Therapeutic Target For Triple-negative Breast Cancer Research Articles

S100A14 is a novel independent prognostic biomarker in the triple-negative breast cancer subtype.

Triple-negative breast cancer (TNBC) represents a heterogeneous subgroup with generally poor outcome and lack of an effective targeted therapy. Prognostic or predictive biomarkers to guide treatment decisions for this group of patients are needed. To evaluate the potential of S100A14 protein as a novel biomarker in TNBC, the protein expression of S100A14 was correlated with clinical outcomes in a Pilot Sample set and a Danish cohort of predominantly TNBC patients. Kaplan-Meier analysis identified a prognostic impact of S100A14 on disease-free survival and overall survival, showing that tumors with high S100A14 protein expression levels were significantly correlated with poor outcome in TNBC patients (p = 0.017; p = 0.038), particularly those in the basal-like subgroup (p = 0.006; p = 0.037). Importantly, TNBC patients with high S100A14 expression, but tumor-negative axillary lymph nodes (N-), had equally poor outcomes as those with tumor-positive axillary lymph nodes (N+), while TNBC/N- patients with low S100A14 expression had a significantly better disease free survival (p = 0.013). Multivariate analysis revealed that S100A14 is an independent prognostic factor for TNBC patients (p = 0.024; p = 0.05). At the cellular level, S100A14 was found to be expressed in epithelial-like, but not in mesenchymal-like, TNBC cells in vitro. S100A14 is an independent prognostic factor in TNBC and a novel potential therapeutic target in 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.

Abstract P4-05-04: MECP2 is a frequently amplified oncogene and potential therapeutic target in TNBC

Abstract Background: To identify new oncogenes that drive cancer development, we conducted an unbiased genome-scale screen for genes that can substitute for activated RAS in oncogenic transformation. We focused attention on one of the new potential oncogenes identified in this screen, Methyl CpG Binding Protein 2 (MECP2), which has no previously described role in malignancy and is amplified across ∼20% of all human cancers, and ∼30% of Triple-Negative Breast Cancer (TNBC). MECP2 is an X-linked gene known to bind methylated cytosines, and can act as a transcriptional repressor in this context. Recent studies show that it also acts as a transcriptional activator, likely through binding to another epigenetic modification of DNA, 5-hydroxymethylcytosine (5hmC). Results: MECP2 is as potent as activated RAS in conferring anchorage independent growth upon primary human mammary epithelial cells (hMECs) previously transduced with the SV40 early region and hTERT (N−RAS hMECs). MECP2 partially rescues the growth inhibition of RAS-addicted human cancer cell lines after the shRNA-mediated suppression of RAS. MECP2 expresses two spliced isoforms; experiments showed the short isoform activates the MAPK pathway, while the long isoform activates the PI3K pathway. Neither isoform alone can cause growth of N−RAS hMECs as a xenograft in nude mice; together, they can. The transformation and growth factor pathway induction activities of MECP2 absolutely require its DNA-binding activity. A number of TNBC cell lines have amplified, overexpressed MECP2, and of the first 13 TNBC patient-derived xenografts examined, 4 have MECP2 overexpression. Several TNBC cell lines that have amplified, overexpressed MECP2 show significant growth inhibition after shRNA-mediated downregulation of MECP2 (MECP2 addiction), while a breast cancer cell line without MECP2 overexpression showed no such inhibition. N−RAS hMECs transformed with MECP2 are an order of magnitude more sensitive to either of the DNA methylation inhibitors 5-azacytidine or decitabine than isogenic cells transformed by activated RAS, or isogenic cells without an additional transforming gene. Further, we find that combined treatment with the DNA methylation inhibitor 5-azacytidine and the HDAC inhibitor Trichostatin A is synergistic in our hMEC experimental system. Conclusion: MECP2 is a commonly amplified and overexpressed oncogene whose two splicing isoforms together recapitulate the major oncogenic functions of activated RAS. Because MECP2 requires DNA binding to methylated or hydroxymethylated cytosines for its tumor-promoting activities, DNA methylation inhibition with FDA-approved drugs may be therapeutic for tumors overexpressing MECP2. Citation Format: Daniel P Silver, Manish Neupane, Allison P Clark, Marc Vidal, David E Hill. MECP2 is a frequently amplified oncogene and potential therapeutic target in TNBC [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-04.

