Tumor Suppressor In Breast Cancer Research Articles

Epithelial PBLD attenuates intestinal inflammatory response and improves intestinal barrier function by inhibiting NF-\u03baB signaling

Intestinal barrier function defects and dysregulation of intestinal immune responses are two key contributory factors in the pathogenesis of ulcerative colitis (UC). Phenazine biosynthesis-like domain-containing protein (PBLD) was recently identified as a tumor suppressor in gastric cancer, hepatocellular carcinoma, and breast cancer; however, its role in UC remains unclear. Therefore, we analyzed colonic tissue samples from patients with UC and constructed specific intestinal epithelial PBLD-deficient (PBLDIEC−/−) mice to investigate the role of this protein in UC pathogenesis. We found that epithelial PBLD was decreased in patients with UC and was correlated with levels of tight junction (TJ) and inflammatory proteins. PBLDIEC−/− mice were more susceptible to dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid-induced colitis compared with wild-type (WT) mice. In DSS-induced colitis, PBLDIEC−/− mice had impaired intestinal barrier function and greater immune cell infiltration in colonic tissue than WT mice. Furthermore, TJ proteins were markedly reduced in PBLDIEC−/− mice compared with WT mice with colitis. Nuclear factor (NF)-κB activation was markedly elevated and resulted in higher expression levels of downstream effectors (C–C motif chemokine ligand 20, interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) in colonic epithelial cells isolated from PBLDIEC−/− mice than WT mice with colitis. PBLD overexpression in intestinal epithelial cells (IECs) consistently inhibited TNF-α/interferon-γ-induced intestinal barrier disruption and TNF-α-induced inflammatory responses via the suppression of NF-κB. In addition, IKK inhibition (IKK-16) rescued excessive inflammatory responses induced by TNF-α in PBLD knockdown FHC cells. Co-immunoprecipitation assays showed that PBLD may interact with IKKα and IKKβ, thus inhibiting NF-κB signaling, decreasing inflammatory mediator production, attenuating colonic inflammation, and improving intestinal barrier function. Modulating PBLD expression may provide a novel approach for treatment in patients with UC.

Regulation of Wnt/PCP signaling through p97/VCP-KBTBD7-mediated Vangl ubiquitination and endoplasmic reticulum-associated degradation.

The four-pass transmembrane proteins Vangl1 and Vangl2 are dedicated core components of Wnt/planar cell polarity (Wnt/PCP) signaling that critically regulate polarized cell behaviors in many morphological and physiological processes. Here, we found that the abundance of Vangl proteins is tightly controlled by the ubiquitin-proteasome system through endoplasmic reticulum-associated degradation (ERAD). The key ERAD component p97/VCP directly binds to Vangl at a highly conserved VCP-interacting motif and recruits the E3 ligase KBTBD7 via its UBA-UBX adaptors to promote Vangl ubiquitination and ERAD. We found that Wnt5a/CK1 prevents Vangl ubiquitination and ERAD by inducing Vangl phosphorylation, which facilitates Vangl export from the ER to the plasma membrane. We also provide in vivo evidence that KBTBD7 regulates convergent extension during zebrafish gastrulation and functions as a tumor suppressor in breast cancer by promoting Vangl degradation. Our findings reveal a previously unknown regulatory mechanism of Wnt/PCP signaling through the p97/VCP-KBTBD7-mediated ERAD pathway.

CBFB cooperates with p53 to maintain TAp73 expression and suppress breast cancer.

The CBFB gene is frequently mutated in several types of solid tumors. Emerging evidence suggests that CBFB is a tumor suppressor in breast cancer. However, our understanding of the tumor suppressive function of CBFB remains incomplete. Here, we analyze genetic interactions between mutations of CBFB and other highly mutated genes in human breast cancer datasets and find that CBFB and TP53 mutations are mutually exclusive, suggesting a functional association between CBFB and p53. Integrated genomic studies reveal that TAp73 is a common transcriptional target of CBFB and p53. CBFB cooperates with p53 to maintain TAp73 expression, as either CBFB or p53 loss leads to TAp73 depletion. TAp73 re-expression abrogates the tumorigenic effect of CBFB deletion. Although TAp73 loss alone is insufficient for tumorigenesis, it enhances the tumorigenic effect of NOTCH3 overexpression, a downstream event of CBFB loss. Immunohistochemistry shows that p73 loss is coupled with higher proliferation in xenografts. Moreover, TAp73 loss-of-expression is a frequent event in human breast cancer tumors and cell lines. Together, our results significantly advance our understanding of the tumor suppressive functions of CBFB and reveal a mechanism underlying the communication between the two tumor suppressors CBFB and p53.

