Profiling Of Breast Cancer Cells Research Articles

Gene Expression Profiling of Maslinic Acid-treated MCF-7 Breast Cancer Cells Using Nanostring nCounter Pancancer Pathway Panel

Breast cancer remains a significant global health concern, impacting millions of women every year. Maslinic acid (MA), a pentacyclic triterpene has been found to exert promising anticancer effect in various cancers, including breast cancer, yet the underlying mechanisms remain unclear. This study aims to elucidate the anticancer properties of MA via gene expression profiles in breast cancer cells. Cytotoxicity assay results revealed that MCF-7 exerts the highest sensitivity after 72 h of MA treatment followed by T-47D and MDA-MB-231. MCF-7 were then selected for in-depth analysis using the Nanostring nCounter Pancancer Pathway Panel to analyze the differential expression of genes (DEGs). Across three time points (24, 48, and 72 h), 20 significant DEGs were identified, of which 5 were upregulated and 15 were downregulated. In silico analysis indicated that these DEGs were involved in Pathway of Cancer, Focal Adhesion-PI3K-mTOR Signaling Pathway, PI3K-Akt, and Ras Signaling Pathway. The regulation of these DEGs contributes to several cellular activities such as apoptosis, inhibition of cell proliferation, cell cycle and survival, reduction of glycolysis, angiogenesis, and DNA repair. Additionally, the unfolded protein response emerged as a noteworthy biological process in this study. This study unravels the molecular mechanisms underpinning the therapeutic potential of MA against breast cancer.

Exploring Anticancer Properties of Medicinal Plants against Breast Cancer by Downregulating Human Epidermal Growth Factor Receptor 2.

Plants have a history of being employed in managing breast cancer. However, no scientific evidence supports the idea that these plants can effectively reduce the level of HER2 expression. In this study, extracts from 10 medicinal plants were evaluated for their anticancer properties against HER2-positive breast cancer cells through various methods, including the SRB assay, comet assay, annexin V-FITC dual staining, and immunoblotting. All extracts exerted antiproliferative activity against HER2-positive breast cancer cells. Furthermore, Terminalia chebula (T. chebula), Berberis aristata (B. aristata), and Mucuna pruriens (M. pruriens) reduced HER2 expression in tested cell lines. In addition, an increased Bax/Bcl-2 ratio was observed after the treatment. A comparative proteomics study showed modulation in the proteome profile of breast cancer cells after treatment with T. chebula, B. aristata, Punica granatum, M. pruriens, and Acorus calamus. Metabolic profiling of lead plants revealed the existence of multiple anticancer compounds. Our study demonstrates the considerable potential of the mentioned plants as innovative therapies for HER2-positive breast cancer.

Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation.

More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.

Mapping metabolic perturbations induced by glutathione activatable synthetic ion channels in human breast cancer cells

Ion channels and transporters play key roles in various biological processes, including cell proliferation and programmed cell death. Recently, we reported that 2,4-dinitrobenzene-sulfonyl-protected N1,N3-dihexy-2-hydroxyisophthalamide (1) forms ion channels upon activation by glutathione (GSH) and results in the induction of apoptosis by depleting the intracellular GSH reservoir in cancer cells. However, the detailed molecular events leading to the induction of apoptosis by these synthetic transport systems in cancer cells still need to be uncovered. Along these lines, we investigated the alterations in cellular metabolites and the associated metabolic pathways by performing untargeted global metabolic profiling of breast cancer cells – MCF-7 – using 1H NMR-based metabolomics. The evaluation of spectral profiles from MCF-7 cells exposed to 1 and their comparison with those corresponding to untreated (control) cells identified 14 significantly perturbed signature metabolites. These metabolites belonged mostly to antioxidant defence, energy metabolism, amino acid biosynthesis, and lipid metabolism pathways and included GSH, o-phosphocholine, malate, and aspartate, to name a few. These results would help us gain deeper insights into the molecular mechanism underlying 1-mediated cytotoxicity of MCF-7 cells and eventually help identify potential novel therapeutic targets for more effective cancer management.

