Tumor Suppressor Gene In Breast Cancer Research Articles

Molecular Characterization and Subtyping of Breast Cancer Cell Lines Provide Novel Insights into Cancer Relevant Genes.

Continuous cell lines are important and commonly used in vitro models in breast cancer (BC) research. Selection of the appropriate model cell line is crucial and requires consideration of their molecular characteristics. To characterize BC cell line models in depth, we profiled a panel of 29 authenticated and publicly available BC cell lines by mRNA-sequencing, mutation analysis, and immunoblotting. Gene expression profiles separated BC cell lines in two major clusters that represent basal-like (mainly triple-negative BC) and luminal BC subtypes, respectively. HER2-positive cell lines were located within the luminal cluster. Mutation calling highlighted the frequent aberration of TP53 and BRCA2 in BC cell lines, which, therefore, share relevant characteristics with primary BC. Furthermore, we showed that the data can be used to find novel, potential oncogenic fusion transcripts, e.g., FGFR2::CRYBG1 and RTN4IP1::CRYBG1 in cell line MFM-223, and to elucidate the regulatory circuit of IRX genes and KLF15 as novel candidate tumor suppressor genes in BC. Our data indicated that KLF15 was activated by IRX1 and inhibited by IRX3. Moreover, KLF15 inhibited IRX1 in cell line HCC-1599. Each BC cell line carries unique molecular features. Therefore, the molecular characteristics of BC cell lines described here might serve as a valuable resource to improve the selection of appropriate models for BC research.

Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells.

Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear. We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9-Tet1CD-based CDO1-targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated. CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p<.05). The CRISPR/dCas9-based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non-invasive biomarker for early diagnosis of BC. CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.

Expression of BRCA1 by immunohistochemistry and its association with ER, PR, Her2neu status in infiltrative ductal carcinoma of breast.

Breast cancer is a heterogeneous disease, which differs in its clinical behaviors and responses to treatment and outcome. The prognosis of breast cancer depends on histopathological parameters and molecular subtypes. Among more than 300 genes, which are involved in the pathogenesis of breast cancer tumor suppressor gene such as BRCA1 is known to play a significant role in hereditary cancers. However, its role in sporadic cases of infiltrating ductal carcinoma is yet to be established. To evaluate the expression of BRCA1 in infiltrative ductal carcinoma and to analyze the association of BRCA1 with histopathological parameters and estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor2 (Her2) neu expression. This was a laboratory-based exploratory study in which 56 patients with infiltrative ductal carcinoma who underwent radical mastectomy from October 2019 to July 2021 were included. Patients with chemotherapy and radiotherapy, trucut biopsies, and incomplete patient details were excluded. Immunostaining for BRCA1 was performed. Individual clinicopathological parameters were compared with the BRCA1 mutation. Statistical analysis was done using SPSS 22. A P value of < 0.05 was considered statistically significant. Among 56 cases of IDC, 18 cases (32.1%) showed BRCA1 mutation. BRCA1 mutation was associated with postmenopausal age, larger tumor size, lower tumor grade, and higher tumor staging. When we analyzed the biomarkers with BRCA1 mutation, it showed a negative association with ER, PR, and Her2 neu and a high Ki67 proliferation index. No family history of breast carcinoma was seen in 34/56 patients where history was available. Our study showed BRCA1 mutation in 32.1% and associated with postmenopausal age group, larger tumor size, and higher staging and negative hormonal status of breast carcinoma.

Uncovering Oncogenic Mechanisms of Tumor Suppressor Genes in Breast Cancer Multi-Omics Data.

