Breast Cancer Tissue Specimens Research Articles

Downregulation of Ras GTPase‑activating protein 1 is associated with poor survival of breast invasive ductal carcinoma patients.

Ras GTPase‑activating protein 1 (RASA1) functions to inactivate Ras‑GTPase and inhibit the mitogenic signal. Reduction or loss of RASA1 expression occurs during human cancer development and progression. This study investigated RASA1 expression in normal and breast cancer tissue specimens to determine the association with prognosis of breast cancer patients. Two sets of patient samples (45 fresh tissues and 373 paraffin‑embedded tissues) were analyzed for RASA1 expression using RT‑qPCR and immunohisto-chemistry. The results showed that the expression of RASA1 mRNA was lower in breast cancer tissues than in the corresponding normal tissues (P<0.001). Additionally, RASA1 expression was reduced in 60.6% (226/373) of breast cancer tissues. The reduced RASA1 expression was significantly associated with tumor lymph node metastasis (P=0.002), advanced TNM stages (P=0.017), estrogen receptor (ER) expression (P=0.002), Ki‑67 (P=0.009), higher histological grade (P<0.001), and triple‑negative breast cancer (P=0.041). Moreover, the reduced RASA1 expression was associated with shorter disease‑free survival (P=0.036) and overall survival (P<0.001) of breast cancer patients. RASA1 expression, together with tumor lymph‑node metastasis, TNM stage, Her‑2 expression, and triple‑negative breast cancer were independent factors in predicting survival of breast cancer patients. In conclusion, RASA1 expression is frequently reduced in breast cancer tissues, and the reduced RASA1 expression is associated with breast cancer progression and poor survival and disease‑free survival of patients.

Abstract 1972: The sodium channel auxiliary subunit SCN1B promotes breast tumor growth and metastasis

Abstract Voltage-gated Na+ channels (VGSCs) are heteromeric proteins composed of pore-forming α subunits and smaller β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). β1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. β1 is also expressed in breast cancer cell lines, where it regulates adhesion and migration in vitro. Using the Oncomine database, we found that SCN1B mRNA was up-regulated in breast cancer, compared with normal breast. Similarly, β1 protein was up-regulated in a cohort of breast cancer tissue specimens compared with surrounding normal tissue. Over-expression of β1 in MDA-MB-231 cells substantially increased tumor growth and metastasis from orthotopic xenografts in immune-deficient mice. β1 had no effect on the density of Ki67-positive cycling cells in the primary tumors. However, β1 increased the density of CD31-positive vessel structures and reduced the density of apoptotic cells expressing activated caspase-3. β1-expressing MDA-MB-231 cells had an elongate morphology in tumors in vivo and in 2D culture in vitro. β1 potentiated the outgrowth of processes from MDA-MB-231 cells and MCF-7 cells co-cultured with fibroblasts, via trans-homophilic adhesion. Both the src family tyrosine kinase inhibitor PP2 and siRNA targeting fyn kinase blocked β1-mediated process extension in breast cancer cells. Thus, β1-mediated process outgrowth in breast cancer cells requires the presence and activity of fyn kinase. This recapitulates the mechanism by which β1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumors, β1 drives pathological growth and cellular dissemination by recapitulating its well-defined role in CNS ontogeny. This study is the first demonstration of a functional role for β1 as a CAM in tumor growth and metastasis. We propose that targeting β1-mediated adhesion interactions may have potential as a novel anti-cancer therapy. Citation Format: Michaela Nelson, Rebecca Millican-Slater, Lorna C. Forrest, William J. Brackenbury. The sodium channel auxiliary subunit SCN1B promotes breast tumor growth and metastasis. [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 1972. doi:10.1158/1538-7445.AM2014-1972