USP2 promotes cell migration and invasion in triple negative breast cancer cell lines.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is often associated with a poor prognosis. The aim of our study was to identify biomarkers predictive of TNBC progression. Primary TNBC breast tissue samples including four with metastasis and six without metastasis were subjected to Affymetrix GeneChip® analysis (human genome U133). Ubiquitin-specific protease 2 (USP2) was identified as an upregulated gene in the metastatic group, and its expression was analyzed by immunohistochemistry in 121 primary breast cancers, 13 paired normal tissues, and 13 paired metastatic lesions. Survival analysis was performed using the log-rank test and Cox regression hazard model. Matrigel migration and invasion assays in USP2-silenced and USP2-overexpressed breast cancer cell lines were used to investigate the mechanisms of USP2 in vitro. Positive immunostaining for USP2 was detected in breast tumors and was correlated with estrogen receptor (ER) and progesterone receptor (PR) statuses and TNBC subtype. USP2 was overexpressed in distant metastatic lesions compared with primary breast cancers. Survival analyses demonstrated that positive USP2 is a poor prognostic factor for disease-free survival. Silencing of USP2 expression decreased migration and invasion in LM2-4175 and SCP46 cells in association with the downregulation of matrix metalloproteinase-2 (MMP2) expression, whereas overexpression of USP2 in MDA-MB-468 and MDA-MB-231 cells enhanced migration and invasion and upregulated the expression of MMP2. The present study showed that USP2 expression is associated with TNBC cell line's invasiveness and poor survival of breast cancer patients and may serve as a prognostic biomarker and therapeutic target for TNBC.

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.

Silencing of CXCR4 sensitizes triple-negative breast cancer cells to cisplatin.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer for which there is no effective treatment. Previously, we and others demonstrated that CXCR4 surface expression is an independent prognostic factor for disease relapse and survival in breast cancer. In this study, we investigated the effects of CXCR4 gene silencing on cisplatin chemosensitivity in human triple-negative breast cancer cell lines. We found that CXCR4 silencing significantly inhibited cell growth, decreased colony formation, and enhanced cisplatin sensitivity while overexpression of CXCR4 rendered cells more resistant to cisplatin. Moreover, the percentage of apoptosis and cell cycle arrest at the G2/M phase of cisplatin-treated CXCR4 knockdown cells was significantly higher than control cells. Furthermore, we demonstrated CXCR4 knockdown cells showed lower levels of mutant p53 and Bcl-2 protein than the control group, while also having higher levels of caspase-3 and Bax. However overexpression of CXCR4 had the reverse effect. In vivo experiments confirmed that downregulation of CXCR4 enhanced cisplatin anticancer activity in tumor-bearing mice, and that this enhanced anticancer activity is attributable to tumor cell apoptosis. Thus, this study indicates that CXCR4 can modulate cisplatin sensitivity in TNBC cells and suggests that CXCR4 may be a therapeutic target for TNBC.

Abstract 531: MiR-199a-5p is a biomarker for and regulator of epithelial-mesenchymal transition in triple-negative breast cancer patients

Abstract Objectives: Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer, and is characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor. The lack of targeted therapy is a major obstacle for treating against this aggressive subtype, hence the search of specific biomarkers may use as potential diagnostic marker and perhaps a therapeutic target for TNBC. Methods: Plasma samples from patients with TNBC and non-TNBC were recruited for miRNA profiling. By using miRCURY LNA array containing 730 miRNAs, we compared the differential miRNA expressions in plasma of patient with TNBC (n=5) and non-TNBC (n=5), as well as healthy controls (n=5). Selected miRNAs were further validated in an independent cohort (n=250) using real-time RT-PCR. Functional study of miRNA was also carried out in the cell lines. Results: Microarray data revealed miR-16, miR-21 and miR-199a-5p have differentially expression between TNBC and non-TNBC. We found that miR-16, miR-21 and miR-199a-5p were underexpressed in TNBC cases when compared with healthy controls. Moreover, post-operative expression levels of miR-16, miR-21 and miR-199a-5p were significantly increased than that of pre-operative plasma of TNBC. We then stratified the patients by TNM stage and associate with miRNA expression level. Plasma miR-199a-5p expression in TNBC patients had significant difference when compared with healthy controls (stage I, p<0.002; stage II, p<0.001; stage III, p<0.011). Transfection of miR-199a-5p mimic significantly reduced cell proliferation and restored the epithelial-mesenchymal transition (EMT) markers (E-cadherin, ZEB1 and TWIST2). Conclusions: Our data implicate that miR-199a-5p is a potential marker for discriminating TNBC cases and non-TNBC cases. The association of miR-199a-5p with EMT markers uncovers an important pathway in metastatic breast cancer. This study provides insights for the potential use of miRNA as diagnostic marker and targeted therapy for the treatment of TNBC. Citation Format: Vivian Y. Shin, Man T. Siu, John C. Ho, Ava Kwong. MiR-199a-5p is a biomarker for and regulator of epithelial-mesenchymal transition in triple-negative breast cancer patients. [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 531. doi:10.1158/1538-7445.AM2014-531

