Phenotype Of Breast Cancer Cells Research Articles

Discrete functional and mechanistic roles of chromodomain Y-like 2 (CDYL2) transcript variants in breast cancer growth and metastasis

Rationale: Chromodomain Y-like 2 (CDYL2) is a member of the CDY gene family involved in spermatogenesis, but its role in human cancer has not been reported. Analyses of publicly available databases demonstrate that CDYL2 is abundantly expressed in breast tumors. However, whether CDYL2 is involved in breast cancer progression remains unknown.Methods: Quantitative real-time PCR and immunoblotting assays were used to determine the expression levels of CDYL2 transcript variants in breast cancer cell lines and primary breast tumors. The effect of CDYL2 transcript variants on the malignant phenotypes of breast cancer cells was examined through in vitro and in vivo assays. Immunofluorescent staining, RNA-seq, ATAC-seq, and ChIP-qPCR were used to investigate the underlying mechanisms behind the aforementioned observations.Results: Here we show that CDYL2 generated four transcript variants, named CDYL2a-CDYL2d. CDYL2a and CDYL2b were the predominant variants expressed in breast cancer cell lines and breast tumors and exerted strikingly discrete functions in breast cancer growth and metastasis. CDYL2a was upregulated in the majority of the breast cancer cell lines and tumors, and promoted breast cancer cell proliferation, colony formation in vitro, and tumorigenesis in xenografts. In contrast, CDYL2b was mainly expressed in luminal- and HER2-positive types of breast cancer cell lines and tumors, and suppressed the migratory, invasive, and metastatic potential of breast cancer cells in vitro and in vivo. Mechanistically, CDYL2a partially localized to SC35-positive nuclear speckles and promoted alternative splicing of a subset of target genes, including FIP1L1, NKTR, and ADD3 by exon skipping. Elimination of full-length FIP1L1, NKTR, and ADD3 rescued the impaired cell proliferation through CDYL2a depletion. In contrast, CDYL2b localized to heterochromatin and transcriptionally repressed several metastasis-promoting genes, including HPSE, HLA-F, and SELL. Restoration of HPSE, HLA-F, or SELL expression in CDYL2b-overexpressing cells attenuated the ability of CDYL2b to suppress breast cancer cell migration and invasion.Conclusions: Collectively, these findings establish an isoform-specific function of CDYL2 in breast cancer development and progression and highlight that pharmacological inhibition of the CDYL2a, but not the CDYL2b, isoform may be an effective strategy for breast cancer therapy.

Acetylation of cedrelone increases its cytotoxic activity and reverts the malignant phenotype of breast cancer cells in 3D culture

Cedrelone is a limonoid isolated from the plant Trichilia catigua (Meliaceae). Previous studies have demonstrated that cedrelone (1) has several damaging effects on triple negative breast tumor (TNBC) cell line MDA-MB-231. In this work we investigated two new derivatives of cedrelone, the acetate (1a) and the mesylate (1b), to examine whether their effects are improved in comparison to the lead molecule. Cedrelone acetate (1a) was the most cytotoxic compound on TNBC cells and was chosen for additional analyses in traditional two-dimensional (2D) monolayer cultures and three-dimensional (3D) assays. In 2D, 1a induced cell cycle arrest, apoptosis and inhibited essential steps of the metastasis process of the MDA-MB-231 cells, in vitro. Moreover, 1a was able to revert the malignant phenotype of the T4-2 cells in 3D. These effects were concomitant with the downregulation of EGFR, β1-integrin and phospho-Akt, which could have resulted in a decrease of NFκB levels and MMP9 activity. These results suggest that 1a could be used as an important model for the design of a new drug to be applied in cancer treatment and be further studied in vivo for its antitumor and antimetastatic effects.

Inhibition of the histone demethylase, KDM5B, directly induces re-expression of tumor suppressor protein HEXIM1 in cancer cells

BackgroundThe tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA.MethodsWe identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin–NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays.ResultsWe have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis.ConclusionHMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.