Abstract 3908: Focal adhesion kinase (FAK) inhibitor VS-6063 (defactinib) preferentially targets cancer stem cells in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is defined phenotypically as the lack of ER, PR and HER2 expression, and comprises a heterogeneous group of breast cancers. Cancer stem cells (CSCs), identified as either ALDH1+ or CD44hi/CD24lo subpopulations in breast cancer, have been shown to be resistant to standard chemotherapy and associated with poor clinical outcome. In recent reports, the presence of ALDH1+ (but not ALDH1-) cells in axillary lymph nodes following neo-adjuvant chemotherapy was associated with poor overall survival. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors. In addition to regulating the proliferation, survival, invasion and metastasis of cancer cells, FAK also plays a critical role in the self-renewal and survival of CSCs. VS-6063 (defactinib) is a potent, selective, and orally bioavailable FAK inhibitor with demonstrated tolerability and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in phase I clinical trials. We report here that VS-6063 preferentially targets CSCs in preclinical models of TNBC. The effect of VS-6063 on CSCs was assessed in a panel of orthogonal assays. Treatment of human TNBC cells with VS-6063 in 3D matrigel reduced the percentage of Aldefluor+ CSCs, Hoechst dye-excluding side population (SP) CSCs, and tumorsphere-forming efficiency in a serial tumorsphere passaging assay, suggesting that VS-6063 preferentially inhibits CSCs. VS-6063 also reduced the proportion of Aldefluor+ CSCs in primary breast cancer tissue specimens cultured ex vivo. Consistent with the concept that the effect of VS-6063 on CSCs is mediated by FAK inhibition, siRNA-mediated knockdown of FAK in SUM159 TNBC cells recapitulated the inhibitory effect of VS-6063 on CSCs. In contrast, the standard-of-care (SoC) chemotherapeutic agents paclitaxel and doxorubicin enriched the percentage of CSCs, suggesting that these SoC agents preferentially target bulk tumor cells relative to CSCs. When administered in combination, VS-6063 attenuated the chemotherapy-induced enrichment of CSCs. Following oral administration of VS-6063 in the MDA-MB-231 human TNBC xenograft model in vivo, reduction of CSCs in tumors was evidenced by a decrease in secondary tumorsphere-forming efficiency. Importantly, cells dissociated from VS-6063-treated tumors displayed reduced tumor-initiating capability upon re-implantation into immunodeficient mice in limiting dilutions. In summary, our results indicate that the FAK inhibitor VS-6063 preferentially targets TNBC CSCs and support the clinical development of VS-6063 in combination with SoC agents, such as paclitaxel, to potentially improve clinical outcomes for patients with TNBC through simultaneous targeting of both CSCs and bulk tumor cells. Citation Format: Qunli Xu, Vihren N. Kolev, Quentin G. Wright, Jennifer E. Ring, Christian M. Vidal, Irina M. Shapiro, David T. Weaver, Mahesh V. Padval, Jonathan A. Pachter. Focal adhesion kinase (FAK) inhibitor VS-6063 (defactinib) preferentially targets cancer stem cells 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 3908. doi:10.1158/1538-7445.AM2014-3908

B-cell lymphoma 6 protein stimulates oncogenicity of human breast cancer cells.

BackgroundB-cell lymphoma 6 (BCL6) protein, an evolutionarily conserved zinc finger transcription factor, showed to be highly expressed in various human cancers in addition to malignancies in the lymphoid system. This study investigated the role of BCL6 expression in breast cancer and its clinical significance in breast cancer patients.MethodsExpression of BCL6 protein was assessed using in situ hybridization and immunohistochemistry in 127 breast cancer patients and 50 patients with breast benign disease as well as in breast cell lines. Expression of BCL6 was restored or knocked down in two breast cancer cell lines (MCF-7 and T47D) using BCL6 cDNA and siRNA, respectively. The phenotypic change of these breast cancer cell lines was assessed using cell viability MTT, Transwell invasion, colony formation, and flow cytometry assays and in a xenograft mice model. Luciferase reporter gene, immunoblot, and qRT-PCR were used to investigate the molecular events after manipulated BCL6 expression in breast cancer cells.ResultsBCL6 protein was highly expressed in breast cancer cell lines and tissue specimens and expression of BCL6 protein was associated with disease progression and poor survival of breast cancer patients. In vitro, the forced expression of BCL6 results in increased proliferation, anchorage-independent growth, migration, invasion and survival of breast cancer cell lines, whereas knockdown of BCL6 expression reduced these oncogenic properties of breast cancer cells. Moreover, forced expression of BCL6 increased tumor growth and invasiveness in a nude mouse xenograft model. At the gene level, BCL6 was a target gene of miR-339-5p. Expression of BCL6 induced expression of CXCR4 and cyclinD1 proteins.ConclusionsThe current study demonstrated the oncogenic property of BCL6 in breast cancer and further study could target BCL6 as a novel potential therapeutic strategy for breast cancer.

Increased MTHFD2 expression is associated with poor prognosis in breast cancer.

