Evaluation of genetic variation in tumor suppressor miRNA encoding and their target genes in breast cancer; focus on miRNA interaction and expression analysis

: Breast cancer requires estrogen for its initiation and maintenance before progressing into a more aggressive stage. We proposed to study the role of estrogen receptor (ER) target genes in the promotion of breast cancer. To achieve this goal we generated a regulable repressor KEDPK to directly turn-off the expression of ER target genes. The KEDPK contains an ER-DBD that recognizes the ERE elements of ER target genes, a KRAB repressive domain which can silence target genes when tethered to the promoter region and a mutated PR-LED which responds only to exogenous ligand. The KEDPK shows dose- dependent inhibition of ER target genes in transient transfections. The inhibitory activity of KEDPK is under the control of its ligand RU486 and is specific to ER target genes. Currently we have devoted our effort to generate stable cell lines expressing KEDPK and determine its ability to suppress the endogenous ER target genes. The success of developing these repressors will allow us to study the role of ER target genes in breast cancer progression. Results obtained in these studies will be highly relevant to efforts in optimization of current hormone therapy and gene therapy for breast cancer.

: Estrogen receptor (ER) is ligand-dependent transcription factor that has an important role in the development and progression of breast cancer. In this study, we developed a chimeric repressor to turn off ER target genes with the aim to directly investigate the role of ER target genes in tumor progression. The chimeric repressor contains the ER DNA-binding domain, a Krupple-Associated Box (KRAB) repressor domain and a truncated progesterone ligand-binding domain. The ability of the chimeric repressor to block ER mediated transcription was assessed in transient transfection assays. ER-induced reporter activity was inhibited by the repressor in a dose-dependent manner, with the maximum effect of more than 80% reduction. The inhibitory activity of the chimeric repressor was tightly under the control of RU486. Though we successfully generated regulable repressors to inhibit the ER reporter genes in vitro in transient transfection, these repressors fail to suppress endogenous ER target gene expression in breast cancer cell lines. More efforts need to be focus on in vivo gene expression. The success in creating such a repressor will provide a useful tool to study the role of ER target genes in breast cancer progression, and it may be potentially useful for gene therapy of breast cancer.

BackgroundE2F transcription factors are crucial in various biological processes, including cell proliferation, differentiation, and apoptosis. However, the exact role of E2F target genes in breast cancer (BC), as well as their influence on survival and immune response, remains poorly understood.MethodsTo investigate the differential expression of E2F target genes and their relationship with patient prognosis and immune cell infiltration, transcriptomic data from the Cancer Genome Atlas database were analyzed. A risk model was developed to identify genes associated with survival. BC samples were clustered into high-expression (C1) and low-expression (C2) groups of E2F target genes. The correlation between gene expression and factors such as survival, immune cell infiltration (CD4 + and CD8 + T cells), and immune checkpoint inhibitors (PD-L1 and PD-L2) was analyzed. We analyzed the link between clusters and clinical characteristics using the chi-squared test. For further investigation, single-cell data from GSE243526 were utilized. For validation, the expression levels of JPT1 and TBRG4 were assessed using RT-qPCR in clinical samples.ResultsGenes targeting E2F, such as AURKB, JPT1, TBRG4, and KIF4A, showed increased expression linked to poor patient prognosis, regardless of clinical features. Kaplan-Meier survival analysis revealed that elevated expression of these genes correlated significantly with decreased survival rates and heightened mortality risk. Single-cell data confirmed that candidate genes exhibited higher expression in tumor-associated epithelial cells than healthy ones. Furthermore, samples from group C1 exhibited a lower survival rate than C2. Immune cell infiltration analysis determined that high expression of E2F target genes in the C1 subgroup was associated with diminished T cell infiltration and increased PD-L1 and PD-L2 expression. A strong and significant association was also identified between triple-negative breast cancer and the C1 cluster. RT-qPCR validation confirmed a significant elevation of JPT1 and TBRG4 expression levels relative to adjacent healthy tissues in BC.ConclusionThese findings suggest that E2F target genes, including JPT1 and TBRG4, may act as prognostic biomarkers and contribute to immune evasion in BC. E2F target genes can also offer good potential for classifying and treating patients.