WDR5 inhibition halts metastasis dissemination by repressing the mesenchymal phenotype of breast cancer cells

BackgroundDevelopment of metastases and drug resistance are still a challenge for a successful systemic treatment in breast cancer (BC) patients. One of the mechanisms that confer metastatic properties to the cell relies in the epithelial-to-mesenchymal transition (EMT). Moreover, both EMT and metastasis are partly modulated through epigenetic mechanisms, by repression or induction of specific related genes.MethodsWe applied shRNAs and drug targeting approaches in BC cell lines and metastatic patient-derived xenograft (PDX) models to inhibit WDR5, the core subunit of histone H3 K4 methyltransferase complexes, and evaluate its role in metastasis regulation.ResultWe report that WDR5 is crucial in regulating tumorigenesis and metastasis spreading during BC progression. In particular, WDR5 loss reduces the metastatic properties of the cells by reverting the mesenchymal phenotype of triple negative- and luminal B-derived cells, thus inducing an epithelial trait. We also suggest that this regulation is mediated by TGFβ1, implying a prominent role of WDR5 in driving EMT through TGFβ1 activation. Moreover, such EMT reversion can be induced by drug targeting of WDR5 as well, leading to BC cell sensitization to chemotherapy and enhancement of paclitaxel-dependent effects.ConclusionsWe suggest that WDR5 inhibition could be a promising pharmacologic approach to reduce cell migration, revert EMT, and block metastasis formation in BC, thus overcoming resistance to standard treatments.

A systematic comparison of lipopolymers for siRNA delivery to multiple breast cancer cell lines: In vitro studies

Small interfering RNA (siRNA) therapy is a promising approach for treatment of a wide range of cancers, including breast cancers that display variable phenotypic features. To explore the general utility of siRNA therapy to control aberrant expression of genes in breast cancer, we conducted a detailed analysis of siRNA delivery and silencing response in vitro in 6 separate breast cancer cell models (MDA-MB-231, MDA-MB-231-KRas-CRM, MCF-7, AU565, MDA-MB-435 and MDA-MB-468 cells). Using lipopolymers for siRNA complexation and delivery, we found a large variation in siRNA delivery efficiency depending on the specific lipopolymer used for siRNA complexation and delivery. Some lipopolymers were effective in all cell types used in this study, indicating the possibility of universal carriers for siRNA therapy. The delivery efficiency for effective lipopolymers was not correlated with dextran uptake in the cells tested, which indicated a receptor-mediated internalization for siRNA complexes with lipopolymers, unlike fluid-phase transfer associated with dextran uptake. Consistent with this, specific inhibitors involved in clathrin- and caveolin-mediated endocytosis significantly (>50%) reduced the internalization of siRNA complexes in all cell types. Using JAK2 and STAT3 silencing in MDA-MB-231 and MDA-MB-468 cells, a general correlation between the uptake and silencing efficiency at the mRNA level was evident, but it appeared that the choice of the target rather than the cell type was more critical for consistent silencing. We conclude that siRNA therapy with lipopolymers can be undertaken in multiple breast cancer cell phenotypes with similar efficiency, indicating the general applicability of non-viral RNAi in clinical management of molecularly heterogeneous breast cancers. Statement of significanceThe manuscript investigated the efficacy of siRNA carriers across multiple breast cancer cell lines. The lipopolymeric carriers were capable of delivering effective dose of siRNA to a range of breast cancer cells. Despite some differences in uptake efficiency among cell types, the mechanism of delivery was similar, with CME and CvME significantly involved in the internalization of polyplexes, while fluid-phase endocytosis was not significant. Specific target silencing was correlated to delivery efficiency, but we did notice the presence of lipopolymers that achieved high silencing with minimal siRNA delivery. Silencing specific targets in different cell types were more uniformly achieved as compared to targeting different targets in the same cells. Our studies enhance the feasibility of delivering siRNA to different types of breast cancer cells.

Cancer: Context Is Key for E-cadherin in Invasion andMetastasis.

A recent paper demonstrates how tissue context impacts the breast cancer cell phenotype. Loss of the E-cadherin tumor suppressor protein enhanced cell invasion, but inhibited multiple steps in metastatic spread due to the accumulation of reactive oxygen species and induction of apoptosis.

Long non-coding RNA PRNCR1 has an oncogenic role in breast cancer.

