Development Of Triple-negative Breast Cancer Research Articles

PSMB5 is associated with proliferation and drug resistance in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by advanced disease stage and poor prognosis. Moreover, due to the lack of therapeutic markers, TNBC patients can't benefit fully from currently available targeted therapies. To fully understand the molecular basis of TNBC, we used gene set enrichment analysis (GSEA) to screen out the most altered functional module in TNBC, from publicly available microarray data and studied the association of the candidate gene with TNBC development. We found that the proteasome was significantly activated in TNBC. As compared with other breast cancer subtypes and normal tissue, proteasome subunit beta 5 (PSMB5), the key regulator of proteasome function, was overexpressed in TNBC tissue and predictive of poor prognosis. Moreover, we also found that PSMB5 knockdown induced TNBC apoptosis and significantly enhanced cancer cell sensitivity to the chemotherapeutic agents bortezomib and paclitaxel. Our results suggest a potential role for PSMB5 as a biomarker and therapeutic target for TNBC.

Estrogen receptor-α36 is involved in icaritin induced growth inhibition of triple-negative breast cancer cells

A sub-class of ER-negative breast cancer that is negative for ER, PR and HER2 expression known as triple-negative breast cancer (TNBC) is highly malignant and lacks effective treatment. Recently, it has been reported that an isoform of estrogen receptor-alpha ER-α36 is expressed and plays a critical role in development of TNBC. ER-α36 forms a positive regulatory loop with epidermal growth factor receptor (EGFR), which promotes malignant growth of TNBC cells. Thus, ER-α36 has been proposed as an important target for development of novel drugs for TNBC. In this study, we evaluated the effects of icaritin, a prenylflavonoid derivant purified from Epimedium Genus, on growth of TNBC cells and examined the possible underlying mechanisms. Our study demonstrated that icartin decreased both ER-α36 and EGFR protein expression, and induced apoptosis in TNBC MDA-MB-231 and MDA-MB-453 cells. We also found that icaritin inhibited ER-α36-mediated MAPK/ERK pathway and cyclin D1 induction by estrogen. Our results thus indicated that icaritin has a potential to be developed into a novel therapeutic agent for human TNBC.

Protein kinase C inhibitor chelerythrine selectively inhibits proliferation of triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targeted therapy currently. Recent studies imply that protein kinase C may play important roles in TNBC development and could be a specific target. In this study, we evaluated the anti-proliferative activity of PKC inhibitor chelerythrine on a panel of breast cancer cell lines. Chelerythrine selectively inhibited the growth of TNBC cell lines compared to non-TNBC cell lines as demonstrated by in vitro cell proliferation assay and colony formation assay, as well as evidenced by in vivo xenograft assay. The selective anti-proliferative effect of chelerythrine was associated with induction of apoptosis in TNBC cell lines. We further demonstrated that PKN2, one of the PKC subtypes, was highly expressed in TNBC cell lines, and knocking down PKN2 in TNBC cells inhibited colony formation and xenograft growth. This indicates that PKN2 is required for the survival of TNBC cells, and could be the target mediates the selective activity of chelerythrine. Finally, combination of chelerythrine and chemotherapy reagent taxol showed synergistic/additive effect on TNBC cell lines. Our results suggest chelerythrine or other PKC inhibitors may be promising regimens for TNBC tumors.

Regulation of miRNA-29c and its downstream pathways in preneoplastic progression of triple-negative breast cancer.

Little is understood about the early molecular drivers of triple-negative breast cancer (TNBC), making the identification of women at risk and development of targeted therapy for prevention significant challenges. By sequencing a TNBC cell line-based breast cancer progression model we have found that miRNA-29c is progressively lost during TNBC tumorigenesis. In support of the tumor suppressive role of miRNA 29c, we found that low levels predict poor overall patient survival and, conversely, that ectopic expression of miRNA-29c in preneoplastic cell models inhibits growth. miRNA-29c exerts its growth inhibitory effects through direct binding and regulation of TGFB-induced factor homeobox 2 (TGIF2), CAMP-responsive element binding protein 5 (CREB5), and V-Akt murine thymoma viral oncogene homolog 3 (AKT3). miRNA-29c regulation of these gene targets seems to be functionally relevant, as TGIF2, CREB5, and AKT3 were able to rescue the inhibition of cell proliferation and colony formation caused by ectopic expression of miRNA-29c in preneoplastic cells. AKT3 is an oncogene of known relevance in breast cancer, and as a proof of principle we show that inhibition of phosphoinositide 3-kinase (PI3K) activity, a protein upstream of AKT3, suppressed proliferation in TNBC preneoplastic cells. We explored additional opportunities for prevention of TNBC by studying the regulation of miRNA-29c and identified DNA methylation to have a role in the inhibition of miRNA-29c during TNBC tumorigenesis. Consistent with these observations, we found 5 aza-cytadine to relieve the suppression of miRNA-29c. Together, these results demonstrate that miRNA-29c loss plays a key role in the early development of TNBC.

