MCF10A Breast Epithelial Cells Research Articles

Exposure to Selenomethionine and Selenocystine Induces Redox-Mediated ER Stress in Normal Breast Epithelial MCF-10A Cells.

Selenium is an essential trace element co-translationally incorporated into selenoproteins with important biological functions. Health benefits have long been associated with selenium supplementation. However, cytotoxicity is observed upon excessive selenium intake. The aim of this study is to investigate the metabolic pathways underlying the response to the selenium-containing amino acids selenomethionine and selenocysteine in a normal human breast epithelial cell model. We show that both selenomethionine and selenocystine inhibit the proliferation of non-cancerous MCF-10A cells in the same concentration range as cancerous MCF-7 and Hela cells, which results in apoptotic cell death. Selenocystine exposure in MCF-10A cells caused a severe depletion of free low molecular weight thiols, which might explain the observed upregulation of the expression of the oxidative stress pathway transcription factor NRF2. Both selenomethionine and selenocystine induced the expression of target genes of the unfolded protein response (GRP78, ATF4, CHOP). Using a redox-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we show that both selenoamino acids shifted the ER redox balance towards an even more oxidizing environment. These results suggest that alteration of the redox state of the ER may disrupt protein folding and cause ER stress-induced apoptosis in MCF-10A cells exposed to selenoamino acids.

Evaluation of anticancer activity of novel platinum(II) bis(thiosemicarbazone) complex against breast cancer

The study aims to synthesize a novel bis(thiosemicarbazone) derivative based on platinum (thioPt) and evaluate its anticancer properties against MFC-7 and MDA-MB-231 breast cancer cells. A new platinum complex was synthesised by reacting K2PtCl4 with 2,2′-(1,2-diphenylethane-1,2-diylidene)bis(hydrazine-1-carbothioamide) in ethanol in the presence of K2CO3. In the obtained complex, the platinum atom is coordinated by a conjugated system = N-NC-S-The structures of the new compound were characterised using NMR spectroscopy, HR MS, IR, and X-ray structural analysis. The obtained results of the cytotoxicity assay indicate that compound thioPt had potent anticancer activity (MCF-7: 61.03 ± 3.57 µM, MDA-MB-231: 60.05 ± 5.40 µM) with less toxicity against normal MCF-10A breast epithelial cells, even compared to the reference compound (cisplatin). In addition, subsequent experiments found that thioPt induces apoptosis through both an extrinsic (↑caspase 8 activity) and intrinsic (↓ΔΨm) pathway, which ultimately leads to an increase in active caspase 3/7 levels. The induction of autophagy and levels of proteins involved in this process (LC3A/B and Beclin-1) were examined in MCF-7 and MDA-MB-231 breast cancer cells exposed to tested compounds (thio, thioPt, cisPt) at a concentration of 50 µM for 24 h. Based on these results, it can be concluded that thio and thioPt do not significantly affect the autophagy process. This demonstrates their superiority over cisplatin, which can stimulate cancer cell survival through its effect on stimulation of autophagy.

YAP1-induced RBM24 promotes the tumorigenesis of triple-negative breast cancer through the β-catenin pathway.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and refractory to current treatments. RBM24 is an RNA-binding protein and shows the ability to regulate tumor progression in multiple cancer types. However, its role in TNBC is still unclear. In this study, we analyzed publicly available profiling data from TNBC tissues and cells. Loss- and gain-of-function experiments were performed to determine the function of RBM24 in TNBC cells. The mechanism for RBM24 action in TNBC was investigated. RBM24 was deregulated in TNBC tissues and TNBC cells with depletion of SIPA1, YAP1, or ARID1A, three key regulators of TNBC. Compared to MCF10A breast epithelial cells, TNBC cells had higher levels of RBM24. Knockdown of RBM24 inhibited TNBC cell proliferation, colony formation, and tumorigenesis, while overexpression of RBM24 promoted aggressive phenotype in TNBC cells. YAP1 overexpression induced the expression of RBM24 and the RBM24 promoter-driven luciferase reporter. YAP1 was enriched at the promoter region of RBM24. Overexpression of RBM24 increased β-catenin-dependent transcriptional activity. Most importantly, knockdown of CTNNB1 rescued RBM24 aggressive phenotype in TNBC cells. Collectively, the YAP1/RBM24/β-catenin axis plays a critical role in driving TNBC progression. RBM24 may represent a novel therapeutic target for TNBC treatment.