PCID2 Impacts Centrosome Duplication by Regulating the Nuclear Export of BARD1 mRNA in Breast Cancer Cells

The nuclear export of proteins and other macromolecules out of the nucleus to different intracellular locations is a significant and crucial function of the cell that affects cellular processes such as DNA synthesis, RNA transcription, protein processing, the cell cycle, and apoptosis. Chromosome region maintenance (CRM1) is the major export receptor within the cell, regulating the localization of most proteins. One nuclear export cargo of CRM1 is BARD1, which dimerizes with BRCA1 to regulate DNA repair in the nucleus, while the pair also function in the cytoplasm as negative regulators of centrosome amplification during cell cycle. Despite cooperation at both of these locations BARD1 and BRCA1 are independently exported from the nucleus to the cytoplasm. Our laboratory is interested characterizing the protein PCID2, which is involved in CRM1-mediated protein export and localizes to the centrosome, where it potentially regulates centrosome duplication. We previously demonstrated that PCID2 helps mediate CRM1 dependent export of BRCA1 from the nucleus to the centrosome in Hs578T Breast Cancer cells. The present work aims to determine the role of PCID2 in BARD1 nuclear export and localization in these same cells. PCID2 siRNA knockdowns were used and confirmed via western blot, and BARD1 localization was viewed via immunofluorescence. PCID2 knockdown caused a twenty percent decrease in BARD1 protein in the nucleus and a thirty percent decrease in BARD1 protein at the centrosome. The loss of BARD1 at centrosomes was tied to a fifteen percent increase in Hs578T cells demonstrating excess centrosomes duplication. The loss of BARD1 at both locations in the absence of PCID2 suggests this protein is not involved in the CRM1 mediated export of BARD1 protein from nucleus to centrosomes, and instead changes in cellular localization of BARD1 may be due to an observed 50% decrease in total BARD1 protein with PCID2 knockdown. Interestingly, PCID2 is also a part of the TREX-2 mRNA nuclear export complex; therefore loss of PCID2 may have instead impacted the nuclear export, and translation of BARD1 mRNA. Indeed, PCID2 siRNA knockdown led to a two-fold increase in nuclear BARD1 mRNA as observed by in situ hybridization. Nuclear accumulation of BARD1 mRNA likely contributed the overall decrease in BARD1 expression. The correlation between loss of PCID2 and centrosome amplification suggests PCID2 plays impacts centrosome duplication by regulating both BARD1 mRNA export and BRCA1 protein export; with the loss of PCID2 creating an overall decrease in these negative regulators at the centrosome. Understanding PCID2’s effect on centrosome duplication helps to determine it's impact on cell cycle regulation and define it's potential role as tumor suppressor in Breast Cancer.

The contradictory functions of NLRX1 in breast cancer allow for suppression or promotion of tumor growth and metastasis in a cell-specific manner.

Abstract Breast cancer is a deadly malignancy that is responsible for 15% of all cancer-related deaths in women. Examining the innate immune system in breast cancer has revealed the importance of pattern recognition receptors, such as NLRX1, in disease progression. NLRX1 has been implicated as a tumor suppressor because it suppresses inflammation by many mechanisms, including by inhibiting the NF-κB pathway. Here, we investigated the potential of NLRX1 as a tumor suppressor in breast cancer when it is expressed either by healthy host cells or cancer cells. To determine the role of NLRX1 in healthy host cells, we used the 4T1 breast cancer model in WT and Nlrx1−/− (KO) mice. KO mice exhibited increased lung metastasis and tumor size compared to WT mice. Gene expression data revealed increased expression of proliferation, survival, growth, invasion, and metastatic genes in tumors from KO mice compared to WT mice. Thus, NLRX1 in healthy host cells is protective against breast cancer tumor growth and metastasis. Next, we investigated the role of NLRX1 in breast cancer cells. 4T1 cells were transduced to overexpress NLRX1 and injected into WT (WTOE) and KO(KOOE) mice. Transduction controls were also generated and injected into WT (WTOE-CTL) and KO (KOOE-CTL) mice. KOOE mice developed larger tumors than KOOE-CTL mice. KOOE mice also developed more lung metastasis compared to WTOE, WTOE-CTL, and KOOE-CTL mice. This suggests NLRX1 in breast cancer cells contributes to tumor growth and metastasis, which directly contradicts the function of NLRX1 in healthy host cells and demonstrates a cell-specific function of NLRX1 in breast cancer. These cell-specific differences are critical foundations for the development of NLR-based breast cancer therapies.