Abstract 750: JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative breast cancer

Abstract Background: Patients with triple-negative breast cancer (TNBC) have a very poor prognosis due to metastasis and limited targeted therapies. Basal-like TNBC has hyperactivated JNK (c-Jun N-terminal kinase). JNK plays a vital role in malignant transformation in different cancers and self-renewal and maintenance of glioma cancer stem cells (CSCs). However, it is not known whether JNK signaling is a clinically relevant target in TNBC. We tested the hypotheses that JNK signaling plays a fundamental role in TNBC tumorigenesis and metastasis by promoting CSCs and that suppressing JNK signaling using JNK-IN-8 inhibits tumorigenicity and metastasis of TNBC cells. Methods: We examined the effects of JNK knockdown or overexpression on cellular functions of TNBC cells and CSC subpopulations in vitro. We also evaluated the anti-TNBC efficacy of JNK-IN-8, an ATP-directed covalent small molecule inhibitor of JNK that specifically binds the Cys116 on JNK. Results: In patients with TNBC (n=79), low c-Jun mRNA level was associated with better disease-free survival (DFS) (P=0.025), and there was a tendency (P=0.167) toward better DFS with low JNK1 mRNA level. No similar trend was observed for JNK2. These results suggest that JNK1 and c-Jun may have clinical significance in TNBC. Knockdown of JNK1 or JNK2 led to reductions in mammosphere formation, migration, and invasion in HCC70 and SUM149 cells. In contrast, overexpression of constitutively active MKK7-JNK1 or -JNK2 enhanced these cellular activities and increased the ALDH1+ subpopulation. These data indicate a potential role of JNK signaling in regulating TNBC tumorigenesis and metastasis, possibly by promoting CSCs. JNK-IN-8 suppressed proliferation, anchorage-independent growth, migration, and invasion in HCC70 and SUM149 cells. In addition, JNK-IN-8 also reduced mammosphere formation and the CD44+/CD24- and ALDH1+ subpopulations, suggesting that JNK-IN-8 can inhibit JNK signaling-mediated CSC self-renewal and maintenance. Furthermore, overexpression of mutant JNK1- or JNK2-C116S, in which Cys116 was replaced with Ser116, partially abolished the inhibitory effects of JNK-IN-8 on cellular functions, suggesting that JNK-IN-8 executes antitumor activity by specifically targeting JNK. Lastly, JNK inhibition by JNK-IN-8 led to down-regulation of Notch1, suggesting involvement of Notch1 in JNK signaling-mediated CSC regulation. Conclusion: JNK signaling regulates TNBC tumorigenesis and metastasis by promoting CSCs partly via Notch signaling. Our data demonstrate that JNK is an important therapeutic target in TNBC, providing a rationale for further preclinical and clinical development of a JNK-targeted therapy for TNBC. JNK-IN-8 is an effective inhibitor targeting JNK signaling in TNBC. Our studies justify searching for JNK isoform selective inhibitors since there is evidence that JNK1 and JNK2 can have opposing roles in certain settings. Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupunganti, Tinghu Zhang, Takahiro Kogawa, Gaurav B. Chauhan, Nathanael S. Gray, Chandra Bartholomeusz, Kevin N. Dalby, Naoto T. Ueno. JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative breast cancer. [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 750. doi:10.1158/1538-7445.AM2014-750

PD-L1 expression in triple-negative breast cancer.

