Target For Breast Cancer Patients Research Articles

Let-7d Inhibits Growth and Metastasis in Breast Cancer by Targeting Jab1/Cops5

Background/Aims: MicroRNAs (miRNAs) regulate the expressions of cancer-related genes, and are involved in the development and progression of various human cancers. Here, we performed further analyses to determine whether let-7d is functionally linked to Jab1 in breast cancer. Methods: In situ hybridization and immunohistochemical analyses were used to determine the level of let-7d and Jab1 in breast cancer clinical specimens and its correlation with clinicopathological data. Let-7d overexpressing breast cancer cell lines combined with mouse models bearing cell-derived xenografts were used to assess the functional role of let-7d both in vitro and in vivo. Results: In this study, we found that let-7d was downregulated in breast cancer tissues, coupled with the elevations of Jab1 protein expressions, compared with paired adjacent noncancerous breast tissues. Let-7d overexpression significantly suppressed the proliferation and invasion in MCF-7 and MDA-MB-231 cells. Dual luciferase reporter assay indicated that Jab1 was the direct target of let-7d. Stepwise studies from in vitro and in vivo experiments indicated that let-7d overexpression inhibited cell growth and decreased Jab1 expressions in breast cancer cells and nude mice tumor tissues. Statistical analyses demonstrated that breast cancer patients with low levels of let-7d or high levels of Jab1 had a significant correlation with worse prognosis. Conclusion: These findings provide novel insights into molecular mechanism of let-7d and Jab1 in tumor development and progression of breast cancer, and thus let-7d/Jab1 are novel potential therapeutic targets for breast cancer patients.

Hypoxia-Induced TPM2 Methylation is Associated with Chemoresistance and Poor Prognosis in Breast Cancer

Background/Aims: Tropomyosin-2 (TPM2) plays important roles in functions of the cytoskeleton, such as cytokinesis, vesicle transport, cell proliferation, migration and apoptosis,and these functions imply that TPM2 also plays a role in cancer development. Indeed, it has been shown that TPM2 plays a critical role in some cancers. However, the role of TPM2 in breast cancer is still poorly characterized. Thus, we explored the role of TPM2 in breast cancer. Methods: We analysed TPM2 expression and its correlation with the clinicopathological features in breast cancer. Then, we examined the influence of hypoxia on TPM2 expression and methylation status using bisulfite sequencing PCR. Furthermore, we performed TPM2-mediated migration and invasion assays in the context of hypoxia and examined changes in matrix metalloproteinase-2 (MMP2) expression. Finally, we detected the influence of TPM2 on survival and chemotherapy drug sensitivity. Results: We found that TPM2 expression is down-regulated in breast cancer cells compared to that in normal breast cells. The data from TCGA supported these results. Promoter methylation of TPM2, which could be induced by hypoxia, was responsible for its low expression. Hypoxia might regulate cell invasiveness partly by TPM2 down-regulation-mediated changes of MMP2 expression. Importantly, low TPM2 expression was correlated with lymph node metastasis (P=0.031), tumour node metastasis stage (P=0.01), histological grade (P=0.037), and shorter overall survival (P=0.028). Univariate and multivariate analyses indicated that TPM2 was an independent predictor in breast cancer patients. Paclitaxel chemotherapy did not benefit patients with low TPM2 expression (P<0.0001). TPM2 knockdown significantly reduced cell sensitivity to paclitaxel. Conclusion: TPM2 is a potential novel tumour suppressor gene in breast cancer. TPM2 is associated with poor survival and chemoresistance to paclitaxel in breast cancer, and TPM2 may represent a promising therapeutic gene target for breast cancer patients with chemoresistance.

Deficiency of Follistatin-Like Protein 1 Accelerates the Growth of Breast Cancer Cells at Lung Metastatic Sites.

