Primary Breast Cancer Tissues Research Articles

Risk of breast cancer recurrence associated with carbohydrate intake and tissue expression of IGFI receptor.

The insulin-like growth factor-I (IGFI) receptor is a potential target for breast cancer treatment and may be influenced by dietary intake. Nested, case-control study of 265 postmenopausal breast cancer survivors; primary breast cancer tissue was stained to determine IGFI receptor status. Change in carbohydrate intake from baseline to year 1 of study was estimated from 24-hour dietary recalls. Breast cancer recurrence cases (91) were matched to two controls (n = 174) on disease and study characteristics and counter matched on change in carbohydrate intake. Weighted conditional logistic regression models fit the risk of recurrence on IGFI receptor status and dietary change. Half of the tumors were IGFI receptor positive. Increased risk of recurrence was associated with IGFI receptor-positive status [HR 1.7; 95% confidence interval (CI), 1.2-2.5] and, separately, with a stable/increased intake of carbohydrates (HR 2.0; 95% CI, 1.3-5.0). There was a borderline significant interaction between those two variables (P = 0.11). Specifically, carbohydrate intake had no significant impact on risk of recurrence among women who were receptor negative, yet increased the risk of recurrence by more than 5-fold among women who were receptor positive (HR 5.5; 95% CI, 1.8-16.3). Among women whose tumor tissue is positive for the IGFI receptor, reducing carbohydrate intake after diagnosis could reduce the risk of breast cancer recurrence. These findings need replication in a larger sample. This is the first study to suggest that it may be possible to personalize dietary recommendations for breast cancer survivors based on molecular characteristics of their primary tumor tissue. .

Expression of stemness genes in primary breast cancer tissues: the role of SOX2 as a prognostic marker for detection of early recurrence.

The events leading to breast cancer (BC) progression or recurrence are not completely understood and new prognostic markers aiming at identifying high risk-patients and to develop suitable therapy are highly demanded. Experimental evidences found in cancer cells a deregulated expression of some genes involved in governance of stem cell properties and demonstrated a relationship between stemness genes overexpression and poorly differentiated BC subtypes. In the present study 140 primary invasive BC specimens were collected. The expression profiles of 13 genes belonging to the OCT3/SOX2/NANOG/KLF4 core circuitry by RT-PCR were analyzed and any correlation between their expression and the BC clinic-pathological features (CPfs) and prognosis was investigated. In our cohort (117 samples), NANOG, GDF3 and SOX2 significantly correlated with grade 2, Nodes negative status and higher KI67 proliferation index, respectively (p=0.019, p=0.029, p= 0.035). According to multivariate analysis, SOX2 expression resulted independently associated with increased risk of recurrence (HR= 2,99; p= p=0,004) as well as Nodes status (HR=2,44; p=0,009) and T-size >1 (HR=1,77; p=0,035). Our study provides further proof of the suitable use of stemness genes in BC management. Interestingly, a prognostic role of SOX2, which seems to be a suitable marker of early recurrence irrespective of other clinicopathological features.

Prostate specific membrane antigen (PSMA) expression in primary gliomas and breast cancer brain metastases

BackgroundPrimary and secondary brain cancers are highly treatment resistant, and their marked angiogenesis attracts interest as a potential therapeutic target. Recent observations reveal that the microvascular endothelium of primary high-grade gliomas expresses prostate specific membrane antigen (PSMA). Breast cancers express PSMA and they frequently form secondary brain tumors. Hence we report here our pilot study addressing the feasibility of PSMA targeting in brain and metastatic breast tumors, by examining PSMA levels in all glioma grades (19 patients) and in breast cancer brain metastases (5 patients).MethodsTumor specimens were acquired from archival material and normal brain tissues from autopsies. Tissue were stained and probed for PSMA, and the expression levels imaged and quantified using automated hardware and software. PSMA staining intensities of glioma subtypes, breast tumors, and breast tumor brain metastases were compared statistically versus normals.ResultsNormal brain microvessels (4 autopsies) did not stain for PSMA, while a small proportion (<5%) of healthy neurons stained, and were surrounded by an intact blood brain barrier. Tumor microvessels of the highly angiogenic grade IV gliomas showed intense PSMA staining which varied between patients and was significantly higher (p < 0.05) than normal brain. Grade I gliomas showed moderate vessel staining, while grade II and III gliomas had no vessel staining, but a few (<2%) of the tumor cells stained. Both primary breast cancer tissues and the associated brain metastases exhibited vascular PSMA staining, although the intensity of staining was generally less for the metastatic lesions.ConclusionsOur results align with and extend previous data showing PSMA expression in blood vessels of gliomas and breast cancer brain metastases. These results provide a rationale for more comprehensive studies to explore PSMA targeted agents for treating secondary brain tumors with PSMA expressing vasculature. Moreover, given that PSMA participates in angiogenesis, cell signaling, tumor survival, and invasion, characterizing its expression may help guide later investigations of the poorly understood process of low grade glioma progression to glioblastoma.