The aim of this study was to investigate the expression levels of methylenetetrahydrofolate dehydrogenase (NADP + -dependent) 2 (MTHFD2) and the associated clinical implications in breast cancer. MTHFD2 expression was measured by Western blot and immunohistochemistry in 698 tissue sections taken from breast cancer patients. The relationship between MTHFD2 expression, clinicopathological parameters, and the prognosis of breast cancer was subsequently determined. In comparison with para-carcinoma tissue specimens, an enhanced expression of MTHFD2 was observed in breast cancer tissue specimens (P < 0.05). In total, 41.12 % (287/698) of breast cancer tissue specimens had high levels of MTHFD2. After universal and Spearman regression correlation analyses, MTHFD2 expression was found to correlate with tumor size, histological grade, lymph node metastasis, and distant metastases (P = 0.001, 0.002, 0.001, and 0.001, respectively). Furthermore, patients with MTHFD2-expressing tumors had a significantly poorer prognosis than those with no or low MTHFD2 expression. (P = 0.002). Using the Cox regression test, MTHFD2 was identified as an independent prognostic factor (P = 0.001). MTHFD2 was differentially expressed in breast cancer tissue. Therefore, this protein may be an independent prognostic factor and a potential therapeutic target for future breast cancer treatments.

Expression of ATP-binding cassette superfamily G member 2 and β-tubulin-III in human breast carcinomas and clinical significance

Objective To investigate the correlations of ATP-binding cassette superfamily G mem-ber 2 (ABCG2) and β-tubulin-Ⅲ (TUBB3)mRNA expressions with formation and development of breast carcinomas.Methods 196 cases of invasive breast cancer tissue specimens and specimens with normal breast tissue were collected.Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expressions of ABCG2 and TUBB3 gene.The relationship between gene expression and clinicopatholog-ical features was analyzed.Results The ABCG2 gene expression rate in 196 samples of invasive breast cancer was 58.7％ and the electrophoretic bands grayscale was 0.685 ± 0.126,and those in adjacent normal breast tissue were 30.1％ and 0.271 ±0.143,respectively (P ＜0.05).The TUBB3 gene expression rate was 67.9％ and electrophoretic bands grayscale was 0.793 ± 0.115 in the samples of invasive breast cancer,and those in adjacent normal breast tissue were 23.0％ and 0.463 ± 0.127,respectively (P ＜0.05).ABCG2 gene expression in tumor tissue was correlated with estrogen receptor (ER) and progester-one receptor (PR) (P ＜ 0.05),but not with human epidermal growth factor receptor-2 (Her-2),clinical stage,vascular invasion,lymph node metastasis,tumor size and proliferation cell nuclear antigen (Ki-67)(P ＞0.05).The TUBB3 expression in tumor tissue was correlated with axillary lymph node metastasis,vascular invasion,tumor size,histological grade,clinical stage,Ki-67 and Her-2 (P ＜ 0.05),but not with ER and PR (P ＞ 0.05).Conclusion The abnormal expression of ABCG2 and TUBB3 genes may play important roles in drug resistance and development of breast cancer. Key words: Breast carcinomas ; Resistance gene ; ATP-binding cassette superfamily G member 2 ; Molecular typing

MiR-99a Antitumor Activity in Human Breast Cancer Cells through Targeting of mTOR Expression

MicroRNAs (miRNAs) play an important role in human tumorigenesis as oncogenes or tumor suppressors. miR-99a has been reported as a tumor suppressor gene in various cancers in humans. However, only limited information about the function of miR-99a in human breast cancers is available. Here we investigated the expression of miR-99a in breast cancer tissue specimens and its antitumor activity in breast cancer cells. We initially identified that the expression of miR-99a was significantly reduced in four breast cancer cell lines. More importantly, we found downregulation of miR-99a in breast cancer specimens from ten different patients. We then analyzed the mechanism of miR-99a in inhibiting tumorigenesis. Cell-based assays that showed overexpression of miR-99a not only reduced breast cancer cell viability by inducing accumulation of cells at sub-G1 phase and cell apoptosis, but also inhibited tumorigenicity in vivo. As a critical miR-99a target, we have shown that the function of mammalian target of rapamycin (mTOR) was greatly inhibited by miR-99a-based Luciferase report assay; overexpression of miR-99a reduced the expression of mTOR and its downstream phosphorylated proteins (p-4E-BP1 and p-S6K1). Similar to restoring miR-99a expression, mTOR downregulation suppressed cell viability and increased cell apoptosis, whereas restoration of mTOR expression significantly reversed the inhibitory effects of miR-99a on the mTOR/p-4E-BP1/p-S6K1 signal pathway and the miR-99a antitumor activity. In clinical specimens and cell lines, mTOR was commonly overexpressed and its protein levels were statistically inversely correlated with miR-99a expression. Taken together, these results have demonstrated that miR-99a antitumor activity is achieved by targeting the mTOR/p-4E-BP1/p-S6K1 pathway in human breast cancer cells. This study suggests a potential therapeutic strategy to effectively control breast cancer development.