PurposeFollistatin-like protein 1 (FSTL1) is a secreted glycoprotein that has been shown to play a role in various types of cancer. However, the clinical significance and function of FSTL1 in breast cancer have not been reported. We investigated the role of FSTL1 in breast cancer in this study.MethodsEnzyme-linked immunosorbent assays, western blot analysis, and reverse transcription polymerase chain reaction were used to monitor the expression of FSTL1 in breast cancer tissue and in serum samples from breast cancer patients. We employed a 4T1 breast cancer model and Fstl1+/− mice for in vivo studies. Hematoxylin and eosin staining, western blot analysis, and RNA sequencing were used to analyze the effect of FSTL1 on primary tumor growth and lung metastasis.ResultsWe demonstrated that the expression of FSTL1 is reduced in both the breast cancer tissue and the serum of breast cancer patients. We showed that reduced levels of FSTL1 in serum correlate with elevated expression of Ki-67 and epidermal growth factor receptor (EGFR) in cancer tissues. Moreover, lowered expression of FSTL1 was associated with decreased survival in breast cancer patients. Experiments on the Fstl1+/− mouse model established that FSTL1 deficiency had no effect on primary tumor growth, but increased the lung metastases of breast cancer cells, resulting in reduced survival of tumor-bearing mice. RNA sequencing found significantly reduced expression of Egln3 and increased expression of EGFR in Fstl1+/− mice. Thus, our results suggest that FSTL1 may affect the expression of EGFR through Egln3, inhibiting the proliferation of breast cancer cells at lung metastatic sites.ConclusionIn conclusion, we suggest a suppressor role of FSTL1 in breast cancer lung metastasis. Furthermore, FSTL1 may represent a potential prognostic biomarker and a candidate therapeutic target in breast cancer patients.

SF3B1 mutation is a poor prognostic indicator in luminal B and progesterone receptor-negative breast cancer patients.

The purpose of this study was to explore the relationship between SF3B1 mutations and the prognoses of patients with breast cancer. Clinical and SF3B1 mutation data from The Cancer Genome Atlas were analyzed. SF3B1 mutations were evaluated as prognostic factors in all breast cancer patients and specific subgroups through Cox regression and Kaplan-Meier analyses. We also investigated the relationship between traditional parameters and SF3B1 mutations. Receiver operating characteristics curves were used to analyze common risk factors for their sensitivity and specificity in predicting SF3B1 mutations. SF3B1 mutations were a poor prognostic factor in luminal B and progesterone receptor (PR)-negative breast cancer (P < 0.01). Age at diagnosis and estrogen receptor (ER) status were associated with SF3B1 mutations in all breast cancers (P < 0.01) and in luminal B and PR-negative subgroups (P < 0.01). The age at diagnosis and ER status combined had a higher sensitivity and specificity for predicting SF3B1 mutations than each factor alone. SF3B1 mutations are a poor prognostic factor in luminal B and PR-negative breast cancer patients. These mutations are significantly associated with age at diagnosis and ER status. SF3B1 mutations may therefore be a novel therapeutic target for breast cancer patients.

Abstract 2843: DNMT3B mediated DNA methylation and epigenetic reprogramming of metastatic cancer cells

Abstract Approximately 90% of cancer patients die from metastasis. In many cases, current therapies are designed based on the primary tumor characterization and are ineffective against metastatic disease. It is not clear whether metastatic cancer cells acquire additional genetic and epigenetic alterations, if so, what are the underlying mechanisms. Using several tumor models, we demonstrated that DNMT3B, de nova DNA methyltransferase, was increased in metastatic nodules in comparison to that from the primary tumors. DNMT3B knockdown or overexpression significantly inhibited or increased metastatic colonization of cancer cells. To understand the underlying molecular mechanisms of DNMT3B regulation, we performed DNMT3B ChIP-seq, RNA-seq, and DNA methylation-seq comparing lung metastases with primary tumors and 4T1 cells. We identified genes that are differentially methylated and expressed. Ingenuity Pathway Analysis revealed DNMT3B targeting multiple oncogenic signaling pathways. Bioinformatics analysis of human databases demonstrated significantly higher expression levels of DNMT3B in metastases than primary tumors in breast, prostate and melanoma cancer patients. In summary, our study demonstrated that DNMT3B as a key epigenetic regulator which plays critical roles for tumor metastasis. Our work suggests DNMT3B as potential therapeutic targets for breast cancer patients with metastatic disease. Citation Format: Jae Young So, Nicolas Skrypek, Xiang Wang, Anand Merchant, Howard Yang, Wei-Dong Chen, Gangqing Hu, Bhagelu R Achyut, Meggie Cam, Keji Zhao, Maxwell Lee, Li Yang. DNMT3B mediated DNA methylation and epigenetic reprogramming of metastatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2843. doi:10.1158/1538-7445.AM2017-2843

Cigarette Smoke Regulates Calcium-Independent Phospholipase A2 Metabolic Pathways in Breast Cancer

Phospholipase A2 (PLA2)-dependent pathways are important in the regulation of cell proliferation, differentiation, motility, and immune responses, and can be dysregulated during tumor development and progression. We show herein, for the first time, that cigarette smoking leads to an increase in platelet-activating factor (PAF) content and PAF receptor expression in human breast cancer cells and tissue. PAF production could be abrogated in triple-negative breast cancer cells by inhibition of calcium-independent PLA2 (iPLA2). We also demonstrate that cigarette smoke induces the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 and reduces 15-hydroxyprostaglandin dehydrogenase, resulting in prostaglandin E2 release in human breast cancer. Increased cyclooxygenase-2 expression and prostaglandin E2 release could be abrogated in metastatic breast cancer cells by inhibition of iPLA2. These studies indicate that iPLA2-dependent metabolic pathways play an important role in tumor initiation or progression in smokers, representing novel therapeutic targets for breast cancer patients who smoke.

