Silenced Breast Cancer Research Articles

Knockdown of PAIP1 Inhibits Breast Cancer Cell Proliferation by Regulating Cyclin E2 mRNA Stability.

Polyadenylate-binding protein-interacting protein 1 (PAIP1) is a protein that modulates translation initiation in eukaryotic cells. Studies have shown that PAIP1 was overexpressed in various type of cancers, and drives cancer progression by promoting cancer cell proliferation, invasion, and migration. In our previous study, we identified that PAIP1 was overexpressed in breast cancer, and the expression was correlated with poor prognosis. However, the biological function of PAIP1 in breast cancer has not been clearly understood. In this study, we constructed PAIP1 specifically silenced breast cancer cells. Then, cell proliferation, cell cycle distribution, and apoptosis were detected in PAIP1 knockdown cells. RNA-seq analysis was performed to predict the downstream target of PAIP1, and the molecular mechanism was explored. As a results, we found that knockdown of PAIP1 repressed cell proliferation, induced cell cycle arrest, and triggers apoptosis. Xenograft mouse model showed that knockdown of PAIP1 inhibits cell growth in vivo. RNA-seq predicted that CCNE2 mRNA was one of the downstream targets of PAIP1. In addition, we identified that knockdown of PAIP1-inhibited cell proliferation through modulating cyclin E2 expression. Mechanically, knockdown of PAIP1 reduces the expression of cyclin E2 by regulating the mRNA stability of cyclin E2. Moreover, in breast cancer tissues, we found that the expression of PAIP1 was positively correlated with cyclin E2. Taken together, our findings establish the role and mechanism of PAIP1 in breast cancer progression, indicating that PAIP1 would be a new therapeutic target for breast cancer treatment.

Pharmacological Activation of SIRT3 Modulates the Response of Cancer Cells to Acidic pH.

Cancer cells modulate their metabolism, creating an acidic microenvironment that, in turn, can favor tumor progression and chemotherapy resistance. Tumor cells adopt strategies to survive a drop in extracellular pH (pHe). In the present manuscript, we investigated the contribution of mitochondrial sirtuin 3 (SIRT3) to the adaptation and survival of cancer cells to a low pHe. SIRT3-overexpressing and silenced breast cancer cells MDA-MB-231 and human embryonic kidney HEK293 cells were grown in buffered and unbuffered media at pH 7.4 and 6.8 for different times. mRNA expression of SIRT3 and CAVB, was measured by RT-PCR. Protein expression of SIRT3, CAVB and autophagy proteins was estimated by western blot. SIRT3-CAVB interaction was determined by immunoprecipitation and proximity ligation assays (PLA). Induction of autophagy was studied by western blot and TEM. SIRT3 overexpression increases the survival of both cell lines. Moreover, we demonstrated that SIRT3 controls intracellular pH (pHi) through the regulation of mitochondrial carbonic anhydrase VB (CAVB). Interestingly, we obtained similar results by using MC2791, a new SIRT3 activator. Our results point to the possibility of modulating SIRT3 to decrease the response and resistance of tumor cells to the acidic microenvironment and ameliorate the effectiveness of anticancer therapy.

Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

BackgroundBreast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy.ResultsIn this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression.ConclusionsOur results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients’ outcome.

HER2/neu Oncogene Silencing in a Breast Cancer Cell Model Using Cationic Lipid-Based Delivery Systems.

The overexpression of the human epidermal growth factor 2 (HER2/neu) oncogene is predictive of adverse breast cancer prognosis. Silencing the HER2/neu overexpression using siRNA may be an effective treatment strategy. Major requirements for siRNA-based therapy are safe, stable, and efficient delivery systems to channel siRNA into target cells. This study assessed the efficacy of cationic lipid-based systems for the delivery of siRNA. Cationic liposomes were formulated with equimolar ratios of the respective cholesteryl cytofectins, 3β-N-(N', N'-dimethylaminopropyl)-carbamoyl cholesterol (Chol-T) or N, N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09), with the neutral helper lipid, dioleoylphosphatidylethanolamine (DOPE), with and without a polyethylene glycol stabilizer. All cationic liposomes efficiently bound, compacted, and protected the therapeutic siRNA against nuclease degradation. Liposomes and siRNA lipoplexes were spherical, <200 nm in size, with moderate particle size distributions (PDI < 0.4). The siRNA lipoplexes exhibited minimal dose-dependent cytotoxicity and effective HER2/neu siRNA transfection in the HER2/neu overexpressing SKBR-3 cells. The non-PEGylated Chol-T-siRNA lipoplexes induced the highest HER2/neu silencing at the mRNA (10000-fold decrease) and protein levels (>111.6-fold decrease), surpassing that of commercially available Lipofectamine 3000 (4.1-fold reduction in mRNA expression). These cationic liposomes are suitable carriers of HER2/neu siRNA for gene silencing in breast cancer.