MicroRNA-761 induces aggressive phenotypes in triple-negative breast cancer cells by repressing TRIM29 expression.

Despite advances that have been made in systemic chemotherapy, the prognosis of advanced triple-negative breast cancer (TNBC) patients is still poor. The identification of key factors governing TNBC development is considered imperative for the development of novel effective therapeutic approaches. Previously, it has been reported that microRNA (miR)-761 may act as either a tumor suppressor or as an oncogene in different types of cancer. Here, we aimed at assessing the biological role of this miRNA in TNBC. First, we measured the expression of miR-761 in primary breast cancer tissues and breast cancer-derived cell lines using qRT-PCR. Subsequently, over-expression and silencing experiments were performed to determine the role of miR-761 in TNBC cell proliferation, colony formation, migration and invasion in vitro. The in vivo role of miR-761 in TNBC growth and metastasis was determined in mouse models. Bioinformatics analyses, dual-luciferase reporter assays, Western blot analyses and rescue experiments were performed to identify miR-761 target gene(s). We found that miR-761 was up-regulated in primary breast cancer tissues and its derived cell lines and, particularly, in TNBC tissues and cell lines. We also found that exogenous miR-761 over-expression augmented in vitro TNBC cell proliferation, colony formation, migration and invasion, whereas miR-761 down-regulation impaired these features. In vivo, we found that miR-761 over-expression facilitated TNBC growth and lung metastasis. Mechanistically, miR-761 was found to negatively regulate the expression of tripartite motif-containing 29 (TRIM29) in TNBC cells by binding to the 3'-untranslated region of its mRNA. In conformity with these results, a significant negative correlation between miR-761 expression and TRIM29 protein expression was noted in primary TNBC tissues (r=-0.452, p=0.0126). We also found that exogenous TRIM29 over-expression reversed the proliferative and invasive capacities of TNBC cells. Our data indicate that miR-761 acts as an oncogene in TNBC. This mode of action can, at least partially, be ascribed to the down-regulation of its target TRIM29. We suggest that miR-761 may serve as a promising therapeutic target for TNBC.

Triple negative breast cancer development can be selectively suppressed by sustaining an elevated level of cellular cyclic AMP through simultaneously blocking its efflux and decomposition.

Triple negative breast cancer (TNBC) has the highest mortality among all breast cancer types and lack of targeted therapy is a key factor contributing to its high mortality rate. In this study, we show that 8-bromo-cAMP, a cyclic adenosine monophosphate (cAMP) analog at high concentration (> 1 mM) selectively suppresses TNBC cell growth. However, commonly-used cAMP-elevating agents such as adenylyl cyclase activator forskolin and pan phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) are ineffective. Inability of cAMP elevating agents to inhibit TNBC cell growth is due to rapid diminution of cellular cAMP through efflux and decomposition. By performing bioinformatics analyses with publically available gene expression datasets from breast cancer patients/established breast cancer cell lines and further validating using specific inhibitors/siRNAs, we reveal that multidrug resistance-associated protein 1/4 (MRP1/4) mediate rapid cAMP efflux while members PDE4 subfamily facilitate cAMP decomposition. When cAMP clearance is prevented by specific inhibitors, forskolin blocks TNBC's in vitro cell growth by arresting cell cycle at G1/S phase. Importantly, cocktail of forskolin, MRP inhibitor probenecid and PDE4 inhibitor rolipram suppresses TNBC in vivo tumor development. This study suggests that a TNBC-targeted therapeutic strategy can be developed by sustaining an elevated level of cAMP through simultaneously blocking its efflux and decomposition.