The prognostic significance of Cdc6 and Cdt1 in breast cancer

DNA replication is a critical step in cell proliferation. Overexpression of MCM2-7 genes correlated with poor prognosis in breast cancer patients. However, the roles of Cdc6 and Cdt1, which work with MCMs to regulate DNA replication, in breast cancers are largely unknown. In the present study, we have shown that the expression levels of Cdc6 and Cdt1 were both significantly correlated with an increasing number of MCM2-7 genes overexpression. Both Cdc6 and Cdt1, when expressed in a high level, alone or in combination, were significantly associated with poorer survival in the breast cancer patient cohort (n = 1441). In line with this finding, the expression of Cdc6 and Cdt1 was upregulated in breast cancer cells compared to normal breast epithelial cells. Expression of Cdc6 and Cdt1 was significantly higher in ER negative breast cancer, and was suppressed when ER signalling was inhibited either by tamoxifen in vitro or letrozole in human subjects. Importantly, breast cancer patients who responded to letrozole expressed significantly lower Cdc6 than those patients who did not respond. Our results suggest that Cdc6 is a potential prognostic marker and therapeutic target in breast cancer patients.

Abstract P4-21-29: HER2-associated lipogenic phenotype as a potential therapeutical target in breast cancer patients

Abstract Lipogenic phenotype (genetic program activation involved inde novo fatty acid –FA-synthesis and key metabolic regulators) is associated to oncogenic transformation and malignancy. We hypothesize that in HER2 overexpressing breast cancer cells, HER2 function is affected by cell membrane lipid profile, mainly lipid rafts, necessary to HER2 location and activation within the cell membrane. Therefore, metabolic lipid imbalance could be a potential factor and marker associated to HER2 expression. To evaluate if cell lipogenesis and membrane lipid composition are affected by HER2 overexpression, we used an oncogenic transformed cell line engineered to overexpress HER2 receptor (HB4aC5.2), but identical to their parental strain (HB4a) in all other aspects. Several lipid related molecules including synthesis-FASN, uptake-CD36, transport-FABP4 and FA storage-DGAT by RT-PCR; and lipogenic regulatory pathways (mTOR, DEPTOR, SREBP1 and PPARγ) were evaluated in both cells. Lipogenic contribution to lipid rafts formation was evaluated by gas chromatography and confocal microscopy. The influence of HER2 overexpression and lipogenic phenotype on proteins activated by HER2 (AKT, ERK1/2 and FASN) was analyzed by western blot. Next, HB4a and HB4aC5.2 cells were treated, alone or in combination with DHA (omega-3 polyunsaturated fatty acid), by Trastuzumab (anti-HER2), and GW9662 (anti-PPARγ) for 72h, and all above experiments were repeated. Cell death was analyzed by flow cytometry. In vitro results were confirmed in Primary tumor and adjacent tissue samples from a cohort of 182 European breast cancer patients through metabolomics analysis. Statistical analysis was performed by MetaboAnalyst software (p &amp;lt;0.05). In HB4aC5.2 cells, the HER2 overexpression was associated with a lipogenic phenotype, which increase in cholesterol and saturated FA synthesis for the lipid rafts formation, suggesting that large amounts of HER2 require cell membrane changes to favor rafts saturation and formation, when compared to HB4a. Lipid rafts were associated with survival and proliferation signals hiper activation (p&amp;lt;0.001). Cell lipogenesis was independent ofmTOR/SREBP/PPARγpathway and occur concomitantly to DEPTOR (mTOR inhibitor) increased expression. Moreover, GW9662 or Trastuzumab treatment did not influence lipogenesis in cell culture. In HB4aC5.2 cells, only DHA treatment decreased the lipogenic phenotype, DEPTOR expression, saturated FA synthesis, disrupted lipid raft, inhibited HER2 signaling, and induced apoptosis (p&amp;lt;0.001). In addition, when added to Trastuzumab DHA potentiated cell death. In tumor tissue samples an increased FA synthesis was observed, such as palmitic acid, sphingolipids and phosphatidylcholines saturated FA enriched, synthesis of cholesterol, suggesting an increase of lipid rafts when compared to normal breast adjacent tissue. Our results indicate that lipogenic phenotype is associated with HER2 superexpression in breast cancer cells, and it seems important to promote survival and proliferation, through lipid rafts increase. DHA modulation increased death in tumor cells, suggesting that cell membrane lipogenesis and lipid composition may represent a treatment target. Lipogenic phenotype confirmation in tumor tissue samples strengthens this hypothesis. Citation Format: Ravacci GR, Santos JR, Brentani MM, Tortelli T, Dale I, Logullo AF, Waitzberg DL. HER2-associated lipogenic phenotype as a potential therapeutical target in breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-29.