Surface Proteome of Extracellular Vesicles and Correlation Analysis Reveal Breast Cancer Biomarkers

Breast cancer (BC) is the second most frequently diagnosed cancer and accounts for approximately 25% of new cancer cases in Canadian women. Using biomarkers as a less-invasive BC diagnostic method is currently under investigation but is not ready for practical application in clinical settings. During the last decade, extracellular vesicles (EVs) have emerged as a promising source of biomarkers because they contain cancer-derived proteins, RNAs, and metabolites. In this study, EV proteins from small EVs (sEVs) and medium EVs (mEVs) were isolated from BC MDA-MB-231 and MCF7 and non-cancerous breast epithelial MCF10A cell lines and then analyzed by two approaches: global proteomic analysis and enrichment of EV surface proteins by Sulfo-NHS-SS-Biotin labeling. From the first approach, proteomic profiling identified 2459 proteins, which were subjected to comparative analysis and correlation network analysis. Twelve potential biomarker proteins were identified based on cell line-specific expression and filtered by their predicted co-localization with known EV marker proteins, CD63, CD9, and CD81. This approach resulted in the identification of 11 proteins, four of which were further investigated by Western blot analysis. The presence of transmembrane serine protease matriptase (ST14), claudin-3 (CLDN3), and integrin alpha-7 (ITGA7) in each cell line was validated by Western blot, revealing that ST14 and CLDN3 may be further explored as potential EV biomarkers for BC. The surface labeling approach enriched proteins that were not identified using the first approach. Ten potential BC biomarkers (Glutathione S-transferase P1 (GSTP1), Elongation factor 2 (EEF2), DEAD/H box RNA helicase (DDX10), progesterone receptor (PGR), Ras-related C3 botulinum toxin substrate 2 (RAC2), Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), Aconitase 2 (ACO2), UTP20 small subunit processome component (UTP20), NEDD4 binding protein 2 (N4BP2), Programmed cell death 6 (PDCD6)) were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. In total, 846 surface proteins were identified from the second approach, of which 11 were already known as BC markers. This study supports the proposition that Evs are a rich source of known and novel biomarkers that may be used for non-invasive detection of BC. Furthermore, the presented datasets could be further explored for the identification of potential biomarkers in BC.

Orai1α and Orai1β support calcium entry and mammosphere formation in breast cancer stem cells

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1β, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1β support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1β in spheroid forming efficiency and self-renewal in breast cancer stem cells.

THU498 Establishing The Link Between Glucocorticoid Signaling And The Circadian Regulator DEC1 In Breast Cancer