GSTP1 silencing in breast cancer is a redox vulnerability that redirects the flux of cysteine oxidation in the proteome

GSTP1 silencing in breast cancer is a redox vulnerability that redirects the flux of cysteine oxidation in the proteome

Methylation-Mediated Silencing of RBP7 Promotes Breast Cancer Progression through PPAR and PI3K/AKT Pathway.

Retinoid-binding protein7 (RBP7) is a member of the cellular retinol-binding protein (CRBP) family, which is involved in the pathogenesis of breast cancer. The study aims to illustrate the prognostic value and the potential regulatory mechanisms of RBP7 expression in breast cancer. Bioinformatics analysis with the TCGA and CPTAC databases revealed that the mRNA and protein expression levels of RBP7 in normal were higher compared to breast cancer tissues. Survival analysis displayed that the lower expression of RBP7, the worse the prognosis in ER-positive (ER+) breast cancer patients. Genomic analysis showed that low expression of RBP7 correlates with its promoter hypermethylation in breast cancer. Functional enrichment analysis demonstrated that downregulation of RBP7 expression may exert its biological influence on breast cancer through the PPAR pathway and the PI3K/AKT pathway. In summary, we identified RBP7 as a novel biomarker that is helpful for the prognosis of ER+ breast cancer patients. Promoter methylation of RBP7 is involved in its gene silencing in breast cancer, thus regulating the occurrence and development of ER+ breast cancer through the PPAR and PI3K/AKT pathways.

Zinc-finger protein 382 antagonises CDC25A and ZEB1 signaling pathway in breast cancer

Our previous studies found that Zinc-finger protein 382 (ZNF382) played as a tumor suppressor gene in esophageal and gastric cancers, and a positive correlation between the high expression of ZNF382 and better outcome in breast cancer patients. However, the biological roles and mechanisms of ZNF382 in breast cancer remains unclear. We detected ZNF382 expression by reverse-transcription PCR (RT-PCR) and real-time quantitative PCR (qRT-PCR) in breast cancer cells and tissues, and explored the impacts and mechanisms of ectopic ZNF382 expression in breast cancer cells in vitro and in vivo, respectively. Our results revealed that ZNF382 was significantly down-regulated in breast cancer tissues compared with adjacent non-cancer tissues. Restoration of ZNF382 expression in silenced breast cancer cells not only inhibited tumor cell colony formation, viability, migration and invasion, and epithelial-mesenchymal-transition (EMT), but also induced apoptosis and G0/G1 arrest. In conclusion, ZNF382 could induce G0/G1 cell cycle arrest through inhibiting CDC25A signaling, and, inhibit cell migration, invasion and EMT by antagonizing ZEB1 signaling in breast cancer cells.

LncRNA SNHG1 promotes tumor progression and cisplatin resistance through epigenetically silencing miR-381 in breast cancer

ABSTRACT The long-non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) is a known cause of tumorigenesis. Nevertheless, it’s yet unclear how lncRNA SNHG1 influences breast cancer. Herein, we explored the mechanisms through which SNHG1 modulates breast cancer tumor progression. Our findings demonstrated that SNHG1 is significantly upregulated in breast cancer tissues and cells. High SNHG1 levels were closely linked to reduced survival rates in breast cancer patients. SNHG1 silencing has been shown to inhibit the proliferative, migratory, and invasive activity of breast cancer cells. Moreover, SNHG1 silencing enhanced cisplatin (DDP) sensitivity of these cells through improving DDP-induced cell apoptosis. Mechanistically, SNHG1 was found to interact with enhancer of zeste homolog 2 (EZH2), recruiting EZH2 to trigger trimethylation of histone H3 lysine 27 (H3K27me3), thus epigenetically inhibiting miR-381 transcription in these cells. Overexpression of miR-381 inhibited tumor progression and sensitized cells to the chemotherapeutic reagent DDP. More importantly, rescue experiments demonstrated that miR-381 inhibition could inverse the tumor-suppressive effect of SNHG1 silencing in breast cancer. In summary, SNHG1 silencing suppressed tumor progression and overcame breast cancer cell DDP resistance via the epigenetic suppression of miR-381 expression. Our study revealed that SNHG1 served as a novel therapeutic target for breast cancer chemoresistance.