Long non-coding RNAs (lncRNAs) have important roles in the development and progression of various types of human cancer. However, the expression and function of the lncRNA prostate cancer-associated non-coding RNA 1 (PRNCR1) in breast cancer remains unclear. Reverse transcription-quantitative PCR was performed to measure the levels of mRNA expression. Cell counting kit-8, flow cytometry, wound healing and Transwell assays were also performed to study cell proliferation, cell cycle, migration and invasion, respectively. The results of the present study revealed that PRNCR1 expression levels were higher in breast cancer tissues compared with adjacent normal tissues in a patient study. It was also determined that high expression of PRNCR1 was significantly associated with advanced clinical stage, positive metastasis and poor prognosis for patients with breast cancer. In vitro experiments determined that PRNCR1 was significantly upregulated in the breast cancer cell lines BT-549, MCF-7, SK-BR-3 and MDA-MB-231 compared with the normal human breast cell line, MCF-10A. Silencing of PRNCR1 significantly inhibited the proliferation, colony formation, cell cycle progression, migration and invasion of SK-BR-3 and BT-549 cells, while cell apoptosis was induced. In addition, knockdown of PRNCR1 suppressed epithelial-mesenchymal transition in SK-BR-3 and BT-549 cells. In summary, the present results demonstrated that lncRNA PRNCR1 was significantly upregulated in breast cancer and was associated with cancer progression and poor patient prognosis. In vitro experiments determined that knockdown of PRNCR1 inhibited the malignant phenotypes of breast cancer cells. Taken together, the results indicated that PRNCR1 may be used as a potential therapeutic target for patients with breast cancer.

The MicroRNA-382-5p/MXD1 Axis Relates to Breast Cancer Progression and Promotes Cell Malignant Phenotypes

BackgroundMicroRNA (miRNA)-382-5p functions as an oncogenic miRNA in breast cancer. MXD1 was demonstrated to be one of its direct targets. However, the involvement of miRNA-382-5p/MXD1 axis in breast cancer remains unknown. The aim of this study was to investigate the expression pattern, clinical significance, and potential functions of miRNA-382-5p/MXD1 axis in breast cancer. Materials and methodsQuantitative polymerase chain reaction was performed to detect the expression levels of miRNA-382-5p and MXD1 messenger RNA (mRNA) in 96 pairs of breast cancer and matched noncancerous breast tissue samples from the same patients. Relationships between miRNA-382 expression, MXD1 expression, and combined miRNA-382-5p and MXD1 expression, and various clinicopathological characteristics of breast cancer were statistically evaluated, and their roles in breast cancer cell proliferation and invasion were also examined. ResultsCompared with noncancerous breast tissues, miRNA-382-5p expression was upregulated but MXD1 mRNA expression was downregulated in breast cancer tissues (both P < 0.01). High miRNA-382 expression, MXD1 expression, and combined miRNA-382-5p and low MXD1 expression were significantly associated with advanced tumor stage and the presence of lymph node metastasis (all P < 0.05). Overexpression of miRNA-382-5p dramatically reduced MXD1 mRNA and protein expression levels in breast cancer cells. miRNA-382-5p upregulation markedly enhanced breast cancer cell proliferation and invasion, while its downregulation inhibited these malignant phenotypes of breast cancer cells in vitro. Notably, overexpressed MXD1 reversed the effects of upregulated miRNA-382-5p on cell proliferation and invasion in vitro. ConclusionsThe dysregulation of miRNA-382-5p-MXD1 axis may be involved in the development and aggressive progression of breast cancer. miRNA-382-5p may target MXD1, leading to cell invasion and proliferation in breast cancer cells in vitro, implying its potentials as a therapeutic target for this type of cancer.

Microporous scaffolds loaded with immunomodulatory lentivirus to study the contribution of immune cell populations to tumor cell recruitment in vivo.

Metastases are preceded by stochastic formation of a hospitable microenvironment known as the premetastatic niche, which has been difficult to study. Herein, we employ implantable polycaprolactone scaffolds as an engineered premetastatic niche to independently investigate the role of interleukin-10 (IL10), CXCL12, and CCL2 in recruiting immune and tumor cells and impacting breast cancer cell phenotype via lentiviral overexpression. Lentivirus delivered from scaffolds in vivo achieved sustained transgene expression for 56 days. IL10 lentiviral expression, but not CXCL12 or CCL2, significantly decreased tumor cell recruitment to scaffolds in vivo. Delivery of CXCL12 enhanced CD45+ immune cell recruitment to scaffolds while delivery of IL10 reduced immune cell recruitment. CCL2 did not alter immune cell recruitment. Tumor cell phenotype was investigated using conditioned media from immunomodulated scaffolds, with CXCL12 microenvironments reducing proliferation, and IL10 microenvironments enhancing proliferation. Migration was enhanced with CCL2 and reduced with IL10-driven microenvironments. Multiple linear regression identified populations of immune cells associated with tumor cell abundance. CD45+ immune and CD8+ T cells were associated with reduced tumor cell abundance, while CD11b+Gr1+ neutrophils and CD4+ T cells were associated with enhanced tumor cell abundance. Collectively, biomaterial scaffolds provide a tool to probe the formation and function of the premetastatic niche.