NF-κB Activation-Induced Anti-apoptosis Renders HER2-Positive Cells Drug Resistant and Accelerates Tumor Growth

Breast cancers with HER2 overexpression are sensitive to drugs targeting the receptor or its kinase activity. HER2-targeting drugs are initially effective against HER2-positive breast cancer, but resistance inevitably occurs. We previously found that NF-κB is hyperactivated in a subset of HER2-positive breast cancer cells and tissue specimens. In this study, we report that constitutively active NF-κB rendered HER2-positive cancer cells resistant to anti-HER2 drugs and cells selected for lapatinib resistance upregulated NF-κB. In both circumstances, cells were antiapoptotic and grew rapidly as xenografts. Lapatinib-resistant cells were refractory to HER2 and NF-κB inhibitors alone but were sensitive to their combination, suggesting a novel therapeutic strategy. A subset of NF-κB-responsive genes was overexpressed in HER2-positive and triple-negative breast cancers, and patients with this NF-κB signature had poor clinical outcome. Anti-HER2 drug resistance may be a consequence of NF-κB activation, and selection for resistance results in NF-κB activation, suggesting that this transcription factor is central to oncogenesis and drug resistance. Clinically, the combined targeting of HER2 and NF-κB suggests a potential treatment paradigm for patients who relapse after anti-HER2 therapy. Patients with these cancers may be treated by simultaneously suppressing HER2 signaling and NF-κB activation. The combination of an inhibitor of IκB kinase (IKK) inhibitor and anti-HER2 drugs may be a novel treatment strategy for drug-resistant human breast cancers.

Significance of ERβ expression in different molecular subtypes of breast cancer

PurposeThis study is to investigate the estrogen receptor β (ERβ) expression in molecular subtypes of breast cancer and clinic significance of ERβ expression.MethodThe ERβ expression was detected in 730 cases of breast cancer tissue specimens by immunohistochemistry. Twenty-one patients were censored during 2–10 years follow-up. The difference in ERβ expression was analyzed by Pearson Chi-square Test. Its correlation with estrogen receptor α (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her-2) was analyzed by Spearman rank correlation. The accumulative tumor-free survival rate was calculated by Kaplan-Meier method and difference in survival rate was analyzed by Log-rank test. Cox regression was used for multi-factor analysis.ResultThe ERβ expression was significantly different among the molecular subtypes of breast cancer (P < 0.05). The ERβ expression in breast cancer was positively correlated with Her-2 (P < 0.05) while it had no correlation with ERα and Her-2. The expression of ERα was negatively correlated with Her-2 (P < 0.01) whereas positively correlated with PR (P < 0.01). The expression of PR was negatively correlated with Her-2 (P < 0.05). The tumor-free survival rate in patients with positive ERβ expression was significantly lower than that in patients with negative ERβ expression.ConclusionPositive ERβ expression is a poor prognostic factor of breast cancer.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1084557586106833

Chromatin-associated CSF-1R binds to the promoter of proliferation-related genes in breast cancer cells

The colony-stimulating factor-1 (CSF-1) and its receptor CSF-1R physiologically regulate the monocyte/macrophage system, trophoblast implantation and breast development. An abnormal CSF-1R expression has been documented in several human epithelial tumors, including breast carcinomas. We recently demonstrated that CSF-1/CSF-1R signaling drives proliferation of breast cancer cells via ‘classical' receptor tyrosine kinase signaling, including activation of the extracellular signal-regulated kinase 1/2. In this paper, we show that CSF-1R can also localize within the nucleus of breast cancer cells, either cell lines or tissue specimens, irrespectively of their intrinsic molecular subtype. We found that the majority of nuclear CSF-1R is located in the chromatin-bound subcellular compartment. Chromatin immunoprecipitation revealed that CSF-1R, once in the nucleus, binds to the promoters of the proliferation-related genes CCND1, c-JUN and c-MYC. CSF-1R also binds the promoter of its ligand CSF-1 and positively regulates CSF-1 expression. The existence of such a receptor/ligand regulatory loop is a novel aspect of CSF-1R signaling. Moreover, our results provided the first evidence of a novel localization site of CSF-1R in breast cancer cells, suggesting that CSF-1R could act as a transcriptional regulator on proliferation-related genes.