Abstract Disclosure: M.I. Castro: None. W.S. Ybañez: None. P.D. Bagamasbad: None. Epidemiological studies have linked altered circadian rhythms due to irregular shift work, chronic jet lag, and heightened night-time light exposure to increased breast cancer (BCa) pathogenesis. Dysregulated glucocorticoid (GC; CORT) signaling in BCa may further promote chronodisruption by directly regulating the cyclic expression of clock genes found in the breast. The differentiated embryo-chondrocyte expressed gene 1 (DEC1; also known as STRA13/SHARP2/ BHLHB2) is a transcription factor involved in the regulation of circadian rhythmicity and tumor progression of several cancer types including the breast and associated with hormone receptor signaling in BCa. In this study, we explored the involvement of DEC1 in the crosstalk between GC signaling and the circadian axis in BCa. To establish DEC1 expression pattern in BCa tumor samples, we assessed publicly available RNA-seq datasets from the Genotype-Tissue Expression Project and found no significant difference in DEC1 expression in tumors when compared to normal tissue controls. However, when tumor samples were stratified into molecular subtypes based on transcriptomic data from the Cancer Genome Atlas, DEC1 expression was significantly downregulated in triple-negative BCa (TNBC) in comparison to luminal subtypes. This pattern of expression was reflected in the BCa cell lines where we observed higher DEC1 expression in the estrogen receptor-positive MCF7 cells relative to nontumorigenic triple-negative MCF10A cells and the highly aggressive TNBC MDA-MB-231 cell line. To determine if DEC1 is regulated by GCs in the breast, we treated non-tumorigenic breast epithelial MCF10A cells and the TNBC MDA-MB-231 cells with increasing concentrations of CORT and observed that DEC1 mRNA expression decreases in response to increasing concentrations of CORT. Treatment of MCF10A and MDA-MB-231 cells with GC receptor (GR)- specific agonist RU486 abolished the GC-dependent repression of DEC1 expression and this pattern of DEC1 expression persisted even in the presence of the protein synthesis inhibitor cycloheximide, suggesting that DEC1 is a direct transcriptional target of GR in mammary epithelial cells. Through in silico analysis of publicly available ChIP-sequencing datasets from BCa cell lines, we identified multiple CORT-responsive cis-regulatory elements in the DEC1 locus that could mediate the CORT-dependent repression of DEC1 expression. Lentiviral knockdown of DEC1 in three breast epithelial cell lines was generated and will be used in cancer hallmark assays on proliferation, apoptosis, survival, and migration to assess the functional role of DEC1 in BCa. Taken together, our data will contribute to understanding the combined effect of GC signaling and circadian gene regulation towards BCa progression. Presentation: Thursday, June 15, 2023

SELENOF Controls Proliferation and Cell Death in Breast-Derived Immortalized and Cancer Cells.

SELENOF expression is significantly lower in aggressive breast tumors compared to normal tissue, indicating that its reduction or loss may drive breast tumorigenesis. Deletion of SELENOF in non-tumorigenic immortalized breast epithelial MCF-10A cells resulted in enhanced proliferation, both in adherent culture and matrix-assisted three-dimmensional (3D) growth. Modulation of SELENOF in vitro through deletion or overexpression corresponded to changes in the cell-cycle regulators p21 and p27, which is consistent with breast tumor expression data from the METABRIC patient database. Together, these findings indicate that SELENOF affects both proliferation and cell death in normal epithelial and breast cancer cells, largely through the regulation of p21 and p27. In glandular cancers like breast cancer, the filling of luminal space is one of the hallmarks of early tumorigenesis. Loss of SELENOF abrogated apoptosis and autophagy, which are required for the formation of hollow acini in MCF-10A cells in matrix-assisted 3D growth, resulting in luminal filling. Conversely, overexpression of SELENOF induced cell death via apoptosis and autophagy. In conclusion, these findings are consistent with the notion that SELENOF is a breast tumor suppressor, and its loss contributes to breast cancer etiology.

A Novel Cell Membrane-Associated RNA Extraction Method and Its Application in the Discovery of Breast Cancer Markers.

Cell membrane-associated RNA (mem-RNA) has been demonstrated to be cell-specific and disease-related and are considered as potential biomarkers for disease diagnostics, drug delivery, and cell screening. However, there is still a lack of methods specifically designed to extract mem-RNA from cells, limiting the discovery and applications of mem-RNA. In this study, we propose the first all-in-one solution for high-purity mem-RNA isolation based on two types of magnetic nanoparticles, named MREMB (Membrane-associated RNA Extraction based on Magnetic Beads), which achieved ten times enrichment of cell membrane components and over 90% recovery rate of RNA extraction. To demonstrate MREMB's potential in clinical research, we extracted and sequenced mem-RNA of typical breast cancer MCF-7, MDA-MB-231, and SKBR-3 cell lines and non-neoplastic breast epithelial cell MCF-10A. Compared to total RNA, sequencing results revealed that membrane/secreted protein-encoding mRNAs and long noncoding RNAs (lncRNAs) were enriched in the mem-RNA, some of which were significantly overexpressed in the three cancer cell lines, including extracellular matrix-related genes COL5A1 and lncRNA TALAM1. The results indicated that MREMB could enrich membrane/secreted protein-coding RNA and amplify the expression differences of related RNAs between cancer and non-neoplastic cells, promising for cancer biomarker discovery.

Mesenchymal Stem Cell Membrane-Coated TPCS2a-Loaded Nanoparticles for Breast Cancer Photodynamic Therapy.