Tumor suppressor genes (TSGs) are essential genes in the development of cancer. While they have many roles in normal cells, mutation and dysregulation of the TSGs result in aberrant molecular processes in cancer cells. Therefore, understanding TSGs and their roles in the oncogenic process is crucial for prevention and treatment of cancer. In this research, multi-omics breast cancer data were used to identify molecular mechanisms of TSGs in breast cancer. Differentially expressed genes and differentially coexpressed genes were identified in four large-scale transcriptomics data from public repositories and multi-omics data analyses of copy number, methylation and gene expression were performed. The results of the analyses were integrated using enrichment analysis and meta-analysis of a p-value summation method. The integrative analysis revealed that TSGs have a significant relationship with genes of gene ontology terms that are related to cell cycle, genome stability, RNA processing and metastasis, indicating the regulatory mechanisms of TSGs on cancer cells. The analysis frame and research results will provide valuable information for the further identification of TSGs in different types of cancers.

GLAD-PCR assay of DNA methylation sites in regulatory regions of some tumor-suppressor genes in breast cancer

Hypermethylation of the RcgY sites is shown for many cancer diseases. such aberrant methylation, suppressing the gene activity, occurs at early stages of carcinogenesis. Recently, using glad-pcR assay, we have detected aberrantly methylated RcgY sites, which can be considered to be epigenetic markers of colorectal, lung, and gastric cancers. in breast cancer, methylation of the regulatory regions of ALX4, BMP2, CCND2, CDH13, CDX1, FOXA1, GALR1, GATA5, GREM1, HIC1, HMX2, HS3ST2, HOXC10, ICAM5, LAMA1, RARB, RASSF1A, RUNX3, RXRG, RYR2, SFRP2, SOX17, TERT, and ZNF613 tumor-suppressor genes is reported. in the present work, we determined aberrantly methylated RcgY sites in the regulatory regions of these genes in dNa preparations from breast cancer tissues. the study of dNa samples from 30 tumor and 22 normal mammary tissue samples demonstrates a high diagnostic potential of selected R(5mc)gY sites in regulatory regions of CCND2, BMP2, GALR1, SOX17, HMX2, and HS3ST2 genes with total index of sensitivity and specificity for R(5mc)gY detection in tumor dNa 90.0 % and 100.0 %, respectively.

Implications of targeted next-generation sequencing for bladder cancer: report of four cases

BackgroundBladder cancer is considered heterogeneous diseases with two major subgroups: non-muscle- invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). It is a major healthcare problem, and it is one of the leading causes of mortality worldwide. Genetic mutations are not only a cause for carcinogenesis but are also a way for treatment strategy. The present study aimed to investigate breast cancer (BRCA genes) tumor suppressor gene mutations in bladder cancer tissue and combined blood samples for patients who developed secondary tumor after or during trimodal therapy. Fresh tissue samples and their matched blood samples were collected from four patients with bladder cancer. The objective regions for the examined genes (BRCA1 and BRCA2) were sequenced using next-generation sequencing (NGS); generated BAM files were uploaded to the cloud-based Ionreporter server, and the Oncomine BRCA-specific plugin was used to analyze the paired normal and tumor sample for each patient using the default plugin parameters. ResultsIntronic BRCA1 mutation c.5050-104 C >T was reported among the four investigated bladder cancer patients, and three somatic mutations were reported as follows: two of them were found to be benign rs1064793056 and rs28897679 on the Clinivar database and one nonsense pathogenic variant rs80357006. BRCA 2 gene mutation reported an exonic synonymous mutation rs397507876 in the tissue and germline DNA. Patients were treated with trimodal; however, three bladder cancer patients who reported BRCA mutations developed secondary tumors. ConclusionIdentification of mutational BRCA changes in bladder cancer is a promising marker for better treatment strategy. Further studies are encouraged on a large cohort of bladder cancer patients to confirm our findings.

DNAJB4 identified as a potential breast cancer marker: evidence from bioinformatics analysis and basic experiments