Bioinformatic analysis of the expression and prognosis of ZNF589 in human breast cancer.

BackgroundZinc finger protein 589 (ZNF589) is a member of the zinc finger protein (ZNF) family and plays an important role in the differentiation of haemopoietic system stem cells. However, its effects on tumorigenesis and progression have not yet been reported. The purpose of this study was to explore the prognosis and underlying mechanism of ZNF589 in breast cancer (BRCA) through a bioinformatic analysis.MethodsZNF589 transcription levels in a TCGA BC cohort were analysed and then validated using Oncomine and UALCAN. The prognostic value of ZNF589 was determined based on the overall survival (OS) and relapse-free survival (RFS) based on The Cancer Genome Atlas (TCGA) cohort and Kaplan-Meier (K-M) database. LinkedOmics and STRING were carried out to explore the potential co-expressed genes and interactive proteins as well as corresponding enrichment analysis. A Gene Set Enrichment Analysis (GSEA) was performed between two gene matrices separated by the median cut-off value of ZNF589. The methylation levels of the ZNF589 promoter were analysed using UALCAN.ResultsZNF589 was downregulated in breast tumours, and lower expression was associated with poor OS (P=0.047) and RFS (P=0.0043) according to TCGA. A subgroup analysis showed that the downregulation of ZNF589 was significantly associated with poor OS in stage 3–4 patients (P=0.0249) and progesterone receptor (PR)-negative patients (P=0.0002). Consistently, lower ZNF589 predicted poor RFS in stage 3–4 patients (P=0.0090), hormone receptor-negative patients [oestrogen receptor (ER)–, P=0.0129; PR–, P=0.0130; human epidermal growth factor receptor 2 (HER2)–, P<0.0001] and triple-negative BRCA patients (P=0.0052). Univariate and multivariate Cox regressions indicated that ZNF589 could act as an independent prognostic biomarker for OS based on age and TNM stage. Functional enrichment analysis of co-expressed genes and a protein-protein interaction (PPI) network both suggested that ZNF589 expression was negatively correlated with cell cycle progression at the transcriptional and protein-interactive levels. Finally, we found that the downregulation of ZNF589 correlated with promoter hypermethylation, and the corresponding subgroup analysis presented similar results.ConclusionsOur study highlighted that ZNF589 could act as a potential prognostic biomarker and a tumour suppressor in BRCA. A functional enrichment analysis suggested that ZNF589 may participate in multiple cancer-related pathways, including the cell cycle. Epigenetic factor promoter methylation could contribute to the downregulation of ZNF589 expression. However, deeper research about its function and underlying mechanism in cancer progression is required.

Naturally-occurring spinosyn A and its derivatives function as argininosuccinate synthase activator and tumor inhibitor

Argininosuccinate synthase (ASS1) is a ubiquitous enzyme in mammals that catalyzes the formation of argininosuccinate from citrulline and aspartate. ASS1 genetic deficiency in patients leads to an autosomal recessive urea cycle disorder citrullinemia, while its somatic silence or down-regulation is very common in various human cancers. Here, we show that ASS1 functions as a tumor suppressor in breast cancer, and the pesticide spinosyn A (SPA) and its derivative LM-2I suppress breast tumor cell proliferation and growth by binding to and activating ASS1. The C13-C14 double bond in SPA and LM-2I while the Cys97 (C97) site in ASS1 are critical for the interaction between ASS1 and SPA or LM-2I. SPA and LM-2I treatment results in significant enhancement of ASS1 enzymatic activity in breast cancer cells, particularly in those cancer cells with low ASS1 expression, leading to reduced pyrimidine synthesis and consequently the inhibition of cancer cell proliferation. Thus, our results establish spinosyn A and its derivative LM-2I as potent ASS1 enzymatic activator and tumor inhibitor, which provides a therapeutic avenue for tumors with low ASS1 expression and for those non-tumor diseases caused by down-regulation of ASS1.

Cystatin M/E (Cystatin 6): A Janus-Faced Cysteine Protease Inhibitor with Both Tumor-Suppressing and Tumor-Promoting Functions.