Despite substantial improvements in breast cancer (BC) treatment there is still an urgent need to find alternative treatment options to improve the outcomes for patients with advanced-stage disease. Photodynamic therapy (PDT) is gaining a lot of attention as a BC therapeutic option because of its selectivity and low off-target effects. However, the hydrophobicity of photosensitizers (PSs) impairs their solubility and limits the circulation in the bloodstream, thus representing a major challenge. The use of polymeric nanoparticles (NPs) to encapsulate the PS may represent a valuable strategy to overcome these issues. Herein, we developed a novel biomimetic PDT nanoplatform (NPs) based on a polymeric core of poly(lactic-co-glycolic)acid (PLGA) loaded with the PS meso-tetraphenylchlorin disulfonate (TPCS2a). TPCS2a@NPs of 98.89 ± 18.56 nm with an encapsulation efficiency percentage (EE%) of 81.9 ± 7.92% were obtained and coated with mesenchymal stem cells-derived plasma membranes (mMSCs) (mMSC-TPCS2a@NPs, size of 139.31 ± 12.94 nm). The mMSC coating armed NPs with biomimetic features to impart long circulation times and tumor-homing capabilities. In vitro, biomimetic mMSC-TPCS2a@NPs showed a decrease in macrophage uptake of 54% to 70%, depending on the conditions applied, as compared to uncoated TPCS2a@NPs. Both NP formulations efficiently accumulated in MCF7 and MDA-MB-231 BC cells, while the uptake was significantly lower in normal breast epithelial MCF10A cells with respect to tumor cells. Moreover, encapsulation of TPCS2a in mMSC-TPCS2a@NPs effectively prevents its aggregation, ensuring efficient singlet oxygen (1O2) production after red light irradiation, which resulted in a considerable in vitro anticancer effect in both BC cell monolayers (IC50 < 0.15 µM) and three-dimensional spheroids.

Similar deamination activities but different phenotypic outcomes induced by APOBEC3 enzymes in breast epithelial cells.

APOBEC3 (A3) enzymes deaminate cytosine to uracil in viral single-stranded DNA as a mutagenic barrier for some viruses. A3-induced deaminations can also occur in human genomes resulting in an endogenous source of somatic mutations in multiple cancers. However, the roles of each A3 are unclear since few studies have assessed these enzymes in parallel. Thus, we developed stable cell lines expressing A3A, A3B, or A3H Hap I using non-tumorigenic MCF10A and tumorigenic MCF7 breast epithelial cells to assess their mutagenic potential and cancer phenotypes in breast cells. The activity of these enzymes was characterized by γH2AX foci formation and in vitro deamination. Cell migration and soft agar colony formation assays assessed cellular transformation potential. We found that all three A3 enzymes had similar γH2AX foci formation, despite different deamination activities in vitro. Notably, in nuclear lysates, the in vitro deaminase activity of A3A, A3B, and A3H did not require digestion of cellular RNA, in contrast to that of A3B and A3H in whole-cell lysates. Their similar activities in cells, nonetheless, resulted in distinct phenotypes where A3A decreased colony formation in soft agar, A3B decreased colony formation in soft agar after hydroxyurea treatment, and A3H Hap I promoted cell migration. Overall, we show that in vitro deamination data do not always reflect cell DNA damage, all three A3s induce DNA damage, and the impact of each is different.

Microclaw Array Fabricated by Single Exposure of Femtosecond Airy Beam and Self-Assembly for Regulating Cell Migratory Plasticity.

The highly aligned extracellular matrix of metastatic breast cancer cells is considered to be the "highway" of cancer invasion, which strongly promotes the directional migration of cancer cells to break through the basement membrane. However, how the reorganized extracellular matrix regulates cancer cell migration remains unknown. Here, a single exposure of a femtosecond Airy beam followed by a capillary-assisted self-assembly process was used to fabricate a microclaw-array, which was used to mimic the highly oriented extracellular matrix of tumor cells and the pores in the matrix or basement membrane during cell invasion. Through the experiment, we found that metastatic breast cancer MDA-MB-231 cells and normal breast epithelial MCF-10A cells exhibit three major migration phenotypes on microclaw-array assembled with different lateral spacings: guidance, impasse, and penetration, whereas guided and penetrating migration are almost completely arrested in noninvasive MCF-7 cells. In addition, different mammary breast epithelial cells differ in their ability to spontaneously perceive and respond to the topology of the extracellular matrix at the subcellular and molecular levels, which ultimately affects the cell migratory phenotype and pathfinding. Altogether, we fabricated a microclaw-array as a flexible and high-throughput tool to mimic the extracellular matrix during invasion to study the migratory plasticity of cancer cells.

