Invasive Ductal Carcinoma Tissues Research Articles

Roles of Ras Homolog A in Invasive Ductal Breast Carcinoma.

Breast cancer has a poor prognosis owing to tumor cell invasion and metastasis. Although Ras homolog (Rho) A is involved in tumor cell invasion, its role in breast carcinoma is unclear. Here, RhoA expression was examined in invasive ductal carcinoma (IDC), with a focus on its relationships with epidermal-mesenchymal transition (EMT) and collective cell invasion. Forty-four surgical IDC tissue samples and two normal breast tissue samples were obtained. RhoA, E-cadherin, vimentin, and F-actin protein expression were analyzed by immunohistochemistry. RhoA, ROCK, mTOR, AKT1, and PIK3CA mRNA expression were conducted using laser microdissection and semi-nested quantitative reverse transcription-polymerase chain reaction. RhoA expression was stronger on the tumor interface of IDCs than the tumor center (P<0.001). RhoA expression was correlated with ROCK expression only in HER2-subtype IDC (P<0.05). In IDCs co-expressing RhoA and ROCK, F-actin expression was stronger on the tumor interface, particularly at the edges of tumor cells, than it was in ROCK-negative IDCs (P<0.0001). In conclusion, RhoA expression was not correlated with EMT in IDC, but enhanced F-actin expression was localized on the edge of tumor cells that co-expressed ROCK. RhoA/ROCK signaling may be associated with collective cell invasion, particularly in HER2-subtype IDC.

Associations between cruciferous vegetable intake and selected biomarkers among women scheduled for breast biopsies.

To examine the relationship between dietary cruciferous vegetable intake and selected tumour biomarkers for histone acetylation (H3K9ac, H3K18ac, HDAC3 and HDAC6), proliferation (Ki-67) and cell-cycle regulation (p21) from breast tissue. The study used baseline data of women recruited to participate in a clinical trial of sulforaphane supplement. Dietary cruciferous vegetable intake was collected through a validated Arizona Cruciferous Vegetable Intake Questionnaire. Breast tissue was obtained from biopsy samples. Spearman correlations were calculated between intake of specific cruciferous vegetables and biomarkers. Tissue biomarkers were log2-transformed to obtain approximate normality. Linear regression analyses were conducted to examine associations between cruciferous vegetable intake and biomarkers adjusting for age and use of non-steroidal anti-inflammatory drugs. False discovery rate (FDR) was used to account for multiple comparisons. Clinical trial baseline. Fifty-four women who had abnormal mammogram findings and were scheduled for breast biopsy. Mean intake of total cruciferous vegetables from all food sources was 81·7 (sd 57·3) g/d. Mean urinary total sulforaphane metabolites was 0·08 (sd 0·07) µm/mm creatinine. Total cruciferous vegetable intake was inversely associated with Ki-67 protein expression in breast ductal carcinoma in situ (DCIS) tissue (β=-0·004; se=0·001; FDR q value=0·03), but not in benign or invasive ductal carcinoma (IDC) tissue. No association was found for other biomarkers measured (HDAC3, HDAC6, H3K9, H3K18 and p21) in all tissues examined (benign, DCIS and IDC). The present study sought to provide additional evidence for the potential role of sulforaphane in histone acetylation and cell proliferation. Here, we report that total cruciferous vegetable intake is associated with decreased cell proliferation in breast DCIS tissue.

Reduction of global 5-hydroxymethylcytosine is a poor prognostic factor in breast cancer patients, especially for an ER/PR-negative subtype.

DNA methylation at the 5 position of cytosine (5 mC) is an epigenetic hallmark in cancer. The 5 mC can be converted to 5-hydroxymethylcytosine (5 hmC) through a ten-eleven-translocation (TET). We investigated the impact of 5 mC, 5 hmC, TET1, and TET2 on tumorigenesis and prognosis of breast cancer. Immunohistochemistry was used to assess the levels of 5 mC, 5 hmC, TET1, and TET2 in the corresponding tumor adjacent normal (n = 309), ductal carcinoma in situ (DCIS, n = 120), and invasive ductal carcinoma (IDC, n = 309) tissues for 309 breast ductal carcinoma patients. 5 mC, 5 hmC, TET1-n, and TET2-n were significantly decreased during DCIS and IDC progression. In IDC, the decrease of 5 hmC was correlated with the cytoplasmic mislocalization of TET1 (p < 0.001) as well as poor disease-specific survival (DSS) (adjusted hazard ratio [AHR] 1.95, p = 0.003) and disease-free survival (DFS) (AHR 1.91, p = 0.006). The combined decrease of 5 mC and 5 hmC was correlated with worse DSS (AHR 2.19, p = 0.008) and DFS (AHR 1.99, p = 0.036). Stratification analysis revealed that the low level of 5 mC was associated with poor DSS (AHR 1.89, p = 0.044) and DFS (AHR 2.02, p = 0.035) for the ER/PR-positive subtype. Conversely, the low level of 5 hmC was associated with worse DSS (AHR 2.77, p = 0.002) and DFS (AHR 2.69, p = 0.006) for the ER/PR-negative subtype. The decreases of 5 mC, 5 hmC, TET1-n, and TET2-n were biomarkers of tumor development. The global reduction of 5 hmC was a poor prognostic factor for IDC, especially for ER/PR-negative subtype.