Breast cancer (BC) is the leading cause of tumor-related death in women worldwide, but its pathogenesis is not clear. The efficient screening of new therapeutic targets for BC through bioinformatics and biological experimental techniques has become a hot topic in BC research. The bioinformatics method was used to analyze the gene chips and obtain the hub genes, playing an important role in the development of BC. The biological processes (BP) involved in the hub genes were analyzed by Bingo, and the impact of each hub gene on disease-free survival (DFS) and overall survival (OS) in BC patients was evaluated in the Kaplan-Meier Plotter database. The expression of DNAJB4, the hub gene with the greatest degree and having an effect on the prognosis of BC patients, was detected in BC cell lines and clinicopathological specimens. And DNAJB4 was selected for further biological experiments and clinical prognosis verification. Ten hub genes including DNAJB4, the greatest degree genes, were found by bioinformatics analysis of BC gene chips. DNAJB4 expressions in both BC cell lines and clinicopathological specimens were detected and the results showed that DNAJB4 was significantly down-regulated in BC cell lines and tissues. After interfering with the expression of DNAJB4, it was found that the invasion and migration ability of MDA-MB-231 cell line was significantly enhanced in vitro. The clinical survival data of BC patients showed that patients with high DNAJB4 expression had longer DFS. DNAJB4 may be a tumor suppressor gene in BC as it could regulate invasion and migration of BC cells and its expression level is related to the prognosis of BC patients. Nevertheless, further researches are still necessary to verify its role in BC so as to provide evidences for clinical guidance regarding diagnosis and treatment.

Circular RNA hsa_circ_0068033 Acts as a Diagnostic Biomarker and Suppresses the Progression of Breast Cancer Through Sponging miR-659.

PurposeRecently, dysregulated circular RNAs (circRNAs) have been associated with the progression of numerous malignant tumors. However, the mechanism through which circRNAs participate in breast cancer (BC) remains unclear. This study was designed to illustrate the role of hsa_circ_0068033 in BC.MethodsWe detected the expression levels of hsa_circ_0068033 in BC tissues using quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). A series of functional experiments were conducted to assess the function of hsa_circ_0068033 in BC development and the underlying mechanisms.ResultsThe results suggested that the expression of hsa_circ_0068033 was downregulated in BC tissues, and its expression was markedly correlated with tumor size (P=0.021), and the Tumor, Node, and Metastasis stage (P=0.023). Receiver operating characteristic analysis showed that hsa_circ_0068033 testing yielded an area under the curve value of 0.8480 in discriminating BC from non-tumor controls. Functionally, in-vitro experiments demonstrated that overexpression of hsa_circ_0068033 could inhibit the growths, clone formation, invasion and migration of MCF-7 and MDA-MB-231 cells while activating the intrinsic apoptotic pathway to induce apoptosis. The xenograft experiment revealed that exogenous hsa_circ_0068033 is able to evidently reduce the tumorigenic ability of MDA-MB-231 cells in nude mice. Rescue assays further proved that hsa_circ_0068033 exerts biological functions by sponging miR-659.ConclusionCollectively, this study revealed for the first time that hsa_circ_0068033 acts as a tumor suppressor gene in BC, and the hsa_circ_0068033/miR-659 axis participates in the progression of BC.

Prognostic significance of KN motif and ankyrin repeat domains 1 (KANK1) in invasive breast cancer

BackgroundKN motif and ankyrin repeat domains 1 (KANK1) plays an important role in cytoskeleton maintenance and contributes to the regulation of cell proliferation, adhesion and apoptosis. KANK1 is involved in progression of a variety of solid tumours; however, its role in invasive breast cancer (BC) remains unknown. This study aims to evaluate the clinicopathological and prognostic value of KANK1 expression in operable BC.MethodsKANK1 expression was assessed at the transcriptomic level using multiple BC cohorts; the Molecular Taxonomy of BC International Consortium cohort (METABRIC; n = 1980), The Cancer Genome Atlas BC cohort (TCGA; n = 949) and the publicly available BC transcriptomic data hosted by BC Gene-Expression Miner (bc-GenExMiner v4.0) and Kaplan–Meier plotter?. The Nottingham BC cohort (n = 1500) prepared as tissue microarrays was used to assess KANK1 protein expression using immunohistochemistry (IHC). The association between clinicopathological variables and patient outcome was investigated.ResultsIn the METABRIC cohort, high expression of KANK1 mRNA was associated with characteristics of good prognosis including lower grade, absence of lymphovascular invasion and HER2 negativity (all; p < 0.001) and with better outcome [p = 0.006, Hazards ratio, (HR) 0.70, 95% CI 0.54–0.91]. High KANK1 protein expression was correlated with smaller tumour size and HER2 negativity, and better outcome in terms of longer breast cancer-specific survival [p = 0.013, HR 0.7, 95% CI 0.536–0.893] and time to distant metastasis [p = 0.033, HR 0.65, 95% CI 0.51–0.819].ConclusionThese results supported that upregulation of KANK1 works as a tumour suppressor gene in BC and is associated with improved patients’ outcomes.