The effects of MYC on exosomes derived from cancer cells in the context of breast cancer.

MYC amplification and overexpression in breast cancer occur 16% and 22%, respectively, and MYC has a linchpin role in breast carcinogenesis. Emerging evidence has started to shed light on central role of MYC in breast cancer progression. On the contrary, tumor-derived exosomes and their cargo molecules are required for the modulation of the tumor environment and to promote carcinogenesis. Still, how MYC regulates tumor-derived exosomes is still a matter of investigation in the context of breast cancer. Here, we investigated for the first time how MYC affects the biological functions of normal breast cells cocultured with exosomes derived from MYC-expression manipulated breast cancer cells. Accordingly, exosomes were isolated from MCF-7 and MDA-MB-231 cells that MYC expression was manipulated through siRNAs or lentiviral vectors by using exosome isolation reagent. Then, normal breast epithelial MCF-10A cells were treated with breast cancer cell-derived exosomes. The cellular activity of MCF-10A was investigated by cell growth assay, wound healing assay, and transwell assay. Our results suggested that MCF-10A cells treated with exosomes derived from MYC-overexpressing breast cancer cells demonstrated higher proliferation and migration capability compared with nontreated cells. Likewise, MCF-10A cells treated with exosomes derived from MYC-silenced cancer cells did not show high proliferation and invasive capacity. Overall, MYC can drive the functions of exosomes secreted from breast cancer cells. This may allow exploring a new mechanism how tumor cells regulate cancer progression and modulate tumor environment. The present study clears the way for further researches as in vivo studies and multi-omics that clarify exosomal content in an MYC-dependent manner.

Scutebarbatine A induces ROS-mediated DNA damage and apoptosis in breast cancer cells by modulating MAPK and EGFR/Akt signaling pathway

Scutebarbatine A (SBT-A), a diterpenoid alkaloid, has exerted cytotoxicity on hepatocellular carcinoma cells in our previous works. Here, the antitumor activity of SBT-A in breast cancer cells and the underlying mechanism were explored. The anti-proliferative effect of SBT-A was measured by trypan blue staining, 5-ethynyl-2′-deoxyuridine (EdU) incorporation and colony formation assay. DNA double-strand breaks (DSBs) were evaluated by observing the nuclear focus formation of γ-H2AX. Cell cycle distribution was assessed by flow cytometry. Apoptosis was determined by a TUNEL assay. Intracellular reactive oxygen species (ROS) generation and superoxide production were measured with 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE) staining, respectively. The results indicated that SBT-A showed a dose-dependent cytotoxic effect against breast cancer cells while revealing less toxicity toward MCF-10A breast epithelial cells. Moreover, SBT-A remarkably induced DNA damage, cell cycle arrest and apoptosis in both MDA-MB-231 and MCF-7 cells. SBT-A treatment increased the levels of ROS and cytosolic superoxide production. Pretreatment with N-acetyl cysteine (NAC), a ROS scavenger, was sufficient to block viability reduction, DNA damage, apoptosis and endoplasmic reticulum (ER) stress caused by SBT-A. By exposure to SBT-A, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) was upregulated, while the phosphorylation of extracellular signal-regulated kinase (ERK) was downregulated. In addition, SBT-A inhibited the EGFR signaling pathway by decreasing EGFR expression and phosphorylation of Akt and p70S6K. As mentioned above, SBT-A has a potent inhibitory effect on breast cancer cells through induction of DNA damage, apoptosis and ER stress via ROS generation and modulation of MAPK and EGFR/Akt signaling pathway.