Abstract P1-08-13: The clinical importance of nuclear wild-type p53-induced phosphatase 1(Wip1) expression in breast cancer patients

Abstract Backgrounds; The wild-type p53-induced phosphatase 1(Wip1) is a member of the serine/threonine protein phosphatases, and plays an important role in the nucleus as one of the key components in the DNA damage response network. Wip1 is encoded by the protein phosphatase magnesium dependent 1 delta (PPM1D), sited on locus 17q23. PPM1D gene amplification and/or Wip1 expression have been observed in numerous tumors, including breast cancer. PPM1D is referred to as oncogene, as Wip1 inhibits phosphorylation of p53 and work as a negative regulator in cell death. Inhibition of Wip1 may have an important therapeutic role in suppressing tumor growth and evolution. Aims; We evaluated the expression of Wip1 mRNA, Wip1 protein and PPM1D DNA copy number to clarify the relationship between Wip1 expression and the clinicopathological features and prognosis to determine the biological significance of Wip1. Materials and Methods; Breast cancer cell lines (MCF7, T47D, MDA-MB231, HCC1937, HS578T, BT20 and SKBr3) were used for Wip1 expression analysis and copy number analysis. The specimens were obtained from Japanese breast cancer patients who underwent surgery without neoadjuvant chemotherapy or endocrine therapy in our department. Wip1 mRNA expression was evaluated in 140 cases by quantitative RT-PCR and Wip1 protein expression was evaluated in 192 cases by immunohistochemistry (IHC). The PPM1D DNA copy number was analyzed by genomic PCR in 33 breast cancer cases and by SNP-CGH array (Illumina, Human Omni 2.5-8) in 12 cases. The effects on the cell growth of the Wip1 inhibitor (GSK2830371) were analyzed by the viability assay in MCF7. Results; Wip1 mRNA expression was significantly higher in MCF7, luminal type cell line. There was no significant correlation between Wip1 mRNA expression and prognosis. In IHC, positive nuclear Wip1 protein expression was detected in 21 cases (10.9%). There was no significant correlation between Wip1 mRNA expression and Wip1 protein expression. There was no significant association between the Wip1 protein expression levels and the clinicopathological factors and the prognosis. PPM1D DNA copy number significantly correlated with Wip1 protein expression (p=0.0035). Copy number gain at 17q23 was detected in 6 cases by SNP-CGH array, and all of these six cases showed positive nuclear Wip1 expression. PPM1D copy number gain was not observed in Wip1 negative cases. In the cell viability assay, the MCF7 cell growth was suppressed by Wip1 inhibitor administration. Conclusions;Wip1 protein expression in nucleus is important as oncogene, and its expression may be regulated by PPM1D copy number gain. Wip1 is considered to be the new therapeutic target for breast cancer patients. Citation Format: Inoue Y, Yamashita N, Tokunaga E, Kitao H, Tanaka K, Saeki H, Oki E, Maehara Y. The clinical importance of nuclear wild-type p53-induced phosphatase 1(Wip1) expression in breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-13.

Clinicopathological characteristics and prognostic value of cancer stem cell marker CD133 in breast cancer: a meta-analysis.

