Novel Therapeutic Target In Triple-negative Breast Cancer Research Articles

CircPLK1 sponges miR-296-5p to facilitate triple-negative breast cancer progression.

Aim: To investigate the role of circRNAs in triple-negative breast cancer (TNBC) and the underlying mechanisms. Materials & methods: We performed circRNA microarrays to explore the expression profiles of TNBC cell lines. Experiments in vitro and in vivo were conducted to explore the effects of circPLK1 on tumor proliferation and metastasis as well as the interaction between circPLK1, miR-296-5p and PLK1 in TNBC. Results & conclusion: CircPLK1 was significantly upregulated in TNBC and associated with poor survivals. CircPLK1 knockdown inhibited cell growth and invasion in vitro as well as tumor occurrence and metastasis in vivo. CircPLK1-miR-296-5p-PLK1 axis regulates tumor progression by ceRNA mechanism in TNBC, indicating that circPLK1 may serve as a prognostic factor and novel therapeutic target for TNBC.

Association of p53 expression with poor prognosis in patients with triple-negative breast invasive ductal carcinoma.

TP53 gene is mutated in approximately 80% of triple-negative breast cancer (TNBC). However, the prognostic significance of immunohistochemical (IHC)-detected p53 protein expression remains controversial in TNBC. In this study, we retrospectively analyzed the association between IHC-detected p53 expression and the prognosis in a cohort of 278 patients with stage I-III triple-negative breast invasive ductal carcinoma (IDC), who received surgery at the department of breast surgery in the Fourth Hospital of Hebei Medical University from 2010–01 to 2012–12. We found a positive expression ratio of IHC-detected p53 in triple-negative breast IDC of 58.6% (163/278). Furthermore, levels of expression were significantly associated with vessel tumor emboli and higher histologic grade (P = .038, P = .043, respectively), with the highest expression level observed in G3 breast cancer (64.7%). Additionally, Kaplan-Meier analysis showed that p53 expression indicated worse overall survival (OS) in the whole cohort (79.6% vs 89.6%, Log-rank test P = .025) as well as in stratified prognostic stage II patients (90.8% vs 100%, Log-rank test P = .027). The mortality risk of p53 expression patients was 2.22 times higher than that of p53 negative patients (HR: 2.222; 95%CI: 1.147–4.308). In addition, p53 expression was also associated with poor disease-free survival (DFS) (76.7% vs 86.8%, P = .020). Cox proportional hazard ratio model showed p53 expression was an independent risk factor for OS (P = .018) and DFS (P = .018) after controlling for tumor size, lymph node status, and vessel tumor emboli. Altogether, our data showed that IHC-detected p53 expression is a promising prognostic candidate for poor survival in triple-negative breast IDC patients. However, more studies are needed to determine if p53 may be applied to clinical practice as a biomarker and/or novel therapeutic target for TNBC.

Abstract P2-06-20: CDC20 is a novel therapeutic target in triple negative breast cancer

Abstract Background: Approximately 15% of breast cancers lack expression of Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER-2), and are referred to as Triple Negative Breast Cancer (TNBC). Clinical outcomes in patients with TNBC continue to be poor due to inherently aggressive biology of the disease and paucity of targeted therapies. In this study, we identified Cell Division Cycle protein 20 (CDC20), a key regulatory protein that mediates sister chromatid separation in mitosis, as a novel therapeutic target in TNBC. Methods: To uncover new drivers of TNBC, we nominated genes that are significantly overexpressed in patients with TNBC, compared to non-TNBCs. After identifying CDC20 as a top hit in this analysis, we used expression microarrays, RNA-seq analyses and Western blotting to assess CDC20 expression levels in breast cancer patients and cell lines. Kaplan-Meier analyses were performed to study the correlation between CDC20 expression levels and clinical outcomes, including recurrence-free survival, metastasis-free survival and overall survival. shRNA-mediated knockdown of CDC20 in TNBC cell lines was employed to study the role of CDC20 in TNBC proliferation, invasion, migration and xenograft growth in mice. Results: CDC20 was significantly overexpressed in TNBCs (compared to non-TNBCs) in The Cancer Genome Atlas (TCGA) breast dataset, and in several other independent breast cancer datasets, including Curtis, Stickeler and Kao. CDC20 expression was also significantly higher in TNBC cell lines compared to non-TNBC cell lines. Furthermore, expression levels of CDC20 were highly significantly correlated with clinical outcomes, including recurrence-free survival, metastasis-free survival and overall survival. shRNA-mediated knockdown of CDC20 expression in TNBC cell lines, MDA-MB-231 and MDA-MB-468, resulted in decreased proliferation, invasion and migration. Our preliminary findings suggest that CDC20 knockdown also results in inhibition of cell line-derived xenograft growth in mice. Conclusion: We identified CDC20 is a novel therapeutic target in TNBC. Our findings support development of pharmacologic strategies to inhibit CDC20 function as a potential targeted therapy against TNBC. Citation Format: Sharma J, Raicu I, Nguyen J, Udden N, Wang Q, Alluri P. CDC20 is a novel therapeutic target in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-20.