Abstract 342: Breast cancer adhesion cascade is influenced by Galectin-1 and E-selectin receptor-ligand interactions

Abstract Invasion of other tissues during bloodborne metastasis requires the adhesion of cancer cells to the endothelium by specific fluid shear-dependent receptor-ligand interactions. It is hypothesized that ligands are shared between the endothelial adhesion molecule known as E-selectin, a transmembrane glycoprotein, and Galectin-1 (Gal-1), a secretory dimer protein, both of which are upregulated during inflammation and cancer. Our previous data strongly indicate that this shear-dependent interaction involves the same or closely related cancer cell-expressed ligands, such as the glycoprotein Mac-2BP. Initial investigation using immuno-staining methods to locate E-selectin and Gal-1 ligands and determine relative proximity to one another revealed that E-selectin and Gal-1 ligands on ZR-75-1 breast cancer cells and breast cancer tissue microarrays had a high percentage of colocalization, as high as 75% in some cases. These results allude to the possibility that these reactive molecules are in close proximity or are in fact the same molecule. Subsequently, E-selectin microspheres were perfused over Gal-1-treated ZR-75-1 cell monolayers and breast cancer tissue microarrays to determine the functional relationship, if any, between Gal-1 and E-selectin ligands during adhesion from flow. Flow assay data show that Gal-1 treatment enhances E-selectin microsphere interactions to a breast cancer monolayer in a shear-dependent manner, with a shear stress of 0.5 dynes/cm2 having an almost 300% increase in binding compared to the untreated monolayer. This amount of binding at a physiological shear stress gives credence to the hypothesis that Gal-1 influences E-selectin receptor-ligand interactions via the same or similar ligands during breast cancer cell adhesion. Dynamic biochemical tissue analysis (DBTA), a novel flow-based tissue interrogation technique developed in our lab, was then implemented to test human invasive ductal breast carcinoma tissue sections. Similar to ZR-75-1 cell line flow assay data, a 142% increase in shear-dependent binding interactions between the E-selectin microspheres and the Gal-1 treated breast cancer tissue sections at 0.5 dynes/cm2 shear stress was observed. Colocalization of reactive signals and increased shear-dependent flow interactions indicate that metastatic binding in breast cancer is aided through E-selectin to Gal-1 interaction via a shared ligand intermediate. Ascertaining the mechanism behind this interaction would lead to a better understanding of the circulating breast cancer cells’ adhesion cascade during metastasis. Citation Format: Nathan M. Reynolds, Claire R. Hall, Sean E. Thomas, Monica M. Burdick. Breast cancer adhesion cascade is influenced by Galectin-1 and E-selectin receptor-ligand interactions. [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 342. doi:10.1158/1538-7445.AM2015-342

Abstract 2141: Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells

Abstract In this study we investigated cellular and molecular mechanisms for breast cancer signaling by EGFR and Ras proteins, independent of activating mutations. Epidermal Growth Factor (EGF) ligation to its receptor, EGFR, at the plasma membrane (PM) activates Ras small GTPases, propagating Ras mitogenic signaling. However, activating Ras mutations are not associated with breast cancers. EGFR and palmitoylated (Pa-) Ras localize to lipid rafts at the PM. Pa-Ras isotypes (H- and N-Ras, but not K-Ras) cycle through vesicle trafficking pathways between the Golgi and PM. We have found that anterograde H-Ras vesicles are sorted in the endocytic recycling compartment (ERC) into vesicles which contain the epithelial-specific transport mediator Rab25 (Rab11c), and Rab11/FIP1C (Rab-coupling protein, RCP). RCP couples Rab25 to vesicles to mediate vesicle anterograde transport. Rab25 and RCP expression are highly correlated with breast cancer progression, aggressiveness, and invasion and metastasis. Rab25, unique among the large family of human Rab genes, has been identified as a major marker and potential stimulator of breast cancers. RCP was recently shown to be a breast cancer driver oncogene, supporting the suggestion that RCP and Rab25 status may be significant clinical markers. Moreover, RCP stimulates H-Ras activation. Meta-analysis of human breast cancer cell line panels shows that while most do not harbor H- or N-Ras mutations, both RCP and Rab25 are highly over-expressed and levels correlate with resistance to EGF and Ras signaling inhibitors. We demonstrate that Rab25, RCP, H-Ras and EGFR co-localized in the ERC and in transport vesicles in MCF10A mammary epithelial cells, and that dominant negative (DN) Rab25 sequestered H-Ras and EGFR from the plasma membrane. DN Rab25 or Rab25 knockdown by shRNA inhibited EGF-induced EGFR phosphorylation, H-Ras activation, and ERK phosphorylation (ppERK), demonstrating a functional link between Rab25 activity and EGFR and H-Ras signaling. Both ER/PR-positive and -negative human breast invasive ductal carcinoma tissues showed elevated ppERK correlated with elevated Rab25 expression over normal breast tissue, whereas HER2+ tumor tissues had elevated ppERK even with low Rab25 expression. Thus, Rab25 and RCP regulate EGFR and Ras trafficking to the plasma membrane, required for mitogenic signaling and associated with tumorigenic effects, even in Ras (WT) cells. Citation Format: Allison B. Herman, Jeremy G.T. Wurtzel, Lawrence E. Goldfinger. Rab25 and Rab-coupling protein (RCP) coordinate H-Ras and EGFR post-Golgi vesicle trafficking, plasma membrane targeting, and function in mammary epithelial cells. [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 2141. doi:10.1158/1538-7445.AM2015-2141