BackgroundThe association of CD133 overexpression with clinicopathological significance and prognosis in patients with breast cancer remains controversial. We thus performed a meta-analysis to evaluate the role of CD133 expression in the development and prognosis of breast cancer.MethodsThe databases PubMed, Embase, and Cochrane Library (updated to August 1, 2016) were searched. Pooled odds ratios (ORs) or hazard ratios (HRs) with 95% confidence intervals (95% CI) were used to evaluate the impact of CD133 expression on clinicopathological features, overall survival, and disease-free survival.ResultsA total of 1,734 patients from 13 studies were subject to final analysis. The results showed a significant association between overexpression of CD133 and estrogen receptor status (OR 0.35, 95% CI 0.18–0.70), progesterone receptor status (OR 0.56, 95% CI 0.43–0.74), human epidermal growth factor-2 status (OR 1.81, 95% CI 1.33–2.45), lymph node metastasis (OR 1.98, 95% CI 1.34–2.92), and tumor histological grade (OR 1.79, 95% CI 1.26–2.54) in breast cancer. However, no significant correlation was found between upregulation of CD133 expression and onset age (OR 1.03, 95% CI 0.70–1.53) or tumor size (OR 1.29, 95% CI 0.80–2.09). Moreover, CD133-positive breast cancer patients had a higher risk of mortality (HR 1.91, 95% CI 1.21–3.03) and disease progression (HR 2.70, 95% CI 1.05–6.95).ConclusionThis meta-analysis suggested that CD133 might be a predictor of clinical outcomes as well as prognosis and could be a potentially new gene therapy target for breast cancer patients.

Prognostic value of reduced E-cadherin expression in breast cancer: a meta-analysis.

The prognostic value of E-cadherin expression in patients with breast cancer has been studied for years, yet results remain controversial. We thus performed a comprehensive evaluation of the association between E-cadherin expression and prognosis through a meta-analysis. The databases PubMed, Embase and Cochrane Library were searched. A total of 7,353 patients from 33 studies were subject to final analysis. The results showed there was a significant association between reduced expression of E-cadherin and overall survival (OS) (HR 1.79, 95% CI 1.41–2.27) and disease-free survival (DFS) (HR 1.62, 95% CI 1.31–1.99) in breast cancer. Downregulated expression of E-cadherin significantly correlated with tumor histological grade (OR 1.44, 95% CI 1.06–1.96), TNM stage (OR 2.44, 95% CI 1.75–3.41), tumor size (OR 1.38, 95% CI 1.18–1.60), lymph node status (OR 1.55, 95% CI 1.15–2.10), and progesterone receptor status (OR 1.44, 95% CI 1.10–1.88).This meta-analysis suggested that reduced E-cadherin expression might be a predictor of a poorer prognosis and could be a potentially new gene therapy target for breast cancer patients.

Overexpression of PTK6 predicts poor prognosis in bladder cancer patients.

Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase and works as an oncogene in various cancers. Recently, PTK6 has been used as a therapeutic target for breast cancer patients in a clinical study. However, the prognostic value of PTK6 in bladder cancer (BC) remains vague. Therefore, we retrieved 3 independent investigations of Oncomine database and found that PTK6 is highly expressed in BC tissues compared with corresponding normal controls. Similar results were also observed in clinical specimens at both mRNA and protein levels. Immunohistochemical analysis indicated that PTK6 overexpression was highly related to the T classification, N classification, grade, recurrence, and poor prognosis of BC patients. Furthermore, we demonstrated that when PTK6 expression was knocked down by siRNAs, cell proliferation and migration were considerably inhibited in BC cell lines T24 and EJ. By these approaches, we are intended to elucidate PTK6 may be a reliable therapeutic target in BC and might benefit from PTK6 inhibitors in the future.

Abstract 3483: ESR1 gene expression and expression ratio of ESR1 to ESR2 are associated with worse prognosis in papillary thyroid carcinoma

Abstract Introduction Expression of estrogen receptor (ER) and progesterone receptor (PR) in breast cancer have been widely studied and used as prognostic markers or therapeutic targets in breast cancer patients. However, clinical and pathologic roles of these hormone receptor genes have not been clearly established in thyroid cancer. Materials and methods To evaluate the relationship between sex hormone receptor gene expressions and clinicopathologic variables, we analyzed ESR1, ESR2, ESR ratio (ESR1/ESR2) and PGR mRNA expression data from The Cancer Genome Atlas (TCGA) RNA sequencing experiment in papillary thyroid carcinoma (PTC). To validate results from TCGA analysis, we additionally analyzed a public microarray data (GSE 54598) from the gene expression omnibus (GEO) database. Results In TCGA data analysis, ESR1, ESR ratio and PGR expression were significantly higher in PTC tissue than in normal thyroid tissue (Mean value of normal versus tumor were 264.045 vs 659.427 in ESR1, 19.064 vs 92,017 in ESR ratio and 58.514 vs 119.792 in PGR, respectively). In female patients, ESR1 and ESR ratio was negatively correlated with advanced age (t-value was -2.257 and -2.949 in linear regression analysis). Expression of ESR1 and ESR ratio were higher in classic type PTC, presentation of lymphovascular invasion and BRAF V600E mutation. Higher expression group of ESR1 and ESR ratio showed worse overall survival in female PTC patients (Hazard ratio was 6.348 in ESR1 and 4.031 in ESR ratio, respectively). In microarray analysis, ESR1 and ESR ratio expressions were higher in tumor than normal, classic than follicular variant and BRAF V600E than wild type. Conclusion Expression of ESR1 and ESR ratio were associated with aggressive prognostic factors and worse overall survival in female PTC patients. These results suggest that higher expression of ESR1 and ESR ratio can be used as prognostic markers to predict survival in female patients. Citation Format: Jin Wook Yi, Ra-Yeong Song, Hyeong Won Yu, Joon-Hyop Lee, Hyungju Kwon, Young Jun Chai, June Young Choi, Su-jin Kim, Kyu-Eun Lee. ESR1 gene expression and expression ratio of ESR1 to ESR2 are associated with worse prognosis in papillary thyroid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3483.