PI3K/AKT/mTOR pathway activation in primary and corresponding metastatic breast tumors after adjuvant endocrine therapy

Both preclinical and clinical data suggest that activation of the PI3K/AKT/mTOR pathway in response to hormonal therapy results in acquired endocrine therapy resistance. We evaluated differences in activation of the PI3K/AKT/mTOR pathway in estrogen receptor α (ERα) positive primary and corresponding metastatic breast cancer tissues using immunohistochemistry for downstream activated proteins, like phosphorylated mTOR (p-mTOR), phosphorylated 4E Binding Protein 1 (p-4EBP1) and phosphorylated p70S6K (p-p70S6K). For p-mTOR and p-4EBP1, the proportion of immunostained tumor cells (0–100%) was scored. Cytoplasmic intensity (0–3) was assessed for p-p70S6K. The difference between expression of these activated PI3K/AKT/mTOR proteins- in primary and metastatic tumor was calculated and tested for an association with adjuvant endocrine therapy. In patients who had received endocrine therapy (N = 34), p-mTOR expression increased in metastatic tumor lesions compared to the primary tumor (median difference 45%), while in patients who had not received adjuvant endocrine therapy (N = 37), no difference was found. Similar results were observed for p-4EBP1 and p-p70S6K expression. In multivariate analyses, adjuvant endocrine therapy was significantly associated with an increase in p-mTOR (p = 0.01), p-4EBP1 (p = 0.03) and p-p70S6K (p = 0.001), indicating that compensatory activation of the PI3K/AKT/mTOR pathway might indeed be a clinically relevant resistance mechanism resulting in acquired endocrine therapy resistance.What's new?Inhibitors of the PI3K/AKT/mTOR pathway can overcome the resistance to estrogen-depletion therapy that often develops in metastatic breast cancer. In this study, the authors compared primary and metastatic tumors; their results suggest that activation of the PI3K/AKT/mTOR pathway in patients who receive adjuvant endocrine therapy is a clinically relevant mechanism of acquired hormone resistance. For identification of companion diagnostics for PI3K/AKT/mTOR inhibitors, the authors conclude that analyzing primary tumor tissue may often fail to predict treatment response in metastatic breast cancer.

GPER-mediated proliferation and estradiol production in breast cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) are crucial co-mediators of breast cancer progression. Estrogen is the predominant driving force in the cyclic regulation of the mammary extracellular matrix, thus potentially affecting the tumor-associated stroma. Recently, a third estrogen receptor, estrogen (G-protein-coupled) receptor (GPER), has been reported to be expressed in breast CAFs. In this study, GPER was detected by immunohistochemical analysis in stromal fibroblasts of 41.8% (59/141) of the primary breast cancer samples. GPER expression in CAFs isolated from primary breast cancer tissues was confirmed by immunostaining and RT-PCR analyses. Tamoxifen (TAM) in addition to 17β-estradiol (E2) and the GPER agonist G1 activated GPER, resulting in transient increases in cell index, intracellular calcium, and ERK1/2 phosphorylation. Furthermore, TAM, E2, and G1 promoted CAF proliferation and cell-cycle progression, both of which were blocked by GPER interference, the selective GPER antagonist G15, the epidermal growth factor receptor (EGFR) inhibitor AG1478, and the ERK1/2 inhibitor U0126. Importantly, TAM as well as G1 increased E2 production in breast CAFs via GPER/EGFR/ERK signaling when the substrate of E2, testosterone, was added to the medium. GPER-induced aromatase upregulation was probably responsible for this phenomenon, as TAM- and G1-induced CYP19A1 gene expression was reduced by GPER knockdown and G15, AG1478, and U0126 administration. Accordingly, GPER-mediated CAF-dependent estrogenic effects on the tumor-associated stroma are conceivable, and CAF is likely to contribute to breast cancer progression, especially TAM resistance, via a positive feedback loop involving GPER/EGFR/ERK signaling and E2 production.

Aberrant PTPRO methylation in tumor tissues as a potential biomarker that predicts clinical outcomes in breast cancer patients.