Ziyuglycoside II suppresses the aggressive phenotype of triple negative breast cancer cells through regulating Src/EGFR-dependent ITGB4/FAK signaling

Triple negative breast cancer (TNBC), an aggressive form of breast cancer, has high rate of metastasis and which is the main cause of poor outcomes for such disease. The acquisition of invasive properties such as epithelial mesenchymal transition (EMT) and anoikis resistance is estimated to be the critical step in TNBC metastasis. Therefore, targeting these metastatic processes may contribute to the control of TNBC metastasis. In this study, investigations were conducted into the effect and mechanism of Ziyuglycoside II (Ziyu II), the main compound extracted from Sanguisorba Officinails L, on EMT, anoikis resistance as well as cell migration and invasion in human triple negative breast carcinoma MDA-MB-231 cells. The results showed that the treatment of Ziyu II could inhibit EMT, reverse anoikis resistance and subsequently suppress cell migration and invasion in MDA-MB-231 cells, which was associated with the inactivation of Src/EGFR-dependent ITGB4/FAK signaling and subsequent Akt and p38MAPK signaling pathways. These findings provide the new evidence of the anti-metastatic activity of Ziyu II and suggest the possibility of using this compound for the control of TNBC metastasis.

Mitochondrial fragmentation, elevated mitochondrial superoxide and respiratory supercomplexes disassembly is connected with the tamoxifen-resistant phenotype of breast cancer cells

Tamoxifen resistance remains a clinical obstacle in the treatment of hormone sensitive breast cancer. It has been reported that tamoxifen is able to target respiratory complex I within mitochondria. Therefore, we established two tamoxifen-resistant cell lines, MCF7 Tam5R and T47D Tam5R resistant to 5 μM tamoxifen and investigated whether tamoxifen-resistant cells exhibit mitochondrial changes which could help them survive the treatment. The function of mitochondria in this experimental model was evaluated in detail by studying i) the composition and activity of mitochondrial respiratory complexes; ii) respiration and glycolytic status; iii) mitochondrial distribution, dynamics and reactive oxygen species production. We show that Tam5R cells exhibit a significant decrease in mitochondrial respiration, low abundance of assembled mitochondrial respiratory supercomplexes, a more fragmented mitochondrial network connected with DRP1 Ser637 phosphorylation, higher glycolysis and sensitivity to 2-deoxyglucose. Tam5R cells also produce significantly higher levels of mitochondrial superoxide but at the same time increase their antioxidant defense (CAT, SOD2) through upregulation of SIRT3 and show phosphorylation of AMPK at Ser 485/491. Importantly, MCF7 ρ0 cells lacking functional mitochondria exhibit a markedly higher resistance to tamoxifen, supporting the role of mitochondria in tamoxifen resistance. We propose that reduced mitochondrial function and higher level of reactive oxygen species within mitochondria in concert with metabolic adaptations contribute to the phenotype of tamoxifen resistance.

ADAM15 mediates upregulation of Claudin-1 expression in breast cancer cells

A Disintegrin and Metalloproteinase-15 (ADAM15) is a transmembrane protein involved in protein ectodomain shedding, cell adhesion and signalling. We previously cloned and characterised alternatively spliced variants of ADAM15 that differ in their intracellular domains and demonstrated correlation of the expression of specific variants with breast cancer prognosis. In this study we have created isogenic cell panels (MDA-MB-231 and MCF-7) expressing five ADAM15 variants including wild-type and catalytically inactive forms. The expression of ADAM15 isoforms in MDA-MB-231 cells led to cell clustering to varying degree, without changes in EMT markers vimentin, slug and E-cadherin. Analysis of tight junction molecules revealed ADAM15 isoform specific, catalytic function dependent upregulation of Claudin-1. The expression of ADAM15A, and to a lesser degree of C and E isoforms led to an increase in Claudin-1 expression in MDA-MB-231 cells, while ADAM15B had no effect. In MCF-7 cells, ADAM15E was the principal variant inducing Claudin-1 expression. Sh-RNA mediated down-regulation of ADAM15 in ADAM15 over-expressing cells reduced Claudin-1 levels. Additionally, downregulation of endogenous ADAM15 expression in T47D cells by shRNA reduced endogenous Claudin-1 expression confirming a role for ADAM15 in regulating Claudin-1 expression. The PI3K/Akt/mTOR pathway was involved in regulating Claudin-1 expression downstream of ADAM15. Immunofluorescence analysis of MDA-MB-231 ADAM15A expressing cells showed Claudin-1 at cell-cell junctions, in the cytoplasm and nuclei. ADAM15 co-localised with Claudin-1 and ZO1 at cell-cell junctions. Immunoprecipitation analysis demonstrated complex formation between ADAM15 and ZO1/ZO2. These findings highlight the importance of ADAM15 Intra Cellular Domain-mediated interactions in regulating substrate selection and breast cancer cell phenotype.