Breast cancer is the leading type of cancer among females. However, the association between microRNAs (miRNAs) and target genes in breast tumorigenesis is poorly studied. The original data set GSE26659 was downloaded from the Gene Expression Omnibus, and then the differentially expressed miRNAs among 77 breast cancer patients and 17 controls were identified using the Limma package in R software. Furthermore, breast cancer-related differentially expressed miRNAs were selected from a human miRNA disease database and their target genes were selected from five miRNA databases. Then, functional analysis was performed for the target genes followed by construction of a miRNA-target gene network. A total of 34 differentially expressed miRNAs were identified, including 13 breast cancer-related miRNAs. Moreover, the target genes of the 13 miRNAs were significantly enriched in regulation of transcription (P=7.43E-09) and pathways related to cancer (P=3.33E-11). Finally, eight upregulated miRNAs (including hsa-miR-425) and five downregulated miRNAs (including hsa-miR-143, hsa-miR-145 and hsa-miR-125b) were identified in the miRNA-target gene network. In conclusion, using bioinformatics approaches, we demonstrate that the changes in regulation of transcription and cancer pathways may play significant roles in the process of breast cancerogenesis. Differentially expressed miRNAs and their target genes may be new targets for breast cancer therapy.

Breast cancer is one of the most common malignant cancers among women with increasing number of patients. Gene regulatory network and identifying target genes for cancer treatment, and reducing breast cancer death rates is of great importance medically. This study aims to model gene regulatory network of breast cancer using hidden Markov model which greatly aids doctors in early diagnosis and faster treatment of breast cancer using identification of target genes. In this study, gene expressions of $206$ patients diagnosed with four subtypes of breast cancer including, Basal, Her2, LumA, LumB, were obtained from the Cancer Genome Atlas (TCGA). $8$ genes with the verified interaction among them were investigated by hidden Markov model of gene regulatory network and target genes. with the results of transition probability matrix, FADD, TNFRSF10B, CASP8 are the target genes in the mentioned cancer subtypes so that genes that their transmit probabilities are more than an initial value of $0.125$ are regulatory genes and transmit matrix identifies the probability of the mentioned cancers regarding gene expression level.

Both cyclin D1 and c-myc are key molecules in breast cancer carcinogenesis, and their transcriptional level and stability are regulated through several signaling pathways, including the Wnt signaling pathway. We performed immunohistochemical and mutational analyses of Wnt signaling components to investigate the association of Wnt signaling alterations with breast cancer carcinogenesis using 49 surgically resected primary breast cancer samples. Positive staining of cyclin D1 and c-myc was observed in 55.1% and 30.6% of the 49 breast cancer samples, respectively. Aberrant cytoplasmic expression of beta-catenin, which indicates the existence of alterations in the Wnt signaling pathway, was observed in 38.8% of breast cancer samples, though no mutation was found in the beta-catenin and Axin 1 genes. Reduced expression of APC was observed in 34.7% of samples. Statistical analysis revealed strong correlations between overexpression of beta-catenin and that of cyclin D1 and c-myc (p=0.0001 and 0.0117, respectively). Furthermore, overexpression of beta-catenin was significantly correlated with reduced expression of APC (p=0.0127). Wnt signaling alterations were frequently observed in breast cancer from the results of beta-catenin immunohistochemistry, although no mutation in the components of the Wnt signaling pathway was found in the present study. Based on the statistical analyses, we speculated that reduced expression of APC leads to overexpression of beta-catenin, and aberrant expression of cyclin D1 and c-myc mainly depends on alterations in the Wnt signaling pathway in breast cancer.

Breast cancer (BC) is both an inherited and environmental-based disease which is the leading cause of death among women. Early detection of BC can prevent invasion and metastasis in patients. Currently, researchers endeavor to find non-invasive biological markers from body fluids. Circulating non-coding RNAs such as microRNAs (miRNAs) can potentially be valuable prognostic and detective biomarkers. To identify novel miRNA-based biomarkers, we utilized bioinformatic tools. To reach this goal, the miRNA expression profiles of GSE31309, GSE 44,281, GSE98181, and GSE118782 were analyzed through a limma package of R. Target gene prediction of differentially expressed miRNAs, called differentially expressed miRNAs (DEMs), between samples of healthy individuals and BC patients was implemented through Multimir package of R. Functional enrichment analysis of predicted target genes through Enrich R (online database) revealed that most of the genes are enriched in the mitochondrial outer membrane for cellular component, intrinsic apoptotic signaling regulations for biological processes, transcription co-receptor activity for molecular functions, and dopaminergic synapse pathway. Furthermore, our survival analysis results revealed that miR-29c and mir-361 have the potential to serve as prognostic biomarkers.