LINCS gene expression signature analysis revealed bosutinib as a radiosensitizer of breast cancer cells by targeting eIF4G1.

Radioresistance is the predominant cause for radiotherapy failure and disease progression, resulting in increased breast cancer‑associated mortality. Using gene expression signature analysis of the Library of Integrated Network‑Based Cellular Signatures (LINCS) and Gene Expression Omnibus (GEO), the aim of the present study was to systematically identify potential candidate radiosensitizers from known drugs. The similarity of integrated gene expression signatures between irradiated eukaryotic translation initiation factor 4γ1(eIF4G1)‑silenced breast cancer cells and known drugs was measured using enrichment scores(ES). Drugs with positive ES were selected as potential radiosensitizers. The radiosensitizing effects of the candidate drugs were analyzed in breast cancer cell lines (MCF‑7, MX‑1 and MDA‑MB‑231) using CCK‑8 and colony formation assays following exposure to ionizing radiation. Cell apoptosis was measured using flow cytometry. The expression levels of eIF4G1 and DNA damage response (DDR) proteins were analyzed by western blotting. Bosutinib was identified as a promising radiosensitizer, as its administration markedly reduced the dosage required both for the drug and for ionizing radiation, which may be associated with fewer treatment‑associated adverse reactions. Moreover, combined treatment of ionizing radiation and bosutinib significantly increased cell killing in all three cell lines, compared with ionizing radiation or bosutinib alone. Among the three cell lines, MX‑1 cells were identified as the most sensitive to both ionizing radiation and bosutinib. Bosutinib markedly downregulated the expression of eIF4G1 in a dose‑dependent manner and also reduced the expression of DDR proteins (including ATM, XRCC4, ATRIP, and GADD45A). Moreover, eIF4G1 was identified as a key target of bosutinib that may regulate DNA damage induced by ionizing radiation. Thus, bosutinib may serve as a potential candidate radiosensitizer for breast cancer therapy.

Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer

Ring1b is a core subunit of polycomb repressive complex 1 (PRC1) and is essential in several high-risk cancers. However, the epigenetic mechanism of Ring1b underlying breast cancer malignancy is poorly understood. In this study, we showed increased expression of Ring1b promoted metastasis by weakening cell–cell adhesions of breast cancer cells. We confirmed that Ring1b could downregulate E-cadherin and contributed to an epigenetic rewiring via PRC1-dependent function by forming distinct complexes with DEAD-box RNA helicases (DDXs) or epithelial-mesenchymal transition transcription factors (EMT TFs) on site-specific loci of E-cadherin promoter. DDXs-Ring1b complexes moderately inhibited E-cadherin, which resulted in an early hybrid EMT state of epithelial cells, and EMT TFs-Ring1b complexes cooperated with DDXs-Ring1b complexes to further repress E-cadherin in mesenchymal-like cancer cells. Clinically, high expression of Ring1b with DDXs or EMT TFs predicted low levels of E-cadherin, metastatic behavior, and poor prognosis. These findings provide an epigenetic regulation mechanism of Ring1b complexes in E-cadherin expression. Ring1b complexes may be potential therapeutic targets and biomarkers for diagnosis and prognosis in invasion breast cancer.

HER-2/neu and MYC gene silencing in breast cancer: therapeutic potential and advancement in nonviral nanocarrier systems.

Globally, breast cancer is the second leading cause of cancer-related mortality among women, with approximately 1.4 million new cases diagnosed annually. Associated genetic perturbations are emerging in the face of intense scientific enquiry, facilitating its classification, prognostication and treatment. RNAi, utilizing siRNA, is a powerful treatment strategy to silence disease-causing genes. However, therapeutic siRNA instability and poor cellular uptake have limited its clinical application, necessitating the use of nanocarriers. In this review, we highlight the RNAi mechanism, HER-2/neu and MYC as breast cancer gene targets, and nonviral nanocarriers as potentially safe and efficient delivery systems.