Early-phase trials targeting the T-cell inhibitory molecule programmed cell death ligand 1 (PD-L1) have shown clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-negative breast cancer (TNBC) and to investigate the loss of PTEN as a mechanism of PD-L1 regulation. The Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene in TNBC (n = 120) compared with non-TNBC (n = 716; P < 0.001). Breast tumor tissue microarrays were evaluated for PD-L1 expression, which was present in 19% (20 of 105) of TNBC specimens. PD-L1(+) tumors had greater CD8(+) T-cell infiltrate than PD-L1(-) tumors (688 cells/mm vs. 263 cells/mm; P < 0.0001). To determine the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN short hairpin RNA (shRNA). PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, suggesting transcriptional regulation. Moreover, phosphoinositide 3-kinase (PI3K) pathway inhibition using the AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K signaling to PD-L1 regulation. Coculture experiments were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to decreased T-cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBCs, suggesting PD-L1 as a therapeutic target in TNBCs. Because PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses.

Integrated analysis of expression profiling data identifies three genes in correlation with poor prognosis of triple-negative breast cancer

Triple-negative breast cancer (TNBC) shows more aggressive clinical behavior and poorer outcome than non-triple-negative breast cancer (NTNBC), and cannot be treated either via endocrine therapy or by Trastuzumab. For TNBC, chemotherapy is currently the mainstay of systemic medical treatment, the lack of more efficient options of treatment has been a problem in breast cancer prevention. In this study, we aimed to find genes related to prognosis in TNBC by bioinformatic analysis and to provide therapeutic candidates for TNBC treatment. We compared the differences in gene expression levels between cancer patients and healthy individuals across five breast cancer microarray databases to generate a gene cohort specifically upregulated in the NTNBC subtype, whose expression levels are ≥2-fold higher in TNBC compared to NTNBC and healthy individuals. Another two databases with clinical information were applied for following Kaplan-Meier analysis, and high expression of BIRC5, CENPA and FAM64A in this cohort were found to be related to poor survival (OS, DMFS, DFS and RFS). This correlation was also seen in patients at early stages and grades. On the other hand, the outcome of patients with synchronous upregulation of these three genes was the worst, while those with synchronous low gene level was the best. In conclusion, BIRC5, CENPA and FAM64A are specifically upregulated in TNBC, and the high expression of these three genes is associated with poor breast cancer prognosis, suggesting their clinical implication as therapeutic targets in TNBC.

Chk1 as a new therapeutic target in triple-negative breast cancer

ObjectivesBioinformatics analyses of pathways and genes differentially expressed between malignant and benign lesions could allow discovering new therapeutic targets. Here, we identified Checkpoint kinase 1 (Chk1) as a potent therapeutic target in triple-negative breast cancer (TNBC). Materials and methodsDifferential gene expression between TNBC, other malignant and benign lesions was performed on two breast cancer datasets. Chk1 was targeted using RNA interference or chemical inhibitor in several TNBC cell lines. ResultsDNA repair pathway was identified as one mostly deregulated pathway in TNBC as compared to benign lesions. Chk1 was identified as candidate target among the 35 genes included in this pathway. Gene expression analysis revealed that Chk1 gene was significantly overexpressed in TNBC as compared to non-TNBC and benign lesions. Depletion of Chk1 protein expression induced a marked reduction of cell viability and led to mitotic catastrophe in TNBC cells. Chemical Chk1 inhibitor decreased survival in TNBC cells, and transcriptome analyze revealed a modulation of gene expression profile in response to Chk1 treatment. ConclusionThese findings suggest that Chk1 may represent a therapeutic target in TNBC, and provide a rationale to evaluate Chk1 inhibitors in breast cancer patients.

Elevated Protein Kinase D3 (PKD3) Expression Supports Proliferation of Triple-negative Breast Cancer Cells and Contributes to mTORC1-S6K1 Pathway Activation

Here, we show that the expression of the Golgi-localized serine-threonine kinase protein kinase D3 (PKD3) is elevated in triple-negative breast cancer (TNBC). Using an antibody array, we identified PKD3 to trigger the activation of S6 kinase 1 (S6K1), a main downstream target of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Accordingly, PKD3 knockdown in TNBC cells led to reduced S6K1 phosphorylation, which was associated with impaired activation of mTORC1 at endolysosomal membranes, the accumulation of the mannose 6-phosphate receptor in and the recruitment of the autophagy marker light chain 3 to enlarged acidic vesicles. We further show that PKD3 depletion strongly inhibited cell spreading and proliferation of TNBC cells, identifying this kinase as a potential novel molecular therapeutic target in TNBC. Together, our data suggest that PKD3 in TNBC cells provides a molecular connection between the Golgi and endolysosomal compartments to enhance proliferative mTORC1-S6K1 signaling.

