Proteins In Breast Cancer Cells Research Articles

The Breast Cancer Protein Co-Expression Landscape.

Simple SummaryProteins are among the most fundamental building blocks and molecular players behind the functions of cells and tissues. Their abundance and interaction patterns shape, to a large extent, what happens at the cellular and organ levels. This is also true regarding tumor tissues. In this work, we explored the patterns of abundance and co-occurrence of a large number of proteins in breast cancer cells and their healthy counterparts. We discovered the main differences and tried to see whether those differences may be associated with relevant aspects of the biology of these tumors. Our final goal is to provide information to empower cancer clinicians and pharmacologists to develop better diagnostic, prognostic, and therapeutic tools.Breast cancer is a complex phenotype (or better yet, several complex phenotypes) characterized by the interplay of a large number of cellular and biomolecular entities. Biological networks have been successfully used to capture some of the heterogeneity of intricate pathophenotypes, including cancer. Gene coexpression networks, in particular, have been used to study large-scale regulatory patterns. Ultimately, biological processes are carried out by proteins and their complexes. However, to date, most of the tumor profiling research has focused on the genomic and transcriptomic information. Here, we tried to expand this profiling through the analysis of open proteomic data via mutual information co-expression networks’ analysis. We could observe that there are distinctive biological processes associated with communities of these networks and how some transcriptional co-expression phenomena are lost at the protein level. These kinds of data and network analyses are a broad resource to explore cellular behavior and cancer research.

Polymer Nanoparticles Overcome Drug Resistance by a Dual-Targeting Apoptotic Signaling Pathway in Breast Cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activating therapy has received wide attention due to its capacity to precisely induce cancer cell apoptosis. However, drug resistance and the poor pharmacokinetic properties of TRAIL protein are obstacles in TRAIL-based therapy for cancer. Herein, a strategy is developed to remotely control and specifically initiate TRAIL-mediated apoptotic signaling to promote TRAIL-resistant cancer cell apoptosis using near-infrared (NIR) light-absorbing conjugated polymer nanoparticles (CPNs). Upon 808 nm laser excitation, the promoter 70 kilodalton heat shock protein (HSP70) initiates transcription of the TRAIL gene in response to heat shock, thereby expressing TRAIL protein in breast cancer cells, which activates the TRAIL-mediated apoptosis signaling pathway. Simultaneously, the CPNs locally release W-7, which targets calmodulin (CaM) and further promotes caspase-8 cleavage and enhances cancer cell apoptosis. Both in vitro and in vivo results demonstrate that CPNs/W-7/pTRAIL produces an excellent synergistic therapeutic effect on breast cancer upon near-infrared light with low toxicity. Therefore, this work provides a strategy for overcoming drug resistance through dual-targeting TRAIL-mediated apoptotic signaling in breast cancer.

Circular RNA circ_0048764 promotes the development of breast cancer by regulating microRNA-1296-5p/tripartite motif containing 14 axis

ABSTRACT Breast cancer (BC) is one of the leading causes of cancer-related deaths in females. Circular RNA (circRNA), as reported, is involved in the progression of BC. This work focuses on clarifying the biological function of circ_0048764 in BC and its hidden mechanism. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expressions of circ_0048764, microRNA-1296-5p (miR-1296-5p), and tripartite motif containing 14 (TRIM14) in BC tissues and cell lines. Besides, the status of proliferation, migration, invasion and apoptosis of BC cells was probed by cell counting kit-8 (CCK-8), EdU, transwell and flow cytometry assays. Western blot was adopted to examine the level of TRIM14 protein in BC cells. In addition, dual-luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay were conducted to corroborate the targeting relationships between miR-1296-5p and circ_0048764 or TRIM14. It was revealed that circ_0048764 expression was remarkably up-regulated in BC tissues and cells, and circ_0048764 expression was associated with TNM stage and tumor size. Functionally, overexpression of circ_0048764 significantly promoted BC cell proliferative, migrative and invasive abilities and inhibited apoptosis, while circ_0048764 knockdown exerted the opposite effects. Mechanistically, circ_0048764 directly targeted miR-1296-5p and could negatively modulate its expression in BC cells. Besides, miR-1296-5p could reverse the influence of circ_0048764 on BC viability, migration, invasion and apoptosis. Moreover, TRIM14 was confirmed to be a downstream target of miR-1296-5p. Circ_0048764 positively regulated TRIM14 expression in BC cells via targeting miR-1296-5p. Collectively, it is concluded that circ_0048764 promotes the development of BC via modulating the miR-1296-5p/TRIM14 axis.