Effects of cisplatin on the proliferation, invasion and apoptosis of breast cancer cells following β‑catenin silencing.

Resistance to the chemotherapeutic drug cisplatin has been documented in various types of cancer, while the increased expression of β-catenin has been observed in cisplatin-resistant ovarian cancer. However, the involvement of β-catenin in cisplatin resistance is unclear. The present study investigated the antitumor effect of cisplatin on the proliferation, invasion and apoptosis of breast cancer (BC) cells following β-catenin silencing in BC, which is the most frequent type of malignancy among women. The expression of β-catenin in BC tissues and cell lines was measured by reverse transcription-quantitative polymerase chain reaction, and the association between expression levels and clinical characteristics was statistically analyzed. The viability of BC cell lines treated with siR-β-catenin or with siR-β-catenin and cisplatin in combination was determined using a Cell Counting Kit-8 assay. The migratory and invasive abilities of BC cells treated with both siR-β-catenin and cisplatin were examined with Transwell assays. The CD44 antigen/intercellular adhesion molecule 1 expression ratio, cell cycle distribution and apoptosis levels of BC cells treated with siR-β-catenin and cisplatin in combination were detected by flow cytometry. The expression levels of apoptosis-associated proteins, including caspase-3/9, in the BC cells treated with both siR-β-catenin and cisplatin were investigated by western blot analysis. The levels of apoptosis in the BC cells following combined treatment with siR-β-catenin and cisplatin was further quantified by Hoechst 33342 staining. β-catenin was identified to be highly expressed in BC tissues and cell lines and was associated with pathological stage and lymph node status. Following knockdown of β-catenin expression, cisplatin treatment suppressed the viabilities, and the migratory and invasive capabilities of the T47D and MCF-7 cells, and induced extensive apoptosis. β-catenin knockdown upregulated caspase-3/9 levels following cisplatin treatment and induced the apoptosis of T47D and MCF-7 cells. In conclusion, β-catenin may be of value as a therapeutic target during cisplatin treatment in patients with BC treated with cisplatin.

Aquaporin expression in breast cancer and their involvement in bleb formation, cell motility and invasion in endocrine resistant variant cells.

Estrogen receptor (ER)‑silenced breast cancer cell lines exhibit endocrine resistance and morphological changes from an epithelial to a mesenchymal phenotype. These cells also display increased motility and invasive properties that are further accentuated by exposure to an alkaline pH, exhibiting dynamic plasma membrane blebbing and cytoplasmic streaming. These latter morphological changes are hypothesized to involve substantial water movement across the plasma membrane, contributing to bleb formation; this may involve aquaporin channel proteins(AQPs). AQP1, 3, 4 and5 expression/localization was examined via reverse transcription‑quantitative PCR, western blotting and confocal microscopy in endocrine‑sensitive(YS1.2) and ‑resistant (pII and MDA‑MB‑231) breast cancer cells, as well as normal breast epithelial cells(MCF10A). The effects of osmotic changes on bleb formation were examined via live cell imaging. AQP3 protein expression was knocked down by small interfering RNA(siRNA) transfection, and the effect of its reduced expression on bleb formation, cell motility and invasion were determined via immunofluorescence, scratch and Cultrex assays, respectively. Expression of the four AQPs varied across the different cell lines, and exhibited nuclear, cytoplasmic and membranous localization. Osmotic changes affected the formation of blebs. In pIIcells exposed to alkaline pH, AQP3 was observed to be redistributed from the nucleus into the newly formed blebs. siRNA‑mediated knockdown of AQP3 in pIIcells significantly reduced cellular blebbing induced by alkaline pH, as well as motility and invasion. These data suggested that AQP3, and potentially other aquaporins, may participate in the processes leading to blebbing of endocrine‑resistant cells which is proposed to be a mechanism that drives tumor metastasis.

Genistein Inhibits Proliferation of BRCA1 Mutated Breast Cancer Cells: The GPR30-Akt Axis as a Potential Target.