Identification of FOXM1 as a specific marker for triple‑negative breast cancer.

The present study aimed to identify the therapeutic role of the forkhead box M1 (FOXM1)-associated pathway in triple-negative breast cancer (TNBC). Using a Cancer Landscapes-based analysis, a gene regulatory network model was constructed. The present results demonstrated that FOXM1 occupies a key position in gene networks and is a critical regulatory gene in breast cancer. Using breast carcinoma gene expression data from The Cancer Genome Atlas, it was identified that FOXM1 expression was increased in the basal-like breast cancer subtype compared with other breast cancer subtypes. RNA-sequencing analysis of MDA-MB-231 cells treated with 4 and 10 µl/ml Thiostrepton identified 662 and 5,888 significantly differentially expressed genes, respectively. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses demonstrated that FOXM1 was highly associated with multiple biological processes and was markedly associated with metabolic pathways in TNBC. The use of Search Tool for the Retrieval of Interacting Genes/Proteins provided a critical assessment and integration of protein-protein interactions, and demonstrated the multiple important functions of FOXM1 in TNBC. Real-time cell analysis, reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining were used to assess the anti-tumor activity of Thiostrepton in TNBC cells in vitro. The present results identified that suppression of FOXM1 using Thiostrepton inhibited MDA-MB-231 cell proliferation and the expression of cell cycle-associated genes, including cyclin A2, cyclin B2, checkpoint kinase 1, centrosomal protein 55 and polo like kinase 1. Immunofluorescence staining analysis demonstrated that vimentin, filamentous actin and zinc finger E-box-binding homeobox 1 were all decreased following treatment with Thiostrepton. Furthermore, a BALB/C nude mouse subcutaneous xenograft model was used to verify the function of FOXM1 in vivo. The present results demonstrated that FOXM1 inhibition significantly suppressed MDA-MB-231 cell tumorigenesis in vivo. Overall, the present results suggested that FOXM1 is a key gene that serves important roles in multiple biological processes in TNBC and that it may serve as a novel therapeutic target in TNBC.

Targeting LRP8 inhibits breast cancer stem cells in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most difficult subtype of breast cancer to treat due to a paucity of effective targeted therapies. Many studies have reported that breast cancer stem cells (BCSCs) are enriched in TNBC and are responsible for chemoresistance and metastasis. In this study, we identify LRP8 as a novel positive regulator of BCSCs in TNBC. LRP8 is highly expressed in TNBC compared to other breast cancer subtypes and its genomic locus is amplified in 24% of TNBC tumors. Knockdown of LRP8 in TNBC cell lines inhibits Wnt/β-catenin signaling, decreases BCSCs, and suppresses tumorigenic potential in xenograft models. LRP8 knockdown also induces a more differentiated, luminal-epithelial phenotype and thus sensitizes the TNBC cells to chemotherapy. Together, our study highlights LRP8 as a novel therapeutic target for TNBC as inhibition of LRP8 can attenuate Wnt/β-catenin signaling to suppress BCSCs.

MiR‑30a inhibits epithelial‑mesenchymal transition and metastasis in triple‑negative breast cancer by targeting ROR1.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. In the present study, we revealed that the expression of miR-30a was significantly decreased in TNBC, and TNBC patients with low expression of miR-30a were associated with high histological grade and more lymph node metastasis. Moreover, we found that miR-30a suppressed TNBC cell epithelial-mesenchymal transition (EMT), as demonstrated by the overexpression of miR-30a which increased the expression of epithelial marker E-cadherin but decreased the expression of mesenchymal markers N-cadherin and vimentin. Furthermore, we demonstrated that overexpression of miR-30a significantly suppressed TNBC cell invasion and migration, as well as inhibited tumor growth and metastasis in vivo. More importantly, RTK-like orphan receptor 1 (ROR1) was predicted as the direct target of miR-30a, which was subsequently confirmed by luciferase assays. Forced expression of miR-30a in TNBC cells decreased ROR1 expression, whereas the overexpression of ROR1 reversed the suppressive effects of miR-30a in TNBC cell migration and invasion. Collectively, this study indicated that miR-30a functions as a tumor-metastasis suppressor miRNA in TNBC by directly targeting ROR1 and that miR-30a may serve as a novel therapeutic target for TNBC.