BackgroundAberrant hypermethylation of gene promoter regions is a primary mechanism by which tumor suppressor genes become inactivated in breast cancer. Epigenetic inactivation of the protein tyrosine phosphatase receptor-type O gene (PTPRO) has been described in several types of cancer.ResultsWe screened primary breast cancer tissues for PTPRO promoter hypermethylation and assessed potential associations with pathological features and patient outcome. We also evaluated its potential as a breast cancer biomarker. PTPRO methylation was observed in 53 of 98 (54%) breast cancer tissues but not in adjacent normal tissue. Among matched peripheral blood samples from breast cancer patients, 33 of 98 (34%) exhibited methylated PTPRO in plasma. In contrast, no methylated PTPRO was observed in normal peripheral blood from 30 healthy individuals. PTPRO methylation was positively associated with lymph node involvement (P = 0.014), poorly differentiated histology (P = 0.037), depth of invasion (P = 0.004), and HER2 amplification (P = 0.001). Multivariate analysis indicated that aberrant PTPRO methylation could serve as an independent predictor for overall survival hazard ratio (HR): 2.7; 95% CI: 1.1-6.2; P = 0.023), especially for patients with HER2-positive (hazard ratio (HR): 7.5; 95% CI: 1.8-31.3; P = 0.006), but not in ER + and PR + subpopulation. In addition, demethylation induced by 5-azacytidine led to gene reactivation in PTPRO-methylated and -silenced breast cancer cell lines.ConclusionsHere, we report that tumor PTPRO methylation is a strong prognostic factor in breast cancer. Methylation of PTPRO silences its expression and plays an important role in breast carcinogenesis. The data we present here may provide insight into the development of novel therapies for breast cancer treatment. Additionally, detection of PTPRO methylation in peripheral blood of breast cancer patients may provide a noninvasive means to diagnose and monitor the disease.

Abstract P3-07-01: Family history of breast cancer in a population-based breast cancer cohort: No association with PAM50 intrinsic subtype or prognosis

Abstract Basal-like intrinsic subtype and poor prognosis are characteristic of breast cancers in BRCA1 families, but other genes also confer inherited predisposition to breast cancer, and subtype associations may differ. The objective of this study was to examine whether family history of breast cancer is associated with basal-like subtype and/or poor prognosis in a population-based sample of women with breast cancer. METHODS: The study population was the Life After Cancer Epidemiology (LACE) cohort, diagnosed with breast cancer in 1996-2000, and the Pathways cohort, diagnosed with breast cancer in 2006-2008. History of breast cancer among first-degree relatives was obtained by self-report at study enrollment, and women were followed for breast cancer recurrence and mortality. Primary breast cancer tissue was obtained for a case-cohort sample. Intrinsic subtypes were classified based on RT-PCR assay of 50 genes (PAM50). Sample-weighted subtype distributions were compared by family history. Hazard ratios (HR) for recurrence and mortality were estimated from Cox proportional hazards models. RESULTS: From 4,256 women in the parent cohorts who responded to the family history question, 20.5% reported a first-degree family history of breast cancer. Among women with family history, 80.6% were diagnosed at ages 50 and older, compared with 76.3% of women without family history, p = 0.007. Among cases with family history, 79.2% were non-Hispanic whites, compared with 72.3% of cases without family history, p &amp;lt; 0.001. Within the subcohort with PAM50 results (n = 1,319), cases with and without family history had 8.5% and 10.2% prevalence of basal-like tumors, respectively, p = 0.27. For cases diagnosed before age 50, the prevalences of basal-like subtype for women with and without family history were 18.6% and 15.9%, respectively, p = 0.62. Women with family history had similar recurrence risk to those without, HR 0.82 (95% CI 0.61, 1.11; age- and race-adjusted). Women with family history had a suggestive reduced risk of death from all causes, HR 0.75 (95% 0.55, 1.02). Family history was not significantly associated with recurrence or survival within any intrinsic subtype group. DISCUSSION: Although BRCA1 is known to predispose to basal-like subtype, we found no association between family history of breast cancer and basal-like subtype or worse prognosis in this population-based cohort. Mutation status for BRCA1 and other cancer susceptibility genes are unknown for cohort members. First-degree family history of breast cancer may represent the presence of one of the several inherited breast cancer susceptibility genes, or chance aggregation of sporadic cases. Particularly for a woman diagnosed at an older age, her mother and sisters would also have reached ages when sporadic breast cancer incidence is high. Among women diagnosed before age 50, family history was associated with a small and non-significant excess of basal-like tumors. Implications of this study are that for women from the general population diagnosed with breast cancer, a first-degree family history of breast cancer indicates neither a higher probability of a basal-like tumor nor worse prognosis. Support: NIH CA129059 and CA105274. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-07-01.

Abstract P6-09-01: Central Ki67 analysis as a predictor for adjuvant capecitabine efficacy in early breast cancer (EBC) subtypes in US oncology trial 01062