The Effect of Tau and Taxol on Polymerization of MCF7 Microtubules In Vitro.

Overexpression of Tau protein in breast cancer cells is identified as an indicator for potential resistance to taxane-based therapy. As reported findings have been obtained mostly from clinical studies, the undetermined underlying mechanism of such drug resistance needs to be thoroughly explored through comprehensive in vitro evaluations. Tau and Taxol bind to the beta tubulin site in microtubules’ structure. This is of particular interest in breast cancer, as microtubules of these cancer cells are structurally distinct from some other microtubules, such as neuronal microtubules, due to their unique beta tubulin isotype distribution. The observed changes in the in vitro polymerization of breast cancer microtubules, and the different function of some molecular motors along them, leave open the possibility that the drug resistance mechanism can potentially be associated with different responses of these microtubules to Tau and Taxol. We carried out a series of parallel experiments to allow comparison of the in vitro dual effect of Tau and Taxol on the polymerization of MCF7 microtubules. We observed a concentration-dependent demotion-like alteration in the self-polymerization kinetics of Tau-induced MCF7 microtubules. In contrast, microtubules polymerized under the simultaneous effects of Tau and Taxol showed promoted assembly as compared with those observed in Tau-induced microtubules. The analysis of our data obtained from the length of MCF7 microtubules polymerized under the interaction with Tau and Taxol in vitro suggests that the phenomenon known as drug resistance in microtubule-targeted drugs such as Taxol may not be directly linked to the different responses of microtubules to the drug. The effect of the drug may be mitigated due to the simultaneous interactions with other microtubule-associated proteins such as Tau protein. The observed regulatory effect of Tau and Taxol on the polymerization of breast cancer microtubules in vitro points to additional evidence for the possible role of tubulin isotypes in microtubules’ functions.

Precision Targeting of Mutant PI3Kα in Cancer by Selective Degradation.

PIK3CA, which encodes the p110α catalytic subunit of PI3Kα, is one of the most frequently genetically activated kinases in solid tumors. In this issue of Cancer Discovery, Song and colleagues report that the related PI3Kα inhibitors taselisib and inavolisib trigger receptor tyrosine kinase (RTK)-dependent degradation of the mutant p110α protein in breast cancer cells that are positive for HER2 RTK, limiting feedback-mediated drug resistance and potentially widening the therapeutic index of PI3Kα inhibition.See related article by Song et al., p. 204.

PER1 as a Tumor Suppressor Attenuated in the Malignant Phenotypes of Breast Cancer Cells.

BackgroundCircadian clock genes play a crucial role in physiological and pathological processes, and their aberrant expressions were involved in various human cancers. The objective of this study was to investigate the expression level of Period circadian regulator 1 (PER1), an important circadian clock gene, and its biological roles in the development and progression of breast cancer.MethodsThe expression level of PER1 in breast cancer samples was analyzed using the Oncomine database, and the correlation between PER1 expression and clinicopathologic parameters was assessed by bc-GenExMiner v4.5. In addition, Kaplan–Meier plotter database was used to determine the prognostic significance of PER1 expression for breast cancer patients. The expressions of PER1 in breast cancer tissues and cells were validated by Western blot. The loss-or-gain assay of PER1 was conducted to investigate the effects of its expression on cell proliferation, migration and invasion of breast cancer. The relationship between PER1 expression and epigenetic modifications was further explored by Western blot.ResultsThe results of the bioinformatics analysis revealed that the expression level of PER1 was markedly reduced in breast cancer tissues (P<0.001), and patients with high expression of PER1 had a better overall survival (HR:0.78, 95% CI:0.63–0.97, P=0.026) and recurrence-free survival (HR:0.83, 95% CI:0.75–0.93, P=0.001) than those with low expression. The assay of gene loss-or-gain indicated that downregulation of PER1 expression markedly promoted cell proliferation, migration and invasion (P<0.05), whereas these malignant phenotypes of breast cancer cells were inhibited by PER1 overexpression (P<0.05). Further studies showed that trichostatin A (TSA), a histone deacetylase inhibitor, induced the expression of PER1 protein in breast cancer cells (P<0.05).ConclusionPER1 functions as a tumor suppressor in the development and progression of breast cancer, and its expression silencing might be regulated by epigenetic modifications.