Transcriptomic Profiling of Breast Cancer Cells Induced by Tumor-Associated Macrophages Generates a Robust Prognostic Gene Signature.

Breast cancer, one of the most prevalent neoplasms in the world, continues attracting worldwide attention. Macrophage, as the most abundant non-malignant cell in tumor, plays critical roles in both immune surveillance and tumorigenesis and has become a cell target of immunotherapy. Among all macrophages, tumor-associated macrophage (TAM) is regarded as the main force to promote tumorigenesis. To get an overall view of its impact on breast cancer, we employed a simplified and indirect coculturing cell model followed by RNA-sequencing to detect cancer cell's transcriptomic response induced by TAM and a prognostic gene signature was constructed based on it. Evidence from both cell models and clinical samples strengthened TAM's full-dimensional impact on breast cancer, involved in almost all known signal pathways dysregulated during tumorigenesis from transcription, translation and molecule transport to immune-related pathways. Consequently, the gene signature developed from these genes was tested to be powerful in prognostic prediction and associated with various clinical and biological features of breast cancer. Our study presented a more complete view of TAM's impact on breast cancer, which strengthened its role as an important therapy target. A 45-gene signature from the TAM-regulated genes was developed and shown potential in clinical application.

Heterogeneous microenvironmental stiffness regulates pro-metastatic functions of breast cancer cells

Besides molecular and phenotypic variations observed in cancer cells, intratumoral heterogeneity also occurs in the tumor microenvironment. Correlative stiffness maps of different intratumor locations in breast tumor biopsies show that stiffness increases from core to periphery. However, how different local ECM stiffness regulates key functions of cancer cells in tumor progression remains unclear. Although increased tissue stiffness is an established driver of breast cancer progression, conclusions from 2D cultures do not correspond with newer data from cancer cells in 3D environments. Many past studies of breast cancer in 3D culture fail to recapitulate the stiffness of a real breast tumor or the various local stiffnesses present in a tumor microenvironment. In this study, we developed a series of collagen/alginate hybrid hydrogels with adjustable stiffness to match the core, middle, and peripheral zones of a breast tumor. We used this hydrogel system to investigate effects of different local stiffness on morphology, proliferation, and migration of breast cancer cells. RNA sequencing of cells in hydrogels with different stiffness revealed changes in multiple cellular processes underlying cancer progression, including angiogenesis and metabolism. We discovered that tumor cells in a soft environment enriched YAP1 and AP1 signaling related genes, whereas tumor cells in a stiff environment became more pro-angiogenic by upregulating fibronectin 1 (FN1) and matrix metalloproteinase 9 (MMP9) expression. This systematic study defines how the range of environmental stiffnesses present in a breast tumor regulates cancer cells, providing new insights into tumorigenesis and disease progression at the tumor-stroma interface. Statement of significanceApplied a well-defined hybrid hydrogel system to mimic the tumor microenvironment with heterogeneous local stiffness. Breast cancer cells tended to proliferate in soft core environment while migrate in stiff peripheral environment. Breast cancer cells shift from glycolysis to OXPHOS and fatty acid metabolism responding to stiff matrix microenvironment. The transcriptomic profile of breast cancer cells altered due to microenvironmental stiffness changes.

Variation of Long Non-Coding RNA And mRNA Profiles in Breast Cancer Cells With Influences of Adipocytes.