Abstract 2776: Codon 72 and Intron-3 polymorphisms in TP53 are risk factors for breast cancer

Abstract Purpose: A single nucleotide polymorphism (SNP) in exon-4 of the TP53 gene (Arg72Pro) has been associated with an increased risk of developing various human malignancies. Since a 16-base pair polymorphism (insertion/duplication) intron-3 (PIN3) is in linkage disequilibrium with Arg72Pro of TP53, we assessed whether these two polymorphisms serve as breast cancer risk alleles for African Americans (AAs) and non-Hispanic Caucasians (CAs). Methods: Genomic DNA was extracted from formalin-fixed, paraffin-embedded invasive ductal carcinoma tissues [cases, n = 120 (AAs = 55 and CAs = 65)] and from blood of healthy women [controls, n = 152 (AA = 74 and CAs = 78)]. Exon-4 of TP53 was amplified by PCR using exon-specific primers, followed by Sanger sequencing. Identification of SNP was done by using DNASTAR software. TP53 PIN3 polymorphism was detected by amplifying genomic DNA using specific primers followed by gel electrophoresis. The A1 allele (no duplication) resulted in 162 bp fragment and the A2 allele (with 16 bp duplication) resulted in 178 bp fragment. The phenotypes of Arg72Pro (Arg/Arg, Arg/Pro, and Pro/Pro) were correlated with PIN3 status, clinicopathologic features, race/ethnicity, and patient survival using the χ2 test and Kaplan-Meier analyses. A p value of &amp;lt;0.05 was considered statistically significant. Results: In cases, the Arg/Arg/, Pro/Arg, and Pro/Pro phenotypes were 15%, 31%, and 55% in AAs and 57%, 29%, and 14% in CAs (p&amp;lt;0.001), respectively. A similar distribution was observed in controls (16%, 42%, and 42% in AAs and 56%, 37%, and 6% in CAs) (p&amp;lt;0.001). When compared to ER positive, a higher frequency of Arg/Pro or Pro/Pro was noted in estrogen-negative breast cancers (p = 0.03) in both racial groups. However, no significant survival difference in these racial groups was observed when Pro carriers were compared with Arg/Arg cases (log rank, p = 0.565). Frequencies of PIN3 polymorphisms (A1A1, A1A2, and A2A2) were similar in both cases and controls; however, overall A1A1 frequency was higher than the other two genotypes of PIN3. The phenotype Pro/Pro, was strongly associated with A1A1 polymorphism in both racial groups of breast cancer patients (p&amp;lt;0.000078). Conclusion: These preliminary findings suggest that Arg72Pro polymorphism and PIN3 in TP53 are related with higher breast cancer susceptibility for both African American and non-Hispanic Caucasian patients. This work was supported by a pilot grant from UAB Breast SPORE grant of the National Institutes of Health/National Cancer Institute (5P50CA089019). Citation Format: Trafina Jadhav, Jesus Salazar-Gonzalez, Shantel Hebert-Magee, Michael Behring, Balananda-Dhurjati Putcha, Jeehyun Helen Bae, Andra Frost, Isam-Eldin Eltoum, Sejong Bae, Upender Manne. Codon 72 and Intron-3 polymorphisms in TP53 are risk factors for breast cancer. [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 2776. doi:10.1158/1538-7445.AM2015-2776

Abstract P2-05-10: Explore the differences and relationships between normal and ADH, DCIS and IDC tissues in breast based on Raman spectroscopy