Characterization of green synthesis, in silico docking and biological activity studies of N, N′-BIS (3-aminopropyl) piperazine derivatives

Novel bioactive piperazine derivatives are synthesised and charecterised by IR, NMR, UV, and elemental analysis techniques. The biological activity of the synthesised materials is investigated by using In vitro and docking experiments. The novel imines were prepared in quantitative yield using the microwave technique. The compound 2a and 3a are charecterised IR, NMR, UV, and elemental analysis techniques and confirm the formation of the desired product. The antimicrobial activity of the synthesised compounds showed excellent activity with compound 2a and 3a having the highest activity of about 6 µg/ml against Enterococcus Faecalis and Pseudomonas aeruginosa, respectively. The antioxidant properties of the synthesised compounds were investigated and the compound 2a has the smallest LC50 value (9 µg/ml). In silico docking of the BRCA1 protein in breast cancer cells confirmed an excellent binding affinity value of −7.10 kcal/mol. Similarly, invitro anticancer analysis on the MDA MB-231 breast cancer cell line indicate that the drug-treated cells have a uniform structure with few abnormalities.

Study of novel bioactive n, n'-bis (3-aminopropyl) piperazine derivatives: Green synthesis, spectral characterization, in silico docking and biological activity

Bioactive properties of piperazine derivatives are well known for its biological applications. In general, synthesis of bioactive compound is laborious and time consuming process. In the present investigation, the Microwave was used to synthesise two imines compounds using piperazine and various aldehydes in a quantitative yield. IR, NMR, UV, and elemental analysis techniques were used to characterise the compound 2a and 3a. In vitro method and docking experiments were used to investigate biological activities novel piperazines compounds. The antimicrobial activity of the synthesised compounds showed better results against Pseudomonas aeruginosa, with a MIC of 11.9 µg/ml. The antioxidant properties of the synthesised compounds were found to be high with the compound 2a has the smallest LC50 value (15 µg/ml). In silico docking of the BRCA1 protein in breast cancer cells confirmed an excellent binding affinity value of −7.10 kcal/mol. The findings of the anticancer analysis on the MDA MB-231 breast cancer cell line indicate that the drug-treated cells have a uniform structure with few abnormalities. Therefore, the synthesised compounds can be used in different biological applications.

Anti-proliferative activity of disulfiram through regulation of the AKT-FOXO axis: A proteomic study of molecular targets