CD73 promotes anthracycline resistance and poor prognosis in triple negative breast cancer

Using gene-expression data from over 6,000 breast cancer patients, we report herein that high CD73 expression is associated with a poor prognosis in triple-negative breast cancers (TNBC). Because anthracycline-based chemotherapy regimens are standard treatment for TNBC, we investigated the relationship between CD73 and anthracycline efficacy. In TNBC patients treated with anthracycline-only preoperative chemotherapy, high CD73 gene expression was significantly associated with a lower rate of pathological complete response or the disappearance of invasive tumor at surgery. Using mouse models of breast cancer, we demonstrated that CD73 overexpression in tumor cells conferred chemoresistance to doxorubicin, a commonly used anthracycline, by suppressing adaptive antitumor immune responses via activation of A2A adenosine receptors. Targeted blockade of CD73 enhanced doxorubicin-mediated antitumor immune responses and significantly prolonged the survival of mice with established metastatic breast cancer. Taken together, our data suggest that CD73 constitutes a therapeutic target in TNBC.

A Targeted RNAi Screen of the Breast Cancer Genome Identifies KIF14 and TLN1 as Genes That Modulate Docetaxel Chemosensitivity in Triple-Negative Breast Cancer

To identify biomarkers within the breast cancer genome that may predict chemosensitivity in breast cancer. We conducted an RNA interference (RNAi) screen within the breast cancer genome for genes whose loss-of-function enhanced docetaxel chemosensitivity in an estrogen receptor-negative, progesterone receptor-negative, and Her2-negative (ER-, PR-, and Her2-, respectively) breast cancer cell line, MDA-MB-231. Top candidates were tested for their ability to modulate chemosensitivity in 8 breast cancer cell lines and to show in vivo chemosensitivity in a mouse xenograft model. From ranking chemosensitivity of 328 short hairpin RNA (shRNA) MDA-MB-231 cell lines (targeting 133 genes with known somatic mutations in breast cancer), we focused on the top two genes, kinesin family member 14 (KIF14) and talin 1 (TLN1). KIF14 and TLN1 loss-of-function significantly enhanced chemosensitivity in four triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, HCC38, HCC1937, and Hs478T) but not in three hormone receptor-positive cell lines (MCF7, T47D, and HCC1428) or normal human mammary epithelial cells (HMEC). Decreased expression of KIF14, but not TLN1, also enhanced docetaxel sensitivity in a Her2-amplified breast cancer cell line, SUM190PT. Higher KIF14 and TLN1 expressions are found in TNBCs compared with the other clinical subtypes. Mammary fat pad xenografts of KIF14- and TLN1-deficient MDA-MB-231 cells revealed reduced tumor mass compared with control MDA-MB-231 cells after chemotherapy. KIF14 expression is also prognostic of relapse-free and overall survival in representative breast cancer expression arrays. KIF14 and TLN1 are modulators of response to docetaxel and potential therapeutic targets in TNBC.

Targeting triple-negative breast cancer through the somatostatin receptor with the new cytotoxic somatostatin analogue AN-162 [AEZS-124

Previously, we have shown that the targeted cytotoxic somatostatin (sst) analogue AN-162 [AZSE-124] inhibits the growth of MDA-MB-231 human breast cancers xenografted into nude mice. In this study, we examined the trafficking of AN-162 into the cell, the expression of the somatostatin receptors (sstr) in specimens of human triple-negative breast cancers (TNBC), and the effect of AN-162 on HCC 1806 human TNBC xenografts. The expression of sstr in TNBC tumor samples was investigated by immunohistochemical staining. The expression of sstr in HCC 1806 was evaluated by reverse transcription PCR. Internalization studies with I-labeled AN-162 were carried out and the autofluorescence sign of doxorubicin moiety in the cell nucleus after incubation with AN-162 was measured using a fluorescence assay. The effects of AN-162 on the growth of HCC 1806 xenografted into nude mice were studied. A fluorescence microscopy cytotoxicity assay in vitro to detect cell death after treatment with AN-162 was also carried out. About 28% of TNBC tumor specimens showed a positive staining for sstr subtype 2a. HCC 1806 expresses all five subtypes of sstr. In the fluorescence cytotoxicity assay, dead HCC 1806 cells were found 24 h after incubation with AN-162. The growth of HCC 1806 tumors in nude mice was significantly inhibited by treatment with AN-162. AN-162 was internalized into the HCC 1806 cells and doxorubicin moiety was detected in the cell nuclei. This study is the first to show that the trafficking of the cytotoxic sst analogue AN-162 into the cell is mediated by sstr. Our work shows that the growth of xenografted HCC 1806 TNBCs can be effectively inhibited in vivo with AN-162. This investigation provides information on the mechanism of action and efficacy of this new targeted cytotoxic sst analogue and identifies in this relation the sstr as a favorable therapeutic target in 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.