PTP1B markedly promotes breast cancer progression and is regulated by miR-193a-3p.

The protein tyrosine phosphatase PTP1B, which is encoded by PTPN1, is a ubiquitously expressed nonreceptor protein tyrosine phosphatase. PTP1B has long been known to negatively regulate insulin and leptin receptor signalling. Recently, it was reported to be aberrantly expressed in cancer cells and to function as an important oncogene. In this study, we found that PTP1B protein levels are dramatically increased in breast cancer (BC) tissues and that PTP1B promotes the proliferation, and suppresses the apoptosis, of both HER2-positive and triple-negative BC cell lines. Bioinformatics analysis identified that the miRNA, miR-193a-3p, might potentially target PTP1B. We demonstrate that miR-193a-3p regulates PTP1B in BC cells and that it regulates the proliferation and apoptosis of BC cells by targeting PTP1B, both in vitro and in vivo. In conclusion, this study confirms that PTP1B acts as an oncogene in BC and demonstrates that miR-193a-3p can serve as a tumour suppressor gene in BC by targeting PTP1B.

ARID1A and CEBPα cooperatively inhibit UCA1 transcription in breast cancer.

As one of the primary members of SWI/SNF chromatin remodeling complexes, ARID1A contains frequent loss-of-function mutations in many types of cancers. However, the molecular mechanisms underlying ARID1A deficiency in cancer biology remain to be investigated. Using breast cancer as a model, we report that silencing ARID1A significantly increased cellular proliferation and migration. Mechanistically, primarily functioning as a transcriptional repressor, loss of ARID1A profoundly alters histone modifications and the transcriptome. Notably, ARID1A inhibited the expression of a long non-coding RNA, UCA1, by regulating chromatin access of the transcription factor CEBPα. Restoration experiments showed that UCA1 mediates the functions of ARID1A that induces loss of cellular proliferation and migration. Together, our findings characterize ARID1A as a key tumor-suppressor gene in breast cancer through cooperation with CEBPα, and loss-of-function mutations of ARID1A activates UCA1.

PO-389 Epigenomic and mutation determinants of carcinogen-driven primary epithelial cell immortalization

IntroductionBased on cancer genome sequencing, almost 600 genes have been implicated in cancer development to date, and recently, chromatin structural changes have emerged as a key mechanism for translating exogenous cues, such as carcinogen exposures, to the deregulation of biological processes implicated in tumorigenesis. However, the functional relationship between genetic and epigenetic alterations in the context of early events of carcinogen-driven cell transformation is not well understood.Material and methodsWe employ an in vitro carcinogen exposure system using primary human mammary epithelial cells (HMEC), which utilises a biological barrier bypass to mimic early steps of cell transformation, to introduce genetic and epigenetic alterations and to investigate their functional interplay during cell immortalization. A temporal, integrated (epi)genomic analysis is applied to benzo[a]pyrene (B[a]P)-exposed, immortalised HMEC, involving whole genome sequencing (WGS), analyses of chromatin structure (ChIP-seq, ATAC-seq), and patterns of gene regulation (DNA methylome 850K array, RNA-seq).Results and discussionsWGS of sequentially derived post-stasis and immortalised clones, as well as tumorigenic clones (generated by TP53 insertional mutagenesis of post-stasis lines), identified a prominent B[a]P mutational signature across all clones. While all sequenced clones had comparable SBS counts, we observed a gradual increase of structural variants, most notably copy number variations, from post-stasis to immortal and tumorigenic clones. Reproducibly distinct methylome patterns also correlated closely with the individual phenotypic stages of cell transformation. Chromatin accessibility analysis further showed changes characteristic to individual as well as multiple exposed clones, such as in the breast cancer tumour suppressor gene BRCA2 or the promoter region of PCDH18, a gene frequently inactivated by promoter methylation in colorectal cancer. Cell type-matched histone modification and RNA expression patterns are being established to enable a thorough integrated analysis of genetic and epigenetic determinants of carcinogen-driven HMEC transformation.ConclusionWe propose that in-depth, integrated (epi)genomic analysis of this innovative model system can reveal novel molecular determinants of carcinogen-driven cellular immortalization, thus improving our understanding of cancer causes, development and progression.Funding INCa-INSERM Plan Cancer 2015 Grant.