BackgroundBRCA1 mutated breast cancer cells exhibit the elevated cell proliferation and the higher metastatic potential. G protein-coupled receptor 30 (GPR30) has been shown to regulate growth of hormonally responsive cancers, such as ovarian and breast cancers, and high expression of GPR30 is found in estrogen receptor (ER)-negative breast cancer cells. ER-negative breast cancer patients often have a mutation in the tumor suppressor gene, BRCA1. This study explored antiproliferative effects of genistein, a chemopreventive isoflavone present in legumes, and underlying molecular mechanisms in triple negative breast cancer cells with or without functionally active BRCA1.MethodsExpression of BRCA1, GPR30 and Nrf2 was measured by Western blot analysis. Reactive oxygen species (ROS) accumulation was monitored by using the fluorescence-generating probe, 2’,7’-dichlorofluorescein diacetate. The effects of genistein on breast cancer cell viability and proliferation were assessed by the MTT, migration and clonogenic assays.ResultsThe expression of GPR30 was dramatically elevated at both transcriptional and translational levels in BRCA1 mutated breast cancer cells compared to cells with wild-type BRCA1. Notably, there was diminished Akt phosporylation in GPR30 silenced cells. Treatment of BRCA1 silenced breast cancer cells with genistein resulted in the down-regulation of GPR30 expression and the inhibition of Akt phosphorylation as well as the reduced cell viability, migration and colony formation. Genistein caused cell cycle arrest at the G2/M phase in BRCA1-mutant cells through down-regulation of cyclin B1 expression. Furthermore, BRCA1-mutant breast cancer cells exhibited higher levels of intracellular ROS than those in the wild-type cells. Genistein treatment lowered the ROS levels through up-regulation of Nrf2 expression.ConclusionsLack of functional BRCA1 activates GPR30 signaling, thereby stimulating Akt phosphorylation and cell proliferation. Genistein induces G2/M phase arrest by down-regulating cyclin B1 expression, which is attributable to its suppression of GPR30 activation and Akt phosphorylation in BRCA1 impaired breast cancer cells.

ZMYND10, an epigenetically regulated tumor suppressor, exerts tumor-suppressive functions via miR145-5p/NEDD9 axis in breast cancer

BackgroundRecent studies suggested that ZMYND10 is a potential tumor suppressor gene in multiple tumor types. However, the mechanism by which ZMYND10 inhibits breast cancer remains unclear. Here, we investigated the role and mechanism of ZMYND10 in breast cancer inhibition.ResultsZMYND10 was dramatically reduced in multiple breast cancer cell lines and tissues, which was associated with promoter hypermethylation. Ectopic expression of ZMYND10 in silenced breast cancer cells induced cell apoptosis while suppressed cell growth, cell migration and invasion in vitro, and xenograft tumor growth in vivo. Furthermore, molecular mechanism studies indicated that ZMYND10 enhances expression of miR145-5p, which suppresses the expression of NEDD9 protein through directly targeting the 3'-untranslated region of NEDD9 mRNA.ConclusionsResults from this study show that ZMYND10 suppresses breast cancer tumorigenicity by inhibiting the miR145-5p/NEDD9 signaling pathway. This novel discovered signaling pathway may be a valid target for small molecules that might help to develop new therapies to better inhibit the breast cancer metastasis.

Role of TMPRSS4 Modulation in Breast Cancer Cell Proliferation.

Background:TMPRSS4 is a novel Type II transmembrane serine protease found at the surface of the cells and is involved in the development and cancer progression. However, TMPRSS4 functions in breast cancer remain poor understand. The present study investigated the function of TMPRSS4 in the breast cancer cells and the potential mechanistic action underling. Materials and Methods:The lentiviral vectors causing TMPRSS4 down-regulation and over-expression were established and transfected in MDA-MB-468 and MCF-7 cells, respectively. By using the CCK-8 assay, cell proliferation was analyzed. Moreover, western blot was used to detect the expression of certain proteins related to cell apoptosis (Bax and Bcl2) signaling pathway and telomere maintenance (POT1, TPP1, and UBE2D3). Cell cycle and cell apoptosis were also analyzed by using the Flow cytometry analysis. TMPRSS4 expression was detected at the mRNA level and protein level by performing qPCR and western blot technique, respectively. Results:TMPRSS4 expression is inhibited in stable transfected MDA-MB-468-shTMPRSS4 cells compared to the control MDA-MB-468-NC and its expression is up-regulated in stable transfected MCF-7-TMPTSS4 compared to its control MCF-7-NC. Moreover, TMPRSS4 silencing in breast cancer reduces cells proliferation by promoting cell cycle arrest in G2/M phase, cell apoptosis, and telomere maintenance impairment while the TMPRSS4 overexpression increases cells proliferation through cell apoptosis reduction and telomere maintenance reinforcement associated with insignificant change in cell cycle progression. Conclusion:TMPRSS4 plays important roles in cancer progression and may be considered as a good therapeutic target for cancer gene therapy especially breast cancer.