Abstract Background: USON 01062 (O’Shaughnessy J, et al. Proc SABCS, 2010, abst S4-2) showed no improvement in the primary endpoint of disease-free survival (DFS) (median FU 5 yrs: HR 0.84, 95% CI: 0.67-1.05; p = 0.125) with the addition of capecitabine (X) to standard adjuvant chemotherapy, but showed improvement in OS (HR 0.68, 95% CI: 0.51-0.92; p = 0.011). Exploratory analysis of local pathology-assessed Ki67 suggested benefit from adjuvant X in pts with more highly proliferative cancers with Ki67 ≥ 10% (Pippen J et al. Proc ASCO, 2011, abst 500). The objective of this study is to determine whether centrally-performed Ki67 IHC results corroborate or refute this finding. Methods: 2610 pts with resected high risk EBC were randomized to receive 4 cycles of AC (doxorubicin 60mg/m2 and cyclophosphamide 600mg/m2) IV every 3 wks for 4 cycles followed by either docetaxel 100mg/m2 IV or docetaxel 75mg/m2 IV plus X 825mg/m2 PO bid for 14 days every 3 wks for 4 cycles. Archival primary breast cancer tissue was collected on 2000 pts for predictive biomarker analyses. Central Ki67 IHC was performed using the anti-Ki67 monoclonal antibody SP6 and was read by one pathologist (HK) according to published recommendations (Dowsett M, et al. JNCI 103:1-9, 2011). Results: Central Ki67 IHC has been performed on 1440 pts who had centrally-validated informed consents. The distribution of% Ki67-positive cells by locally-assessed ER/HER2 subtype is shown below. 45% of HR+ HER2- BCs had a Ki67 ≤ 10%, while 24% had a Ki67 11% to 20%, and 31% had a Ki67 &amp;gt; 20%. The concordance between the local vs central Ki67 results was low at 46% for Ki67 &amp;lt;10%, 49% for Ki67 10%-20%, and 76% for Ki67 &amp;gt; 20%. The central Ki67 results tended to be higher than the local testing results. Central mRNA classifiers were developed for ER, PR, HER2 and Ki67 using Fluidigm Microfluidics Dynamic Arrays and correlate highly with central IHC assessment of these markers. Conclusions: HR+ HER2- EBC is enriched for cancers with a low proliferative rate, a group of pts unlikely to benefit from the cell cycle-specific cytotoxic agent, capecitabine. Analyses of the impact of adjuvant X added to AC/T in EBC pts according to ER status, and according to Ki67 (analyzed as a binary and continuous variable) will be performed prior to SABCS, 2013. Number of Patients% Ki67 Pos CellsTotal *HR+TNHER2+/HR+HER2+/ HR-0-104163622222711-151391066151016-20126871615821-3018411539201031-1005751403423555Total144081042510790*Totals do not equal sum of subtype categories due to missing HER2 information Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-09-01.

Abstract PD4-2: Whole exome and transcriptome sequencing of 120 primary breast cancer to discover novel therapeutic target

Abstract Introduction: Many somatic mutations, structural alterations, and gene expression changes are causally implicated in oncogenesis and tumor progression, and as a result, affect clinical outcome. Although majority of breast cancer patients have benefits from therapeutics targeting tumor biology, such as estrogen receptor and HER-2, still many patients suffer from disease recurrence and metastasis. More kind of specific target therapies are needed, especially for hormone-resistant tumor and triple-negative breast cancer. Materials and Method: To find novel therapeutic target in breast cancer, here we examine the both whole exome and whole transcriptome of fresh-frozen primary breast cancer tissues from 120 patients whose clinical, pathological, and survival data are available. Patients with Stage IV disease or who received neoadjuvant chemotherapy were excluded. 36 patients had distant metastasis within 5 years from surgery, and 84 patients were NED at least 5 years. RNA and DNA were extracted and qualities were assessed in all samples. Exome and transcriptome sequencing were done using NGS technology (Illumina HiSeq 2000). As a control, exome sequencing was done for 93 normal DNA from matched patients. Single nucleotide variations (SNV) identified in cancer samples on exonic region, nonsynonymous SNV or stop gain/loss, whose quality ≥20, and not found in 93 normal samples were included. SNVs registered in dbSNP135_common or 1000 genome allele frequency &amp;gt;0.001 were excluded.Results and Discussion: We identified 11,684 putative somatic mutations in 7,373 genes. Of them, 6,547 were deleterious or damaging mutation by Provean or SIFT analysis. Mutations were found in potential drug target genes, such as PIK3CA(25), PTEN(3), AKT1(3), ALK(3), ROS1(2), FGFR4(3), FGFR3(2), ERBB2(2), and IDH1(1) etc. In a pathway analysis, mutations in insulin signaling pathway were most dominant. We hypothesized that driver gene and therapeutic target has to have recurrent mutation and gene expression at least more than average expression. We calculated expression “Volume” according to the median normalized FPKM value of individual gene's RNA-seq data. With a cut-off of 3 or more mutations in each gene, 1,116 genes were selected. After the filtering of Volume&amp;lt;0.3, 696 genes were selected. Finally, 55 genes were selected which are druggable or potentially druggable using drug database (DrugBank, TOCRIS, Ingenuity) and Pubmed. DriverNet analysis result was also considered for the selection. All 342 tumor suppressor genes were filtered out. Interestingly, 18 of the 55 were genes involved in metabolism (fatty acid, glucose, amino acids). 12 were kinases and 4 were involved in insulin pathway. Excluding the previously confirmed therapeutic target, PIK3CA, AKT1, and NOTCHs, and considering the patients’ clinical data, our primary candidates for hormone-resistant breast cancer were NQO2, CELSR1, GLUD2, MYH9, PSMD2, NADK, IRS2, MAP3K5, and for triple-negative breast cancer were HSPG2, PHGDH, MYLK, etc. Validation with Sanger sequencing and functional study is on-going. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-2.