Simple SummaryCystatin M/E is a low molecular mass protein and a potent inhibitor of proteolytic enzymes (cathepsins B, L, V, and legumain). Cystatin M/E participates in epidermal homeostasis and its deregulation is associated with several skin diseases. Cystatin M/E was initially identified as a tumor suppressor in breast cancer. Its gene is epigenetically downregulated and its methylation status holds a prognostic significance. Moreover, it may also serve as a biomarker for clinical diagnosis. This tumor-suppressing role was documented in cutaneous carcinoma, melanoma, lung, cervical, brain, prostate, gastric and renal cancers. Conversely, increased levels of cystatin M/E in triple-negative breast cancer tissues correlate with a higher risk of metastasis and a lower disease-free survival rate. Beside its orthodox tumor-suppressing role, cystatin M/E may operate as a tumor-promoting effector as reported for thyroid, oral and pancreatic cancer and hepatocellular carcinoma. Given its seemingly contradictory results, in-depth analysis of the regulatory mechanisms of the expression and activity of cystatin M/E during tumorigenesis have to be expanded. Likewise, the possible involvement of cystatin M/E in signaling pathways, beside its protease inhibitor function, remains to be scrutinized.Alongside its contribution in maintaining skin homeostasis and its probable involvement in fetal and placental development, cystatin M/E (also known as cystatin 6) was first described as a tumor suppressor of breast cancer. This review aims to provide an update on cystatin M/E with particular attention paid to its role during tumorigenesis. Cystatin M/E, which is related to type 2 cystatins, displays the unique property of being a dual tight-binding inhibitor of both legumain (also known as asparagine endopeptidase) and cysteine cathepsins L, V and B, while its expression level is epigenetically regulated via the methylation of the CST6 promoter region. The tumor-suppressing role of cystatin M/E was further reported in melanoma, cervical, brain, prostate, gastric and renal cancers, and cystatin M/E was proposed as a biomarker of prognostic significance. Contrariwise, cystatin M/E could have an antagonistic function, acting as a tumor promoter (e.g., oral, pancreatic cancer, thyroid and hepatocellular carcinoma). Taking into account these apparently divergent functions, there is an urgent need to decipher the molecular and cellular regulatory mechanisms of the expression and activity of cystatin M/E associated with the safeguarding homeostasis of the proteolytic balance as well as its imbalance in cancer.

An integrated bioinformatics analysis to investigate the targets of miR-133a-1 in breast cancer.

BackgroundThe microRNA (miRNA) miR-133a-1 has been identified as a tumor suppressor in breast cancer. However, the underlying mechanisms of miR-133a-1 in breast cancer have not been fully elucidated. This study aimed to explore the targets of miR-133a-1 in breast cancer using an integrated bioinformatics approach.MethodsHuman SKBR3 breast cancer cells were transfected with miR-133a-1 or a miRNA negative control (miRNA-NC) for 48 hours. The RNA-seq sequencing technique was performed to identify the differential expression of genes induced by miR-133a-1 overexpression. Functional enrichment analysis was conducted to determine the target genes and pathways involved in breast cancer.ResultsBreast cancer patients with high levels of miR-133a-1 expression commonly showed longer overall survival compared to patients with a low level of miR-133a-1 expression. Using Cuffdiff, we identified 1,216 differentially expressed genes induced by miR-133a-1 overexpression, including 653 upregulated and 563 downregulated genes. MOCS3 was the most upregulated gene and KRT14 was the most downregulated gene. The top 10 pathways related to the differentially expressed genes were identified through Gene Ontology (GO) enrichment analysis. Sex-determining region Y-box 9 (SOX9) demonstrated the highest semantic similarities among the differentially expressed genes. Since SOX9 and CD44 were hub nodes in the protein-protein interaction network, the SOX9 gene may be a target of miR-133a-1 in breast cancer.ConclusionsThis report provides useful insights for understanding the underlying mechanisms in the pathogenesis of breast cancer.

Regulation of ZMYND8 to Treat Cancer.