BackgroundIt is well known that obesity is one of the risks for incurrence and development in breast cancer patients. Long non-coding RNAs (lncRNAs) are reported to participate in the composition of tumor microenvironment and to regulate breast cancer cell metabolic activities. However, there was rare study focused on the lncRNAs in breast cancer with the influences of adipocytes. The study aimed to investigate lncRNAs expression profiles and discover potential biomarkers to predict the incidence and progression of adipocyte-associated-breast cancer.MethodsWe co-cultured adipocytes with breast cancer cells and profiled the expression of lncRNAs as well as mRNAs by using the RNA-sequencing method. Wound Healing, Migration assays and Invasion assays were applied to verify the invasion and metastasis of cancer cells.ResultsMDA-MB-231/Hpa-V and SK-BR-3/Hpa-V cells showed elevated migration and invasiveness compared to the control group. A sum of 371 mRNAs (181 upregulated and 190 downregulated) and 850 lncRNAs(414 upregulated and 436 downregulated) were differentially expressed in MDA-MB-231/Hpa-V comparing to MDA-MB-231(P < 0.05; |log2 (fold change)|>1.2). GO enrichment, KEGG pathway and interaction networks demonstrated that differentially expressed lncRNAs were involved in functional categories, such as material metabolism, which might lead to the progression of breast cancer.ConclusionOur study detected a lncRNA profile in breast cancer cells affecting by adipocytes and provided a better understanding of the tumor microenvironment. LncRNAs may be helpful to predict the therapeutic responses and prognosis of obese breast cancer patients.

Evaluation of the anticancer effect of violacein, phycocyanin and phycocyanobilin on apoptotic genes expression and glycan profiles in breast cancer cells.

Background: Cancer researchers have been concentrated on finding the best therapeutic strategies to decline cancer mortality rates. Marine natural products with pharmacological activities have potent anticancer activities. Aim: This study investigated the anticancer effect of violacein, phycocyanin, and phycocyanobilin in MCF-7 cells. Additionally, the elucidation of combined therapy efficiency (violacein-phycocyanin) on enhancing the anticancer activity of monotherapy. Materials and Methods: The cultured untreated, and treated cells by etoposide, violacein, phycocyanin, phycocyanobilin, and combined therapy were subjected to molecular studies, and glycan profiles by real time-PCR, and MALDI-TOF respectively. Results: Bax, Bcl-2, caspase-3 genes expression, and Bax/Bcl-2 ratio in all groups were significantly upregulated compared to the untreated cells except for a significant decrease in Bcl-2 by etoposide or combined therapy and an insignificant difference in Bax/Bcl-2 by PC or PCB treatment. The intensity of tetra-, tri-sialylated N-glycan, and di-sialylated O-glycan was significantly decreased in violacein, and combined-treated cells when compared to untreated or PC-treated cells. Mono-sialylated O-glycan was also decreased in combined therapy compared to the treated or untreated cells. Conclusion: Cotreatment with violacein/PC generated synergistic, antiproliferative, and pro-apoptotic effects on MCF-7 cells. Therefore, these compounds may be promising chemotherapeutic agents for breast cancer

Self‐Assembly of Cholesterol‐Doxorubicin and TPGS into Prodrug‐Based Nanoparticles with Enhanced Cellular Uptake and Lysosome‐Dependent Pathway in Breast Cancer Cells

AbstractDeveloping new easy‐to‐prepare functional drug delivery nanosystems with good storage stability, low hemotoxicity, as well as controllable drug delivery property, has attracted great attention in recent years. In this work, a cholesterol‐based prodrug nanodelivery system is prepared by self‐assembly of cholesterol‐doxorubicin prodrug conjugates (Chol‐Dox) and tocopherol polyethylene glycol succinate (TPGS) using thin‐film hydration method. The Chol‐Dox/TPGS assemblies (molar ratio 2:1, 1:1, and 1:2) are able to form nanoparticles with average hydrodynamic diameter of ≈140–214 nm, surface zeta potentials of ≈−24.2–−0.3 mV, and remarkable solution stability in 0.1 m PBS, 16 days). The Chol‐Dox/TPGS assemblies show low hemotoxicity and different cytotoxicity profiles in breast cancer cells (MCF‐7 and MDA‐MB‐231), which are largely dependent on the molar ratio of Chol‐Dox and TPGS. The Chol‐Dox/TPGS assemblies tend to enter into MCF‐7 and MDA‐MB‐231 cells through non‐Clathrin‐mediated multiple endocytosis and lysosome‐dependent uptake pathways, moreover, these nanoassemblies demonstrate lysosome‐dependent intracellular localization, which is different from that of free DOX (nuclear localization). The results demonstrate that the Chol‐Dox/TPGS assemblies are promising cholesterol‐based prodrug nanomaterials for breast cancer chemotherapy.Practical Applications: This work demonstrates a lipid prodrug‐based nanotherapeutic system. Herein the Chol‐Dox/TPGS nanoassemblies could serve as promising and controllable cholesterol‐based prodrug nanomaterials/nano‐formulations for potential breast cancer chemotherapy.