Alcohol promotes migration and invasion of triple‐negative breast cancer cells through activation of p38 MAPK and JNK

Although alcohol is an established breast cancer risk factor, the underlying mechanisms remain unclear. Previous studies examined the general association between alcohol consumption and breast cancer risk; however, the risk for different breast cancer subtypes has been rarely reported. Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking hormone receptors and HER2 expression, and having poor prognosis. Understanding the molecular mechanisms of TNBC etiology remains a significant challenge. In this study, we investigated cellular responses to alcohol in two TNBC cell lines, MDA-MB-231 and MDA-MB-468. Our results showed that alcohol at low concentrations (0.025-0.1% v/v) induced cell proliferation, migration, and invasion in 1% FBS-containing medium. Molecular analysis indicated that these phenotypic changes were associated with alcohol-induced reactive oxygen species production and increased p38 and JNK phosphorylation. Likewise, p38 or JNK inhibition attenuated alcohol-induced cell migration and invasion. We revealed that alcohol treatment activated/phosphorylated NF-κB regulators and increased transcription of NF-κB-targeted genes. While examining the role of acetaldehyde, the major alcohol metabolite, in alcohol-associated responses in TNBC cells, we saw that acetaldehyde induced cell migration, invasion, and increased phospho-p38, phospho-JNK, and phospho-IκBα in a pattern similar to alcohol treatment. Taken together, we established that alcohol promotes TNBC cell proliferation, migration, and invasion in vitro. The underlying mechanisms involve the induction of oxidative stress and the activation of NF-κB signaling. In particular, the activation of p38 and JNK plays a pivotal role in alcohol-induced cellular responses. These results will advance our understanding of alcohol-mediated development and promotion of TNBC. © 2016 Wiley Periodicals, Inc.

Bisphenol A Increases the Migration and Invasion of Triple‐Negative Breast Cancer Cells via Oestrogen‐related Receptor Gamma

Triple-negative breast cancer (TNBC) is characterized by great metastasis and invasion capability. Our study revealed that nanomolar bisphenol A (BPA), one of the most ubiquitous endocrine disruptors, can increase wound closure and invasion of both MDA-MB-231 and BT-549 cells. BPA treatment can increase protein and mRNA expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, while had no effect on the expression of vimentin (Vim) and fibronectin (FN) in TNBC cells. The expression of G-protein-coupled receptor (GPER), which has been suggested to mediate rapid oestrogenic signals, was not varied in BPA-treated MDA-MB-231 and BT-549 cells. Its inhibitor G15 also had no effect on BPA-induced MMPs expression and cell invasion. Interestingly, BPA treatment can significantly increase the mRNA and protein expressions of oestrogen-related receptor γ (ERRγ), but not ERRα or ERRβ, in both MDA-MB-231 and BT-549 cells. The knock-down of ERRγ can markedly attenuate BPA-induced expression of MMP-2 and MMP-9 in TNBC cells. BPA treatment can activate both ERK1/2 and Akt in TNBC cells. Both inhibitors of ERK1/2 (PD98059) and Akt (LY294002) can attenuate BPA-induced ERRγ expression and cell invasion of MDA-MB-231 cells. Collectively, our data revealed that BPA can increase the expression of MMPs and in vitro motility of TNBC cells via ERRγ. Both activation of ERK1/2 and Akt participated in this process. Our study suggests that more attention should be paid to the roles of xenoestrogens such as BPA in the development and progression of TNBC.

Abstract B17: High expression of the transcription factor Kaiso correlates with aggressive breast cancers (BC) and racial disparity in BC outcomes in women of African ancestry