Zinc finger myeloid, nervy, and deformed epidermal autoregulatory factor 1-type containing 8 (Zinc finger MYND-type containing 8, ZMYND8) is a transcription factor, a histone H3-interacting protein, and a putative chromatin reader/effector that plays an essential role in regulating transcription during normal cellular growth. Mutations and altered expression of ZMYND8 are associated with the development and progression of cancer. Increased expression of ZMYND8 is linked to breast, prostate, colorectal, and cervical cancers. It exerts pro-oncogenic effects in breast and prostate cancers, and it promotes angiogenesis in zebrafish, as well as in breast and prostate cancers. In contrast, downregulation of ZMYND8 is also reported in breast, prostate, and nasopharyngeal cancers. ZMYND8 acts as a tumor suppressor in breast and prostate cancers, and it inhibits tumor growth by promoting differentiation; inhibiting proliferation, cell-cycle progression, invasiveness, and metastasis; and maintaining the epithelial phenotype in various types of cancers. These data together suggest that ZMYND8 is important in tumorigenesis; however, the existing data are contradictory. More studies are necessary to clarify the exact role of ZMYND8 in tumorigenesis. In the future, regulation of expression/activity of ZMYND8 and/or its binding partners may become useful in treating cancer.

CircNR3C2 promotes HRD1-mediated tumor-suppressive effect via sponging miR-513a-3p in triple-negative breast cancer

BackgroundE3 ubiquitin ligase HRD1 (HMG-CoA reductase degradation protein 1, alias synoviolin with SYVN1 as the official gene symbol) was found downregulated and acting as a tumor suppressor in breast cancer, while the exact expression profile of HRD1 in different breast cancer subtypes remains unknown. Recent studies characterized circular RNAs (circRNAs) playing an regulatory role as miRNA sponge in tumor progression, presenting a new viewpoint for the post-transcriptional regulation of cancer-related genes.MethodsExamination of the expression of HRD1 protein and mRNA was implemented using public microarray/RNA-sequencing datasets and breast cancer tissues/cell lines. Based on public RNA-sequencing results, online databases and enrichment/clustering analyses were used to predict the specific combinations of circRNA/miRNA that potentially govern HRD1 expression. Gain-of-function and rescue experiments in vitro and in vivo were executed to evaluate the suppressive effects of circNR3C2 on breast cancer progression through HRD1-mediated proteasomal degradation of Vimentin, which was identified using immunoblotting, immunoprecipitation, and in vitro ubiquitination assays.ResultsHRD1 is significantly underexpressed in triple-negative breast cancer (TNBC) against other subtypes and has an inverse correlation with Vimentin, inhibiting the proliferation, migration, invasion and EMT (epithelial-mesenchymal transition) process of breast cancer cells via inducing polyubiquitination-mediated proteasomal degradation of Vimentin. CircNR3C2 (hsa_circ_0071127) is also remarkably downregulated in TNBC, negatively correlated with the distant metastasis and lethality of invasive breast carcinoma. Overexpressing circNR3C2 in vitro and in vivo leads to a crucial enhancement of the tumor-suppressive effects of HRD1 through sponging miR-513a-3p.ConclusionsCollectively, we elucidated a bona fide circNR3C2/miR-513a-3p/HRD1/Vimentin axis that negatively regulates the metastasis of TNBC, suggesting that circNR3C2 and HRD1 can act as potential prognostic biomarkers. Our study may facilitate the development of therapeutic agents targeting circNR3C2 and HRD1 for patients with aggressive breast cancer.

SCIRT lncRNA Blocks the Shot of Breast Cancer Cells Self-Renewal Mechanism.

The study by Zagorac and colleagues represents an important step forward in the field of breast cancer, explaining a novel molecular mechanism of transition from slowly multiplying tumor-initiating cells (TIC) into their more differentiated version characterized by high proliferation. The mechanism involves the transcription factors SOX2 and EZH2, which directly repress transcription of cell-cycle genes and activate self-renewal genes in breast cancer cells. This mechanism is further controlled by a negative feedback loop mediated by a long noncoding RNA, SCIRT, not described previously, which is upregulated in tumorspheres and inhibits SOX2 and EZH2. SCIRT is an atypical tumor suppressor in breast cancer, being upregulated in cancer cells, but counteracting their aggressive phenotype. At the molecular level, by direct interaction with EZH2, SCIRT inhibits the transcriptional activity of EZH2 and "blocks the shot" of cancer cells' self-renewal. From a translational perspective, activating SCIRT or induction of SCIRT mimetics in breast cancer cells may lead to the dedifferentiation of TICs toward a less protumorigenic phenotype and a therapy-fragile state that could open new therapeutic avenues.See related article by Zagorac et al., p. 580.

Epigenetic Modulation of SPCA2 Reverses Epithelial to Mesenchymal Transition in Breast Cancer Cells.

