macrophages. Taken together, the PDE model system is an outstanding preclinical model to study early tissue‐resident immune responses and effects on epithelial and stromal responses to stimuli found both endogenously in the breast and exogenously as a result of exposures.

Breast-Specific Epigenetic Regulation of DeltaNp73 and Its Role in DNA-Damage-Response of BRCA1-Mutated Human Mammary Epithelial Cells.

The function of BRCA1/2 proteins is essential for maintaining genomic integrity in all cell types. However, why women who carry deleterious germline mutations in BRCA face an extremely high risk of developing breast and ovarian cancers specifically has remained an enigma. We propose that breast-specific epigenetic modifications, which regulate tissue differentiation, could team up with BRCA deficiency and affect tissue susceptibility to cancer. In earlier work, we compared genome-wide methylation profiles of various normal epithelial tissues and identified breast-specific methylated gene promoter regions. Here, we focused on deltaNp73, the truncated isoform of p73, which possesses antiapoptotic and pro-oncogenic functions. We showed that the promoter of deltaNp73 is unmethylated in normal human breast epithelium and methylated in various other normal epithelial tissues and cell types. Accordingly, deltaNp73 was markedly induced by DNA damage in human mammary epithelial cells (HMECs) but not in other epithelial cell types. Moreover, the induction of deltaNp73 protected HMECs from DNA damage-induced cell death, and this effect was more substantial in HMECs from BRCA1 mutation carriers. Notably, when BRCA1 was knocked down in MCF10A, a non-malignant breast epithelial cell line, both deltaNp73 induction and its protective effect from cell death were augmented upon DNA damage. Interestingly, deltaNp73 induction also resulted in inhibition of BRCA1 and BRCA2 expression following DNA damage. In conclusion, breast-specific induction of deltaNp73 promotes survival of BRCA1-deficient mammary epithelial cells upon DNA damage. This might result in the accumulation of genomic alterations and allow the outgrowth of breast cancers. These findings indicate deltaNp73 as a potential modifier of breast cancer susceptibility in BRCA1 mutation carriers and may stimulate novel strategies of prevention and treatment for these high-risk women.

Berberine and Emodin abrogates breast cancer growth and facilitates apoptosis through inactivation of SIK3-induced mTOR and Akt signaling pathway

Salt-inducible kinases 3 (SIK3) belong to the AMPK-related family of kinases, which have been implicated in the regulation of cell metabolism, cell polarity remodelling, and epithelial–mesenchymal transition. Elevated SIK3 expressions in breast cancer cells are shown to contribute to tumorigenesis; however, the underlying mechanism remains to be elucidated. In this study, we demonstrate that SIK3 expression is upregulated and concurrently high expression of SIK3 is associated with poor survival in breast cancer. Specifically, SIK3 knockdown revealed that SIK3 is required for the mTOR/Akt signaling pathway and proliferation of breast cancer cells. Furthermore, our findings showed that Emodin (EMO) combined with Berberine (BBR) significantly inhibited SIK3 activity, leading to reduced cell growth, increased cell cycle arrest and apoptosis in breast cancer cells, but not in non-malignant breast epithelial cell line. Mechanistic studies further reveal that EMO and BBR in combined treatment inhibited SIK3-potentiated mTOR-mediated aerobic glycolysis and cell growth in breast cancer cells. Moreover, combination treatments attenuate Akt signaling, thereby inducing G0/G1 phase cell cycle arrest and apoptosis of breast cancer cells in a SIK3-dependent manner. CRISPR/Cas9 or siRNA-mediated SIK3 knockout/knockdown showed an opposite trend in both the luminal and basal-like breast cancer. Collectively, our findings reveal that combination of EMO and BBR attenuates SIK3-driven tumor growth in breast cancer, and thus, EMO and BBR might be a novel SIK3 inhibitor explored into the prevention of breast cancer.