Prognostic significance of tartrate-resistant acid phosphatase expression in breast cancer.

e12594 Background: Tartrate-resistant acid phosphatase (TRAP) is a metalloproteinase-like protein that is expressed in several primary and metastatic tumors, and its expression is positively correlated with the oncogenic process. Tartrate-resistant acid phosphatase is also a novel product of activated macrophage. We have previously demonstrated the clinical significance of TRAP expression in tumor-infiltrating macrophages and serum TRAP in patients with metastatic breast cancer (BC). Therefore, TRAP protein can potentially be a predictive and prognostic marker to evaluate disease progression and therapeutic response in breast cancer patients with bone metastasis. We aim to investigate the role of TRAP expression in breast cancer metastasis and survival. Methods: RNA-seq expression data were obtained from The Cancer Genome Atlas (TCGA) database. Estrogen receptor (ER)-positive, human epidermal growth factor receptor-2 (HER2)-negative, and TNBC subtypes were included in the analyses. The TRAP-overexpressed and -silenced breast cancer cells (MCF7, 4T1, MDA-MB-231) were used for validation. Survival data was also retrieved from the TCGA database to verify the prognostic biomarker. Results: Through TCGA database analysis, we found that TRAP expression correlated to the Ki-67 expression indicating the cancer cell proliferating activity. Additionally, TRAP expression positively correlated with mesenchymal markers (SNAIL, CDH1, MMP9, Fibronectin), and negatively correlated with epithelial markers (SMAD2, SOX10), implying that the TRAP expression is related to the breast cancer Epithelial-Mesenchymal-Transition process. This phenomenon was validated in TRAP-altered cell and confirmed inferior survival with TRAP-expressed breast cancer patients in TCGA database. Conclusions: Combining clinical TCGA data and cell-based analyses showed that TRAP expression was significantly associated with breast cancer proliferating activity, metastatic potential, and inferior survival. TRAP serves as a breast cancer prognostic biomarker and can be considered as a therapeutic target. Further investigation is warranted.

Abstract P3-05-03: Identification of epigenetically silenced breast cancer driver genes

Abstract Breast cancer is clinically and molecularly complex disease driven by aberrant genetic and epigenetic alterations. Epigenetic alterations in particular DNA methylation changes are one of the most important events involved in breast cancer initiation and progression. Previous reports identified many aberrant DNA methylation signatures associated with molecular subtypes of breast cancer and over 100 candidate genes with promoter hypermethylation in breast cancer. However, it remains elusive which of these genes with promoter hypermethylation play “driver” role in tumorigenesis. In previous studies, the average gain of DNA methylation across all cancer samples compared to the average DNA methylation in normal samples has been the criterion to select for potential targets. However, known tumor suppressor driver genes regulated by methylation are relatively infrequently altered in target cancers. Therefore, we propose the paradoxical hypothesis that identifying hypermethylated cancer drivers require focusing on infrequent rather than frequent events. Hence, to identify these potential driver genes, we developed an algorithm with two unique properties. First, unlike previous studies we focused on targets that gained DNA methylation relatively infrequently (10-40%) and that lost expression in breast cancer. Second, using this algorithm, we distinguished cancer dependent gain of DNA methylation from age-dependent gain of methylation. To discern age dependent and independent DNA methylation changes, we generated DNA methylation sequencing data on 29 normal purified breast epithelium (age range 33-82 years old). Furthermore, to study the biological effects of the overexpression or downregulation of these genes, we generated DNA methylation sequencing data on 6 breast cancer cell lines. We also used DNA methylation and RNA expression datasets (675 cancer, 100 normal) available through the TCGA. Using our algorithm, we identified 53 genes with age independent promoter hypermethylation and loss of expression in TCGA tumor samples. To begin testing the biological effects of these driver genes, we performed canonical pathway enrichment analyses using Ingenuity Pathway Analysis software. We also investigated the mutational status of these genes and their molecular subtype enrichment. Based on these analyses, we picked 12 genes (C10orf125, RUNX3, YOD1, FXYD5, SMOC1, SLC16A5, RNLS, DKK1, PNPLA3, FZD10, RND2, and PLCB1) for further study. We stably overexpressed these potential driver genes in different breast cancer cell lines. Twelve genes out of the 12 tested, slowed cell proliferation and 9 decreased anchorage independent growth. We further validated these driver genes by knocking them out in normal human mammary epithelial cells using CRISPR/Cas9 tool. The loss of these genes, increased cell proliferation rate in normal human mammary epithelial cells compared to the control cells. In conclusion, based on our preliminary data, using bioinformatics tools as well as functional assays, we identified epigenetically altered breast cancer driver genes. Identifying and deciphering true epigenetic cancer drivers could potentially lead to the development of therapeutic drugs targeting these genes and/or targeting pathway dependence. Citation Format: Panjarian S, Madzo J, Slater C, Jelinek J, Chen X, Issa J-P. Identification of epigenetically silenced breast cancer driver genes [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-05-03.