Abstract P5-03-09: Receptor Activator of Nuclear Factor Kappa B (RANK) as a potential therapeutic target in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) has a poorer prognosis in comparison to other breast cancer subtypes because of its high metastatic rate. There is evidence that cancer stem cells (CSCs), known to have an epithelial-to-mesenchymal (EMT) phenotype, may contribute to the metastasis of breast cancer. Denosumab (an anti-RANK ligand antibody) is currently being explored as a treatment for breast cancer in phase II and III clinical trials. RANK is thought to regulate mammary stem cells. Therefore, we hypothesized that RANK promotes tumorigenicity and metastasis of TNBC via the regulation of EMT and enrichment of CSCs. We sought to determine the effects of RANK expression on the tumorigenic and metastatic activities of TNBC cells and whether RANK is a potential therapeutic target in TNBC. Methods: Primary breast tumor specimens from the MD Anderson Cancer Center Tumor Bank were stratified by ER/HER2 status. We analyzed RANK mRNA expression levels, obtained by Affymetrix gene chip array, in ER+/HER2-negative breast tumors (n = 22) and ER−/HER2-negative breast tumors including TNBC tumors (n = 18). To study the effects of RANK suppression in TNBC cells, we generated two stable RANK shRNA MDA-MB-231 TNBC cell lines and a negative shRNA cell line as a control and examined whether knockdown of RANK expression correlated with a decrease in mesenchymal markers, vimentin and snail, and stem cell marker, ALDH1, invasion, migration, and tumorigenicity in TNBC cells. Results: An analysis of RANK mRNA expression levels among human primary breast cancer specimens revealed increased RANK expression in ER−/HER2-negative primary breast tumors including TNBC tumors, compared with ER+/HER2-negative primary breast tumors (p = 0.034). Knocking down RANK in MDA-MB-231 cells significantly inhibited migration (p = 0.0005) and invasion (p = 0.0006) and reduced the stem cell subpopulation, as shown by a decrease in mammospheres (p = 0.0188) and ALDH1 activity. Further, in a three-dimensional (3D) cell culture model, we observed significant inhibition of projections in MDA-MB-231 cells with knockdown of RANK (p &amp;lt; 0.05), which correlated with downregulation of the mesenchymal markers vimentin and snail shown by qRT PCR analysis. Conclusion: Our data demonstrate that RANK may promote the tumorigenesis of TNBC by enriching cancer stem cells. In future studies, we will investigate the effect of RANK inhibition on metastasis in vivo using a TNBC xenograft model. Our long-term goal is to develop RANK-targeted therapy for TNBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-09.

Effect of Notch-1 small interfering RNA on biological function of triple-negative breast cancer cell line MDA-MB-231