Simple SummaryThe triple receptor negative breast cancer subtype, or TNBC, currently has no tailored treatment options. TNBC is highly metastatic, associated with high patient mortality, and disproportionately occurs in Black/African American women where it contributes to racial disparities in health outcomes. Therefore, we focused on new therapeutic approaches to TNBC. We discovered that levels of the Calcium-ATPase SPCA2 are abnormally low in TNBC and that these low levels correlate with poor survival prognosis in patients. Previously, we showed that recombinant SPCA2 prevented TNBC cells from acquiring aggressive “mesenchymal” properties associated with metastasis both in vitro and in vivo. These findings motivated us to search for drugs that turn the SPCA2 gene back on in TNBC cells. In this study, we show that histone deacetylase inhibitors increase SPCA2 levels, activate Ca2+ signaling and convert cancer cells to a less aggressive “epithelial” state. These findings could lead to new treatment options for TNBC.The secretory pathway Ca2+-ATPase SPCA2 is a tumor suppressor in triple receptor negative breast cancer (TNBC), a highly aggressive molecular subtype that lacks tailored treatment options. Low expression of SPCA2 in TNBC confers poor survival prognosis in patients. Previous work has established that re-introducing SPCA2 to TNBC cells restores basal Ca2+ signaling, represses mesenchymal gene expression, mitigates tumor migration in vitro and metastasis in vivo. In this study, we examined the effect of histone deacetylase inhibitors (HDACi) in TNBC cell lines. We show that the pan-HDACi vorinostat and the class I HDACi romidepsin induce dose-dependent upregulation of SPCA2 transcript with concurrent downregulation of mesenchymal markers and tumor cell migration characteristic of epithelial phenotype. Silencing SPCA2 abolished the ability of HDACi to reverse epithelial to mesenchymal transition (EMT). Independent of ATPase activity, SPCA2 elevated resting Ca2+ levels to activate downstream components of non-canonical Wnt/Ca2+ signaling. HDACi treatment led to SPCA2-dependent phosphorylation of CAMKII and β-catenin, turning Wnt signaling off. We conclude that SPCA2 mediates the efficacy of HDACi in reversing EMT in TNBC by a novel mode of non-canonical Wnt/Ca2+ signaling. Our findings provide incentive for screening epigenetic modulators that exploit Ca2+ signaling pathways to reverse EMT in breast tumors.

The Role of STAT1 in T Helper Cell Differentiation during Breast Cancer Progression.

Members of the signal transducer and activator of transcription (STAT) protein family are intracellular transcription factors that facilitate several facets of cellular immunity, proliferation, apoptosis, and differentiation. They are principally stimulated by membrane receptor-associated Janus kinases. Dysregulation of this pathway is often detected in primary tumors and hints at augmented angiogenesis, which enriches tumors persistence and immunosuppression. STAT proteins play indispensable roles in cytokine signaling and T helper (Th) cell differentiation. Among STAT proteins, STAT1 plays a vital role in interferon signaling, which initiates the expression of genes encoding proteins with antitumor and apoptotic roles. STAT1 signaling is essential for Th1 cell differentiation. Several studies have also shown the role of STAT1 as a tumor suppressor in breast cancer, which is the most common intrusive malignancy and the second most common cause of cancer death in women. Herein, we review the intricate STAT1-mediated molecular mechanisms associated with Th cell differentiation and anti-tumor function in breast cancer.

CTEN Inhibits Tumor Angiogenesis and Growth by Targeting VEGFA Through Down-Regulation of β-Catenin in Breast Cancer.

C-terminal tensin-like (CTEN) belongs to the tensin gene family, which encodes proteins that localize to focal adhesions and modulate integrin function. Accumulating studies have reported that CTEN expression can be upregulated or downregulated in different types of cancers, suggesting that CTEN has both oncogenic and tumor suppressor functions. In this study, by analyzing the expression level of CTEN in the human breast cancer (BRCA) samples from the clinically annotated genomic database, The Cancer Genome Atlas, we found that CTEN was downregulated in different BRCA subclasses, including luminal, human epidermal growth factor receptor 2 positive and triple-negative BRCA. Consistently, the protein level of CTEN was also reduced in BRCA based on the Proteomic Tumor Analysis Consortium. In contrast, vascular endothelial growth factor A (VEGFA), a signal protein that stimulates the formation of blood vessels, was upregulated in BRCA. CTEN overexpression in human umbilical vein endothelial cells and MCF7 significantly suppressed the expression of VEGFA, inhibited cell proliferation, migration, and tube formation in vitro. Mechanistically, CTEN bind to casitas B-lineage lymphoma (c-Cbl), an E3 ubiquitin-protein ligase, and decreased the β-catenin expression. In turn, the downregulation of β-catenin reduced the expression of VEGFA. Rescuing β-catenin expression effectively ameliorated the effect of CTEN overexpression in cell proliferation, migration, and tube formation. In conclusion, CTEN inhibited tumor angiogenesis by targeting VEGFA through c-Cbl-mediated down-regulation of β-catenin and may serve as a tumor suppressor in BRCA.