The EZH2- H3K27me3-DNMT1 complex orchestrates epigenetic silencing of the wwc1 gene, a Hippo/YAP pathway upstream effector, in breast cancer epithelial cells

It is well known that epithelial-mesenchymal transition (EMT) can confer cancer cells with invasive and migratory capabilities associated with distant metastasis. As a key upstream factor in the Hippo pathway, Kibra (wwc1 gene) has been shown to suppress EMT in breast cancer cells, and we have found that its expression is reduced or lost completely in both human breast cancer cell lines and clinical tissue samples, particularly in triple negative breast cancer (TNBC). Unfortunately, the molecular mechanisms underlying this progression-associated event remain to be elucidated. Epigenetic gene silencing is one of the most common causes of suppressed expression of tumor suppressor genes. Furthermore, recent studies have demonstrated that EZH2 can recruit DNA methyltransferases, resulting in DNA methylation and subsequent gene silencing in certain circumstances. Thus, we hypothesized that there may exist a link between EZH2 and DNA methylation in association with wwc1 silencing in breast cancer. To test this hypothesis, we performed bisulfite sequencing, shRNA, co-IP, ChIP, MeDIP and ChIP-qPCR. As expected, RG108 or 5-Aza treatment improved the wwc1 gene transcription and Kibra protein expression. Both bisulfite sequencing and MeDIP demonstrated higher CpG methylation of the wwc1 promoter the TNBC cells (MDA-MB-231) than in luminal breast cancer cells (MCF7). It is noteworthy that ChIP and co-IP assays showed that EZH2, H3K27me3 and DNMT1 are enriched at the wwc1 promoter, and there exist physiologically relevant protein-protein interactions between them. We also found that EZH2 knockdown leads to a partial increase in Kibra expression and a considerable reduction in H3K27 and DNMT1 trimethylation. Moreover, ChIP-qPCR revealed more DNA fragments containing the wwc1 promoter in MDA-MB-231 than in MCF7 cells after immunoprecipitation with EZH2, DNMT1 and H3K27me3 antibodies. Collectively, our results reveal crosstalk between H3K27me3 inhibition catalyzed by EZH2 and CpG island methylation mediated by DNMT1 within the wwc1 promoter, which synergistically silence wwc1 gene expression in TNBC. Based on these results, we conclude that EZH2 shows promise as a potential anti-tumor target.

Transcriptomic pathway analysis of urokinase receptor silenced breast cancer cells: a microarray study

Urokinase plasminogen activator receptor (PLAUR) has been implicated in a variety of physiological and pathological conditions. The multi-functionality of PLAUR is due to its capacity to interact with many co-receptors to regulate extracellular proteolysis and intracellular signaling. Recent reports are identifying novel functions of PLAUR which were not evident in the past; however, the molecular mechanisms of PLAUR signaling are not completely understood. Here, we have compared the transcriptomes of silencing control (sicon) and PLAUR silenced (PLAURsi) MDA-MB-231 breast cancer cells on treatment with radiation. We isolated RNA from the cells, synthesized cDNA and measured the gene expression changes by microarray. We identified 24 downregulated and 53 upregulated genes, which were significantly (P-value < 0.005) affected by PLAUR silencing. Our analysis revealed 415 canonical pathways and 743 causal disease networks affected on silencing PLAUR. Transcriptomic changes and predicted pathways supported and consolidated some of the earlier understanding in the context of PLAUR signaling; including our recent observations in DNA damage and repair process. In addition, we have identified several novel pathways where PLAUR is implicated.