RNF144A functions as a tumor suppressor in breast cancer through ubiquitin ligase activity-dependent regulation of stability and oncogenic functions of HSPA2.

Deregulation of E3 ubiquitin ligases is intimately implicated in breast cancer pathogenesis and progression, but the underlying mechanisms still remain elusive. Here we report that RING finger protein 144A (RNF144A), a poorly characterized member of the RING-in-between-RING family of E3 ubiquitin ligases, functions as a tumor suppressor in breast cancer. RNF144A was downregulated in a subset of primary breast tumors and restoration of RNF144A suppressed breast cancer cell proliferation, colony formation, migration, invasion in vitro, tumor growth, and lung metastasis in vivo. In contrast, knockdown of RNF144A promoted malignant phenotypes of breast cancer cells. Quantitative proteomics and biochemical analysis revealed that RNF144A interacted with and targeted heat-shock protein family A member 2 (HSPA2), a putative oncoprotein that is frequently upregulated in human cancer and promotes tumor growth and progression, for ubiquitination and degradation. Notably, the ligase activity-defective mutants of RNF144A impaired its ability to induce ubiquitination and degradation of HSPA2, and to suppress breast cancer cell proliferation, migration, and invasion ascompared with its wild-type counterpart. Moreover, RNF144A-mediated suppression of breast cancer cell proliferation, migration, and invasion was rescued by ectopic HSPA2 expression. Clinically, low RNF144A and high HSPA2 expression in breast cancer patients was correlated with aggressive clinicopathological characteristics and decreased overall and disease-free survival. Collectively, these findings reveal a previously unappreciated role for RNF144A in suppression of breast cancer growth and metastasis, and identify RNF144A as the first, to our knowledge, E3 ubiquitin ligase for HSPA2 in human cancer.

Metformin reverses mesenchymal phenotype of primary breast cancer cells through STAT3/NF-\u03baB pathways

BackgroundBreast cancer currently is the most frequently diagnosed neoplasm and the leading cause of death from cancer in women worldwide, which is mainly due to metastatic disease. Increasing our understanding of the molecular mechanisms leading to metastasis might thus improve the pharmacological management of the disease. Epithelial-mesenchymal transition (EMT) is a key factor that plays a major role in tumor metastasis. Some pro-inflammatory cytokines, like IL-6, have been shown to stimulate phenotypes consistent with EMT in transformed epithelial cells as well as in carcinoma cell lines. Since the EMT is one of the crucial steps for metastasis, we studied the effects of metformin (MTF) on EMT.MethodsCytotoxic effect of MTF was evaluated in eight primary breast cancer cell cultures by crystal violet assay. EMT markers and downstream signaling molecules were measured by Western blot. The effect of MTF on cell proliferation and cell migration were analyzed by MTT and Boyden chamber assays respectively.ResultsWe observed that the response of cultured breast cancer primary cells to MTF varied; mesenchymal cells were resistant to 10 mM MTF and expressed Vimentin and SNAIL, which are associated with a mesenchymal phenotype, whereas epithelial cells were sensitive to this MTF dose, and expressed E-cadherin but not mesenchymal markers. Further, exposure of mesenchymal cells to MTF down-regulated both Vimentin and SNAIL as well as cell proliferation, but not cell migration. In an in vitro IL-6-induced EMT assay, primary breast cancer cells showing an epithelial phenotype underwent EMT upon exposure to IL-6, with concomitant activation of STAT3 and NF-κB; addition of MTF to IL-6-induced EMT reversed the expression of the mesenchymal markers Vimentin and SNAIL, decreased pSTAT3 Y705 and pNF-κB S536 and increased E-cadherin. In addition, downregulation of STAT3·activation was dependent on AMPK, but not NF-κB phosphorylation. Further, MTF inhibited cell proliferation and migration stimulated by IL-6.ConclusionThese results suggest that MTF inhibits IL-6-induced EMT, cell proliferation, and migration of primary breast cancer cells by preventing the activation of STAT3 and NF-κB. STAT3 inactivation occurs through AMPK, but not NF-κB.