Expression of phosphodiesterase 6 (PDE6) in human breast cancer cells

Considerable epidemiological evidence demonstrates a positive association between artificial light at night (LAN) levels and incidence rates of breast cancer, suggesting that exposure to LAN is a risk factor for breast cancer. There is a 30-50% higher risk of breast cancer in the highest LAN exposed countries compared to the lowest LAN countries, and studies showing higher incidence of breast cancer among shift workers exposed to more LAN have led the International Agency for Research on Cancer to classify shift work as a probable human carcinogen. Nevertheless, the means by which light can affect breast cancer is still unknown. In this study we examined established human breast cancer cell lines and patients’ primary breast cancer tissues for expression of genetic components of phosphodiesterase 6 (PDE6), a cGMP-specific PDE involved in transduction of the light signal, and previously thought to be selectively expressed in photoreceptors. By microarray analysis we find highly significant expression of mRNA for the PDE6B, PDE6C, and PDE6D genes in both the cell lines and patients’ tissues, minimal expression of PDE6A and PDE6G and no expression of PDE6H. Using antibody specific for PDE6β, we find expression of PDE6B protein in a wide range of patients’ tissues by immunohistochemistry, and in MCF-7 breast cancer cells by immunofluorescence and Western blot analysis. Considerable expression of key circadian genes, PERIOD 2, CLOCK, TIMELESS, CRYPTOCHROME 1, and CRYPTOCHROME 2 was also seen in all breast cancer cell lines and all patients’ breast cancer tissues. These studies indicate that genes for PDE6 and control of circadian rhythm are expressed in human breast cancer cells and tissues and may play a role in transducing the effects of light on breast cancer.

Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer

Change of DNA cytosine methylation (5mC) is an early event in the development of cancer, and the recent discovery of a 5-hydroxymethylated form (5hmC) of cytosine suggests a regulatory epigenetic role that might be different from 5-methylcytosine. Here, we aimed at elucidating the role of 5hmC in breast cancer. To interrogate the 5hmC levels of the leucine zipper, putative tumor suppressor 1 (LZTS1) gene in detail, we analyzed 75 primary breast cancer tissue samples from initial diagnosis and 12 normal breast tissue samples derived from healthy persons. Samples were subjected to 5hmC glucosyltransferase treatment followed by restriction digestion and segment-specific amplification of 11 polymerase chain reaction products. Nine of the 11 5'LZTS1 fragments showed significantly lower (fold change of 1.61-6.01, P < .05) 5hmC content in primary breast cancer tissue compared to normal breast tissue samples. No significant differences were observed for 5mC DNA methylation. Furthermore, both LZTS1 and TET1 mRNA expressions were significantly reduced in tumor samples (n = 75, P < .001, Student's t test), which correlated significantly with 5hmC levels in samples. 5hmC levels in breast cancer tissues were associated with unfavorable histopathologic parameters such as lymph node involvement (P < .05, Student's t test). A decrease of 5hmC levels of LZTS1, a classic tumor suppressor gene known to influence metastasis in breast cancer progression, is correlated to down-regulation of LZTS1 mRNA expression in breast cancer and might epigenetically enhance carcinogenesis. The study provides support for the novel hypothesis that suggests a strong influence of 5hmC on mRNA expression. Finally, one may also consider 5hmC as a new biomarker.

Expression and clinical significance of activating transcription factor 3 in human breast cancer.

Breast cancer is the most common type of cancer among women worldwide. This study investigated the expression and clinical significance of activating transcription factor 3 (ATF3) in human breast cancer and its relationship with the clinical outcome of breast cancer. Materials and Methods : ATF3 expressions were detected in 114 primary breast cancer tissues and 114 adjacent normal tissues using immunohistochemistry (IHC) assay. Categorical variables were statistically compared by chi-square or Fisher's exact test. Survival curves were evaluated using the Kaplan-Meier method and comparisons of survival rates were tested using a Log-rank test. Results : IHC analysis showed that the positive expression of ATF3 protein was detected in breast cancer tissue with a positive ratio of 76.3%, and the positive ATF3 expression in adjacent normal breast tissue was 13.2%, which is lower than that in breast cancer tissue samples (P<0.01). Furthermore, ATF3 expression showed significant correlation with TNM stage, invasion, lymph node metastasis and number of metastatic lymph nodes (P=0.038, P=0.029, P=0.026, and P=0.039 respectively), and did not correlate with patients' age and tumor size (P>0.05). A significant difference in overall survival rate was found between the patients with positive expression of ATF3 protein and those with negative expression (P=0.041). Conclusion : Increased ATF3 expression participate in the tumorigenesis, invasion and metastasis of breast cancer, and ATF3 may be useful as a new prognostic indicator for breast cancer patients.