Objective To investigate the role of Notch-1 signaling pathway in the growth of triplenegative breast cancer ( TNBC ) and the action mechanism.Methods Notch-1 small interfering RNA (siRNA) was transiently transfected into MDA-MB-231 cells to down-regulate the mRNA expression of Notch-1.The cell proliferation was assessed by methyl thiazol tetrazolium (MTT) assay during 5 days after transfection.And the changes in cell apoptosis and cell cycle were evaluated 48 h after transfection by flow cytometry.Real-time polymerase chain reaction (PCR) was used to detect the mRNA expression of Notch-1,Cyclin D1,bcl-2,bcl-XL and Western blotting was used to measure nuclear factor-κB (NF-κB) level in cell nucleus.Results Notch-1 siRNA could effectively inhibit the expression of Notch-1,with the inhibition rate being 88.6％.The proliferation of MDA-MB-231 cells was markedly inhibited by Notch-1 siRNA with the inhibition rate being 44.7％.The apoptosis rate was increased from 3.67％ to 13.25％ and the percentage of cells in G2 phase was increased from 6.27％ to 14.28％,respectively.Down-regulation of the expression of Notch-1 by siRNA could down-regulate the expression of NF-κB in nucleus,and the expression of Cyclin D1,bcl-2 and bcl-XL in MDA-MB-231 cells.Conclusion Our results suggest that Notch-1 pathway plays a critical role in TNBC.Down-regulation of Notch-1 by siRNA can inhibit the proliferation of MDA-MB-231 cells.Notch-1 may be a novel therapeutic target of TNBC. Key words: Triple-negative breast cancer; Notch-1 ; Nuclear factor-κB; RNA interference

CIP2A is a target of bortezomib in human triple negative breast cancer cells

IntroductionTriple negative breast cancer (TNBC) is very aggressive and currently has no specific therapeutic targets, such as hormone receptors or human epidermal growth factor receptor type 2 (HER2); therefore, prognosis is poor. Bortezomib, a proteasome inhibitor, may exert efficacy in TNBC through its multiple cellular effects. Here, we tested the efficacy of bortezomib and examined the drug mechanism in breast cancer cells.MethodsFive breast cancer cell lines: TNBC HCC-1937, MDA-MB-231, and MDA-MB-468; HER2-overexpressing MDA-MB-453; and estrogen receptor positive MCF-7 were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western Blot. Signal transduction pathways in cells were assessed by Western Blot. Gene silencing was done by small interfering RNA (siRNA). In vivo efficacy of bortezomib was tested in nude mice with breast cancer xenografts. Immunohistochemical study was performed on tumor tissues from patients with TNBC.ResultsBortezomib induced significant apoptosis, which was independent of its proteasome inhibition, in the three TNBC cell lines, but not in MDA-MB-453 or MCF-7 cells. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A), a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of bortezomib. We showed that bortezomib inhibited CIP2A in association with p-Akt downregulation in a dose- and time-dependent manner in all sensitive TNBC cells, whereas no alterations in CIP2A expression and p-Akt were noted in bortezomib-resistant cells. Overexpression of CIP2A upregulated p-Akt and protected MDA-MB-231 and MDA-MB-468 cells from bortezomib-induced apoptosis, whereas silencing CIP2A by siRNA overcame the resistance to bortezomib-induced apoptosis in MCF-7 cells. In addition, bortezomib downregulated CIP2A mRNA but did not affect the degradation of CIP2A protein. Furthermore, bortezomib exerted in vivo antitumor activity in HCC-1937 xenografted tumors, but not in MCF-7 tumors. Bortezomib downregulated CIP2A expression in the HCC-1937 tumors but not in the MCF-7 tumors. Importantly, CIP2A expression is readily detectable in tumor samples from TNBC patients.ConclusionsCIP2A is a major determinant mediating bortezomib-induced apoptosis in TNBC cells. CIP2A may thus be a potential therapeutic target in TNBC.

Identification of New Candidate Therapeutic Target Genes in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a subgroup of breast cancer that is negative for estrogen and progesterone receptor and ERBB2 protein expression. It is characterized by its aggressive behavior and by the lack of targeted therapies. To identify new therapeutic targets in TNBC, we used real-time quantitative RT-PCR to analyze 63 TNBC samples in terms of their mRNA expression of 26 genes coding for the major proteins currently targeted by drugs used to treat other cancers or undergoing clinical trials in breast cancer. Six of the 26 genes tested (VEGFA, SRC, PARP1, PTK2, RAF1, and FGFR3) were significantly upregulated in 13% to 46% of the TNBCs. None of the 6 genes was specifically upregulated in the TNBCs compared with 3 other classical breast tumor subtypes. No association was observed between overexpression of these 6 genes (except for FGFR3) and PIK3CA mutation status. These results confirm the interest of targeting VEGFA and PARP1 in ongoing clinical trials in TNBC patients and also identify new target genes (SRC, PTK2, RAF1, and FGFR3). Clinical trials could be initiated easily with existing drugs. Our results also suggest that these target genes might serve as predictive biomarkers of the TNBC treatment response.