Enhancer of Zeste homolog 2 (EZH2) is a histone methyltransferase which catalyzes the trimethylation of lysine 27 of histone H3 (H3K27me3), an epigenetic mark associated with chromatin compaction and transcriptional repression of target genes. This constitutes its canonical mechanism of action. Several studies have shown that EZH2 is able to activate gene transcription by forming transcriptional complexes through mechanisms that do not involve histone methylation. Indeed, EZH2 can also act in a non-canonical function by regulating transcription independently of its enzymatic activity. We have previously described that progestins induce the interaction between Progesterone Receptor (PR) and EZH2 in breast cancer (BC) cells, which results in the downregulation of tumor suppressor GATA3 and in the increase in cell proliferation. Since EZH2 has been implicated in the progression of several types of cancer, including those of the breast, and our own previous results indicate that EZH2 is required for progestin-induced breast cancer growth, we hypothesized that EZH2 could function as a mediator in the pro-tumorigenic effects of progestins, targeting specific tumor suppressor and differentiating genes to allow ER/PR-positive BC growth. In the present work we found that progestin treatment of T47D cells induced EZH2 mRNA and protein expression, which was abolished by the use of the progestin antagonist RU486 and when PR expression was blocked by siRNAs. We also studied the participation of EZH2 in the regulation of PR-regulated genes TNFalpha (TNFα), cyclin D1 and TIMP2. By ELISA assays, we observed that progestin treatment of T47D cells for 24 h induced TNFα secretion. This effect was abolished when EZH2 expression was inhibited in the presence of EZH2 siRNAs and when EZH2 enzymatic activity was blocked by the use of GSK126. Regarding TIMP2, we observed that EZH2 canonical activity participates in TIMP2 downregulation exerted by progestin treatment. We showed that EZH2 non-canonical function is also involved in progestin modulation of TIMP2 given that blockage of EZH2 activity did not completely revert progestin effect. Finally, we demonstrated that progestin upregulation of cyclin D1 did not require EZH2 activity. Our results prove that EZH2 engagement in PR target genes may involve its canonical or non-canonical function and therefore support further studies on other progestin-regulated cancer genes implicated in tumor growth.Presentation: No date and time listed

HOXB7 encodes a transcription factor that is overexpressed in a number of cancers and encompasses many oncogenic functions. Previous results have shown it to promote cell proliferation, angiogenesis, epithelial–mesenchymal transition, DNA repair and cell survival. Because of its role in many cancers and tumorigenic processes, HOXB7 has been suggested to be a potential drug target. However, HOXB7 binding sites on chromatin and its targets are poorly known. The aim of our study was to identify HOXB7 binding sites on breast cancer cell chromatin and to delineate direct target genes located nearby these binding sites. We found 1,504 HOXB7 chromatin binding sites in BT‐474 breast cancer cell line that overexpresses HOXB7. Seventeen selected binding sites were validated by ChIP‐qPCR in several breast cancer cell lines. Furthermore, we analyzed expression of a large number of genes located nearby HOXB7 binding sites and found several new direct targets, such as CTNND2 and SCGB1D2. Identification of HOXB7 chromatin binding sites and target genes is essential to understand better the role of HOXB7 in breast cancer and mechanisms by which it regulates tumorigenic processes.

Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes

Loss of heterozygosity (LOH) at the distal half of chromosome arm 11q is frequent in a variety of human tumors, including breast cancer, and is often associated with poor prognosis. In an ongoing attempt to locate and characterize the main target genes within this chromosome region, we first looked for aberrations in known genes either suggested to be involved in tumorigenesis or shown to suppress tumor formation. We examined 31 primary breast tumors showing LOH in 11q21-24 for mutations in the MRE11A, CHK1, PPP2R1B, and TSLC1 genes. The absence of intragenic alterations related to cancer led us next to evaluate possible gene silencing resulting from promoter region CpG hypermethylation, using the bisulfite sequencing technique. In addition to the four genes mentioned above, we also analyzed the ATM gene, which had been investigated for certain germline mutations in an earlier study. Only the TSLC1 promoter region exhibited aberrant methylation patterns, and altogether 33% (10/30) of the successfully analyzed tumors showed evidence of elevated levels of TSLC1 CpG methylation. Ten percent (3/30) of the tumors showed significantly increased methylation. Thus, as has been shown in lung and some other forms of cancer, hypermethylation of the TSLC1 promoter region is also frequently a second hit along with LOH in breast cancer.