Analysis of HER2 status in breast carcinoma by fully automated HER2 fluorescence in situ hybridization (FISH): comparison of two immunohistochemical tests and manual FISH

Easy and accurate HER2 testing is essential when considering the prognostic and predictive significance of HER2 in breast cancer. The use of a fully automated, quantitative FISH assay would be helpful to detect HER2 amplification in breast cancer tissue specimens with reduced inter-laboratory variability. We compared the concordance of HER2 status as assessed by an automated FISH staining system to manual FISH testing. Using 60 formalin-fixed paraffin-embedded breast carcinoma specimens, we assessed HER2 immunoexpression with two antibodies (DAKO HercepTest and CB11). In addition, HER2 status was evaluated with automated FISH using the Leica FISH System for BOND and a manual FISH using the Abbott PathVysion DNA Probe Kit. All but one specimen were successfully stained using both FISH methods. When the data were divided into two groups according to HER2/CEP17 ratio, positive and negative, the results from both the automated and manual FISH techniques were identical for all 59 evaluable specimens. The HER2 and CEP17 copy numbers and HER2/CEP17 ratio showed great agreement between both FISH methods. The automated FISH technique was interpretable with signal intensity similar to those of the manual FISH technique. In contrast with manual FISH, the automated FISH technique showed well-preserved architecture due to low membrane digestion. HER2 immunohistochemistry and FISH results showed substantial significant agreement (κ=1.0, p<0.001). HER2 status can be reliably determined using a fully automated HER2 FISH system with high concordance to the well-established manual FISH method. Because of stable signal intensity and high staining quality, the automated FISH technique may be more appropriate than manual FISH for routine applications.

Prognostic significance of let-7b expression in breast cancer and correlation to its target gene of BSG expression

Let-7 microRNAs (miRNAs) are found in a wide range of species, and alterations of let-7 miRNA family member expression levels in humans are associated with various types of cancer. However, few researchers have reported alterations in let-7b levels in breast cancer (BC). Specifically, the use of altered let-7 expression as a prognostic biomarker is of particular interest and significance. The aim of this study was to investigate whether let-7b could be used as a biomarker of tumor progression and patient prognosis in BC and to determine the target gene of let-7b. We retrospectively analyzed the clinical pathological characteristics of 80 BC. We utilized digoxigenin-labeled locked nucleic acid-miRNA probes to detect let-7b expression in 80 BC and 22 benign breast disease (BBD) histologic specimens by in situ hybridization, and also detect the expression of BSG-a potential target gene of let-7b-by immunohistochemistry. We observed that the levels of let-7b expression in BBD were higher than in BC specimens (P < 0.05), indicating that let-7b could inhibit growth and facilitate differentiation of BBD. Also, loss of let-7b expression on BC tissue specimens raised the possibility that let-7b could play a crucial role in the pathogenesis of BC. Furthermore, let-7b expression in breast cancer patients was inversely associated with tumor lymph node metastasis (P = 0.001), patient overall survival (P = 0.027), relapse-free survival (P = 0.016), and BSG protein expression (P = 0.001). Breast cancer patients with low let-7b expression had poor prognoses, indicating let-7b might act as cancer suppressor gene in BC development and progression by inhibiting the expression of BSG.

Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

Expression of the breast cancer resistance protein and 5-fluorouracil resistance in clinical breast cancer tissue specimens

The breast cancer resistance protein (BCRP) is a recently characterized xenobiotic half-transporter protein that acts as an energy-dependent efflux pump and may be associated with the multidrug-resistant phenotype. The aim of this study was to determine the association between BCRP expression and 5-fluorouracil (5-FU) resistance in clinical breast cancer tissue specimens. The BCRP expression was investigated using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) by use of the Master SYBR-Green I reagent and immunohistochemistry (IHC) by use of the BXP-21 anti-BCRP monoclonal antibody in clinical breast cancer tissue specimens. Chemosensitivity to 5-FU for BCRP-positive clinical breast cancer tissue specimens was colorimetrically assessed with the cytotoxicity assay through methyl thiazolyl tetrazolium (MTT) reduction. A total of 37 BCRP-positive clinical breast cancer tissue specimens were identified with quantitative RT-PCR and IHC. There was a significant correlation in BCRP expression between the results of quantitative RT-PCR and IHC in the specimens. The fold resistance to 5-FU was 7–12 compared to sensitivity to paclitaxel as determined by the colorimetric assay through MTT reduction in the 37 specimens. Our study results indicated that 5-FU resistance may be mediated by BCRP expression in clinical breast cancer tissue specimens, which may help optimize the design of breast cancer clinical chemotherapy schemes in BCRP-positive specimens.

Upregulation of miR-195 increases the sensitivity of breast cancer cells to Adriamycin treatment through inhibition of Raf-1

Chemotherapy is an important option for the treatment of advanced breast cancer, but multidrug resistance is one of the major obstacles in the clinical control of breast cancer. The present study investigated the effects of the miR‑195-led gene pathway in the sensitization of breast cancer cells to treatment with the chemotherapeutic drug Adriamycin. Breast cancer cell lines and tissue specimens (obtained from chemotherapy-sensitive or resistant patients) as well as a normal breast cell line were used to assess expression of miR-195, Raf-1, Bcl-2 and P-glycoprotein mRNA and/or mRNA. miR-195 mimics, inhibitor and Raf-1 siRNA were used to transfect breast cancer MCF-7 and MCF-7/ADR cells (an Adriamycin-resistant MCF-7 subline) for cell viability, apoptosis and gene expression analysis. The data showed that miR-195 expression was low in breast cancer cells and multidrug-resistant breast cancer tissues, which was associated with reduced Raf-1 expression invitro and exvivo. Induction of miR-195 expression promoted tumor cell apoptosis and inhibited breast cancer cell viability, but induced the sensitivity of breast cancer cells to Adriamycin treatment and was associated with inhibition of Raf-1 expression in breast cancer cells. Moreover, knockdown of Raf-1 expression had similar effects of miR-195 mimics on breast cancer cells, both of which were able to suppress Bcl-2 and P-glycoprotein expression in breast cancer cells. The data from the current study demonstrated that expression of miR-195 was inversely associated with Raf-1 expression in breast cancer cell lines and tissue specimens, and that Raf-1 is the target gene of miR-195. Thus, expression of miR-195 or knockdown of Raf-1 can similarly reduce tumor cell survival but increase apoptosis through downregulation of Raf-1 and Bcl-2 and P-glycoprotein expression. In conclusion, this gene pathway mediated the sensitivity of breast cancer cells to Adriamycin treatment.

Abstract 4217: A real-time PCR assay for the detection of PIK3CA mutations in formalin-fixed paraffin embedded tissue (FFPET) specimens of breast cancer (BC).

Abstract Somatic mutations in the PIK3CA gene encoding the p110α catalytic subunit of the PI3K protein have been linked to human cancer and have been found to occur at a high frequency in BC (25-40%). Therapeutics that target key nodes in the PI3K signaling pathway are currently being developed and stratifying patients based on their PIK3CA mutation status is becoming more important. We describe the analytical performance of the cobas® PIK3CA Mutation Test (For Research Use Only), a multiplex allele-specific PCR assay designed to detect 17 mutations in exons 1, 4, 7, 9 and 20 in the PIK3CA gene in 3 PCR wells. The test detects ∼94% of known PIK3CA mutations found in BC. The amount of DNA required for the assay (150 ng) can typically be isolated from a single 5 μm FFPET section with automated test results generated within 8 hours. The analytical sensitivity of the test, defined as ≥95% mutation detection rate, was found to be ∼5% mutant allele in BC FFPET tumor sections representing 4 prevalent PIK3CA mutations (H1047R, E545K, E542K, and Q546K). Additionally, the test was able to detect all 17 mutations at ≤ 5% mutant allele in plasmid/gDNA blends. The test was also compared to 2X bi-directional Sanger sequencing using 110 BC FFPET specimens. The PCR test detected mutations in 50 specimens that were Sanger (+) (100% positive agreement). Moreover, the test detected mutations in 4 Sanger (-) specimens and detected a second mutation in 1 Sanger (+) specimen that was not detected by Sanger. All 5 of the PCR (+)/Sanger (-) were confirmed mutant by 454 sequencing. Sanger Sequencing Mutation Detected Mutation Not Detected Total cobas® PIK3CA Mutation Test Mutation Detected 50* 4 54 Mutation Not Detected 0 56 56 Total 50 60 110 *One specimen had an additional mutation. When a panel of BC FFPET specimens was repeatedly tested over multiple days by 2 operators using 2 instruments and 2 reagent lots, a correct call was obtained in 99.5% (191/192) of tests. The results demonstrate that the cobas® PIK3CA Mutation Test is a fast, accurate and reproducible assay that is more sensitive than Sanger sequencing for the detection of PIK3CA mutations in BC tumors. Citation Format: Peter Schlag, Corey Hoeppner, Anona Bristol, Namneet Kaur, Stephen Quashnick, Ramani Ravirala, Joyce Kramer, Minet Negash, Victoria Brophy, Sung Lee, Stephen Soviero. A real-time PCR assay for the detection of PIK3CA mutations in formalin-fixed paraffin embedded tissue (FFPET) specimens of breast cancer (BC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4217. doi:10.1158/1538-7445.AM2013-4217