Abstract The highly metastatic triple negative breast cancers (TNBC) lack expression of estrogen-receptor (ER), progesterone-receptor (PR) and human epidermal growth factor receptor-2 (HER2), and thus have limited patient treatment options and prognosis. Intriguingly TNBC is most prevalent in young women of African Ancestry (WAA) who, despite having lower BC incidence rates than Caucasian women, have higher mortality rates. This racial disparity appears unrelated to socio-economic status and increasing evidence hints at an ancestral genetic predisposition to TNBC development and high mortality rates. Recently, a significant correlation was found between nuclear expression of the transcription factor Kaiso, ERα negativity, basal/TNBCs and invasive ductal carcinoma in African American (AA) women. Emerging evidence from our lab also found that high Kaiso expression is associated with shorter metastasis-free survival in aggressive breast cancers. Based on these findings we hypothesized that Kaiso may play a crucial role in the metastatic spread of advanced breast cancers, and perhaps contribute to the racial disparity in TNBC prevalence and BC outcomes in WAA. To gain insight into Kaiso's role in aggressive breast cancers and the racial disparity associated with BC outcomes, we generated stable Kaiso-depleted TNBC cells (MDA-231) and then performed xenograft studies in immuno-compromised mice. Kaiso-depleted MDA-231 cells exhibited no or negligible metastases to the lungs and liver, compared to control MDA-231 cells, which formed extensive metastases in the lungs and liver of immuno-compromised mice. We also analysed Kaiso expression levels and subcellular localization in a tissue microarray (TMA) comprised of breast tumor tissues from ~100 Caucasian and ~100 AA women. Intriguingly, we observed a significant correlation between high nuclear Kaiso expression and ER(-) status in WAA, but surprisingly we found that high nuclear Kaiso expression is associated with ER(+) status and better overall survival in women of European descent. Collectively our findings implicate high Kaiso expression in breast cancer aggressiveness and suggest a context-dependent role for Kaiso in BC outcomes in women of different ethnicities. Ongoing studies are focused on clarifying the role of Kaiso in TNBC outcomes in WAA, and will incorporate analysis of a clinically annotated TMA comprised of ~200 TNBC tissues obtained from Barbadian and Nigerian women in conjunction with mechanistic studies to elucidate the signaling pathways involved in Kaiso-mediated tumor metastasis. Our findings may unravel a therapeutic role for Kaiso as a useful biomarker for the prognosis of TNBCs especially in WAA. Citation Format: Blessing I. Bassey-Archibong, Lyndsay G. A. Rayner, Kevin Gardner, Juliet Daniel. High expression of the transcription factor Kaiso correlates with aggressive breast cancers (BC) and racial disparity in BC outcomes in women of African ancestry. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B17.

Genetic evaluation of BRCA1 associated a complex genes with triple-negative breast cancer susceptibility in Chinese women.

BackgroundThe tumor suppressor BRCA1 plays a pivotal role in maintaining genomic stability and tumor suppression. The BRCA1-A complex is required for recruitment of BRCA1 to DNA damage sites, DNA repair and cell cycle checkpoint control. Since germline mutations of BRCA1 often lead to breast tumors that are triple-negative breast cancer (TNBC) type, we aimed to investigate whether genetic deficiency in genes of the BRCA1-A complex is associated with risk to TNBC development.ResultsWe found that rs7250266 in the promoter region of NBA1 confers a decreased risk to TNBC development, but not to non-TNBC susceptibility. In addition, the haplotypes containing two polymorphisms rs7250266 and rs2278256 are associated with a lower chance of TNBC development specifically. Our studies also showed that the protective alleles of rs7250266 (C > G) and rs2278256 (T > C) down-regulate promoter activity of NBA1 in mammary epithelial cells.MethodsWe investigated associations between the BRCA1-A complex genes and TNBC developing risk in first case-control study of Chinese Han Women population including 414 patients with TNBC and 354 cancer-free controls. We detected 37 common variants in ABRAXAS, RAP80, BRE, BRCC36 and NBA1/MERIT40 genes encoding the BRCA1-A complex and evaluated their genetic susceptibility to the risk of TNBC. An additional cohort with 652 other types of breast cancer (non-TNBC) cases and 890 controls was used to investigate the associations between TNBC-specific SNPs genotype and non-TNBCs susceptibility.ConclusionsGenetic variants in NBA1 may be an important genetic determinant of TNBC susceptibility. Further investigation and validation of these SNPs in larger cohorts may facilitate in predication and prevention of TNBC and in counseling individuals for risk of TNBC development.

Decreased expression of autophagy protein LC3 and stemness (CD44+/CD24−/low) indicate poor prognosis in triple-negative breast cancer

This study evaluated the prognostic value of expression of autophagy protein light chain 3 (LC3) and the prognostic value of coexpression of LC3 and stemness markers CD44+/CD24-/low in triple-negative breast cancer (TNBC). LC3 and LC3/CD44+/CD24-/low immunophenotypes in tumor tissues were evaluated by immunohistochemistry in 67 TNBC patients. LC3- was expressed in 30 (44.78%) cases. The LC3- phenotype revealed a significant negative association with overall survival in both univariate (P = .0006) and multivariate (P = .0153) analyses. LC3-/CD44+/CD24-/low phenotype was observed in 24 (35.82%) of 67 TNBC patients. According to Kaplan-Meier analysis, prognosis was significantly worse in tumors with LC3-/CD44+/CD24-/low phenotype (P = .0280). Multivariate analysis indicated that LC3-/CD44+/CD24-/low phenotype was a significant independent prognostic indicator of overall survival. These results suggest that LC3 suppresses TNBC in mature tumor cells and cancer stem cells (CSCs). In conclusion, this study suggests that CSCs are linked to progression of autophagy in TNBC. During the progression and development of TNBC, autophagy of CSCs/progenitor cells is low. LC3-/CD44+/CD24-/low immunophenotype indicates a highly aggressive TNBC subgroup associated with a poor prognosis. This study investigated that LC3 deficiency may restrain TNBC in mature tumor cells and CSCs. Therefore, a reasonable inference is that inducing autophagy may be an effective therapeutic strategy in TNBC.