The purpose of this study is to investigate the biologic significance of novel GLI1 splice variants in breast cancer. African American (AA) women suffer a disproportionately high burden of basal-like breast cancer, an aggressive subtype that has no targeted therapy (1). About 15-20% of breast cancers are triple-negative/basal-like, are associated with poor clinical outcomes and show disproportionately higher prevalence in younger women of African descent (2-4). The Hedgehog (Hh)/GLI1 developmental pathway has emerged as a therapeutic target in many cancers, including breast cancer studies from our lab (5, 6). Overexpression of the main Hh transcriptional mediator GLI1, correlates with poor patient prognosis and relapse. The human GLI1 transcript undergoes alternative splicing producing two shorter isoforms, an N-terminal deletion variant (GLI1ΔN) (7) and a truncated GLI1 (tGLI1) (8), which have been reported to have different patterns of tissue expression and function. tGLI1 has been identified as being highly expressed in several cancers, including in breast cancer (9). We performed in silico analysis of the NCBI database for evidence of additional human GLI1 transcripts and identified a novel GLI1 splice variant, GLI1-X2, that has an in-frame deletion of the entire exon 10-zinc finger 5 domain of the GLI1 gene. Our preliminary data show that GLI1-X2 is expressed in basal-like breast cancers at substantially higher levels than GLI1, tGLI1, or GLI1ΔN. To our knowledge, we are the first to demonstrate expression of GLI1-X2 in any model and our findings expand the GLI1 family of zinc finger transcription factors. Our objectives are to examine the expression differences of the GLI1 splice variants in basal-like and luminal breast cancer cell models, AA and White patient samples and to investigate the biologic function of GLI1-X2 in breast cancer. Previous studies report that GLI1ΔN and tGLI1 proteins retain intact functional zinc finger domains. Thus, we hypothesize that the loss of zinc-finger 5 is critical for GLI1-X2's functional role and regulation of downstream target genes in breast cancer. As the biologic significance of GLI1-X2 in normal and cancer biology is unknown, this proposed work is the first to characterize its function and potential role in cancer. Citation Format: Maria S. Dixon, Lhoucine Chdid, David R. Lamson, Michael T. Tarpley, Helen O. Oladapo, Jodie M. Fleming, Jennifer A. Freedman, Gayathri R. Devi, Kevin P. Williams. Identification and functional characterization of a novel GLI1 splice variant in breast cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C100.

Systemic identification of estrogen-regulated genes in breast cancer cells through cap analysis of gene expression mapping

Frequent inactivation of MCC/CTNNBIP1 and overexpression of phospho-beta-cateninY654 are associated with breast carcinoma: Clinical and prognostic significance

FOXA1 is a pioneer transcription factor which has been established as a carcinogenic factor and can regulate the expression of downstream target genes in breast cancer. We hypothesized that genetic variants modulating FOXA1 expression might play a role in the risk of breast cancer. Physical interaction predicted by PreSTIGE analysis and CHIA-PET data integration with cis-expression quantitative trait loci (cis-eQTL) based SNP-FOXA1 analysis were used to identify potentially regulatory variants modulating the expression of FOXA1. Then, we utilized a case-control study consisting of 855 new diagnosed breast cancer cases and 920 controls in the Chinese population to identify breast cancer associated variants. Biological assays were conducted in breast cancer cell lines to illustrate the effects of associated variants on breast cancer risk. We identified that rs7160774 G > A variant was associated with lower risk of breast cancer (OR = 0.77, 95% confidence interval = 0.62-0.96, P = 0.022). Biological experiments indicated that rs7160774[A] allele down-regulated the expression of FOXA1 compared to the G allele by influencing transcription factor binding affinity, thus playing an important role in the development of breast cancer. Our study suggested that the regulatory variant rs7160774 was associated with risk of breast cancer by long-range modulating FOXA1 expression and provided critical insights into pinpoint causal genetic variants.