Abstract Background: Atypical ductal hyperplasia (ADH) is a premalignant breast lesion associated with an increased risk of breast cancer which shares some but not all the features of ductal carcinoma in situ (DCIS). The mechanism that ADH changes to DCIS and even invasive ductal carcinoma (IDC) is still not clear now. There is a pressing need to develop screening techniques that are accurate, minimally invasive and, more sensitive to identify early neoplastic changes and hence improve early breast cancer diagnosis. Our aim was to explore the differences and relationships between normal, ADH, DCIS and IDC lesions of the breast based on biochemical characteristics determined by Raman spectroscopy Methods: Frozen sections were collected from 39 patients (all ladies) who underwent surgical resection or mammotome biopsy at the Department of Breast Surgery, the First Hospital of Jilin University. After operation the samples were immediately frozen at -20-25 ° and two contiguous sections (6 µm thickness) were cut from a sample by freezing microtome. One was stained with haematoxylin and eosin for routine histopathological analysis; the same position of the other section was detected by Microscopic confocal Raman spectrometer (HORIBA JY Lab800, 633nm) with its mirror image (the H&amp;E section). After Raman measurement, the sections were routinely processed, stained with H&amp;E and histologically examined. Support vector machine (SVM) was used to differentiate different breast lesions. Results: A total of 475 Raman spectra were obtained from 9 normal breast tissues, 7 ADH, 8 DCIS, and 15 IDC breast tissues. Pronounced mean Raman spectra differences were observed between normal tissues, ADH, DCIS and IDC tissues. The significant features of normal tissues are 1301, 1438, 1652, and 1743 cm-1, these peak positions are attributed to lipids, and the spectra profiles of normal tissues have no strong protein peaks. Most noticeable was the increased protein and reduced lipid levels of ADH tissues compared to normal tissues. The peak relative intensity of 1158 cm-1 which is attributed to the vibrational modes of C-C stretch of proteins has significantly increased from normal tissues to DCIS and IDC in breast. The major spectra differences in ADH, DCIS and IDC spectra were evidenced by a red shift with a broad peak of CH2, the intensity of the stretching vibration peak of carotenoids, a relatively strong band of amide-I, and the nuclear acid peak. ADH tissues had the largest constituent variations between subjects. During the disease progression, IDC tissues have smaller inter-subject constituent variations than DCIS and ADH tissues. The accuracies of SVM are 93% and 95% in discriminating normal and IDC, while only 50% and 51% in discriminating ADH and DCIS. Conclusion: Malignant transformations in tissue are associated with complex biochemical changes and may provide an effective way to evaluate the malignancy by Raman spectroscopy as it reveals differences between normal tissues, ADH, DCIS and IDC tissues in the breast. Further study to explain the biochemical relationships between these differences will shed more light into a better understanding of the mechanism by which ADH and DCIS convert to IDC. Citation Format: Bing Han, Haipeng Zhang, Chao Zheng, Ye Du, Lijia Liang, Weiqing Xu, Zhimin Fan. Explore the differences and relationships between normal and ADH, DCIS and IDC tissues in breast based on Raman spectroscopy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-10.

Abstract P4-11-28: Clinical significance of adiponectin receptor 1 (AdipoR1) expression in invasive breast cancer

Abstract Background: Recent studies have demonstrated that obesity is associated with an increased risk of breast cancer, but the mechanisms underlying this relationship remain to be fully elucidated. Adiponectin is one of major adipokines secreted from adipose tissue. Adiponectin serum levels have been shown to be inversely correlated with breast cancer risk. This protein is believed to act through AdipoR1 and has been suggested to play an important role in cancer development. The purpose of this study was to quantitatively evaluate the expression of AdipoR1 in invasive breast cancer tissue compared to normal breast tissue. And then, we analyzed clinical significance of AdipoR1 in invasive breast cancer. Materials and Methods: Tissues were obtained from 269 patients who underwent curative surgery with no prior treatment for invasive ductal carcinoma from Jan. 2003 to Dec. 2008 in Hallym Sacred Heart Hospital. A tissue microarray (TMA) containing 269 invasive ductal carcinomas as well as 269 adjacent normal breast tissues was established from paraffin-embedded archived tissue for further analysis. AdipoR1 expression was investigated in epithelium and stroma by immunohistochemistry, using 1:1600 dilution of rabbit anti-adiponectin receptor antibody, and correlated with clinical and pathologic tumor parameters. Results: In 269 patients, median follow-up period was 57 months. AdipoR1 was detected in epithelial and stromal component of both normal breast and invasive ductal carcinoma tissues. In epithelium, immunoreactivity for AdioR1 was much lower in cancer tissue than normal one (24.5% versus 72%). This trend was quite similar in stroma, although the gap between cancer and normal was a bit narrow (48.7% versus 77.6%). AdipoR1 was more expressed in stroma than in epithelium among invasive breast cancer (48.7% versus 24.5%). In clinicopathologic features, mean age at diagnosis of AdipoR1 expression group in both epithelium and stroma was older than negative group. Furthermore, in epithelial component, HER-2/neu overexpression rate was slightly higher in AdipoR1 negative group than in positive one (27.8% versus 15%, p=0.059). And, Ki67 was more expressed in AdipoR1 negative group than in positive one (49.5% versus 34.5%, p=0.051). However, in stroma component, there was no other difference between AdipoR1 expression and clinicopathologic parameters. In survival analysis, AdipoR1 expression group in stroma showed significantly better disease free survival (DFS) than negative group (p=0.02). DFS curve according to AdipoR1 expression in epithelium showed a quite similar trend with ones in stroma (p=0.06). Conclusions: This study showed that AdipoR1 expression was suppressed in both epithelium and stroma of invasive breast cancer tissue compared to normal breast tissue, and AdipoR1 expression group in stroma showed significantly better DFS than negative group. Although it is earlist days yet to make conclusion, loss of AdipoR1 expression in stroma, possibly epithelium either, appears to play a role of independent risk factor to predict disease progression, somehow. Citation Format: Younok Lee, Kang Yool Lee, Young Ah Lim, June Ho Kim, Lee Su Kim. Clinical significance of adiponectin receptor 1 (AdipoR1) expression in invasive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-28.