Due to its potent anti-tumor activity, well-investigated pharmacokinetic properties and safety profile, disulfiram (DSF) has emerged as a promising candidate for drug repurposing in cancer therapy. Although several molecular mechanisms have been proposed for its anti-cancer effects, the precise underlying mechanisms remain unclear. In the present study, we showed that DSF inhibited proliferation of cancer cells by inducing reactive oxygen species (ROS) production, a G1 cell cycle arrest and autophagy. Moreover, DSF triggered apoptosis via suppression of the anti-apoptotic protein survivin.To elucidate the mechanisms for the anti-proliferative activities of DSF, we applied a 2-DE combined with MALDI-TOF-MS/MS analysis to identify differentially expressed proteins in breast cancer cells upon treatment with DSF. Nine differentially expressed proteins were identified among which, three candidates including calmodulin (CaM), peroxiredoxin 1 (PRDX1) and collagen type I alpha 1 (COL1A1) are involved in the regulation of the AKT signaling pathway. The results of western blot analysis confirmed that DSF inhibited p-AKT, suggesting that DSF induces its anti-tumor effects via AKT blockade. Moreover, we found that DSF increased the mRNA levels of FOXO1, FOXO3 and FOXO4, and upregulated the expression of their target genes involved in G1 cell cycle arrest, apoptosis and autophagy. Finally, DSF potentiated the anti-proliferative effects of well-known chemotherapeutic agents such as arsenic trioxide (ATO), doxorubicin, paclitaxel and cisplatin. Altogether, these findings provide mechanistic insights into the anti-growth activities of DSF.

Host-Guest Interaction-Based Dual response core/shell nanoparticles as efficient siRNA carrier for killing breast cancer cells

How to overcome multiple obstacles to achieve the efficient and safe delivery of therapeutic genes is still the key to gene therapy. To address this issue, a cationic carrier consisting of polyamide-amine (HPAA-peptide-Fc) modified by an enzyme-responsive polypeptide as the core and hyperbranched polyglycerol derivative (CD-HPG) as the shell was synthesized by self-assembly. The obtained HPAA-peptide-HPG could form the compact nanocomplex with siPlk1, thus confirming the stable load of genes and subsequent targeted gene delivery. And the nanogenes could significantly induce apoptotic effect via the down-expression of Plk1 protein in breast cancer cells. Moreover, compared to polyethylenimine, HPAA-peptide-HPG exhibited superior biocompatibility through hemolysis and cell viability assays because of the shielding function of CD-HPG, thereby being beneficial to increasing the circulation time of the complex when administrated in vivo. Such an efficient and safe gene delivery complex (HPAA-peptide-HPG) presents a good example of rational design of cationic supramolecular vesicles for stimulus-responsive siRNA transport, which should be encouraged in cancer gene therapy.

Synthesis and intensive characterization for novel Zn(II), Pd(II), Cr(III) and VO(II)-Schiff base complexes; DNA-interaction, DFT, drug-likeness and molecular docking studies

A novel bioactive series was synthesized from Zn(II), Pd(II), Cr(III) and VO(II) ions with a new Schiff base derivative (HNP) [HNP = 1-(Pyrimidin-2-yliminomethyl)-naphthalen-2-ol]. The proposed structures were defined from elemental analysis, molar conductivity, magnetic moment, IR, 1H NMR, UV-Vis, and TGA. Based on analytical and spectroscopic data, suitable geometry has been suggested for all complexes. The HNP ligand acts as a tri-dentate via NNO donors towards the metal ions inside octahedral geometry with Cr(III), square pyramidal with VO(II), tetrahedral with Zn2+ and square planner with Pd2+ ion. The kinetic and thermodynamic parameters of complexes have been determined by using Coats-Redfern method and their values suggested that the activated complexes were more ordered. The binding efficiency of the investigated complexes with calf thymus DNA (ctDNA), was examined by using different methods and the binding feature was suggested to be electrostatic, intercalation, or replacement mode. Moreover, in-vitro antimicrobial and anticancer potency of the compounds were evaluated. The new complexes showed an effective impact on decreasing cell viability of breast carcinoma cells (MCF-7) in a concentration-dependent manner. In-vitro antioxidant activity of all compounds was investigated and the results showed appreciated free radical scavenging activity of Pd(II) complex. Furthermore, Molecular docking inspection has been carried out to explain the binding affinity of the tested compounds towards breast cancer cell-protein (PDB: 3hb5)

Exosomal microRNA-503-3p derived from macrophages represses glycolysis and promotes mitochondrial oxidative phosphorylation in breast cancer cells by elevating DACT2