Facile entry to germanate and stannate complexes [(η6-arene)RuCl(η2-dppm)]+[ECl3]- (E = Ge, Sn) as potent anti-cancer agents

A series of arene Ru(II) salt complexes of the type [(η6-arene)RuCl(η2-dppm)]+[ECl3]- (arene = C6H6,p-cymene, 1,3,5-Me3C6H3; E = Ge, Sn) bearing trichlorogermanate and trichlorostannate anions are reported. Starting from the known complexes: [(η6-C6H6)RuCl2(η1-dppm)] (1), [(η6-p-cymene)RuCl2(η1-dppm)] (3) and the novel complex [(η6-1,3,5-Me3C6H3)RuCl2(η1-dppm)] (7) (dppm = 1,1-bis(diphenylphosphino)methane), reactions with SnCl2 or GeCl2(dioxane) respectively afforded, by halide abstraction at the ruthenium(II) centres in 1, 3 or 7 the salts: [(η6-C6H6)RuCl(η2-dppm)]+ SnCl3− (2), [(η6-p-cymene)RuCl(η2-dppm)]+ SnCl3− (4), [(η6-C6H6)RuCl(η2-dppm)]+ GeCl3− (5), [(η6-p-cymene)RuCl(η2-dppm)]+ GeCl3− (6), [(η6-1,3,5-Me3C6H3)RuCl(η2-dppm)]+ SnCl3− (8) and [(η6-1,3,5-Me3C6H3)RuCl(η2-dppm)]+ GeCl3− (9). The trichlorostannate complexes 2, 4 and 8 are extremely rare examples of ruthenium complexes bearing the SnCl3− counter anion, and the complexes 5, 6 and 9 are the first examples of ruthenium trichlorogermanate complexes to be reported. All compounds were isolated in high yields as air stable materials and were spectroscopically characterized by multinuclear NMR: (1H, 31P{1H}, 13C{1H}), Infra-red (IR), UV–Vis, and high resolution electrospray ionization mass spectrometry (HR-ESI-MS), the latter both in (+) and (−) mode. Additionally, single crystal X-ray diffraction analyses of salts 4 and 6 are reported, revealing pseudotetrahedral Ru(II) centres with η6 bound p-cymene ligands and η2-bound dppm ligands with statistical disorder on the ECl3- anions (E = Ge (6), Sn (4)). Density functional theory calculations (B3LYP with the basis set 6-31 + G(d,p) for H, C, P and Cl atoms; while for Ru, Ge, and Sn atoms DGDZVP basis set) are reported for salts 4 and 6 revealing localization of the LUMOs on the ruthenium-arene rings and some localization on the chloride atom. Finally, MTT in vitro cytotoxicity assays for the MCF-7 and MDA-MB-231 breast cancer cell lines are reported for all complexes and compared to cisplatin. All complexes show remarkable in vitro cytotoxic activity and most are considerably more cytotoxic than cisplatin in both breast cancer cell lines: IC50 values range from 2.25 μM (compound 2) to 5.97 μM (compound 9) (cisplatin = 5.74 μM) in MCF-7 cells; 2.20 μM (compound 2) to 6.39 μM (compound 5) (cisplatin = 13.98 μM) in MDA-MB-231. Moreover, when compared to non-malignant breast epithelial cells (MCF12A), all complexes exhibit promising selectivity indices (SI) with compound 5 having the highest SI in MCF-7 cells at 4.8; and compound 6 at 3.65 in MDA-MB-231, with most of the other compounds also being considerably more selective than cisplatin on both cell-lines (SI = 2.26 on MCF-7 and 0.93 on MDA-MB-231). A clonogenic assay was conducted for salts 5 and 6 and the results reveal that both compounds inhibited long-term (14 days) survival in both breast cancer cell lines tested indicating these drugs are very promising candidates for pre-clinical studies.