Changes of fibroblast immunophenotype and their clinical significance in stromal remodeling of breast tumors

To evaluate the immunophenotype conversion of fibroblasts and its clinical significance in the process of breast tumor stromal remodeling. CD34, FAP-α, p63 and a-SMA were detected by immunohistochemistry in 273 breast biopsies, including 60 normal breast tissues, 46 atypical ductal hyperplasia (ADH), 60 ductal carcinoma in situ (DCIS), 47 DCIS microinvasive carcinoma (DCIS-MI) and 60 invasive ductal carcinoma (IDC). The positive expression rates of CD34, FAP-α and α-SMA in the stromal fibroblasts of normal breast tissues were 93.3%, 6.7% and 18.3%, respectively. Those in the stromal fibroblasts of ADH tissues were 95.7%, 4.3% and 10.9%, respectively. Those in the stromal fibroblasts of DCIS tissues were 95.0%, 8.3% and 15.0%, respectively. Those in the IDC tissues were 35.0%, 85.0% and 93.3%, respectively. The expressions of CD34, α-SMA and FAP-α in the stromal fibroblasts of normal, ASH and DCIS breast tissues did not show significant differences (χ(2) = 1.142, P = 0.896). The main immunophenotype of stromal fibroblasts in the tumor-host interface at the invasive front of ADH and DCIS lesions was CD34(+)α-SMA(+)FAP-α(+). There were statistically significant differences in the expression of CD34, α-SMA and FAP-α between IDC and ADH, DCIS and normal breast tissues (χ(2) = 8.351, P < 0.001). The immunophenotype of stromal fibroblasts in the IDC and DCIS-MI breast tissues was CD34(-) α-SMA(+) FAP-α(+). Immunophenotype conversion from CD34(+) α-SMA(-) FAP-α(-) to CD34(-) α-SMA(+)FAP-α(+) may be a sensitive indicator to judge whether DCIS has microinvasion. Detection of the immunophenotype conversion of stromal fibroblasts may be helpful to determine the presence of microinvasion, and to improve the diagnostic accuracy rate of DCIS.

Intratumoral androgen metabolism and actions in invasive lobular carcinoma of the breast.

Invasive lobular carcinoma (ILC) accounts for approximately 10% of all breast carcinomas and is characterized by higher levels of androgen receptor (AR) compared to invasive ductal carcinoma (IDC). Despite this potentially androgen-responsive environment, the combined importance of AR and androgen metabolism in non-neoplastic lobules and lobular carcinoma remains unknown. Therefore, in this study, we evaluated the status of pivotal androgen-producing enzymes 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5) and 5α-reductase type 1 (5αRed1) in 178 cases of ILC and surrounding histologically non-neoplastic lobular tissue using immunohistochemistry. Androgen receptor prevalence was higher but androgenic enzymes lower in ILC than non-neoplastic lobules. In ILC cases the status of 5αRed1 and 17βHSD5 was inversely correlated with tumor size (P = 0.0053) and nuclear grade (P = 0.0290), and significantly associated with better overall survival of the patients (P = 0.0059). Based on these findings, we hypothesized that androgen signaling could act as a tumor suppressor. As previous studies suggested that androgens might partially act by increasing levels of the estrogen inactivating enzyme 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2) in IDC tissues, this was reasonably considered a potential mechanism of androgen actions. Significantly positive correlation was detected between the status of androgenic enzymes and 17βHSD2 (P < 0.0001) and intratumoral 17βHSD2 was inversely correlated with tumor size in ILC (P = 0.0075). These correlations suggest one protective mode of androgen action could be through modulation of estrogen metabolism. Results of our present study indicated that androgen-producing enzymes could play pivotal protective roles in AR-enriched ILC cases.