Mesenchymal stem cells derived from breast cancer tissue promote the proliferation and migration of the MCF-7 cell line in vitro

Mesenchymal stem cells (MSCs) are critical in promoting cancer progression, including tumor growth and metastasis. MSCs, as a subpopulation of cells found in the tumor microenvironment, have been isolated from several tumor tissues, but have not been isolated from breast cancer tissue to date. Therefore, the purpose of this study was to isolate MSCs from primary human breast cancer tissue, and to study the effect of breast cancer MSCs (BC-MSCs) on the proliferation and migration of the MCF-7 cell line in vitro. MSCs were isolated and identified from primary breast cancer tissue obtained from 9 patients. The MCF-7 cell line was treated with 10 and 20% breast cancer-associated MSC (BC-MSC)-conditioned medium (CM) for 10–48 h, and changes in proliferation and migration were observed. Furthermore, we investigated the migration of 10 and 20% CM concentrations on MCF-7 through a scratch wound assay and a transwell migration assay. We successfully isolated and identified MSCs from primary breast cancer tissues. BC-MSCs showed characteristics similar to those of bone marrow MSCs, and possessed the capability of multipotential differentiation into osteoblasts and adipocytes. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that 10 and 20% CM concentrations increased the proliferation of MCF-7 cells to different levels. The results also revealed a greater increase in different levels compared with the control group. In conclusion, MSCs were confirmed to exist in human breast cancer tissues, and BC-MSCs may promote the proliferation and migration of breast cancer cells.

Abstract A014: A clinically relevant prognostic mRNA signature for triple-negative breast cancer developed from the patient-derived xenograft model

Abstract Triple-negative breast cancer (TNBC) is a unique subtype of breast cancer which lacks effective targeted agent and has a relatively worse prognosis when compared to other subtypes. Identifying a molecular characteristic associated with poor prognosis and classifying TNBC patients accordingly can help the clinicians to provide tailored therapeutic approach. Using a patient-derive xenograft model which use patients' primary breast cancer tissues xenotransplanted into the immunodeficient mice, the xenograft take rates of various molecular subtypes of breast cancer were examined. Also, the mRNA expression profile associated with the xenograft tumor formation was investigated in 13 TNBC patients. The genes showing differential expression according to the xenograft tumor formation were tested in an independent publicly available TNBC mRNA expression datasets to assess the prognostic importance. Among the 81 primary breast cancer tissues used for the xenotransplantation experiments, 19 (23.4%) developed xenograft tumors. Cancer tissues from TNBC patients were more likely to develop xenograft tumors (48%) when compared to other subtypes. The TNBC-derived xenograft tumors showed rapid and exponential tumor growth when compared to luminal type cancers. The genetic characteristics of the primary tumor were maintained after serial xenotransplantation in the immunodeficient mice suggesting the validity of the patient-derived xenograft model for translational research. In 13 TNBC tumors, the mRNA expression profiles were obtained by using microarray chip experiment to identify a genetic characteristic associated with the xenograft tumor formation. Gene involved in immune-reponse, cell migration, angiogenesis and vascular formation were significantly up or downregulated according to the xenograft tumor formation. Among the 13 TNBC patients, the distant metastases were developed only in tumors which formed xenograft tumors. The individual genes showing differential expression, as well as the global expression profile of xenograft tumor formation, was associated with the distant metastasis risk in an independent TNBC mRNA expression datasets. In conclusion, the present study has identified a set of genes that carry prognostic implication in TNBC patients by using the patient-derived xenograft models. Citation Format: Hyeong-Gon Moon, Keunhee Oh, Dong-Sup Lee, Wonshik Han, Dong-Young Noh. A clinically relevant prognostic mRNA signature for triple-negative breast cancer developed from the patient-derived xenograft model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A014.

Nonlinear Growth Kinetics of Breast Cancer Stem Cells: Implications for Cancer Stem Cell Targeted Therapy

Cancer stem cells (CSCs) have been identified in primary breast cancer tissues and cell lines. The CSC population varies widely among cancerous tissues and cell lines, and is often associated with aggressive breast cancers. Despite of intensive research, how the CSC population is regulated within a tumor is still not well understood so far. In this paper, we present a mathematical model to explore the growth kinetics of CSC population both in vitro and in vivo. Our mathematical models and supporting experiments suggest that there exist non-linear growth kinetics of CSCs and negative feedback mechanisms to control the balance between the population of CSCs and that of non-stem cancer cells. The model predictions can help us explain a few long-standing questions in the field of cancer stem cell research, and can be potentially used to predict the efficicacy of anti-cancer therapy.