MicroRNAs (miRNAs) are emerging drivers in tumor progression, while the role of miR-503-3p in breast cancer (BC) remains largely unknown. We aimed to explore the impact of macrophage-derived exosomal miR-503-3p in the development of BC by regulating disheveled-associated binding antagonist of beta-catenin 2 (DACT2). miR-503-3p and DACT2 expression in BC tissues and cells was assessed, and the expression of Wnt/β-catenin signaling pathway-related proteins in BC cells was also evaluated. Macrophages were induced and exosomes were extracted. The screened BC cell lines were, respectively, treated with exosomes, miR-503-3p inhibitor/mimic or upregulated/inhibited DACT2, and then the phenotypes, glucose intake, oxygen consumption rate, and adenosine-triphosphate (ATP) level of BC cells were determined. Cell growth in vivo was also observed. MiR-503-3p was elevated, DACT2 was reduced, and Wnt/β-catenin signaling pathway was activated in BC cells. Macrophage-derived exosomes, upregulated miR-503-3p or inhibited DACT2 promoted malignant behaviors of BC cells, glucose intake, and activity of the Wnt/β-catenin signaling pathway, while repressed oxygen consumption rate and ATP level in BC cells. Reversely, reduced miR-503-3p or upregulated DACT2 exerted opposite effects. This study revealed that reduction of macrophage-derived exosomal miR-503-3p repressed glycolysis and promoted mitochondrial oxidative phosphorylation in BC by elevating DACT2 and inactivating Wnt/β-catenin signaling pathway. Our research may provide novel targets for BC treatment.

Zygote arrest 1 like (ZAR2) and cell cycle in breast cancer cells

ZAR2 is a novel RNA-binding C4 zinc finger containing protein, with cell cycle dependent differential expression and nuclear/ cytosolic localization. We observed that ZAR2 expression is relatively lower in triple negative breast cancer cells (TNBC) compared to non-TNBC and forced expression in TNBC cells inhibit their growth and invasiveness. Knocking down ZAR2 in non-TNBC cells increased their growth and invasiveness. Our studies showed that ZAR2, is inhibited by a c-Myc regulated miRNA, hsa-miR-130a-5p. Studies demonstrate that ZAR2 localizes in the RNA processing P-bodies and regulates the turnover of maternal WEE1 mRNA in developing oocytes. WEE1 is a tyrosine kinase, which regulates G2/M checkpoint in cell cycle, is over-expressed in various cancer types and is associated with resistance development to DNA-damaging agents. WEE1, is identified as a potential molecular target for TNBC. WEE1 inhibition is exclusively toxic for cancer cells and does not harm healthy cells. Major objective of the proposed research is to evaluate the potential of a novel protein ZAR2 as a modulator of G2-M checkpoint and therapeutic target in breast cancer. c-Myc mediated downregulation of ZAR2 expression via hsa-miR-130a-5p, results in increased levels of WEE1 protein in breast cancer cells, increased proliferation and faster progression through cell cycle.