Redox-mediated Anticancer Activity of Anti-parasitic Drug Fenbendazole in Triple-negative Breast Cancer Cells.

An increasing number of studies are reporting anticancer activity of widely used antiparasitic drugs and particularly benzimidazoles. Fenbendazole is considered safe and tolerable in most animal species at the effective doses as an anthelmintic. Little is known about the redox-modulating properties of fenbendazole and the molecular mechanisms of its antiproliferative effects. Our study aimed to investigate the possibility of selective redox-mediated treatment of triple-negative breast cancer cells by fenbendazole without affecting the viability and redox status of normal breast epithelial cells. The experiments were performed on three cell lines: normal breast epithelial cells (MCF-10A) and cancer breast epithelial cells (MCF7 - luminal adenocarcinoma, low metastatic; MDA-MB-231 - triple-negative adenocarcinoma, highly metastatic). Cells were treated with fenbendazole for 48-h and three parameters were analyzed using conventional assays: cell viability and proliferation, level of intracellular superoxide, and level of hydroperoxides. The data demonstrated that MDA-MB-231 cells were more vulnerable to fenbendazole-induced oxidative stress than MCF-7 cells. In normal breast epithelial cells MCF-10A, fenbendazole significantly suppressed oxidative stress compared to untreated controls. These data correlate with the effect of fenbendazole on cell viability and the IC50 values, which is indirect evidence of the potential targeting anticancer effect of the drug, especially in MDA-MB-231 cells. The difference in the levels of oxidative stress induced by fenbendazole in MDA-MB-231 and MCF-7 indicates that the two types of breast cancer respond to the drug through different redox-related mechanisms.

Chronic Exposure to Nitric Oxide Induces P53 Mutations and Malignant-like Features in Human Breast Epithelial Cells.

The small endogenous signaling molecule nitric oxide (NO) has been linked with chronic inflammation and cancer. The effects of NO are both concentration and temporally dependent; under some conditions, NO protects against damage caused by reactive oxygen species and activates P53 signaling. During chronic inflammation, NO causes DNA damage and inhibits repair proteins. To extend our understanding of the roles of NO during carcinogenesis, we investigated the possible effects of chronic NO exposure on MCF10A breast epithelial cells, as defined by changes in cellular morphology, chromosome/genomic stability, RNA, and protein expression, and altered cell phenotypes. Human MCF10A cells were maintained in varying doses of the NO donor DETANO for three weeks. Distinct patterns of genomic modifications in TP53 and KRAS target genes were detected in NO-treated cells when compared to background mutations. In addition, quantitative real-time PCR demonstrated an increase in the expression of cancer stem cell (CSC) marker CD44 after prolonged exposure to 300 μM DETANO. While similar changes in cell morphology were found in cells exposed to 300-500 μM DETANO, cells cultured in 100 μM DETANO exhibited enhanced motility. In addition, 100 μM NO-treated cells proliferated in serum-free media and selected clonal populations and pooled cells formed colonies in soft agar that were clustered and disorganized. These findings show that chronic exposure to NO generates altered breast epithelial cell phenotypes with malignant characteristics.

Ras-mediated activation of mTORC2 promotes breast epithelial cell migration and invasion.

We previously identified the mechanistic target of rapamycin complex 2 (mTORC2) as an effector of Ras for the control of directed cell migration in Dictyostelium. Recently, the Ras-mediated regulation of mTORC2 was found to be conserved in mammalian cells, and mTORC2 was shown to be an effector of oncogenic Ras. Interestingly, mTORC2 has been linked to cancer cell migration, and particularly in breast cancer. Here, we investigated the role of Ras in promoting the migration and invasion of breast cancer cells through mTORC2. We observed that both Ras and mTORC2 promote the migration of different breast cancer cells and breast cancer cell models. Using HER2 and oncogenic Ras-transformed breast epithelial MCF10A cells, we found that both wild-type Ras and oncogenic Ras promote mTORC2 activation and an mTORC2-dependent migration and invasion in these breast cancer models. We further observed that, whereas oncogenic Ras-transformed MCF10A cells display uncontrolled cell proliferation and invasion, disruption of mTORC2 leads to loss of invasiveness only. Together, our findings suggest that, whereas the Ras-mediated activation of mTORC2 is expected to play a minor role in breast tumor formation, the Ras-mTORC2 pathway plays an important role in promoting the migration and invasion of breast cancer cells.