Abstract B48: Clues to the causes of the abundance of triple-negative breast cancer in women of African descent

Abstract Background: Triple negative breast cancer (TNBC) is over-represented in indigenous African women and in women of African descent. The majority of women diagnosed with breast cancer in Nigeria, Uganda and Kenya have TNBC. Studies of TNBC in the US consistently report the percentage of TNBC to be 10-24% greater in African-Americans (AA) than in Caucasians (C). The abundance of TNBCs in both African and African-American women has yet to be explained and the incidence rates suggest that there may be a genetic predisposition to this particularly aggressive form of breast cancer. Methods: Epithelial cell lines were established from normal, healthy breast tissue donated to the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center. Six cell lines from AA donors were matched on the basis of age, menopausal status and Gail-risk score to six cell lines from C donors. RNA was extracted from the cells and gene expression assayed using the Illumina HumanWG6 v3 chip. Significant differences in gene expression were identified using PADE (PAirwise Differential Expression; Expression Analysis, Durham, NC). Results: 1552 genes were differentially expressed (p≤ 0.05). The largest number of genes over-expressed in AAs mapped to the long arm of chromosome 1. Copy number gains have been observed in this region in multiple TNBC data sets. Many of these genes are in the 1q21 region, a region frequently rearranged in epithelial tumors as well as soft tissue and bone. Analysis of differentially expressed genes using MetaCore from Thomson Reuters revealed that the over-expressed genes are responsible for skin development, cell adhesion/cell junctions, and functions of the innate immune system. Pattern recognition is in the vanguard of the innate immune system. The pattern recognition pathways mediated by RAGE (receptor for advanced glycation end products) and the Toll-like receptors are activated in the normal AA epithelium. The expression of genes in these pathways, specifically S100A12, S100A8, S110A9, and TLR5, is known to increase in the presence of Salmonella and Shigella. MDA5 recognizes rotavirus infection. LYST and DUSP4 are active in the control of Leishmania infection. LCN2 disrupts iron acquisition, which protects against a number of pathogens including Salmonella, Klebsiella and mycobacteria. Conclusions: The results of the above gene expression analysis are best understood in light of our recently published findings using these same cells (Sauder et al, BMC Cell Biology, 2014). Epithelial cells grown from mammary explant culture are basal-like and phenotypically plastic. They can assume a wide range of cell types depending on the cues provided by the microenvironment. When grown in basement membrane matrix or on human dermal fibroblasts, these cells produce a stratified squamous epithelium. Although the cells used for the gene expression analysis were grown in plastic tissue culture flasks and grew as a monotonous monolayer, the data suggest that they are, to some extent, transcriptionally “squamous”. For example, they express involucrin and the genes that code for the corneodesmosome proteins: corneodesmin, desmoglein and desmocollin. The skin of humans living in sub-Saharan Africa has evolved to provide optimal barrier protection. It specifically protects against water loss and, pathogenic microorganisms and parasites. Mammary glands are ectodermal appendages of the epithelium and they may share similar immune defense mechanisms. The increased expression of genes involved in innate immunity in AA breast epithelial cells is of significant interest. We hypothesize that a subclinical, chronic inflammatory state is incited within basal breast cells in response to a pathogen that is endemic to Africa and overrepresented in low income communities in the US. This persistent and unresolved innate immunity predates the development of TNBC and can thus participate in its initiation and promotion. Citation Format: Candice A.M. Sauder, Jillian E. Koziel, MiRan Choi, Brittney-Shea Herbert, Susan E. Clare. Clues to the causes of the abundance of triple-negative breast cancer in women of African descent. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B48.