Label‐free subcellular mapping of iron‐bound proteins in breast cancer cells

Dysregulation of iron transport, storage and utilization is at the basis of a variety of human diseases, since free iron can participate in redox reactions that are deleterious to lipid membranes and signaling pathways. As intracellular iron transport occurs in a pH-dependent manner, changes in endosomal pH, often associated with cancer, could lead to alterations in iron homeostasis. Essential to iron transport is transferrin (Tf), which binds iron at a neutral pH for safe transport throughout the body and releases it upon uptake and delivery to a mildly acidified endosome. Excess iron can be stored in cytoplasmic protein ferritin (Ft) or delivered into mitochondria to be incorporated into proteins with heme or iron-sulfur structures. Recently, we have developed a novel label-free Raman hyperspectral microscopy methodology to identify the subcellular distributions and relative quantities of iron-bound Tf, Ft and heme proteins within heterogenous cell populations. Previously, we have shown that Raman hyperspectral imaging could identify endocytic distributions of iron-bound Tf using a specific Raman peak at 1300 cm-1 in intact breast cancer cells. Moreover, we have shown that alterations in the subcellular localization of iron-bound Tf populations correlated with altered endocytic regulation of iron-bound Tf in two distinct breast cancer cell lines. Based on these results, we propose that breast cancer cells display a disrupted iron homeostasis due to iron release delays caused by alterations in the pH or ionic milieu of the early endosomes. Currently, we are testing a putative correlation between pH gradient alterations and iron release delays in different breast cancer cells. To further investigate cellular iron regulation, Ft cell-specific Raman signature was determined via evaluation of cells incubated in the presence or absence of iron-loaded Ft. Since cells may internalize Ft through an endocytic pathway, this approach allowed for identification of Ft Raman signature. However, the subcellular distribution of internalized iron-loaded Ft may not be reflective of normal cytoplasmic Ft in cells. Internalization of iron-loaded Ft results in multiple significantly different Raman spectra peaks at several wavelengths. Importantly, we have used multivariate analysis to show a significant separation of cell Raman spectra from that of cells incubated with Ft. Further we can map a previously known heme protein peak at 1372 cm-1, which is specifically associated with the ferric iron heme. In the future, following further validation of Ft peaks the mapping of Tf, Ft and heme can be evaluated across multiple cell lines and tissue samples. The application of this research has far reaching potential in understanding how dysregulation of endocytic pH may lead to alterations in intracellular iron transport and storage in breast cancer cells.

Abstract OT-03-03: SGNLVA-001: A phase 1 open-label dose escalation and expansion study of SGN-LIV1A administered weekly in breast cancer

Abstract Background: LIV-1 is a highly prevalent transmembrane protein in breast cancer cells. Ladiratuzumab vedotin (LV), SGN-LIV1A, is an investigational antibody-drug conjugate (ADC) that targets LIV-1 via a humanized IgG1 monoclonal antibody conjugated to monomethyl auristatin E (MMAE) by a protease-cleavable linker. LV is internalized when it binds LIV-1 on cell surfaces and MMAE is released, which binds tubulin and induces apoptosis. LV has been shown to be active and tolerable in metastatic breast cancer (mBC) at a recommended dose of 2.5 mg/kg every 21 days (Modi 2017). More frequent, fractionated dosing has improved the activity and/or safety of other ADCs. Thus, this study is actively accruing subjects with metastatic triple negative breast cancer (mTNBC; estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor 2 (HER2) receptor-negative) and endocrine-resistant ER+ or PR+ (hormone receptor [HR+])/HER2-negative mBC to test weekly dosing of LV (Days 1, 8, and 15 of every 3-week cycle). Methods: This study is enrolling up to 82 subjects (42 HR+/HER2-negative and 40 mTNBC) into dose escalation and dose expansion cohorts (NCT01969643). Eligible subjects are females ≥18 years old with pathologically and radiologically confirmed metastatic HR+/HER2-negative or mTNBC with at least 1 measurable lesion per RECIST v1.1. Subjects with HR+/HER2-negative disease must have received no more than 1 prior line of cytotoxic chemotherapy in the locally advanced (LA)/mBC setting, either as single agent or combination therapy. Subjects with mTNBC must have received 1 prior line of cytotoxic chemotherapy in the LA/mBC setting. Progression within 6 months of completion of neoadjuvant or adjuvant therapy is considered an LA/mBC regimen. Subjects must have adequate organ function, ECOG status of ≤1, and no ≥ Grade 2 peripheral neuropathy. Subjects with brain lesions must have received definitive treatment of the lesions. Prior therapy with MMAE-containing agents is not allowed. Dose escalation follows the modified toxicity probability interval method (Ji 2010). Dose expansion cohorts will provide data about activity and tolerability. Tumor assessments will be conducted every 2 cycles per RECIST v1.1 and all subjects will be followed for safety. Pharmacokinetics and markers of pharmacodynamics will be assessed. Primary safety endpoint is the incidence of adverse events and dose-limiting toxicities. Key efficacy endpoints include confirmed overall response rate, duration of response, and progression-free survival. Citation Format: Heather C Beckwith, Diana C Medgyesy, Jame Abraham, Rita Nanda, Katherine HR Tkaczuk, Ian E Krop, Lajos Pusztai, Shanu Modi, Monica M Mita, Jennifer M Specht, Sara A Hurvitz, Hyo S Han, Kevin Kalinsky, Sharon Wilks, Joyce O’Shaughnessy, Lowell L Hart, Hope S Rugo, Zahi I Mitri, Phillip M Garfin, Howard A Burris III. SGNLVA-001: A phase 1 open-label dose escalation and expansion study of SGN-LIV1A administered weekly in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-03-03.