Abstract P6-05-01: Low BRCA2 levels cause epigenetic activation of growth adaptive pathways without inducing recurrent genomic mutations

Abstract The development of therapeutics to prevent cancer onset is hindered by a lack of robust cell systems to study the earliest steps of malignant transformation. Significant barriers to studying cancer-initiating events include intrinsic cellular pathways to prevent cell proliferation in the setting of activating oncogenes or genomic instability. To circumvent this limitation, we studied in vitro transient depletion of breast cancer tumor suppressor genes followed by selective growth conditions to enrich for de novo mutations or stable epigenetic alterations that can confer a growth advantage to non-transformed breast epithelial cells. We report that transient BRCA2 depletion, but not depletion of other breast cancer genes, leads to a selective growth advantage in EGF-free media. This is due to a failure of BRCA2 inactivated cells to enact a mesenchymal-to-epithelial transition upon withdrawal of EGF. Genomic profiling and biochemical approaches indicate that transient loss of BRCA2 induces chromatin remodeling in concert with NF-κB signaling and histone H2B deacetylation to repress genes associated with the epithelial state. Notably, this process did not appear to require recurrent driver mutations, nor DNA methylation changes, but was dependent on ATR signaling. Overall, our work suggests that transiently low BRCA2 levels are sufficient to induce aberrant cell proliferation under certain conditions and that targeting epigenetic modifiers may be an approach to prevent BRCA2-induced malignancy. Citation Format: Gruber JJ, Snyder M. Low BRCA2 levels cause epigenetic activation of growth adaptive pathways without inducing recurrent genomic mutations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-05-01.

CHL1 hypermethylation as a potential biomarker of poor prognosis in breast cancer.

The CHL1 gene encodes a cell-adhesion molecule proposed as being a putative tumour-suppressor gene in breast cancer (BC). However, neither the underlying molecular mechanisms nor the clinical value of CHL1 downregulation in BC has been explored. The methylation status of three CpG sites in the CHL1 promoter was analysed by pyrosequencing in neoplastic biopsies from 142 patients with invasive BC and compared with that of non-neoplastic tissues. We found higher CHL1 methylation levels in breast tumours than in non-neoplastic tissues, either from mammoplasties or adjacent-to-tumour, which correlated with lower levels of protein expression in tumours measured by immunohistochemistry. A panel of five BC cell lines was treated with two epigenetic drugs, and restoration of CHL1 expression was observed, indicating in vitro dynamic epigenetic regulation. CHL1 was silenced by shRNA in immortalized but non-neoplastic mammary cells, and enhanced cell proliferation and migration, but not invasion, were found by real-time cell analysis. The prognostic value of CHL1 hypermethylation was assessed by the log-rank test and fitted in a Cox regression model. Importantly, CHL1 hypermethylation was very significantly associated with shorter progression-free survival in our BC patient series, independent of age and stage (p = 0.001). In conclusion, our results indicate that CHL1 is downregulated by hypermethylation and that this epigenetic alteration is an independent prognostic factor in BC.