Abstract LB-200: Loss of the scaffold protein Kibra in a mouse model of triple-negative breast cancer

Abstract Introduction: Targeted therapies in breast cancer rely on tumor cell expression of Estrogen and/or HER2 receptors. Triple negative breast cancers (TNBCs), lacking these receptors, have no targeted therapies. We have developed a preclinical murine model expressing a naturally occurring oncogenic variant of the Met receptor in the mammary gland combined with loss of function of the tumour suppressor p53 (MMTV-Met;Trp53fl/+;Cre). This model recapitulates many features of TNBC at the level of gene expression, pathological markers and genomic alterations. Notably, this model spontaneously undergoes loss of a genomic region syntenic with human chromosome 5q which is lost in up to 70% of human TNBCs (Turner et al., 2010). This provides an opportunity for us to identify and study potential driver genes for this breast cancer subtype. Experimental procedures: Approximately 80% of MMTV-Met;Trp53fl/+;Cre mammary tumors have a mesenchymal pathology that recapitulates key features of TNBC. Array CGH profiling showed a consistent loss of chromosome 11:31.35-58.2Mb, syntenic with human 5q31.1 and 5q33.1-35.3. Analysis of mouse model expression profiling data confirmed decreased expression of 83 genes within this region. Comparison with human breast cancers within the TCGA dataset further highlighted 14 of these genes with significantly decreased expression in humans with the TNBC subtypes ‘Basal’ and ‘Claudin-low’. One of these genes, WWC1, undergoes heterozygous deletion in 49% of human Basal breast cancers (TCGA, Nature 2012) and has also been associated with Claudin-low cancers (Moleirinho et al., 2013). WWC1, also known as Kibra, encodes a scaffold protein that positively regulates the Hippo tumor suppressor pathway. We used both Kibra knockdown and over-expression to understand the functional consequences of Kibra-loss. Results: MMTV-Met solid carcinoma cells retain both Kibra expression and epithelial characteristics typical of human luminal breast cancers (Ponzo et al., 2009). Kibra depletion by siRNA in these cells led to weakening of cell-cell junctions and colony dispersal, as well as decreased cortical actin and increased membrane protrusions, consistent with epithelial-to-mesenchymal transition (EMT). Mice injected with Kibra shRNA-tumor cells developed secondary lesions at sites distant to the mammary gland. By comparison, re-expression of Kibra in MMTV-Met;Trp53fl/+ cell lines and human TNBC cells led to morphological changes consistent with an EMT reversal, including loss of actin rich protrusions. Furthermore, re-expressing Kibra impaired proliferation in vitro and decreased tumorigenicity in vivo. Globally, our results strongly suggest that Kibra loss plays a significant role in TNBC development and metastasis and is a candidate driver gene for Chr5q loss. Citation Format: Jennifer France Knight, Tina Gruosso, Danielle Angeline de Verteuil, Sadiq Saleh, Robert Lesurf, Hong Zhao, Ryan Davis, Dongmei Zuo, Robert Cardiff, Jeffrey Gregg, Michael Hallett, Morag Park. Loss of the scaffold protein Kibra in a mouse model of triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-200. doi:10.1158/1538-7445.AM2015-LB-200

Microarray expression profiling of dysregulated long non-coding RNAs in triple-negative breast cancer

Triple-negative breast cancer (TNBC) represents a collection of malignant breast tumors that are often aggressive and have an increased risk of metastasis and relapse. Long non-coding RNAs are generally defined as RNA transcripts measuring 200 nucleotides or longer that do not encode for any protein. During the past decade, increasing evidence has shown that lncRNAs play important roles in oncogenesis and tumor suppression; however, the roles of lncRNAs in TNBC are poorly understood. To address this issue, we used Agilent human lncRNA microarray chips and bioinformatics tools, including Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), to assess lncRNA expression in 3 pairs of TNBC tissues. A dysregulated lncRNA expression profile was identified by microarray and verified by qRT-PCR in 48 pairs of breast cancer subtype tissues. Metastasis is the major cause of cancer-related deaths, including those in TNBC, and the presence of dormant residual disseminated tumor cells (DTC) may be a key factor leading to metastasis. ANKRD30A, a potential target for breast cancer immunotherapy, is currently one of the most used DTC markers. Notably, we found the expression levels of the novel intergenic lncRNA LINC00993 to be associated with the expression levels of ANKRD30A. Furthermore, our qRT-PCR data indicated that the expression of LINC00993 was also associated with the expression of the estrogen receptor. In conclusion, our study identified a set of lncRNAs that were consistently aberrantly expressed in TNBC, and these dysregulated lncRNAs may be involved in the development and/or progression of TNBC.