Caveolin-1 signaling-driven mitochondrial fission and cytoskeleton remodeling promotes breast cancer migration.

Mitochondria are highly dynamic organelles that constantly divide and fuse to maintain their proper structure and function. Cancer cells are often accompanied by an imbalance of mitochondrial fusion and fission, cancer progression is greatly affected by this imbalance. Here, we found that high-metastatic breast cancer MDA-MB-231 cells possess higher caveolin-1 (Cav-1) expression compared with low-metastatic breast cancer MCF-7 cells or normal breast epithelial MCF-10A cells. Downregulation of Cav-1 decreases the migratory and invasive abilities of MDA-MB-231 cells. Our results further demonstrated that downregulation of Cav-1 facilitated DRP1 and MFN2 to translocate to mitochondria, increasing the inhibitory phosphorylation level of DRP1 at Ser637 by protein kinase A (PKA), resulting in mitochondria elongation. We also showed that downregulation of Cav-1 significantly reduced the Rac1 activity by affecting intracellular reactive oxygen species (ROS) generation, which then inhibited F-actin formation. Based on these findings, we proposed that Cav-1 mediated mitochondrial fission-affected intracellular ROS generation and activated Rho GTPases, leading to F-actin-dependent formation of lamellipodia and promotion of breast cancer motility.

Pro-Apoptotic and Anti-Migration Properties of a Thiazoline-Containing Platinum(II) Complex in MDA-MB-231 Breast Cancer Cells: The Role of Melatonin as a Synergistic Agent.

Triple-negative breast cancer (TNBC) is an aggressive cancer insensitive to hormonal and human epidermal growth factor receptor 2 (HER2)-targeted therapies and has a poor prognosis. Therefore, there is a need for the development of convenient anticancer strategies for the management of TNBC. In this paper, we evaluate the antitumoral potential of a platinum(II) complex coordinated with the ligand 2-(3,5-diphenylpyrazol-1-yl)-2-thiazoline (DPhPzTn), hereafter PtDPhPzTn, against the TNBC cell line MDA-MB-231, and compared its effect with both cisplatin and its less lipophilic counterpart PtPzTn, the latter containing the ligand 2-(pyrazol-1-yl)-2-thiazoline (PzTn). Then, the putative potentiating actions of melatonin, a naturally occurring antioxidant with renowned antitumor properties, on the tumor-killing ability of PtDPhPzTn were also checked in TNBC cells. Our results show that PtDPhPzTn presented enhanced cytotoxicity compared to both the classical drug cisplatin and PtPzTn. In addition, PtDPhPzTn was able to induce apoptosis, being more selective for MDA-MB-231 cells when compared to non-tumor breast epithelial MCF10A cells. Likewise, PtDPhPzTn produced moderate S phase arrest and greatly impaired the migration ability of MDA-MB-231 cells. Most importantly, the co-stimulation of TNBC cells with PtDPhPzTn and melatonin substantially enhanced apoptosis and markedly improved the anti-migratory action compared to PtDPhPzTn alone. Altogether, our findings provide evidence that PtDPhPzTn and melatonin could be potentially applied to breast cancer treatment as powerful synergistic agents.

Abstract 276: Inflammatory co-regulation of voltage-gated sodium channels and Na,K-ATPases in metastatic breast cancer