Abstract P6-09-01: RAD51B loss-of-function variants confer susceptibility to hereditary breast and ovarian cancers and result in tumors with genomic features of homologous recombination repair defects

Abstract Introduction: Germline pathogenic variants in genes in the homologous recombination (HR) pathway such as BRCA1, BRCA2, ATM, PALB2, RAD51C and RAD51D confer an increased risk of breast and ovarian cancers. Screening for germline variants in these cancer susceptibility genes is critical as prophylactic measures and monitoring can be performed in these individuals to minimize their risk of developing breast and/or ovarian cancers. More recently, it has been recognized that cancers arising in carriers of germline pathogenic (P)/ likely pathogenic (LP) variants in HR genes often show a homologous recombination (HR) deficient (HRD) phenotype and can be targeted with platinum agents or PARP inhibitors. RAD51B is a RAD51 paralog that binds to RAD51C and functions as a heterodimer in the HR pathway. Whilst RAD51B germline variants have been reported in isolated cases of breast and ovarian cancers, it is unclear as to whether RAD51B P/LP germline variants would confer predisposition to these cancer types. Materials and Methods: We screened 9,287 consecutive unselected cancer patients who consented for both tumor and germline testing using the MSK-IMPACT platform for the presence of RAD51B germline truncating variants. Selected RAD51B germline mutant breast cancers identified by MSK-IMPACT were subjected to whole-exome sequencing (WES) analysis to determine the dominant mutational signatures using DeconstructSigs. Functional assays were performed to ascertain the impact of RAD51B loss on HR DNA repair and PARP inhibitor sensitivity using genome editing methods in non-malignant breast epithelial cell lines. Results: Out of the 9,287 cancer patients, we detected likely pathogenic loss of function RAD51B germline variants in 11 patients (0.12%), which was similar to the frequency detected in the gnomAD database (0.09%). All female carriers of RAD51B loss of function variants (n=8) had breast or ovarian cancers (8/1,619 breast or ovarian cancers, 0.5%). The observed carrier frequencies of germline truncating variants in RAD51B was statistically enriched in breast and ovarian cancer patients compared to individuals in the gnomAD database (0.5% vs 0.09% P=0.0003; odds ratio = 5.06 (95% CI: 2.1-10.3)). Although segregation studies were not available, 9/11 of the RAD51B germline loss of function variant carriers had a personal or family history of breast or ovarian cancers. All five breast and ovarian cancers from RAD51B mutation carriers investigated by WES were found to harbor RAD51B bi-allelic inactivation through loss of heterozygosity of the RAD51B wild-type allele. In addition, these five cases were found to display genomic features of HRD including high large-scale state transition scores and a dominant mutational signature 3. CRISPR/Cas9 genome editing of RAD51B in a non-malignant breast epithelial cell model revealed that RAD51B deficient cells display PARP inhibitor sensitivity similar to that reported in BRCA1 and BRCA2 deficient cell lines. Conclusion: RAD51B loss-of-function germline variants confer susceptibility to breast and ovarian cancer development. Breast and ovarian cancers occurring in the context of RAD51B germline mutations harbor bi-allelic inactivation of RAD51B through loss-of-heterozygosity of the wild-type allele, genomic features of HRD and are likely sensitive to PARP inhibition. Albeit rarely germline mutated, RAD51B should be considered as an addition to clinical germline testing panels for hereditary breast and ovarian cancer syndrome patients. <!–EndFragment→ Citation Format: Diana SReis-FilhoReis-Filho Mandelker, Semanti Mukherjee, Jeremy Setton, Pier Selenica, Yelena Kemel, Ozge Ceyhan-Birsoy, Margaret Sheehan, Kaitlyn Tkachuk, David N Brown, Simon Powell, Britta Weigelt, Mark E Robson, Nadeem Riaz, Kenneth Offit, Jorge S Reis-Filho. RAD51B loss-of-function variants confer susceptibility to hereditary breast and ovarian cancers and result in tumors with genomic features of homologous recombination repair defects [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-09-01.