Cell growth analysis and nucleotide excision repair modulation in breast cancer cells submitted to a protocol using doxorubicin and paclitaxel

IntroductionOne of the most used regimens to treat breast cancer is the dose-dense ACT protocol, a combination of anthracycline doxorubicin (DOX) with cyclophosphamide and paclitaxel (PCTX). However, many tumors show resistance to the protocols applied. It is known that the nucleotide excision repair (NER) pathway acts by removing the DOX-generated lesions, and this, together with other DNA repair pathways, can modulate the response to treatment. AimsTo evaluate the in vitro growth profile of breast cancer cells (MCF7), and the modulation of DNA repair genes, submitted to a protocol using DOX and PCTX in a similar regimen to what is used in clinical practice. Main methodsMCF7 cells were treated with repeated cycles of DOX and PCTX and followed-up during and after each of the treatments. The population doubling of the remaining cells was calculated during the complete protocol and DNA repair gene expression was evaluated at different time-points. Key findingsAn increase in all NER genes analyzed after the DOX treatment was observed, but not after the PCTX treatment. MRE11was overexpressed at all evaluated time-points. There was a resumption of NER genes overexpression profile when cells were maintained for follow-up and retook their growth pattern, indicating that DNA repair pathways can modulate their expression during the chemotherapy exposure.

Differential epigenetic profiles induced by sodium selenite in breast cancer cells

ObjectivesSelenium (Se) was a potential anticancer micronutrient with proposed epigenetic effect. However, the Se-induced epigenome in breast cancer cells was yet to be studied. MethodsThe profiles of DNA methylation, microRNA (miRNA), long non-coding RNA (lncRNA), and message RNA (mRNA) in breast cancer cells treated with sodium selenite were examined by microarrays. We verified the epigenetic modifications by integrating their predicted target genes and differentially expressed mRNAs. The epigenetically regulated genes were further validated in a breast cancer cohort by associating with tumor progression. We conducted a series of bioinformatics analyses to assess the biological function of these validated genes and identified the critical genes. ResultsThe Se-induced epigenome regulated the expression of 959 genes, and 349 of them were further validated in the breast cancer cohort. Biological function analyses suggested that these validated genes were enriched in several cancer-related pathways, such as PI3K/Akt and metabolic pathways. Based on the degrees of expression change, hazard ratio difference, and connectivity, NEDD4L and FMO5 were identified as the critical genes. ConclusionsThese results confirmed the epigenetic effects of sodium selenite and revealed the epigenetic profiles in breast cancer cells, which would help understand the mechanisms of Se against breast cancer.

Enhancer RNAs Mediate Estrogen-Induced Decommissioning of Selective Enhancers by Recruiting ERα and Its Cofactor

SUMMARYThe function of enhancer RNAs (eRNAs) in transcriptional regulation remains obscure. By analyzing the genome-wide nascent transcript profiles in breast cancer cells, we identify a special group of eRNAs that are essential for estrogen-induced transcriptional repression. Using eRNAs of TM4SF1 and EFEMP1 as the paradigms, we find that these RNA molecules not only stabilize promoter-enhancer interactions but also recruit liganded estrogen receptor α (ERα) to particular enhancer regions, facilitate the formation of a functional transcriptional complex, and cause gene silencing. Interestingly, ERα is shown to directly bind with eRNAs by its DNA-binding domain. These eRNAs help with the formation of a specific ERα-centered transcriptional complex and promote the association of the histone demethylase KDM2A, which dismisses RNA polymerase II from designated enhancers and suppresses the transcription of target genes. Our work demonstrates a complete mechanism underlying the action of eRNAs in modulating and refining the locus-specific transcriptional program.