The expression of the CEACAM19 gene, a novel member of the CEA family, is associated with breast cancer progression

Breast cancer (BC) continues to affect the lives of millions of women worldwide. Several members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) subfamily are involved in tumor progression. Notably, the CEACAM subfamily harbors the already established cancer biomarker CEA, as well as other potential molecular markers. CEACAM19, a recently identified gene belonging to CEACAM subfamily, was discovered and cloned by members of our research group. The present study analyzes, quantitatively, the expression of CEACAM19 and evaluates its clinical relevance in BC. Total RNA was extracted from 143 cancerous and 89 normal adjacent breast tissue specimens. Following reverse transcription, quantitative analysis of CEACAM19 mRNA expression levels was performed via real-time PCR and the comparative Ct (2-∆∆Ct) method. CEACAM19 expression and detailed clinicopathological data were used for extensive biostatistical analyses. CEACAM19 was found to be overexpressed in breast cancer tissue specimens compared to normal tissue counterparts (p=0.013). CEACAM19 mRNA expression status was also associated with clinicopathological features indicative of aggressive behavior and poor prognosis in BC, such as high tumor grade (p=0.031) and high Ki67 proliferative index (p=0.038). A significant negative association was documented between CEACAM19 expression and tumor ER status (p=0.018) as well as patients' menopausal state (p=0.016). Our results suggest that CEACAM19 mRNA expression represents a promising, novel and clinically useful tissue biomarker for breast cancer management.

Imatinib as a key inhibitor of the platelet‐derived growth factor receptor mediated expression of cell surface heparan sulfate proteoglycans and functional properties of breast cancer cells

Cell surface heparan sulfate proteoglycans (HSPGs), syndecans and glypicans, play crucial roles in the functional properties of cancer cells, such as proliferation, adhesion, migration and invasion. Platelet-derived growth factor (PDGF)/PDGF receptor (PDGF-R) mediated signaling, on the other hand, is highly associated with cancer progression. Specifically, PDGF-Rα and PDGF-Rβ expressions documented in breast cancer tissue specimens as well as breast cancer cell lines are correlated with tumor aggressiveness and metastasis. Imatinib (Glivec(®)) is a tyrosine kinase inhibitor specific for PDGF-Rs, c-ΚΙΤ and BCR-ABL. In this study we evaluated the effects of imatinib on the properties of breast cancer cells as well as on the expression of HSPGs in the presence and absence of PDGF-BB. These studies have been conducted in a panel of three breast cancer cell lines of low and high metastatic potential. Our results indicate that imatinib exerts a significant inhibitory effect on breast cancer cell proliferation, invasion and migration as well as on the cell surface expression of HSPGs even after exposure of PDGF. These effects depend on the aggressiveness of breast cancer cells and the type of HSPG. It is suggested that imatinib may be of potential therapeutic usefulness in breast cancer regimes.