Targeting long non-coding RNA ASBEL with oligonucleotide antagonist for breast cancer therapy

Long non-coding RNAs (lncRNAs) are defined as a class of RNA transcripts longer than 200 nucleotides encoded by mammalian genomes that lack protein-coding potential. LncRNA ASBEL has been identified as an anti-sense transcript of BTG3 (B cell translocation gene 3) gene, which encodes an anti-proliferation protein. Remarkable down-regulation of BTG3 has been reported in triple-negative breast cancer (TNBC). In the present study, a number of single-stranded modified anti-sense DNA oligonucleotides (antago) were designed, synthesized and screened for specific lncRNA ASBEL knockdown. We showed here that anti-ASBEL antago played a significant tumor suppressive role in TNBC by effective down-regulating lncRNA ASBEL, which in turn led to increased BTG3 expression. The obtained data suggest lncRNA ASBEL as a novel therapeutic target in TNBC.

Oncogenic role of SIRT1 associated with tumor invasion, lymph node metastasis, and poor disease-free survival in triple negative breast cancer.

The aim of this study is to evaluate the biological role and clinical implications of silent mating type information regulation 2 homolog 1 (SIRT1) as a novel candidate for target therapy in triple negative breast cancer (TNBC) for which there is no specific agent. 344 patients who received surgical resection for TNBC from January 2003 to December 2006 at Seoul National University Hospital were enrolled, and the role of SIRT1 protein was evaluated via immunohistochemistry on tissue samples. In vivo experiments to evaluate tumor invasiveness were carried out with three human TNBC cell lines following SIRT1-siRNA transfection. Expression of SIRT1 significantly correlated with lymph node metastasis (p=0.008). In multivariate analysis, SIRT1 expression (p=0.011), T stage (p=0.014), and lymphatic invasion (p<0.001) were revealed to be independent predictive factors for lymph node metastasis. Combination of these three parameters revealed predictive performance for lymph node metastasis with an area under the curve (AUC) of 0.689 on receiver operating characteristics (ROC) curves analysis. SIRT1 expression correlated with shorter disease-free survival (P=0.003) but not with overall survival. Inhibition of SIRT1 with small interfering RNA (siRNA) conspicuously suppressed the invasiveness of TNBC cell lines. This study reveals the role of SIRT1 on tumor invasiveness and unfavorable clinical outcomes, and we suggest its potential role as a prognostic indicator as well as a novel therapeutic target in TNBC.

CIB1 depletion impairs cell survival and tumor growth in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with generally poor prognosis and no available targeted therapies, highlighting a critical unmet need to identify and characterize novel therapeutic targets. We previously demonstrated that CIB1 is necessary for cancer cell survival and proliferation via regulation of two oncogenic signaling pathways, RAF-MEK-ERK and PI3K-AKT. Because these pathways are often upregulated in TNBC, we hypothesized that CIB1 may play a broader role in TNBC cell survival and tumor growth. Methods utilized include inducible RNAi depletion of CIB1 in vitro and in vivo, immunoblotting, clonogenic assay, flow cytometry, RNA-sequencing, bioinformatics analysis, and Kaplan-Meier survival analysis. CIB1 depletion resulted in significant cell death in 8 of 11 TNBC cell lines tested. Analysis of components related to PI3K-AKT and RAF-MEK-ERK signaling revealed that elevated AKT activation status and low PTEN expression were key predictors of sensitivity to CIB1 depletion. Furthermore, CIB1 knockdown caused dramatic shrinkage of MDA-MB-468 xenograft tumors in vivo. RNA sequence analysis also showed that CIB1 depletion in TNBC cells activates gene programs associated with decreased proliferation and increased cell death. CIB1 expression levels per se did not predict TNBC susceptibility to CIB1 depletion, and CIB1 mRNA expression levels did not associate with TNBC patient survival. Our data are consistent with the emerging concept of non-oncogene addiction, where a large subset of TNBCs depend on CIB1 for cell survival and tumor growth, independent of CIB1 expression levels. Our data establish CIB1 as a novel therapeutic target for TNBC.