SAT-136 Hypoxia Effect on Cytokine Production by Breast Cancer Cells

Inflammation is a critical component of tumor initiation and progression. Chronic inflammation triggers molecular events that can promote carcinogenesis, tumor vascularization and metastasis. As inflammatory mediators, cytokines play an important role in the interplay between the tumor cells and tumor microenvironment. Cytokines released by the tumor-associated macrophages modulate cancer cell survival, stemness, invasiveness, and tumor vascularization. Breast cancer cells, however, also produce a variety of cytokines, whose role in cancer development is poorly understood. The aim of our study was to characterize the basal cytokine secretory activity in commonly used human breast cancer cell lines (MDA-MB-231, MCF-7, BT-474, and T-47D). Using MILLIPLEX assay, we measured the expression of 41 cytokines, including interleukins, monokines, interferons and growth factors. We also compared cytokine expression profile of breast cancer cells with those of non-tumorigenic human breast epithelial MCF-10A cells. Further, we investigated whether hypoxia modulates cytokine secretion of breast cancer cells. Using cobalt(II) chloride (CoCl2) to mimic hypoxia, we compared the effect of various treatment doses and intervals on cytokine production in the breast cancer cells. Results demonstrated that CoCl2 affects the release of multiple cytokines in a dose- and concentration-dependent manner, thus highlighting the role of cancer cell-derived cytokines on breast tumor progression. Understanding the molecular actions of cytokines in the tumor microenvironment is important for understanding the mechanism of cancer initiation and progression.

MYC-driven regulation of long non-coding RNA profiles in breast cancer cells

AimMYC deregulation contributes to breast cancer development and progression. Deregulated expression levels of long non-coding RNAs (lncRNA) have been demonstrated to be critical players in development and/or maintenance of breast cancer. In this study we aimed to evaluate lncRNA expressions depending on MYC overexpression and knockdown in breast cancer cells. Materials and methodsCells were infected with lentiviral vectors by either knockdown or overexpression of c-MYC. LncRNA cDNA was transcribed from total RNA samples and lncRNAs were evaluated by qRT-PCR. ResultsOur results indicated that some of the lncRNAs having tumor suppressor (GAS5, MEG3, lincRNA-p21) and oncogenic roles (HOTAIR) are regulated by c-MYC. ConclusionWe observed that c-MYC regulates lncRNAs that have important roles on proliferation, cell cycle and etc. Further studies will give us a light to identify molecular mechanisms related to MYC-lncRNA regulatory pathways in breast cancer.

Changes in the transcriptome profile of breast cancer cells grown as spheroids

BackgroundMulticellular tumor spheroids mimic the functional organization of tumors in vivo, providing biological readouts that predict the behavior of cancer cells more accurately. The current study aimed to evaluate the transcriptome (mRNAs and long non-coding RNAs) of multicellular tumor spheroids from breast cancer cells. MethodsMCF-7 cell spheroids were used; the transcriptome was analyzed using RNAseq and RNA microarrays; the secretion of macrophage migration inhibitor (MIF), a cytokine exported by the cholesterol efflux regulatory protein, was measured by ELISA. Linc00052 was inhibited using short-hairpin RNAs (shRNAs). ResultsWe found several differentially regulated mRNAs and lncRNAs in MCF-7 cell spheroids. We also found significant enrichment of the Wnt/B-catenin death receptor and the cholesterol metabolic processes. Interestingly, we also found an increased concentration of MIF. Further, at 12 and 20 days of 3D culture we found 221 and 1146 dysregulated lncRNAs, respectively; including linc00052 (long intergenic non-protein coding RNA 52), which has been involved in breast cancer. Linc00052 knock-down experiments suggest that it could be a key regulator of cholesterol pathways in breast cancer. ConclusionsOur data shows that tumor spheroids can induce changes in the transcriptome of the cultured cells, including both mRNAs and ncRNA. One of the major changes included the deregulation of cholesterol pathways, of which linc00052 is apparently a key regulator.