Abstract 5203: Segregating activities of the estrogen receptor in gene regulation

Abstract Upon stimulation with its ligand estradiol (E2), the estrogen receptor (ER) obviously can induce both transcriptional activation and repression in breast cancer cells. Although the mechanism of gene activation has been extensively scrutinized, how ER targets are turned off is generally overlooked. Some theories have been proposed to explain the trans-suppression process, such as engagement of corepressors, physiological squelch of coactivators, or decommission of RNA Polymerase II, etc. However, none of these models really elucidate the intrinsic and external factors that segregate ER activities in transcriptional regulation. In this study, we identified a functional domain of ER, which is specifically required for E2-mediated gene repression. While the DNA-binding domain (DBD) was essential for the overall response to E2, deletion of AF2 fragment at C-terminus of ER specifically reverted the E2-repressed transcriptional program into E2-activated one. We further demonstrated that RNAs transcribed from these E2-repressed enhancers (eRNAs) act as the exterior determinants of ER activity in gene repression. In addition to stabilizing promoter-enhancer looping structure, these eRNAs recruit ER to particular chromatin loci, and facilitate the hierarchical formation of a functional complex responsible for transcriptional repression. Thus, our findings suggest that by targeting distinct functional domains of ER, we may be able to reprogram expression of a specific subset of ER targets, which play critical roles in conferring malignant phenotypes of breast cancer cells. Meanwhile, as a distinctive class of cis-acting elements besides chromatin regulatory regions, eRNAs help to refine ER-dependent transcriptional program that contributes to the development and progression of breast cancer. Citation Format: Mei Yang, Zhao Zhang, Kexin Xu. Segregating activities of the estrogen receptor in gene regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5203.

Regulation of BS69 Expression in Cancers.

BS69 is encoded by a gene located on chromosome 10, in a region frequently deleted in human cancers and BS69 expression is often down-regulated in human cancers. In addition, BS69 acts as a transcriptional repressor via interaction with transcriptional factors associated with tumorigenesis, such as cellular homolog of the avian myeloblastosis viral oncoprotein, v-ets erythroblastosis virus E26 oncogene homolog 2 oncoprotein, MYC-associated protein X gene-associated protein. Overexpression of BS69 can suppress proliferation of osteosarcoma, breast cancer and glioma cells in vitro; and inhibits tumor growth in xenograft models. Therefore, BS69 may act as a tumor suppressor, and may be a new target for cancer therapy. However, BS69 down-regulation has been found to be involved in cellular senescence and is associated with the reversion of the malignant phenotype of breast cancer cells. Therefore, additional studies are necessary to clarify the role of BS69 in tumor development.

10]-Gingerol Affects Multiple Metastatic Processes and Induces Apoptosis in MDAMB- 231 Breast Tumor Cells.

Triple Negative Breast Cancer (TNBC) represents the approximately 15% of breast cancers that lack expression of Estrogen (ER) and Progesterone Receptors (PR) and do not exhibit amplification of the human epidermal growth factor receptor 2 (HER2) gene, imposing difficulties to treatment. Interactions between tumor cells and their microenvironment facilitate tumor cell invasion in the surrounding tissues, intravasation through newly formed vessels, and dissemination to form metastasis. To treat metastasis from breast and many other cancer types, chemotherapy is one of the most extensively used methods. However, its efficacy and safety remain a primary concern, as well as its toxicity and other side effects. Thus, there is increasing interest in natural antitumor agents. In a previous work, we have demonstrated that [10]-gingerol is able to revert malignant phenotype in breast cancer cells in 3D culture and, moreover, to inhibit the dissemination of TNBC to multiple organs including lung, bone and brain, in spontaneous and experimental in vivo metastasis assays in mouse model. This work aims to investigate the in vitro effects of [10]-gingerol, using human MDA-MB-231TNBC cells, in comparison to non-tumor MCF-10A breast cells, in order to understand the antitumor and antimetastatic effects found in vivo and in a 3D environment. We investigated different steps of the metastatic process in vitro, such as cell migration, invasion, adhesion and MMP activity. In addition, we analyzed the anti-apoptotic and genotoxic effects of [10]-gingerol using PEAnnexin, DNA fragmentation, TUNEL and comet assays, respectively. [10]-gingerol was able to inhibit cell adhesion, migration, invasion and to induce apoptosis more effectively in TNBC cells, when compared to non-tumor cells, demonstrating that these mechanisms can be involved in the antitumor and antimetastatic effects of [10]-gingerol, found both in 3D culture and in vivo. Taken together, results found here are complementary to previous studies of our group and others and demonstrate that additional mechanisms, besides apoptotic cell death, is used by [10]-gingerol to accomplish its antitumor and antimetastatic effects. Our results indicate a potential for this natural compound as an antitumor molecule or as an adjuvant for chemotherapeutics already used in the clinic.