Abstract 5044: CtBP1 regulates olfactory and adhesion pathways in prostate cancer and metabolic syndrome

Abstract Metabolic syndrome (MeS) is a cluster of pathophysiological disorders that comprise at least three of the following factors: abdominal obesity, elevated triglycerides, dyslipidemia, high blood pressure and impaired glucose tolerance. Several studies associated MeS with increased risk for several cancer types, including prostate cancer (PCa). C-terminal binding protein 1 (CtBP1) is a potent transcriptional co-repressor of tumor suppressor genes. This protein is activated by low NAD+/NADH ratio produced by highly energetic environment such as high fat diet (HFD) intake. Previously, we identified CtBP1 as a novel molecular link between MeS and prostate tumor growth. The aim of this work was to assess the CtBP1 related pathways in PCa and MeS. We developed a MeS in vivo model by chronically feeding male nude mice with HFD. Control diet (CD) fed animals were maintained at the same conditions. These mice were inoculated with PC3 stable CtBP1 depleted or control cells. RNA obtained from xenografts was used for Genome-wide expression profiles (Affymetrix) and Gene Set Enrichment Analysis (GSEA). These analyses yielded several important pathways regulated by CtBP1, such as “Cell Adhesion” (COL17A1, PRRS2, CDH3, ITGB4, LCN2, CDH1, GJB5, TGM2 and SPARC) and “Olfactory” (OR4C45, OR5P2, GUCA1C and CLCA2). CtBP1 significantly diminished the capability of PCa cell lines to adhere to a collagen matrix, repressing the epithelial marker CDH1 and inducing the mesenchymal marker VIM expressions. Interestingly, CtBP1 associated to ITGB4 promoter gene, strongly repressing its expression. CtBP1 depletion increased the plasma membrane of the cell attached to the substrate and the number of filopodia. From olfactory pathway, we particularly focus on Chloride Channel Accessory 2 (CLCA2), a reported breast cancer tumor suppressor gene that function inhibiting epithelial to mesenchymal transition (EMT) and invasion processes. We found that CtBP1 regulates the transcription of CLCA2 in xenografts generated on HFD-fed mice. Using a panel of luciferase reporter plasmids with variable length of the CLCA2 promoter region, we determined that CtBP1 represses CLCA2 promoter activity in PC3 cells. Moreover, we established that CtBP1 associates to the CLCA2 proximal promoter region by chromatin immunoprecipitation (ChIP). Finally, several proteins regulate CLCA2 promoter activity independently (p53, ET2 and BRCA1) or dependently of CtBP1 (p300 and HDAC2). Altogether, these results demonstrated a new role for CtBP1 in the regulation of cellular adhesion, EMT and invasion reinforcing a potential function for this protein in cancer progression. Hence, CtBP1 pathway might help to identify new molecular candidates for better prediction of PCa progression in a subset of patients with MeS Citation Format: Guillermo N. Dalton, Juliana Porretti, Cintia Massillo, Georgina Scalise, Paula Lucía Farré, Cristian P. Moiola, Alejandra Paez, Geraldine Gueron, Paola De Luca, Adriana De Siervi. CtBP1 regulates olfactory and adhesion pathways in prostate cancer and metabolic syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5044.

WNT-1 inducible signaling pathway protein-1 enhances growth and tumorigenesis in human breast cancer.

WNT1 inducible signaling pathway protein 1 (WISP1) plays a key role in many cellular functions in a highly tissue-specific manner; however the role of WISP1 in breast cancer is still poorly understood. Here, we demonstrate that WISP1 acts as an oncogene in human breast cancer. We demonstrated that human breast cancer tissues had higher WISP1 mRNA expression than normal breast tissues and that treatment of recombinant WISP1 enhanced breast cancer cell proliferation. Further, ectopic expression of WISP1 increased the growth of breast cancer cells in vitro and in vivo. WISP1 transfection also induced epithelial-mesenchymal-transition (EMT) in MCF-7 cells, leading to higher migration and invasion. During this EMT-inducing process, E-cadherin was repressed and N-cadherin, snail, and β-catenin were upregulated. Filamentous actin (F-actin) remodeling and polarization were also observed after WISP1 transfection into MCF-7 cells. Moreover, forced overexpression of WISP1 blocked the expression of NDRG1, a breast cancer tumor suppressor gene. Our study provides novel evidence that WISP1-modulated NDRG1 gene expression is dependent on a DNA fragment (−128 to +46) located within the human NDRG1 promoter. Thus, we concluded that WISP1 is a human breast cancer oncogene and is a potential therapeutic target.