MiR-491-5p, as a Tumor Suppressor, Prevents Migration and Invasion of Breast Cancer by Targeting ZNF-703 to Regulate AKT/mTOR Pathway.

BackgroundLarge amounts of microRNAs (miRNAs) have been reported to be aberrantly expressed in malignant cancers. MiR-491-5p makes a significant contribution to the inhibition of multiple cancer processes. However, the specific mechanism and function of miR-491-5p and in breast cancer (BC) is still not fully elucidated.MethodsMiR-491-5p and ZNF-703 expressions or gene transfection effects were identified by RT-qPCR or Western blot in BC tissues or cells. And ZNF-703 expression was monitored through immunohistochemistry method. Cellular function was also confirmed using Transwell assay. Besides, AKT/mTOR pathway-related proteins were analyzed using Western blotting analysis. Moreover, the interplay between miR-491-5p and ZNF-703 was verified through dual-luciferase reporter assay.ResultsmiR-491-5p was lowly expressed, ZNF-703 was highly expressed in BC, and miR-491-5p with low expression and ZNF-703 with high expression were associated with poor prognosis of BC patients. Results of cellular function revealed that overexpression of miR-491-5p markedly suppressed BC cell migration and invasion, and knockdown of miR-491-5p had the opposite effect. Besides, mechanism research disclosed that miR-491-5p directly could bind to ZNF-703 and downregulate ZNF-703. Moreover, we proved that ZNF-703 could prominently reverse the influences of miR-491-5p on the migration and invasion of BC cells. More importantly, the data revealed that miR-491-5p repressed AKT/mTOR pathway by ZNF-703 in BC cells.ConclusionMiR-491-5p prominently suppresses the metastasis of BC cells through ZNF-703 to regulate AKT/mTOR pathway, indicating that miR-491-5p and ZNF-703 might be served as the potential therapeutic targets for BC.

MicroRNA-16-1-3p Represses Breast Tumor Growth and Metastasis by Inhibiting PGK1-Mediated Warburg Effect

The Warburg effect (aerobic glycolysis) is a hallmark of cancer and is becoming a promising target for diagnosis and therapy. Phosphoglycerate kinase 1 (PGK1) is the first adenosine triphosphate (ATP)-generating glycolytic enzyme in the aerobic glycolysis pathway and plays an important role in cancer development and progression. However, how microRNAs (miRNAs) regulate PGK1-mediated aerobic glycolysis remains unknown. Here, we show that miR-16-1-3p inhibits PGK1 expression by directly targeting its 3′-untranslated region. Through inhibition of PGK1, miR-16-1-3p suppressed aerobic glycolysis by decreasing glucose uptake, lactate and ATP production, and extracellular acidification rate, and increasing oxygen consumption rate in breast cancer cells. Aerobic glycolysis regulated by the miR-16-1-3p/PGK1 axis is critical for modulating breast cancer cell proliferation, migration, invasion and metastasis in vitro and in vivo. In breast cancer patients, miR-16-1-3p expression is negatively correlated with PGK1 expression and breast cancer lung metastasis. Our findings provide clues regarding the role of miR-16-1-3p as a tumor suppressor in breast cancer through PGK1 suppression. Targeting PGK1 through miR-16-1-3p could be a promising strategy for breast cancer therapy.