Expressions of Twist and hypoxia inducible factor-1α in invasive ductal carcinoma of breast and their relationships with epithelial-mesenchymal transition

Objective To investigate the expression of Twist and hypoxia inducible factor-1α (HIF-1α) in invasive ductal carcinoma of breast and their relationships with epithelial-mesenchymal transition (EMT).Methods Expression levels of Twist,HIF-1α,E-cadherin and Vimentin in 90 cases of invasive ductal carcinoma tissues and corresponding adjacent tissues were detected by immunohistochemical staining of streptavid-in-peroxidase (SP) method.Results The positive expression rate of Twist,HIF-1 α,E-cadherin and Vimentin was 73.3％,68.9％,15.6％ and 64.4％ respectively in breast cancer tissues,and 7.8％,8.9％,75.6％ and 34.4％ respectively in adjacent tissues.The difference in positive rate between breast cancer tissues and adjacent tissues was statistically significant (P ＜ 0.05).The expression levels of Twist and HIF-1α were correlated with histological grade and lymph node metastasis (P ＜ 0.05).There was a positive correlation between expression levels of Twist and HIF-1α.The expression levels of Twist and HIF-1α were closely correlated with low expression of E-cadherin and high expression of Vimentin.Conclusion Twist,HIF-1α and Vimentin in breast cancer tissues showed higher expression,while E-cadherin lower expression.HIF-1α may promote the occurrence of EMT by up-regulating the expression of Twist,which makes it become one of the possible ways to promote the invasion and metastasis of breast cancer. Key words: Breast carcinoma ; Epithelial-mesenchymal transition ; Twist ; Hypoxia inducible factor-1α

Abstract 551: Clinical significance of adiponectin receptor 1 (AdipoR1) expression in invasive breast cancer

Abstract Recent studies have demonstrated that obesity is associated with an increased risk of breast cancer, but the mechanisms underlying this relationship remain to be fully elucidated. Adiponectin is one of major adipokines secreted from adipose tissue. This protein is believed to act through AdipoR1 and has been suggested to play an important role in cancer development. The purpose of this study was to quantitatively evaluate the expression of AdipoR1 in invasive breast cancer tissue compared to normal breast tissue. And then, we analyzed clinical significance of AdipoR1 in invasive breast cancer. Tissues were obtained from 269 patients who were underwent curative surgery with no prior treatment for invasive ductal carcinoma from Jan. 2003 to Dec. 2008 in Hallym Sacred Heart Hospital. A tissue microarray (TMA) containing 269 invasive ductal carcinomas as well as 269 adjacent normal breast tissues was established from paraffin-embedded archived tissue for further analysis. AdipoR1 expression was investigated in epithelium and stroma by immunohistochemistry, using 1:1600 dilution of rabbit anti-adiponectin receptor antibody, and correlated with clinical and pathologic tumor parameters. In 269 patients, median follow-up period was 57 months. AdipoR1 was detected in epithelial and stromal component of both normal breast and invasive ductal carcinoma tissues. In epithelium, immunoreactivity for AdioR1 was much lower in cancer tissue than normal one (24.5% versus 72%). This trend was quite similar in stroma, although the gap between cancer and normal was a bit narrow (48.7% versus 77.6%). AdipoR1 was more expressed in stroma than in epithelium among invasive breast cancer (48.7% versus 24.5%). In clinicopathologic features, mean age at diagnosis of AdipoR1 expression group in both epithelium and stroma was older than negative group. Furthermore, in epithelial component, HER-2/neu overexpression rate was slightly higher in AdipoR1 negative group than in positive one (27.8% versus 15%, p&amp;lt;0.059). And, Ki67 was more expressed in AdipoR1 negative group than in positive one (49.5% versus 34.5%, p&amp;lt;0.051). However, in stroma component, there was no other difference between AdipoR1 expression and clinicopathologic parameters. In survival analysis, AdipoR1 expression group in stroma showed significantly better disease free survival (DFS) than negative group (p&amp;lt;0.02). DFS curve according to AdipoR1 expression in epithelium showed a quite similar trend with ones in stroma (p=0.06). This study showed that AdipoR1 expression was suppressed in both epithelium and stroma of invasive breast cancer tissue, compared to normal breast tissue. Even though there was no significant difference between AdipoR1 expression and many well-known prognostic factors, AdipoR1 expression in stroma as well as epithelium appears to play a role of good prognostic factor to predict disease progression somehow. Citation Format: Lee-Su Kim, Younok Lee, Hae Sung Kim. Clinical significance of adiponectin receptor 1 (AdipoR1) expression in invasive 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 551. doi:10.1158/1538-7445.AM2014-551

MiR-638 mediated regulation of BRCA1 affects DNA repair and sensitivity to UV and cisplatin in triple-negative breast cancer.