2′-Hydroxyflavanone induced changes in the proteomic profile of breast cancer cells

In spite of rapid advances in understanding of signaling networks associated with the incidence and therapeutic-sensitivity, breast cancer (BC) still remains the most commonly diagnosed and prevalent cancer in women. Emergence of resistance to hormonal interventions in estrogen-receptor (ER) positive BC coupled to loss of ER expression and activation of ER-independent growth factor, heat-shock, MYC and WNT pathways along with distinct mechanisms of therapeutic-resistance in HER2 over-expressing and triple-negative subtypes of BC collectively necessitates deeper profiling of the mechanistic networks regulated by potential lead anticancer compounds intended for further development to target BC. A significant part of the search for novel lead anticancer compounds for BC has focused on phytochemicals including flavonoids found in citrus fruits, which have shown promising anticancer activity. Based on the initial studies which revealed the anticancer effect of 2HF in BC, we employed an advanced TMT 10plex labeled proteomic approach to characterize the changes in non-phosphorylated and phosphorylated proteomic profile of ER+ MCF7, triple-negative MDA-MB231 and HER2+ SKBR3 BC cells, and MCF10A normal breast epithelial cells. 2HF induced significant changes in the proteins responsible for BC incidence, metastases and therapeutic sensitivity in BC cells.

Abstract A37: Combined targeting of estrogen receptor alpha and nuclear transport pathways remodels metabolic pathways to induce apoptosis and overcome tamoxifen resistance

Abstract Majority of breast cancer specific deaths occur in women with recurrent, ERα (+), metastatic tumors that are endocrine therapy resistant. There is a critical need for novel therapeutic approaches to resensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and XPO1, a nuclear transport protein in overcoming endocrine resistance. Using Cignalfinder pathway profiling, Seahorse metabolic profiling, and GC/MS whole metabolite profiling, we found that combination of 4-OH-Tam and selinexor (SXR), an XPO1 antagonist being evaluated in multiple later-stage clinical trials in patients with relapsed and /or refractory hematologic and solid tumor malignancies, inhibited Akt phosphorylation by changing the localization of the kinase. Since we observed dramatic changes in Akt activity, we hypothesized that glucose utilization pathways and consequently metabolic profile of breast cancer cells would change in the presence of 4-OH-Tam and SXR. These cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine as the mitochondrial fuel source. In order to examine metabolites that might result in the observed phenotype, we performed whole metabolite profiling and identified proline metabolic pathways to be upregulated when cells were treated with SXR+4-OH-Tam. We demonstrated that combined targeting of XPO1 and ERα rewires metabolic pathways, increases demand on mitochondria, and causes increased production of ROS that would eventually lead to apoptosis. Remodeling metabolic pathways to regenerate new vulnerabilities in endocrine-resistant tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα(+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences. Citation Format: Eylem Kulkoyluoglu Cotul, Kinga Wrobel, Landesman Yosef, Zeynep Madak-Erdogan. Combined targeting of estrogen receptor alpha and nuclear transport pathways remodels metabolic pathways to induce apoptosis and overcome tamoxifen resistance [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A37.