Knockdown of XB130 restrains cancer stem cell-like phenotype through inhibition of Wnt/β-Catenin signaling in breast cancer.

The cancer stem cells (CSCs) is a subset of cancer cells that possess stem cell properties, which plays a crucial role in the occurrence, metastasis, and recurrence of the tumor. XB130 is a novel adapter protein potentially serves as a functional factor in CSCs. To determine the role of CSCs in breast cancer, we focused on the study of XB130. In our study, we found that XB130 expression was significantly upregulated in breast cancer and was closely related to the clinicopathologic characteristics, overall survival and poor prognosis of breast cancer patients. Functionally, we found that knockdown of XB130 was not only played an important role in proliferation, epithelial-mesenchymal transition (EMT), and metastasis in breast cancer cells but also exhibited potent antitumor activity in animal tumor models. Moreover, we demonstrated that silencing endogenous XB130 regulated the cancer stem cell-like properties of breast cancer, including the formation of self-renewing spheres and the proportion of breast cancer SP+ cells. Mechanistically, our studies indicated that downregulation of XB130 restrained the EMT and Wnt/β-catenin signaling, so as to weaken the tumor-initiating cell-like phenotype of breast cancer cells. This study indicates that XB130 plays an important role in maintaining the EMT and stem cell-like characteristics of breast cancer cells, supporting the significance of XB130 as a new potential therapeutic target for early diagnosis and prognosis of breast cancer.

MicroRNA-876 is sponged by long noncoding RNA LINC00707 and directly targets metadherin to inhibit breast cancer malignancy.

Background: MicroRNA-876-5p (miR-876) dysregulation contributes to the aggressiveness of various types of human cancer. This study was aimed at measuring miR-876 expression in breast cancer, determining the specific roles of miR-876 in the progression of breast cancer and understanding the corresponding molecular mechanisms.Materials and methods: miR-876 expression in breast cancer tissues and cell lines was quantified via RT-qPCR. The effect of miR-876 upregulation on the malignant phenotype of breast cancer cells was investigated using CCK-8 assays, flow cytometry, Transwell migration and invasion assays and tumor xenograft experiments. The mechanisms underlying the tumor-suppressive action of miR-876 in breast cancer cells were explored using bioinformatic analysis, luciferase reporter assays, RT-qPCR and Western blot analysis.Results: miR-876 was found to be underexpressed in breast cancer tissues and cell lines. Decreased miR-876 expression notably correlated with lymphatic invasion metastasis, TNM stage and differentiation grade. Overall survival was lower among patients with breast cancer and low miR-876 expression than in patients with high miR-876 expression. Restoration of miR-876 expression decreased breast cancer cell proliferation, migration and invasion in vitro and restricted tumor growth in vivo as well as increased cell apoptosis. Metadherin (MTDH) was identified as a novel target of miR-876 in breast cancer cells. Furthermore, long intergenic nonprotein-coding RNA 707 (LINC00707) acted as a molecular sponge for miR-876, thereby regulating MTDH expression in breast cancer. Finally, silencing miR-876 expression attenuated the influence of a LINC00707 knockdown on the malignancy of breast cancer cells.Conclusion: This study, thus, revealed the vital functions of the LINC00707–miR-876–MTDH pathway in breast cancer and provided attractive targets and markers for its treatment.

A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells.

The most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+ shuttle system in CA IX to facilitate proton efflux through MCT4.