MicroRNA-30a suppresses breast tumor growth and metastasis by targeting metadherin

Accumulating data have shown the involvement of microRNAs in cancerous processes as either oncogenes or tumor suppressor genes. Here, we established miR-30a as a tumor suppressor gene in breast cancer development and metastasis. Ectopic expression of miR-30a in breast cancer cell lines resulted in the suppression of cell growth and metastasis in vitro. Consistently, the xenograft mouse model also unveiled the suppressive effects of miR-30a on tumor growth and distal pulmonary metastasis. With dual luciferase reporter assay, we revealed that miR-30a could bind to the 3'-untranslated region of metadherin (MTDH) gene, thus exerting inhibitory effect on MTDH. Furthermore, we demonstrated that silence of MTDH could recapitulate the effects of miR-30a overexpression, while overexpression of MTDH could partially abrogate miR-30a-mediated suppression. Of significance, expression level of miR-30a was found to be significantly lower in primary breast cancer tissues than in the paired normal tissues. Further evaluation verified that miR-30a was negatively correlated with the extent of lymph node and lung metastasis in patients with breast cancer. Taken together, our findings indicated miR-30a inhibits breast cancer proliferation and metastasis by directly targeting MTDH, and miR-30a can serve as a prognostic marker for breast cancer. Manipulation of miR-30a may provide a promising therapeutic strategy for breast cancer treatment.

Decreased FOXF2 mRNA Expression Indicates Early-Onset Metastasis and Poor Prognosis for Breast Cancer Patients with Histological Grade II Tumor

The transcription factor, FOXF2, plays an important role in tissue development, extracellular matrix synthesis, and epithelial-mesenchymal interactions, implying that it may be associated with the metastatic capabilities of cancer cells. However, the relationship between FOXF2 expression and breast cancer progression, metastasis, and prognosis, remains to be elucidated. In this study, FOXF2 mRNA levels in 305 primary breast cancer tissues were examined using RT-QPCR. Results showed that FOXF2 mRNA levels in primary breast cancer were negatively associated with tumor progression, including tumor size, number of metastatic lymph nodes, and clinical stage. Patients with low FOXF2 mRNA levels had a high risk of relapse and metastasis within three years. Low FOXF2 mRNA levels could predict shorter disease-free survival for those patients with histological grade II and triple-negative breast cancer. Taken together, we conclude that decreased FOXF2 expression indicates the early-onset metastasis and poor prognosis for patients with histological grade II and triple-negative breast cancer.

Abstract 3458: An epigenomic next-generation sequencing approach to identify predictive markers for PARP inhibitor response in breast cancer cells.

Abstract Background: Inhibition of poly(ADP-ribose)-polymerase-1 (PARP1) leads to synthetic lethality in BRCA-associated breast cancer cells, and is a promising novel treatment strategy for hereditary BRCA-mutated tumors. Since BRCA1/2 germline mutations are relatively infrequent, our study aims at a systematic identification of epigenetic aberrations that may predict PARP inhibitor (PARib) response in sporadic breast cancer cells. Methods: Genome-wide DNA methylation was assessed in nine malignant and one non-malignant (MCF10A) breast cell line (BCL) by MBD-isolated genome sequencing (MiGS). Sensitivity to the PARib AG14361 was determined by viability and clonogenicity assays before and after global DNA demethylation in vitro, and confirmed with the PARib AZD-2281 (olaparib). Correlation analyses of DNA methylation with PARib sensitivity identified potentially predictive candidate genes and microRNAs. By incubation with increasing concentrations of AG14361 we generated an isogenic PARib resistant BCL originally sensitive to AG14361. Results: BCL clustered in three sensitivity groups (sensitive, intermediate, resistant) independent of hormone receptor status, but associated with PARP1 level (p=0.042). BRCA1 mutated (MDA-MB-436) and BRCA1 methylated (UACC3199) BCL were most sensitive to PARib. After global DNA demethylation sensitive BCL acquired resistance while resistant BCL were modestly sensitized. MCF10A cells did not shift PARib resistance after DNA demethylation. Correlation of MiGS profiles with PARib sensitivity identified 382 genes and seven microRNAs with exclusive promoter/exon1 methylation in sensitive BCL, while 313 genes and one microRNA were affected in resistant BCL. Artificially mediating resistance to BRCA1 methylated cells did neither hypomethylate the BRCA1 promoter nor induce BRCA1 expression, but significantly reduce cell proliferation. Gene ontology analyses revealed that methylation of WNT, ErbB, SHH, and mTOR signaling genes was significantly enriched in PARib resistant cells, while PARib sensitive cells exhibited methylation in DNA repair genes. Conclusions: Aberrant DNA methylation seems to be involved in PARib sensitivity/resistance in BCL. By epigenomic analyses we identified several potential candidate genes beyond BRCA1 that might modulate PARib sensitivity/resistance in breast cancer, implying that more patients than currently anticipated may benefit from this drug class. Their expression, DNA methylation and net effect on cancer pathways are currently validated in BCL and explored in primary breast cancer tissues. Citation Format: Tim De Meyer, Geert Trooskens, Mascha K. Wibbe, Sebastian B. Bartmann, Manon van Engeland, Wim Van Criekinge, Vivianne C. Tjan-Heijnen, Ruth Knüchel, Jürgen Veeck. An epigenomic next-generation sequencing approach to identify predictive markers for PARP inhibitor response in breast cancer cells. [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 3458. doi:10.1158/1538-7445.AM2013-3458

Abstract 3104: HOXB7 promotes tumor progression via activation of the transformation growth factor-β signaling pathway.