The novel mTORC1/2 dual inhibitor INK128 enhances radiosensitivity of breast cancer cell line MCF-7.

mTOR, a member of the PIKK family, is crucial for cell growth, survival, motility, proliferation, protein synthesis and DNA transcription. Many studies have demonstrated that mTOR inhibitor could enhance radiosensitivity. However, the effect of the novel mTORC1/2 dual inhibitor, INK128, on the radiosensitivity of breast cancer and the underlying mechanisms are still vague. In the present study, the cell viability was estimated using CCK-8 assay, and the dose-survival relationship was analyzed using a clonogenic survival assay. Cell cycle was evaluated by flow cytometry. The staining of γH2AX foci was assessed by immunofluorescence. In addition, we used western blots to verify the downregulating signal protein and to detect the potential related pathway. We found that the exposure of MCF-7 cells to INK128 decreased the cell viability. Exposure of MCF-7 cells to INK128 and combined ionizing radiation greatly reduced the survival rate. INK128 combined radiotherapy significantly induced G2/M arrest, double strand breaks and inhibited its repair. Furthermore, INK128 plus radiation downregulated p-Chk2, p21 and upregulated cleaved PARP, LC3B expression. These findings suggest that mTOR inhibitor could be used as a novel radiosensitizing target for breast cancer patients.

A negative feedback loop between miR-200b and the nuclear factor-κB pathway via IKBKB/IKK-β in breast cancer cells.

MicroRNAs (miRNAs) act as important post-transcriptional regulators of gene expression in diverse signalling pathways. However, the relationship between miR-200b and the nuclear factor-κB (NF-κB) signalling pathway remains poorly understood in breast cancer cells. In the current study, we show that IKBKB is a direct target of miR-200b, and that miR-200b downregulates IKBKB expression via directly binding to its 3'-UTR. miR-200b inhibits IκBα phosphorylation, nuclear p50/p65 expression, NF-κB-binding activity, and the translocation of p65 to the nucleus. In addition, miR-200b also suppresses tumour necrosis factor (TNF)-α-induced NF-κB activation and the expression of NF-κB target genes. Importantly, IKBKB overexpression attenuates the inhibitory roles of miR-200b in NF-κB expression, NF-κB-binding activity, and the nuclear translocation of p65. We also show that NF-κB p65 knockdown reduces the binding of NF-κB to the miR-200b promoter and miR-200b promoter activity. Furthermore, p65 knockdown or inhibition of IκBα phosphorylation suppresses miR-200b expression. Finally, functional studies show that IKBKB overexpression can restore the cell growth and migration that are suppressed by miR-200b. In conclusion, our results demonstrate that miR-200b, a transcriptional target of NF-κB, suppresses breast cancer cell growth and migration, and NF-κB activation, through downregulation of IKBKB, indicating that miR-200b has potential as a therapeutic target in breast cancer patients.

Abstract B205: EphA10 targeted antibody therapy is a potential as a novel option for management of breast cancers including triple negative breast cancer