Cytoplasmic localization of alteration/deficiency in activation 3 (ADA3) predicts poor clinical outcome in breast cancer patients

Transcriptional activation by estrogen receptor (ER) is a key step to breast oncogenesis. Given previous findings that ADA3 is a critical component of HAT complexes that regulate ER function and evidence that overexpression of other ER coactivators such as SRC-3 is associated with clinical outcomes in breast cancer, the current study was designed to assess the potential significance of ADA3 expression/localization in human breast cancer patients. In this study, we analyzed ADA3 expression in breast cancer tissue specimens and assessed the correlation of ADA3 staining with cancer progression and patient outcome. Tissue microarrays prepared from large series of breast cancer patients with long-term follow-ups were stained with anti-ADA3 monoclonal antibody using immunohistochemistry. Samples were analyzed for ADA3 expression followed by correlation with various clinicopathological parameters and patients’ outcomes. We report that breast cancer specimens show predominant nuclear, cytoplasmic, or mixed nuclear + cytoplasmic ADA3 staining patterns. Predominant nuclear ADA3 staining correlated with ER+ status. While predominant cytoplasmic ADA3 staining negatively correlated with ER+ status, but positively correlated with ErbB2, EGFR, and Ki67. Furthermore, a positive correlation of cytoplasmic ADA3 was observed with higher histological grade, mitotic counts, Nottingham Prognostic Index, and positive vascular invasion. Patients with nuclear ADA3 and ER positivity have better breast cancer specific survival and distant metastasis free survival. Significantly, cytoplasmic expression of ADA3 showed a strong positive association with reduced BCSS and DMFS in ErbB2+/EGFR+ patients. Although in multivariate analyses ADA3 expression was not an independent marker of survival, predominant nuclear ADA3 staining in breast cancer tissues correlates with ER+ expression and together serves as a marker of good prognosis, whereas predominant cytoplasmic ADA3 expression correlates with ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Thus, ADA3 cytoplasmic localization together with ErbB2+/EGFR+ status may serve as better prognostic marker than individual proteins to predict survival of patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-012-2363-3) contains supplementary material, which is available to authorized users.

Abstract P1-04-09: mTORC1 and Rictor expression in human breast cancer: correlations with clinicopathological parameters and disease outcome

Abstract BACKGROUND: mTOR (the mammalian target of rapamycin) plays a key role in regulation of cellular metabolism, growth, and proliferation. It forms two multi-protein complexes known as complex 1 (mTORC1) and 2 (mTORC2). Raptor and Rictor are the core proteins for mTORC1 and mTORC2 respectively. There is a growing body of evidence that mTORC1 is up-regulated in many cancers and plays a role in carcinogenesis. The aim of the study was to investigate the mRNA expression of mTOR and Rictor in human breast cancer and examine the relationship between their expression and clinicopathological parameters. METHODS: Specimens of breast cancer (BC) tissues (N = 150) and normal tissues (N = 31) underwent RNA extraction and reverse transcription. mTOR and Rictor transcript levels were determined using real-time quantitative PCR. Expression levels were analyzed against tumor size, grade, nodal involvement, TNM stage, and clinical outcome over a 10 year follow-up period. RESULTS: Significantly higher mRNA transcript levels of mTOR were found in the breast cancer specimens compared to normal glandular tissue (p = 0.0018). The expression of mTOR mRNA was demonstrated to increase with increasing NPI (53 for NPI1 to 219 for NPI3) and tumor grade (37 for grade 2 vs. 159 for grade 3, p = 0.047). mTOR expression was found to be higher in ductal tumors compared with non-ductal tumors (p = 0.0014). The patients who developed recurrent disease or died from breast cancer had higher expression levels than those who had been disease-free after a median follow-up period of 10 years (p = 0.17). Higher expression levels were significantly associated with worse overall survival (p = 0.01). In contrast, higher levels of Rictor mRNA expression were found in normal breast tissue, lower NPI stage (NPI1 vs. 2: p = 0.03) and lower tumor grade (grade 1 vs. grade 3: p = 0.01). Patients with higher Rictor expression had a significantly better overall (p = 0.037) and disease-free (p = 0.048) survival. CONCLUSIONS: mTOR expression was found to be significantly higher in BC specimens compared to normal breast tissue. Higher transcript levels were significantly associated with unfavorable pathological parameters and adverse clinical outcomes. The core protein of the less predominant mTORC2 was associated with favorable pathological parameters and clinical outcome. Taken together these observations are consistent with mTORC1 role in the anti-apoptosis pathway and suggest that selective inhibitors of mTORC1 are likely to be a more effective therapeutic strategy than dual inhibitors in human breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-09.