Abstract 3733: Combined targeting of estrogen receptor alpha and nuclear transport pathways remodel metabolic pathways to induce autophagy and overcome endocrine resistance

Abstract Majority of breast cancer specific deaths in women with ERα (+) tumor occur due to metastases that are resistant to endocrine therapy. There is a critical need for novel therapeutic approaches to resensitize recurrent ERα (+) tumors to endocrine therapies. The objective of this study was to elucidate mechanisms of improved effectiveness of combined targeting of ERα and XPO1, a nuclear transport protein in overcoming endocrine resistance. Selinexor (SXR), an XPO1 antagonist, has been evaluated in multiple later stage clinical trials in patients with relapsed and /or refractory hematological and solid tumor malignancies. Using Cignalfinder to profile kinase signaling pathways, we found that 4-OH-Tam, SXR or their combination induced differential Akt phosphorylation profiles, changing the localization and activity of the kinase. Since we observed dramatic changes in Akt activity we hypothesized that metabolic profile of breast cancer cells would change in the presence of 4-OH-Tam and SXR. Using Seahorse metabolic profiler and cell viability experiments in limited media conditions we showed that tamoxifen resistant cells were more dependent on mitochondria for energy production. Their glucose and fatty acid dependency decreased in the presence of SXR and cells were more dependent on glutamine as the mitochondrial fuel source. In order to examine metabolic pathways that might result in the observed phenotype we performed transcriptomics and GC/MS whole metabolite profiling and identified aminoacid metabolism pathways to be upregulated when cells were treated with SXR+4-OH-Tam. We demonstrated that combined targeting of XPO1 and ERα rewires metabolic pathways and shuts down both glycolytic and mitochondrial pathways that would eventually lead to autophagy. Remodelling metabolic pathways to regenerate new vulnerabilities in endocrine resistant breast tumors is novel, and given the need for better strategies for improving therapy response of relapsed ERα(+) tumors, our findings show great promise for uncovering the role ERα-XPO1 crosstalk plays in reducing cancer recurrences. Citation Format: Zeynep Madak Erdogan, Eylem Cotul-Kulkoyluoglu, Kinga Wrobel, Sunati Sahoo, Barbara Haley, Yosef Landesman. Combined targeting of estrogen receptor alpha and nuclear transport pathways remodel metabolic pathways to induce autophagy and overcome endocrine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3733.

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK4/6 inhibitor palbociclib

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor‐positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib‐induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib‐induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence‐like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse‐free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.

Transcriptomics profiling of breast cancer cells, MDA‐MB‐231, exposed to TNF‐α: focus on pro‐survival and pro‐inflammatory regulated pathways.

Triple‐negative breast cancer (TNBC) is characterized by the absence of estrogen, progesterone and HER2 receptors. TNBC is considered to have a poor prognosis due to the limited treatment options with the exclusion of hormone receptor‐based chemotherapies. Aggressive TNBC growth is accompanied by infiltration of tumor‐associated macrophages which can establish a perpetual state of inflammation rendering sustained release of proinflammatory cytokines such as tumor necrosis factor (TNF)‐α, which can lead to immunological evasion. In this work, we evaluated the effects of TNF‐α on the changes to the whole transcriptome: including mRNAs, long intergenic non‐coding RNA transcripts (lincRNA) in MDA‐MB‐231 cells. The data show enormous upregulation (60‐fold change FC, p&lt;.0001) in CXCL8 chemokine (C‐X‐C motif) ligand 8, with a statistically significant elevation of other transcripts including IL‐6 (+10.47), TNFRSF9 (9.58), TNFAIP3 (+7.52) and CCL2 (+3.5). On the other side, there was a reduction in numerous non‐coding microRNA including MIR29A (−8.8), MIRLET7A2 (−8.7), MIR222 (−4.91), MIR100 (−4.91), MIR27B (−4.68) and MIR181B1 (−7.95) in addition to Glutathione S – transferase (GSTT2) (−5.59). Pathway analysis shows that the most relevant effects of TNF‐α are on driving NF‐kB related survival, cytokines, and inflammatory pathway elements and TNF‐α related pathways.Support or Funding InformationSupported by grants from the National Institute on Minority Health and Health Disparities RCMI grant G12MD007582 and COE grant P20 MD006738.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.