IntroductionTriple-negative breast cancer (TNBC) represents 15 to 20% of all types of breast cancer; however, it accounts for a large number of metastatic cases and deaths, and there is still no effective treatment. The deregulation of microRNAs (miRNAs) in breast cancer has been widely reported. We previously identified that miR-638 was one of the most deregulated miRNAs in breast cancer progression. Bioinformatics analysis revealed that miR-638 directly targets BRCA1. The aim of this study was to investigate the role of miR-638 in breast cancer prognosis and treatment.MethodsFormalin-fixed, paraffin-embedded (FFPE) breast cancer samples were microdissected into normal epithelial and invasive ductal carcinoma (IDC) cells, and total RNA was isolated. Several breast cancer cell lines were used for the functional analysis. miR-638 target genes were identified by TARGETSCAN-VERT 6.2 and miRanda. The expression of miR-638 and its target genes was analyzed by real-time qRT-PCR and Western blotting. Dual-luciferase reporter assay was employed to confirm the specificity of miR-638 target genes. The biological function of miR-638 was analyzed by MTT chemosensitivity, matrigel invasion and host cell reactivation assays.ResultsThe expression of miR-638 was decreased in IDC tissue samples compared to their adjacent normal controls. The decreased miR-638 expression was more prevalent in non-TNBC compared with TNBC cases. miR-638 expression was significantly downregulated in breast cancer cell lines compared to the immortalized MCF-10A epithelial cells. BRCA1 was predicted as one of the direct targets of miR-638, which was subsequently confirmed by dual-luciferase reporter assay. Forced expression of miR-638 resulted in a significantly reduced proliferation rate as well as decreased invasive ability in TNBC cells. Furthermore, miR-638 overexpression increased sensitivity to DNA-damaging agents, ultraviolet (UV) and cisplatin, but not to 5-fluorouracil (5-FU) and epirubicin exposure in TNBC cells. Host cell reactivation assays showed that miR-638 reduced DNA repair capability in post UV/cisplatin-exposed TNBC cells. The reduced proliferation, invasive ability, and DNA repair capabilities are associated with downregulated BRCA1 expression.ConclusionsOur findings suggest that miR-638 plays an important role in TNBC progression via BRCA1 deregulation. Therefore, miR-638 might serve as a potential prognostic biomarker and therapeutic target for breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0435-5) contains supplementary material, which is available to authorized users.

Differential expression of miR-139, miR-486 and miR-21 in breast cancer patients sub-classified according to lymph node status.

Therapeutic decisions in breast cancer are increasingly guided by prognostic and predictive biomarkers. Non-protein-coding microRNAs (miRNAs) have recently been found to be deregulated in breast cancers and, in addition, to be correlated with several clinico-pathological features. One of the most consistently up-regulated miRNAs is miR-21. Here, we specifically searched for differentially expressed miRNAs in high-risk breast cancer patients as compared to low-risk breast cancer patients. In the same patients, we also compared miR-21 expression with the expression of its presumed target PTEN. Both microarray and RT-qPCR techniques were used to assess miRNA expression levels in lymph node-positive and -negative human invasive ductal carcinoma tissues. Simultaneously, PTEN protein expression levels were assessed using immunohistochemistry. miR-486-5p and miR-139-5p were found to be down-regulated in patients with lymph node metastases, whereas miR-21 was found to be up-regulated in patients with a positive lymph node status. miR-21 expression levels were found to significantly correlate with tumour size (r = 0.403, p = 0.009; Spearman's rank), whereas no relation was found between miR-21 and PTEN expression levels (Kruskal-Wallis test). Down-regulation of miR-486-5p and miR-139-5p, in conjunction with up-regulation of miR-21, may represent a useful signature for the identification of high-risk breast cancer patients.

C-X-C motif chemokine 12/C-X-C chemokine receptor type 7 signaling regulates breast cancer growth and metastasis by modulating the tumor microenvironment.