Abstract Introduction: Triple negative breast cancer (TNBC) is defined by the absence of receptors for Her2, estrogen and progesterone and is often referred to as a refractory disease, because it is resistant to treatment with anti-Her2 antibody and anti-hormone drugs. Thus, new targets for therapeutic agents are urgently required. Using a proteomic approach, we have identified EphA10, which is more highly expressed in breast cancer, including TNBC, than in normal breast tissues. Moreover, we previously revealed that EphA10 is expressed in testis, but not in other normal tissues. This suggests that EphA10 could become a promising therapeutic target in breast cancer patients. Here, we describe a novel monoclonal antibody (mAb) against the extracellular region of EphA10 showing tumor accumulation and anti-tumor effects against EphA10-positive breast cancer. Materials and Methods: Tumor accumulation analysis of anti-EphA10 mAb; Alexa647-conjugated anti-EphA10 mAb or control mAb were administrated intravenously and observed daily using an in vivo imaging system. Tumor accumulation was quantified as the fluorescence intensity ratio of the tumor compared to that of the contralateral side. Anti-EphA10 mAb treatment in a mouse xenograft model; A mouse model was constructed by orthotopic transplantation of EphA10 stably expressing MDA-MB435 cells. When the tumor size reached approximately 100 mm3, anti-EphA10 mAb and control mAb were administrated intraperitoneally twice a week. Results and Discussion: Anti-EphA10 mAb we have developed by hybridoma methods displayed an affinity against EphA10 in the nanomolar range and specificity for EphA family proteins. Administration of the fluorescein-labeled mAb to EphA10-positive breast tumor-bearing mice showed that the mAb specifically accumulated in tumors, but not in normal organs. Furthermore, tumor growth was significantly suppressed in the mAb-treated mice in a dose-dependent manner. These data indicate that EphA10 targeted antibody therapy have a potential as a novel option for management of EphA10-positive breast cancer. We are currently generating human anti-EphA10 mAb by phage human antibody library method toward the clinical application. Conclusion: EphA10 targeted antibody therapy is effective for a mouse xenograft model. We hope that these findings will contribute to the development of a novel drug for refractory breast cancer patients. Citation Format: Mori Takahide, Nagano Kazuya, Shin-ichi Tsunoda, Yasuo Tsutsumi. EphA10 targeted antibody therapy is a potential as a novel option for management of breast cancers including triple negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B205.

Clinical and prognostic relevance of EZH2 in breast cancer: A meta-analysis.

The polycomb group protein enhancer of zeste homolog 2 (EZH2) is regarded as a tightly linking oncogene in many types of cancer. However, the prognostic role of EZH2 in breast cancer (BC) still remains controversial. Our study aimed to evaluate the clinical and prognostic relevance of EZH2 in BC patients based on published studies. 11 studies totally containing 2330 patients (1052 EZH2-positive and 1278 EZH2-negative) were included in our meta-analysis. Our data showed that EZH2 over-expression was significantly associated with estrogen receptor (ER) negativity [OR=0.227, 95% CI=0.174-0.297, P=0.000], progesterone receptor (PR) negativity [OR=0.454, 95% CI=0.300-0.687, P=0.000], human epidermal growth factor receptor type 2 (HER-2) positivity [OR=1.846, 95% CI=1.366-2.496, P=0.000], invasive ductal cancer (IDC) [OR=2.237, 95% CI=1.489-3.361, P=0.000], race (Caucasian) [OR=0.707, 95% CI=0.522-0.957, P=0.025], high histological grade [OR=3.177, 95% CI=2.012-5.014, P=0.000] and triple-negative status (TNBCs) [OR=5.380, 95% CI=1.065-27.187, P=0.042], which led to a poor OS rate in BC [RR=2.193, 95% CI=1.495-3.217, P=0.000]. In conclusion, EZH2 participated in the progression of BC as a putative factor, and over-expression of EZH2 was distinctly correlated with a poor patient survival. EZH2 may serve as a prognostic biomarker and target in BC patients.

Abstract 4377: Biodistribution and therapeutic effects of a monoclonal antibody against Eph receptor A10 in a breast cancer xenograft model