Differential profiles of HDAC1 substrates and associated proteins in breast cancer cells revealed by trapping.

Histone deacetylase (HDAC) proteins, which regulate the acetylation state of proteins, are the targets of multiple clinical drugs for cancer treatment. Due to the heterogeneity of tumors, HDAC proteins play different roles in the progression of various cancer types. For example, MDA-MB-468 and MDA-MB-231 cells are both triple negative breast cancer cells but belong to different subtypes that display different response to HDAC inhibitor drugs. To investigate the role of HDAC proteins in breast cancer, the substrate and associated proteins of HDAC1 in MDA-MB-231, MDA-MB-468, and a normal breast epithelial cell line, MCF10A, were analyzed using substrate trapping mutants and proteomics-based mass spectrometry. All three cell lines demonstrated nonoverlapping substrate protein profiles. While both normal MCF10A and cancerous MDA-MB-468 cell lines contained similar HDAC1 associated proteins, including proteins associated with epigenetic and RNA processing mechanisms, the HDAC1 associated protein profile of MDA-MB-231 cells was devoid of expected epigenetic proteins. The variable associated protein profiles of MDA-MB-231 and MDA-MB-468 suggest that HDAC1 plays distinct roles in breast cancer cell biology, which might affect cancer aggressiveness and HDAC inhibitor sensitivity.

Abstract 2605: Cytokeratin 5 promotes endocytosis to remodel cell adhesions in breast cancer

Abstract Breast cancer (BC) is clinically classified according to expression of estrogen receptor, progesterone receptor (ER+ BC), HER2 (HER2+), or absence of these markers (triple negative BC). While TNBC has the inferior prognosis in the short term, ER+ breast cancer has an extended risk of recurrence that lasts up to 20 years and accounts for the most deaths overall. Cytokeratin 5 (CK5) is an intermediate filament protein that is expressed in the majority of TNBC cases and 10-50% of ER+ BC. In CK5+/ER+ BC, there is widespread heterogeneity in the amount of CK5+ tumor cells (~1-50%). The mere presence of CK5+ cells is an indicator of poor prognosis across both subtypes of BC; the reasons for this are poorly understood. We have shown that CK5+ cells within ER+ and TNBC cell lines, and heterogeneous PDX tumor models lose membrane localization of adherens junction proteins β-catenin and E-cadherin. Loss of membrane β-catenin and E-cadherin is associated with poor prognosis in breast cancer and is a precursor to cell invasion. CK5+ cells have been reported to have increased invasive potential by us and others. Thus, identifying the mechanism of CK5-dependent loss of adherens proteins at the membrane could lead to development of therapies targeting this process. β-catenin and E-cadherin localization have been reported to be regulated through endocytosis which is mediated through the small GTPase family of Rab proteins. We performed a screen to identify potential CK5 interacting proteins in breast cancer cells and identified several Rab proteins. Thus, we hypothesize that CK5 acts as a scaffold for Rabs to promote endocytosis of β-catenin and E-cadherin to increase invasive potential.To investigate this, we treated CK5 overexpressing ER+ and TNBC cells with the endocytosis inhibitor Dyngo4a and found membrane β-catenin and E-cadherin localization were restored, suggesting that CK5 may be mediating endocytosis of β-catenin and E-cadherin. Rab5 is responsible for trafficking vesicles from the plasma membrane to the early endosomes. CK5 was confirmed to interact with Rab5 by co-IP in TNBC and CK5 overexpressing ER+ cell lines. In ongoing studies, we are testing whether Rab5 knockdown prevents loss of membrane β-catenin and E-cadherin and decreases cell invasiveness. These experiments will collectively determine whether CK5 regulated endocytosis is a targetable feature in breast cancer cells. Citation Format: Olivia Frances McGinn, Ashley V. Ward, Jessica Finlay-Schultz, Kiran Vinod Paul, Peter Kabos, Carol Sartorius. Cytokeratin 5 promotes endocytosis to remodel cell adhesions in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2605.