AKT survival signaling and phospho-AKT expression are associated with triple negative breast cancer development and clinical outcome

AKT survival signaling and phospho-AKT expression are associated with triple negative breast cancer development and clinical outcome

Deptor Enhances Triple-Negative Breast Cancer Metastasis and Chemoresistance through Coupling to Survivin Expression

Transforming growth factor–β (TGF-β) functions to suppress tumorigenesis in normal mammary tissues and early-stage breast cancers and, paradoxically, acts to promote the metastasis and chemoresistance in late-stage breast cancers, particularly triple-negative breast cancers (TNBCs). Precisely how TGF-β acquires oncogenic characteristics in late-stage breast cancers remains unknown, as does the role of the endogenous mammalian target of rapamycin (mTOR) inhibitor, Dep domain–containing mTOR-interacting protein (Deptor), in coupling TGF-β to TNBC development and metastatic progression. Here we demonstrate that Deptor expression was downregulated in basal-like/TNBCs relative to their luminal counterparts. Additionally, Deptor expression was 1) inversely correlated with the metastatic ability of human (MCF10A) and mouse (4T1) TNBC progression series and 2) robustly repressed by several inducers of epithelial-mesenchymal transition programs. Functional disruption of Deptor expression in 4T07 cells significantly inhibited their proliferation and organoid growth in vitro, as well as prevented their colonization and tumor formation in the lungs of mice. In stark contrast, elevated Deptor expression was significantly associated with poorer overall survival of patients harboring estrogen receptor α–negative breast cancers. Accordingly, enforced Deptor expression in MDA-MB-231 cells dramatically enhanced their 1) organoid growth in vitro, 2) pulmonary outgrowth in mice, and 3) resistance to chemotherapies, an event dependent on the coupling of Deptor to survivin expression. Collectively, our findings highlight the dichotomous functions of Deptor in modulating the proliferation and survival of TNBCs during metastasis; they also implicate Deptor and its stimulation of survivin as essential components of TNBC resistance to chemotherapies and apoptotic stimuli.

MiR-185 suppresses tumor proliferation by directly targeting E2F6 and DNMT1 and indirectly upregulating BRCA1 in triple-negative breast cancer.

Breast cancer is a major public health problem all over the world, and the current treatment strategies are not potent enough for some patients, especially those with triple-negative breast cancer (TNBC). Recent studies have demonstrated that microRNAs (miRNA) play vital roles in the development of TNBC. In this study, we found that miR-185 was strongly downregulated in TNBC tissues and cell lines and that its expression levels were associated with lymph node metastasis, clinical stage, overall survival, and relapse-free survival in TNBC. We also found that ectopic expression of miR-185 inhibited TNBC cell proliferation in vitro and in vivo. We further identified that miR-185 directly targeted DNMT1 and E2F6, which resulted in a marked increase in the expression of BRCA1 at the mRNA and protein levels in TNBC. Our data suggest that miR-185 functions as a tumor suppressor in TNBC development. It is a promising prognostic biomarker and potential therapeutic target for TNBC.

MiR-26a suppresses tumour proliferation and metastasis by targeting metadherin in triple negative breast cancer

It has been reported that miR-26a plays an important role in various cancers. In this study, we found that miR-26a was downregulated in triple-negative breast cancer (TNBC), and its expression levels were associated with lymph node metastasis and overall survival in TNBC. We also found that the ectopic expression of miR-26a inhibited TNBC cell proliferation and metastasis in vitro and in vivo by downregulating MTDH (a miR-26a' target gene) mRNA and protein and that the overexpression of MTDH could partially abrogate miR-26a-mediated suppression. Our data suggest that miR-26a functions as a tumour suppressor in TNBC development and serves as a prognostic marker for breast cancer.

Abstract 820: Inhibition of Rad6 sensitizes triple negative breast cancer cells to platinum-based therapy