Abstract 2962: Loss of the E3 ubiquitin ligase HACE1 results in enhanced Rac1 signaling contributing to breast cancer progression

Abstract The transition from ductal carcinoma in situ (DCIS) to invasive disease is a crucial step in breast cancer progression. The specific alterations that govern this transition have not been elucidated. HER2/neu overexpression is frequently seen (50-60%) at the DCIS stage but is significantly less common in invasive breast cancer (20-30%) suggesting that additional alterations cooperate with HER2/neu to fully transform mammary epithelial cells. We identified novel genes that are capable of cooperating with HER2/neu allowing full malignant transformation, we used a transgenic mouse model that expresses wild-type neu and performed an insertional mutagenesis screen to identify genes whose alterations induce anchorage-independent growth on primary mouse mammary epithelial cells, an in vitro tumor formation assay. We identified the HECT and ankyrin domain containing E3 ligase 1 (HACE1) as a breast cancer tumor suppressor gene whose loss contributes to the transformation process. Loss of HACE1 expression is commonly seen in breast cancer patients and established breast cancer cell lines supporting the role of HACE1 as a breast cancer tumor suppressor gene. HACE1 ubiquitylates the activated GTP-bound form of the Rho family GTPase Rac1, resulting in ubiquitin mediated degradation of Rac1 via the 26S proteasome. The loss of HACE1 results in the accumulation of activated Rac1 leading to enhanced clonogenicity, migration, and invasion of human mammary epithelial cells (HMECs). In addition, the overexpression of HER2 results in activation of Rac1 levels which is further enhanced upon HACE1 loss in HMECs that further enhance colony formation in soft agar, migration, invasion and allows tumor formation in mice. Importantly, knocking down Rac1 using RNA interference or targeting RAC1 using the small molecule inhibitor EHT 1864 can rescue the effects of HACE1 loss mediated transformation. The results establish HACE1 as a breast cancer tumor suppressor gene by attenuating Rac1 signaling. In addition, our work further supports the role of Rac1 as a critical signaling node in breast cancer and that loss of HACE1 leads to enhanced Rac1 signaling that may contribute to breast cancer progression. These results suggest that HACE1 loss may identify precancerous lesions capable of becoming malignant as well as identify cancers susceptible to RAC targeted therapies. Citation Format: Erik T. Goka, Marc E. Lippman. Loss of the E3 ubiquitin ligase HACE1 results in enhanced Rac1 signaling contributing to breast cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2962. doi:10.1158/1538-7445.AM2014-2962

Association of BRCA2 variants with cardiovascular disease in Saudi Arabia.

Abnormalities in the breast cancer tumor suppressor genes (BRCA1 and BRCA2) are associated with breast and ovarian cancer. Recently, two single nucleotide polymorphisms (SNPs; rs11571836 and rs1799943) were identified, both located in untranslated regions of chromosome 13, associated with cardiovascular disease (CVD) in a multi-ethnic population. We examined the association between these BRCA2 polymorphisms and traits of CVD patients from Saudi Arabia. We genotyped rs11571836 and rs1799943 in 159 unrelated CVD patients and 176 healthy controls. The genotype and allele distributions in the overall population revealed a statistically significant association between rs1799943 and CVD (P = 0.01-0.022), whereas no risk association was identified for rs11571836. Additionally, haplotype analysis using both SNPs demonstrated no association between the SNPs and CVD. The genotype distribution of the 2 SNPs in the normal Saudi population deviated significantly (P < 0.000001) from that of the 6 different HapMap populations (CEU, CHB-Han, JPT, YRI, GIH, and MKK), except for the JPT population for rs1799943. This is the first study to examine the association between these SNPs and CVD in a Saudi population. Our results suggest that the increased health risk associated with the rs11571836 genotype is specific to male patients suffering from CVD. Stratification of patients and controls based on gender revealed no association between rs1799943 and the risk of CVD in either gender. These SNPs should be evaluated in larger cohorts in different populations to determine their suitability as screening markers for predicting CVD risk earlier in life to implement necessary preventive measures.