Activating transcription factor-2 (ATF2) is a key determinant of resistance to endocrine treatment in an in vitro model of breast cancer

BackgroundActivating transcription factor-2 (ATF2), a member of the leucine zipper family of DNA binding proteins, has been implicated as a tumour suppressor in breast cancer. However, its exact role in breast cancer endocrine resistance is still unclear. We have previously shown that silencing of ATF2 leads to a loss in the growth-inhibitory effects of tamoxifen in the oestrogen receptor (ER)-positive, tamoxifen-sensitive MCF7 cell line and highlighted that this multi-faceted transcription factor is key to the effects of tamoxifen in an endocrine sensitive model. In this work, we explored further the in vitro role of ATF2 in defining the resistance to endocrine treatment.Materials and methodsWe knocked down ATF2 in TAMR, LCC2 and LCC9 tamoxifen-resistant breast cancer cell lines as well as the parental tamoxifen sensitive MCF7 cell line and investigated the effects on growth, colony formation and cell migration. We also performed a microarray gene expression profiling (Illumina Human HT12_v4) to explore alterations in gene expression between MCF7 and TAMRs after ATF2 silencing and confirmed gene expression changes by quantitative RT-PCR.ResultsBy silencing ATF2, we observed a significant growth reduction of TAMR, LCC2 and LCC9 with no such effect observed with the parental MCF7 cells. ATF2 silencing was also associated with a significant inhibition of TAMR, LCC2 and LCC9 cell migration and colony formation. Interestingly, knockdown of ATF2 enhanced the levels of ER and ER-regulated genes, TFF1, GREB1, NCOA3 and PGR, in TAMR cells both at RNA and protein levels. Microarray gene expression identified a number of genes known to mediate tamoxifen resistance, to be differentially regulated by ATF2 in TAMR in relation to the parental MCF7 cells. Moreover, differential pathway analysis confirmed enhanced ER activity after ATF2 knockdown in TAMR cells.ConclusionThese data demonstrate that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity.

Long non-coding RNA IGF2-AS represses breast cancer tumorigenesis by epigenetically regulating IGF2.

Long non-coding RNAs are a kind of endogenous ncRNAs with a length of more than 200 bp. Accumulating evidence suggests that long non-coding RNAs function as pivotal regulators in tumorigenesis and progression. However, their biological roles in breast cancer remain largely unknown. Here, we found that IGF2 antisense RNA (IGF2-AS) was significantly decreased in breast cancer tissues, cell lines, and plasma. Patients with low IGF2-AS were more likely to develop larger tumor size and later clinical stage. Overexpression of IGF2-AS evidently inhibited the proliferation and induced apoptosis of MCF-7 and T47D cells in vitro, as well as retarded tumor growth in vivo. Further investigation revealed that IGF2-AS inhibited the expression of its sense-cognate gene IGF2 in an epigenetic DNMT1-dependent manner, resulting in the inactivation of downstream oncogenic PI3K/AKT/mTOR signaling pathway. Enforced expression of IGF2 could significantly block the tumor inhibitory effect of IGF2-AS. Importantly, we found that IGF2-AS could be used as an effective biomarker for breast cancer diagnosis and prognosis. Taken together, our study indicates that IGF2-AS is a tumor suppressor in breast cancer, restoration of IGF2-AS may be a promising treatment for this fatal disease.

MicroRNA-623 inhibits tumor progression and is a predictor of poor prognosis of breast cancer.

Dysregulated microRNAs (miRNAs) serve vital roles in the progression and prognosis of breast cancer. miR-623 has been reported to influence the progression of numerous other cancers, such as lung adenocarcinoma and hepatocellular carcinoma, however, its role in breast cancer remains unclear. In the present study, the mRNA expression of miR-623 was studied in 121 pairs of breast cancer and adjacent normal tissues and cultured cell lines by reverse-transcription quantitative PCR. The association between miR-623 expression and clinical characteristics or the overall survival rate of patients was investigated by the χ2 test or Cox regression analysis, respectively. The role of miR-623 in cell proliferation, migration and invasion of breast cancer cells was evaluated by cell transfection to regulate miR-623 expression and the CCK8 and Transwell assays, respectively. miR-623 was downregulated in breast cancer tissues and cell lines compared with normal tissues and breast epithelial cell lines. The χ2 test demonstrated that the downregulation of miR-623 was associated with the tumor node metastasis (TNM) stage of patients with breast cancer. miR-623 and TNM stage were considered as two independent prognostic factors for breast cancer. Additionally, cell proliferation, migration, and invasion of breast cancer cells were promoted by the downregulation of miR-623, while upregulation of miR-623 led to inhibition of the aforementioned processes. Downregulation of miR-623 in breast cancer is associated with the development of breast cancer and indicates a poor prognosis of patients. The downregulation of miR-623 promotes cell proliferation, migration and invasion of breast cancer. The findings of the present study indicate that miR-623 functions as a prognosis biomarker and a tumor suppressor in breast cancer, which provides a potential therapeutic target for patients with breast cancer.