The guanine nucleotide exchange factor VAV3 participates in ERBB4-mediated cancer cell migration

ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4-VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.

Cholesterol and Mevalonate: Two Metabolites Involved in Breast Cancer Progression and Drug Resistance through the ERRα Pathway.

Breast cancer is the second greatest cause of cancer-related death in women. Resistance to endocrine treatments or chemotherapy is a limiting drawback. In this context, this work aims to evaluate the effects of cholesterol and mevalonate during tumor progression and their contribution in the onset of resistance to clinical treatments in use today. In this study, we demonstrated that cholesterol and mevalonate treatments were able to activate the estrogen-related receptor alpha (ERRα) pathway, increasing the expression levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), ERbB2/human epithelial receptor (HER2), tumor protein D52 (TPD52), and NOTCH2 proteins in breast cancer cells. The activation of this pathway is shown to be responsible for intense metabolic switching, higher proliferation rates, sustained motility, the propagation of cancer stem-like cells (CSCs), and lipid droplet formation. All of these events are related to greater tumor propagation, aggressiveness, and drug resistance. Furthermore, the activation and expression of proteins induced by the treatment with cholesterol or mevalonate are consistent with those obtained from the MCF-7/TAMr cell line, which is largely used as a breast cancer model of acquired endocrine therapy resistance. Altogether, our data indicate that cholesterol and mevalonate are two metabolites implicated in breast cancer progression, aggressiveness, and drug resistance, through the activation of the ERRα pathway. Our findings enable us to identify the ERRα receptor as a poor prognostic marker in patients with breast carcinoma, suggesting the correlation between cholesterol/mevalonate and ERRα as a new possible target in breast cancer treatment.

Erastin triggers autophagic death of breast cancer cells by increasing intracellular iron levels.

Erastin is a small molecular compound that induces ferroptosis by binding to voltage-dependent anion-selective channel protein (VDAC)2, VDAC3 and solute carrier family 7 member 5 inhibiting the cystine/glutamate antiporter. However, to the best of our knowledge, the mechanism of erastin-induced breast cancer cell death remains unclear. In present study aimed to explore the underlying mechanisms of the antitumor effects of erastin on breast cancer cells. Cellular viability was assessed using an MTT assay, a lactate dehydrogenase cytotoxicity assay kit was used to determine the cell death rate, the intracellular Fe2+ levels were determined using an iron colorimetric assay kit and western blotting was used to estimate the changes of autophagy-associated proteins levels. The present study demonstrated that erastin inhibited the viability of breast cancer cells and induced breast cancer cell death in a dose-dependent manner. Additionally, autophagy was activated by erastin, as demonstrated by upregulated expression levels of autophagy-associated proteins in breast cancer cells. Bafilomycin A1, 3-methyladenine and knockdown of autophagy related (ATG)5 with small interfering RNA prevented erastin-induced breast cancer cell death and inhibited the erastin-induced changes in the expression levels of the autophagy-associated proteins beclin1, ATG5, ATG12, microtubule-associated proteins 1A/1B light chain 3B (LC3B) and P62. Furthermore, erastin-induced breast cancer cell death was inhibited by an iron chelator, deferoxamine, which inhibited the increases of erastin-induced iron levels and inhibited the erastin-induced changes in the expression levels of the autophagy-related proteins beclin1, ATG5, ATG12, LC3B and P62. In summary, erastin triggered autophagic death in breast cancer cells by increasing intracellular iron levels.