Abstract Treatment of triple negative breast cancers (TNBCs) poses a clinical challenge because they are not treatable with therapies targeting estrogen receptor and Her2/neu as they lack expression of estrogen, progesterone, and Her2/neu receptors. Since TNBCs share several histologic features with BRCA1-related breast cancer, and these breast cancers have aberrant DNA repair, DNA repair pathways are thought to play a role in TNBC development and drug response. Platinum (Pt)-based compounds induce toxic interstrand DNA crosslinks (ICL), the repair of which requires activities of BRCA/Fanconi anemia (FA) network and Rad6 postreplication DNA repair (PRR) pathways. PRR pathway confers tolerance to DNA damage by enabling cells to complete DNA replication in the face of damage and avoid mitotic catastrophe. Rad6 regulates PCNA ubiquitination, a critical event for PRR, and also promotes FancD2 ubiquitination, a critical event for ICL repair. We have recently reported the identification and synthesis of a small molecule inhibitor (SMI) of Rad6 that targets its ubiquitin conjugating activity. We hypothesize that targeting Rad6 will be beneficial to TNBCs treated with Pt-based compounds by preventing acquisition of resistance and overcoming Pt resistance. Treatment with the Rad6 SMI enhanced CDDP sensitivity of BRCA1 wild type MDA-MB-231 TNBC cells that have intrinsic CDDP resistance. Western blot analysis showed that the CDDP-induced DNA damage is accompanied by PCNA and FancD2 monoubiquitination (surrogate markers of Rad6 and FA pathway activation, respectively) and increases in ΓH2AX levels. Treatment of MDA-MB-231 cells with the Rad6 SMI attenuated CDDP-induced PCNA and FancD2 ubiquitination, and ΓH2AX levels. Treatment with Rad6 SMI inhibited formation of CDDP-induced Rad6, PCNA, FancD2, translesion synthesis polymerase eta and ΓH2AX nuclear foci. These data suggest that Rad6 plays an important role in recruitment of repair factors during CDDP-induced DNA damage response. To verify these findings, IdU/CldU double labeling assays were performed to evaluate the impact of Rad6 inhibition on restart of CDDP-induced stalled replication forks. Whereas restarting of CDDP-induced stalled replication forks occurred efficiently in MDA-MB-231 cells treated with nontarget siRNA or vehicle, resumption of stalled forks was severely compromised in MDA-MB-231 cells treated with Rad6B siRNA or Rad6 SMI. Consistent with the in vitro data, orthotopic implantation of MDA-MB-231 cells pretreated with Rad6 SMI or CDDP alone produced significantly smaller tumors compared to vehicle, and tumor growth was further mitigated when cells were pretreated with both CDDP and Rad6 SMI. These data imply a critical role for the Rad6 PRR pathway in repair of Pt-induced DNA damage and that inhibition of Rad6 catalytic activity may be exploited to sensitize TNBCs to Pt therapies. Supported by NIH 1R21CA178117-01. Citation Format: Brittany Haynes, Matthew Sanders, Malathy Shekhar. Inhibition of Rad6 sensitizes triple negative breast cancer cells to platinum-based therapy. [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 820. doi:10.1158/1538-7445.AM2014-820

Characterization of type III TGF-β receptor expression in invasive breast carcinomas: a potential new marker and target for triple negative breast cancer.

Invasive breast carcinomas are heterogeneous and exhibit distinct molecular features and biological behavior. Understanding the underlying molecular events that promote breast cancer progression is necessary to improve treatment and prognostication. TGF-β receptor III (TBR3) is a member of the TGF-β signaling pathway, with functions in cell proliferation and migration in malignancies, including breast cancer. Recent studies propose that TBR3 may function as a tumor suppressor and that its loss may correlate with disease progression. However, there are limited data on the expression of TBR3 in breast cancer in relationship to tumor type, hormonal receptor status and HER-2/neu, and patient outcome. In this study, we investigated the expression of TBR3 in a cohort of 205 primary invasive breast carcinomas in tissue microarrays (TMAs), with comprehensive clinical, pathological and follow- up information. Sections were stained for TBR3 and evaluated for intensity of reactivity based on a 4-tiered scoring system (1 to 4; TBR3 low = scores 1-2; TBR3 high = scores 3-4). Of the 205 invasive carcinomas, 123 were luminal type (95 type A, 28 type B), 8 were HER-2 type, and 62 were triple negative (TN). TBR3 was high in 112 (55%) and low in 93 (45%) cases. Low TBR3 was associated with higher histological grade and worse disease free and overall survival, all features of biologically aggressive breast carcinomas. TBR3 was significantly associated with the subtype of breast cancer, as low TBR3 was detected in 95% of TN compared to 22% of luminal tumors (p < 0.0001). We discovered a significant association between low TBR3 protein expression, TN breast cancer phenotype, and disease progression. These data suggest that TBR3 loss might be linked to the development of TN breast cancers and pave the way to investigating whether restoring TBR3 function may be a therapeutic strategy against TN breast carcinomas.