Abstract Our previous studies have shown that overexpression of Hoxb7 delays tumors onset, but promotes tumor progression and metastasis in oncogene Her2/neu-induced mammary tumorigenesis, which is reminiscent of the dual role of TGF-β in breast cancer. To investigate whether HOXB7 directly or indirectly regulates the TGF-β signaling, we examined the phosphorylation levels of SMAD3, an important mediator of TGF-beta signaling, in primary mammary tumors derived from the Hoxb7 and Her2/neu double-transgenic mice and the Her2/neu mono-transgenic mice. Phosphorylation of SMAD3 is detected in a higher percentage of primary tumor tissues from double transgenic mice than that from Her2/neu mono-transgenic mice (69.2% versus 15.4%, p &amp;lt; 0.05), suggesting that Hoxb7 may activate TGF-β/Smad signaling in breast tumor tissues. In line with this finding, overexpression of HOXB7 induces expression of TGF-β ligands in both mouse and human breast cancer cell lines, leading to increased cell motility and invasiveness. The acquainted phenotypes in HOXB7-overepressing cells can be reversed by pharmacologic inhibition of TGF-β signaling. Promoter deletion analysis and Chromotin-immunprocipiration assay further demonstrated that HOXB7 can directly bind to and activate the promoter of TGFβ2. More importantly, expression of HOXB7 and TGFβ2 is strongly correlated in primary breast cancer tissues and associated with advanced stages of tumor progression and reduced survival rate. Taken together, our results suggest that HOXB7 may promote tumor migration, invasion and possibly metastasis through activation of the TGF-β signaling pathway. Citation Format: Shou Liu, Hongmei Zhang, Hexin Chen. HOXB7 promotes tumor progression via activation of the transformation growth factor-β signaling pathway. [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 3104. doi:10.1158/1538-7445.AM2013-3104 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 1396: Response of primary culture of breast cancer to hypoxia and drug treatment.

Abstract Breast cancer is a heterogeneous disease. Several subtypes of breast cancer have been characterized and corresponding treatment strategies have been devised. However, outcomes are not always satisfactory for a variety of reasons including individual variation in age, race, disease stages, and genetic variations. In order to improve the outcomes of breast cancer treatment, personalized treatment has been developed based on molecular profiling and genetic testing of each individual patient. To compliment the development of personalized treatment based on molecular data from genetic profiling, we hypothesized that culture of primary breast cancer tissue could provide a cell-based model that could be utilized in the evaluation of the response of individual breast cancer to therapeutic agents. In this model, breast cancer samples were cultured in matrigel. To identify the cell types that grew in the matrigel, the cells were subjected to the staining with antibodies against cytokeratin5, cytokeratin14, cytokeratin18, ER, vimentin, E-cadherin, N-cadherin, α-SMA, and β-catenin. The results demonstrated that the cells which grew from breast cancer tissue were heterogeneous, including epithelial and stromal cells. Further analysis of the effect of oxygen indicated that the growth of breast cancer cells increased by 1.4-1.5 fold with 5% O2 compared to growth with 21% O2. Interestingly, hypoxia did not stimulate growth of normal breast tissue. Very intriguingly, growth under hypoxic conditions demonstrated differential responses to several chemotherapeutic agents. In these studies cell proliferation was assayed at 5% and 21% O2 of primary cultures of normal and cancerous breast cells treated with doxorubicin, LY294002, and PHA665752 (c-Met inhibitor). With doxorubicin treatment normal breast cells under hypoxia showed significant drug resistance at 0.1 μM and 1 μM compared to normoxia (P&amp;lt;0.05) while breast cancer cells were similarly responsive to doxorubicin under normoxic and hypoxic conditions. With LY294002 treatment hypoxic breast cancer cells showed an increased drug resistance at 10 and 40μM (P&amp;lt;0.05) while hypoxic normal breast cells showed a significant increase in drug resistance at only 40 μM compared to normoxic cells. The effects of PHA665752 on cell proliferation were not affected by hypoxia for either normal or breast cancer cells. In conclusion, cells grown from breast cancer tissue are heterogeneous in cell type. These primary cells show some differential responses to hypoxia condition and drug treatment. An advantage of this primary cell culture model is that it contains cancer cells in the context of their respective native supportive stromal cells, thus creating a microenvironment that is similar to what obtains in vivo. Therefore, this in vitro primary culture model may have potential applications for the testing of drugs predicted to be effective for personalized treatment of breast cancer. Citation Format: Hong Yin, Patrick Adegboyega, Mylinh Smith Smith, Jonathan Glass. Response of primary culture of breast cancer to hypoxia and drug treatment. [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 1396. doi:10.1158/1538-7445.AM2013-1396