67P - The impact of the uPAR system and its interaction partners as potential therapeutic targets in TNBC

Introduction: Triple-negative breast cancer (TNBC) is characterised as an aggressive type of breast cancer with no or low expression of ER, PR or HER2. Yet, it has been shown that certain components of the urokinase-type plasminogen activator receptor (uPAR) system are highly expressed in TNBC. In this study, uPAR and its interaction partners have been evaluated in TNBC model cell lines and by histological analyses of TNBC clinical specimens to explore the possible therapeutic potential of the uPAR interactome. Methods: Stable RNA interference was employed in MDA-MB-231 and BT549 cells to analyse the long term effects of uPAR and/or uPA depletion, on associated proteins, cell proliferation, cell invasion and migration. Protein expression was analysed by Western blot, in formalin-fixed paraffin-embedded (FFPE) cell line blocks and TNBC tumour specimens (n = 180) by immunohistochemistry (IHC). Additionally, the proximity ligation assay (PLA) was conducted to identify effects on complexes formed by uPAR and its interaction partners. Results: The in vitro analyses showed that 8 weeks after the stable depletion of uPAR and uPA by lentiviral vectors, cell invasion and migration was significantly reduced, supported by the decreased expression of matrix metalloproteinases such as MMP2 that are known to be involved in metastasis processes. Proliferation of the knocked-down cells was significantly diminished which was in accordance with induction of p27. IHC and PLA performed on cell line blocks and tumour samples demonstrated the significant association of uPAR with interaction partners uPA, plasminogen, cathepsin B in this TNBC cohort. Furthermore, our results indicated a regulatory effect of uPAR on the insulin-like growth factor 1 receptor (IGF1R) and in vitro experiments as well as statistical analyses indicate that both enhance malignancy in TNBC. Conclusion: Our findings demonstrate that uPAR and interacting partners are potential therapeutic targets in TNBC since the long-term depletion of uPAR and associated molecules does lead to reduced malignant potential in vitro. In particular, combined inhibition of uPAR and its associated partner IGF1R may be considered as a novel therapeutic target in TNBC. Disclosure: All authors have declared no conflicts of interest.

Identification of dysregulated microRNAs in triple-negative breast cancer (review).

Triple-negative breast cancer (TNBC) is defined by the absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC exhibits a more aggressive phenotype and a poorer clinical outcome compared to other breast cancer subgroups, accounting for 15-20% of total breast cancer cases. To date, the treatment strategies for TNBC are limited to surgery, chemotherapy and radiation, owing to the lack of effective therapeutic targets. Therefore, it is important to identify specific targets for TNBCs. MicroRNAs (miRNAs), a family of small non-coding RNAs regulating gene expression, are an emerging class of regulators of various biological processes, including cell proliferation, invasion, epithelial-mesenchymal transition (EMT) and drug resistance. Actually, miRNAs may serve as a novel therapeutic target in TNBC, and here we review current correlated researches and provide our own profiling results for the miRNAs expressed in TNBC cell lines. The present study offers an additional insight into the potential miRNAs involved in the regulation of TNBC.

Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis

Triple negative breast cancer (TNBC) has a poor outcome due to the lack of beneficial therapeutic targets. To clarify the molecular mechanisms involved in the carcinogenesis of TNBC and to identify target molecules for novel anticancer drugs, we analyzed the gene expression profiles of 30 TNBCs as well as 13 normal epithelial ductal cells that were purified by laser-microbeam microdissection. We identified 301 and 321 transcripts that were significantly upregulated and downregulated in TNBC, respectively. In particular, gene expression profile analyses of normal human vital organs allowed us to identify 104 cancer-specific genes, including those involved in breast carcinogenesis such as NEK2, PBK and MELK. Moreover, gene annotation enrichment analysis revealed prominent gene subsets involved in the cell cycle, especially mitosis. Therefore, we focused on cell cycle regulators, asp (abnormal spindle) homolog, microcephaly-associated (Drosophila) (ASPM) and centromere protein K (CENPK) as novel therapeutic targets for TNBC. Small-interfering RNA-mediated knockdown of their expression significantly attenuated TNBC cell viability due to G1 and G2/M cell cycle arrest. Our data will provide a better understanding of the carcinogenesis of TNBC and could contribute to the development of molecular targets as a treatment for TNBC patients.