RhoBTB2 gene in breast cancer is silenced by promoter methylation

Mutation analysis in breast cancer has failed to explain the inactivation of RhoBTB2, a candidate breast cancer tumor suppressor gene on chromosome 8p. Some breast cancer-related genes in this region become inactivated by hypermethylation, and hypermethylation of RhoBTB2 abrogates its expression in bladder cancers. The aim of the present study was to determine whether RhoBTB2 was silenced by methylation in breast cancer. Nested methylation-specific PCR (nMSP) and quantitative reverse transcription PCR were used to analyze the methylation status and mRNA levels of RhoBTB2 in 50 paired breast cancer and normal tissues and the results were correlated with clinicopathological characteristics. Promoter methylation and the downregulation of RhoBTB2 mRNA was observed in tumor tissues (P<0.001). mRNA levels were decreased in samples with methylation (χ² = 15.751, P<0.001). RhoBTB2 methylation was observed preferentially in progesterone receptor (PR)-negative samples (P<0.05). The results demonstrated that aberrant methylation of RhoBTB2 may be responsible for the suppression of RhoBTB2 mRNA expression in breast cancer, a significant event during the genesis of breast cancer that correlated with PR status.

Abstract P5-04-06: Loss of the E3 ubiquitin ligase HACE1 plays a critical role in transformation of mammary cells and clinical progression of human breast cancer via accumulation of active Rac1

Abstract Invasive ductal breast cancer arises when the immediate precursor, ductal carcinoma in situ (DCIS), breaches the basement membrane of the ductal lumen and invades into the surrounding tissue. Although this transition has been implicated as a crucial step in the progression of breast cancer, the specific alterations responsible for this transition have yet to be identified. HER2/neu, a member of the epidermal growth family receptor (EGFR) family, is frequently overexpressed in non-invasive lesions (50-60%) but is significantly less common in invasive breast cancer (20-30%). This suggests that while HER2/neu may be partially transformative, additional alterations are required for mammary epithelial cells to achieve full malignant transformation. To identify novel genes capable of cooperating with HER2, we performed an insertional mutagenesis screen for genes whose alteration induces anchorage-independent growth on primary mouse mammary epithelial cells isolated from MMTV-neu transgenic mice. We identified HECT domain and ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) as a putative breast tumor suppressor gene whose loss contributes to the transformative process. Loss of HACE1 expression is commonly seen in publicly available breast cancer patient data sets as well as in established breast cancer cell lines supporting the role of HACE1 as a breast cancer tumor suppressor gene. Knockdown of HACE1 in HER2 overexpressing human mammary epithelial cells (HMECs) enhanced colony formation over HER2 overexpressing HMEC controls cells. Moreover, knockdown of HACE1 alone was sufficient to allow anchorage-independent growth in soft agar in the HMECs while the overexpression of HACE1 in breast cancer cell lines diminishes clonogenic capacity in soft agar. We confirmed recent studies that have shown that HACE1 is capable of tagging the Rho GTPase Rac1 for ubiquitin-mediated proteasomal degradation. In mammary epithelial cells, the loss of HACE1 leads to enhanced levels of active Rac1 resulting in increased clonogenicity, migration and invasion. Importantly, we show that targeting Rac1 can rescue the effects of HACE1 loss in mammary epithelial cells. Our results establish HACE1 as a breast cancer tumor suppressor gene by attenuating active Rac1 signaling. Our work supports the role of Rac1 as a critical signaling node in breast cancer and that loss of HACE1 leads to enhanced Rac1 signaling resulting in driving cancer progression. Furthermore, our results suggest the role of HACE1 loss as a biomarker for tumor progression and may identify patients vulnerable to Rac1 or Rac effector targeted therapies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-06.