Abstract Introduction HER2-targeting antibodies and anti-hormone drugs are effective agents for breast cancer patients. However, such treatments are not applicable to triple negative breast cancers (TNBCs) which don't express HER2, estrogen, and progesterone receptors. Thus, effective molecular targets for TNBC are urgently needed. In this respect, we searched for new targets using a proteomics approach and identified Eph receptor A10 (EphA10) to be highly expressed in breast cancer including TNBCs, compared with normal breast tissues. Moreover, we previously found that EphA10 is expressed in testis but not in other normal tissues, thus suggesting that EphA10 could become a promising therapeutic target in breast cancer patients. Here, we developed a novel monoclonal antibody (mAb) against EphA10 and showed that it accumulates in the tumor and has anti-tumor properties in a breast cancer xenograft model. Materials and Methods Characterization of the anti-EphA10 mAb; Specificity of the mAb was evaluated by mAb binding to EphA family proteins (EphA1-A8, A10). Affinity of the mAb was analyzed by surface plasmon resonance method. Tumor accumulation analysis of anti-EphA10 mAb; Alexa647-conjugated anti-EphA10 mAb or control mAb were administrated intravenously and observed daily using an in vivo imaging system. Anti-EphA10 mAb treatment in a mouse xenograft model; A mouse model was generated by orthotopic transplantation of EphA10 expressing MDA-MB-435 (TNBC) cells. When the tumor size reached approximately a volume of 100 mm3, anti-EphA10 mAb and control mAb were administrated intraperitoneally twice a week. Results and Discussion The results showed that the anti-EphA10 mAb had an affinity for EphA10 in the nanomolar range. Moreover, the mAb specifically bound to EphA10 but not to the other EphA family proteins; therefore, our mAb had the same specificity and affinity for EphA10 as other existing antibody drugs. Furthermore, the mAb also displayed cross-reactivity with mouse EphA10, suggesting that we could evaluate the mAb in a mouse model. The administration of the fluorescein-labeled mAb to the mice bearing EphA10-positive breast tumors showed that the anti-EphA10 mAb accumulated in the tumors to a greater extent than the control mAb and tended to be restricted to the tumor. Furthermore, tumor growth was significantly suppressed in the mAb-treated mice in a dose-dependent manner. On the other hand, body weight and biochemical markers of tissue damage did not change between the mAb or control mAb-treated groups. These data indicate that our mAb might become a promising therapeutic tool for treating EphA10-positive breast cancers. Conclusion Our anti-EphA10 mAb mainly accumulated in EphA10-positive tumor cells and showed anti-tumor effects. We believe that these findings will contribute to the development of a novel drug in the treatment of breast cancer patients refractory to traditional therapies. Citation Format: Kazuya Nagano, Yuka Maeda, Takuya Yamashita, Yohei Mukai, Haruhiko Kamada, Kazuma Higashisaka, Yasuo Yoshioka, Yasuo Tsutsumi, Shin-ichi Tsunoda. Biodistribution and therapeutic effects of a monoclonal antibody against Eph receptor A10 in a breast cancer xenograft model. [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 4377. doi:10.1158/1538-7445.AM2015-4377

Downregulation of the long noncoding RNA EGOT correlates with malignant status and poor prognosis in breast cancer.

Eosinophil granule ontogeny transcript (EGOT) is a long noncoding RNA involved in the regulation of eosinophil granule protein transcript expression. However, little is known about the role of EGOT in malignant disease. This study aimed to assess the potential role of EGOT in the pathogenesis of breast cancer. Quantitative real-time polymerase chain reaction was performed to detect the expression levels of EGOT in 250 breast cancerous tissues and 50 adjacent noncancerous tissues. The correlation of EGOT expression with clinicopathological features and prognosis was also analyzed. EGOT expression was lower in breast cancer compared with the adjacent noncancerous tissues (P < 0.001), and low levels of EGOT expression were significantly correlated with larger tumor size (P = 0.022), more lymph node metastasis (P = 0.020), and higher Ki-67 expression (P = 0.017). Moreover, patients with low levels of EGOT expression showed significantly worse prognosis for overall survival (P = 0.040), and this result was further validated in a larger cohort from a public database. Multivariate analysis suggested that low levels of EGOT were a poor independent prognostic predictor for breast cancer patients (HR = 1.857, 95 % CI = 1.032-3.340, P = 0.039). In conclusion, EGOT may play an important role in breast cancer progression and prognosis and may serve as a new potential prognostic target in breast cancer patients.

Association of reduced XRCC2 expression with lymph node metastasis in breast cancer tissues.

The main purpose of this study was to evaluate the association between reduction in XRCC2 gene and involvement of lymph node metastasis in breast cancer. In first part of the study, meta-analysis of 14 published XRCC2 studies was performed to define the role of XRCC2 gene as diagnostic marker and in second part of the study XRCC2 gene expression was observed using real time PCR in study cohort of 100 females (50 breast cancer patients and 50 controls). A statistically significant down regulation of XRCC2 (p < 0.04) and up-regulation of ki-67 (p < 0.05) was observed in breast cancer tissues compared to non-cancerous healthy tissues. In order to explore gene-gene and gene-clinicopathological parameters relationship Spearmen correlation was performed. We observed a significantly negative correlation between XRCC2 and Ki-67 expression (r = -0.376**, p < 0.01). In case of gene-clinicopathological parameters relationship, we observed a significant correlation between XRCC2 expression and lymph node status (r = -0.521***, p < 0.002) and metastatic status (r = -0.303*, p < 0.04) of breast cancer patients. Our data suggests that deregulation of XRCC2 in breast cancer has the potential to predict lymph node metastasis and may serve as a therapeutic target for breast cancer patients at risk of metastasis.