Abstract Changes in membrane potential dramatically impact the progression of cancer, yet little is known as to how ion transport contributes to disease progression. Voltage-gated sodium channels (VGSCs) and Na,K-ATPases are particularly attractive targets as both are upregulated in many cancers and have been correlated specifically with invasiveness in vitro and metastasis and in vivo. However, their precise upstream regulators and downstream effectors remain unclear. Additionally, it has not been determined whether these transporters are co-regulated in cancer, although they display overlap in their downstream effectors and have complementary activity on sodium ions. Such a system has, however, been described in neuronal models of pain, wherein inflammatory injury leads to an upregulation in VGSC expression and a subsequent proportional increase in Na,K-ATPase. We sought to describe a similar phenomenon in cancer cells, which could indicate that this neuronal response to injury may be hijacked as a driver of metastatic development. To elucidate the impact of inflammatory mediators on these ion transporters in cancer cells, we assessed the effects of TNFα and PGE2 on VGSCs and Na,K-ATPases in the human breast epithelial or cancer cells MCF-10a, MCF-7, and MDA-MB-231. RT-qPCR revealed increases in RNA expression for the clinically relevant VGSC subunit Nav1.5. Similar RNA increases were observed in the α1 and α3 subunits of Na,K-ATPase, although in all cases the magnitude and time point of maximal effect differed by cell line. Then a 1hr challenge of TNFα on MDA-MB-231 cells was used to examine whether the targets were independently regulated by inflammatory mediators, co-regulated via the transport of sodium ions, or co-regulated by the expression of one protein influencing the expression of the other protein. Transfection with siRNA against either the α1 or α3 subunit of Na,K-ATPase did not inhibit the inflammatory-mediated increase in RNA for Nav1.5. However, transfection with anti-Nav1.5 siRNA inhibited the upregulation of both Na,K-ATPase subunits. TNFα challenge following pre-incubation of MDA-MB-231 cells in a sodium-free medium completely inhibited the inflammatory-mediated upregulation of Nav1.5, Na,K-ATPase α1 or α3 RNA. Co-incubation of TNFα with the VGSC blocking drug protoxin I inhibited the inflammatory-mediated increase in Na,K-ATPase α1 or α3 RNA, but co-incubation with the Na,K-ATPase blocking drug ouabain did not inhibit the increase in Nav1.5 RNA and indeed may have potentiated the increase. Together these results are evidence that VGSCs and Na,K-ATPase are co-regulated by inflammatory mediators in metastatic breast cancer, that sodium is essential to this mechanism, and that the presence and ion-transporting function of VGSCs is necessary for the inflammatory-mediated effect on Na,K-ATPase but not vice versa. Citation Format: Steven D. Scahill, Harry J. Gould, Dennis Paul. Inflammatory co-regulation of voltage-gated sodium channels and Na,K-ATPases in metastatic breast cancer [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 276.

Abstract 883: Defining the observed mutual exclusivity between SF3B1 and TP53 mutations in cancer

Abstract SF3B1 is the most commonly mutated splicing factor in cancer, occurring in a broad variety of hematologic and solid malignancies, including: breast, melanoma, pancreatic, bladder, colorectal, leukemia, and more. SF3B1 is a protein component of the spliceosome responsible for recognizing intronic splice sites and stabilizing the interaction between the spliceosome and pre-mRNA transcripts. Hotspot mutations in SF3B1 result in a neomorphic protein that causes aberrant splicing of hundreds of transcripts. These transcriptional alterations have several proposed impacts on known cancer pathways; however, the mechanisms by which SF3B1 mutations contribute to tumorigenesis are incompletely understood. Interestingly, systematic analysis of online cancer databases shows SF3B1 mutations and TP53 alterations are mutually exclusive in cancers with predominant SF3B1 hotspots, suggesting either synthetic lethality or overlapping roles in promoting tumorigenesis. To address this question, TP53 was knocked-out using CRISPR-Cas9 in AAV-generated SF3B1 K700E mutant knock-in and wild type MCF-10A breast epithelial cells. Successful generation of TP53 knock-outs in both the SF3B1 mutant and wild type groups suggests the mutations are unlikely to be synthetic lethal. Instead, this finding may direct us to novel mechanisms by which SF3B1 mutations contribute to cancer development and progression. Two overarching areas of interest based on preliminary data include changes in mitochondrial metabolism and metabolic reprogramming and alterations in DNA damage, repair, and genomic stability. Further defining the relationship between mutant SF3B1 and TP53 may reveal new mechanisms of tumorigenesis and reveal therapeutic vulnerabilities that can be additionally leveraged against the large subset of cancers with TP53 mutations. Citation Format: Riley Bergman, Ben Park, Sarah Croessmann. Defining the observed mutual exclusivity between SF3B1 and TP53 mutations in cancer [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 883.