IntroductionAlthough C-X-C motif chemokine 12 (CXCL12) has been shown to bind to C-X-C chemokine receptor type 7 (CXCR7), the exact molecular mechanism regulations by CXCL12/CXCR7 axis in breast tumor growth and metastasis are not well understood. CXCR7 expression has been shown to be upregulated during pathological processes such as inflammation and cancer.MethodsBreast cancer cell lines were genetically silenced or pharmacologically inhibited for CXCR7 and/or its downstream target signal transducer and activator of transcription 3 (STAT3). 4T1 or 4T1 downregulated for CXCR7 and 4T1.2 breast cancer cell lines were injected in mammary gland of BALB/c mice to form tumors, and the molecular pathways regulating tumor growth and metastasis were assessed.ResultsIn this study, we observed that CXCL12 enhances CXCR7-mediated breast cancer migration. Furthermore, genetic silencing or pharmacologic inhibition of CXCR7 reduced breast tumor growth and metastasis. Further elucidation of mechanisms revealed that CXCR7 mediates tumor growth and metastasis by activating proinflammatory STAT3 signaling and angiogenic markers. Furthermore, enhanced breast tumorigenicity and invasiveness were associated with macrophage infiltration. CXCR7 recruits tumor-promoting macrophages (M2) to the tumor site through regulation of the macrophage colony-stimulating factor (M-CSF)/macrophage colony-stimulating factor receptor (MCSF-R) signaling pathway. In addition, CXCR7 regulated breast cancer metastasis by enhancing expression of metalloproteinases (MMP-9, MMP-2) and vascular cell-adhesion molecule-1 (VCAM-1). We also observed that CXCR7 is highly expressed in invasive ductal carcinoma (IDC) and metastatic breast tissue in human patient samples. In addition, high CXCR7 expression in tumors correlates with worse prognosis for both overall survival and lung metastasis-free survival in IDC patients.ConclusionThese observations reveal that CXCR7 enhances breast cancer growth and metastasis via a novel pathway by modulating the tumor microenvironment. These findings identify CXCR7-mediated STAT3 activation and modulation of the tumor microenvironment as novel regulation of breast cancer growth and metastasis. These studies indicate that new strategies using CXCR7 inhibitors could be developed for antimetastatic therapy.

Complexity in Regulation of microRNA Machinery Components in Invasive Breast Carcinoma

Altered expression of microRNA (miRNA) machinery components may play an important role in breast cancer progression. The objective of the current study was to evaluate Drosha, the DiGeorge syndrome critical region gene 8 (DGCR8), Dicer, and Argonaute 2 (AGO2) mRNA expression in invasive breast carcinoma (IBC) and to assess the value of clinical parameters on their expression. By using quantitative real-time PCR, we examined the expression of the four miRNA machinery components in 52 breast tumor tissues which are diagnosed as invasive ductal carcinoma and adjacent non-neoplastic tissues. In the present study, decreased mRNA expression levels of major miRNA machinery components were observed in IBC. The altered mRNA expression levels of DGCR8 and AGO2 are positively correlated with to each other. This study revealed for the first time that expression alterations of DGCR8 are significantly associated with estrogen receptor and Ki-67 status in IBC. Moreover, AGO2 mRNA expression level was significantly correlated with N stage. These results provided evidences that down-regulated the four miRNA machinery components may play an important role in breast pathobiology and that DGCR8 and AGO2 might be associated with important clinical factors.

An N-terminal splice variant of human Stat5a that interacts with different transcription factors is the dominant form expressed in invasive ductal carcinoma

We have identified a new variant of human Stat5a, found at higher ratios to full-length Stat5a in invasive ductal carcinoma versus contiguous normal tissue. The variant, missing exon 5, inhibits p21 and Bax production and increases cell number. After prolactin stimulation, only full-length Stat5a interacts with the vitamin D and retinoid X receptors, whereas only Δ5 Stat5a interacts with activating protein 1–2 and specificity protein 1. Prolactin also oppositely regulates interaction of the two Stat5a forms with β-catenin. We propose that a change in splicing leading to upregulation of this new isoform is a pathogenic aspect of invasive ductal carcinoma.

Prognostic significance of RSPO1, WNT1, P16, WT1, and SDC1 expressions in invasive ductal carcinoma of the breast

BackgroundTo better understand the mechanisms of the SDC1 expression in invasive ductal carcinoma, we studied the correlations between SDC1 expression and related gene expressions (RSPO1, WNT1, WT1, and P16).MethodsUsing 100 cases of invasive ductal carcinoma tissue, we screened expressions of RSPO1, WNT1, WT1, P16, and SDC1 using immunohistochemistry. We analyzed the association between the immunoreactivities and clinicopathological parameters.ResultsWT1 expression was associated with tumor grade. RSPO1 expression was associated with progesterone receptor expression. Expressions of RSPO1, WT1, and P16 were significantly associated with disease-free survival. RSPO1 and P16 showed statistically significant hazard ratios. SDC1 ectodomain expression was significantly associated only with P16 expression. Immunoreactivity of SDC1 cytoplasmic domain was associated with WT1 and WNT1. However, WNT1 expression failed to show a significant association with disease-free survival.ConclusionsRSPO1 and P16 immunoreactivity was found to be an independent prognostic indicator in invasive ductal cancer. Cytoplasmic expression of SDC1 is positively correlated with tumor-prone proteins (WT1 and WNT1) and membranous expression of SDC1 is positively correlated with the tumor suppressor (P16).

Transient receptor potential melastatin 7 is involved in oestrogen receptor-negative metastatic breast cancer cells migration through its kinase domain

Oestrogen receptor negative (ER−) invasive ductal carcinoma (IDC) represents a significant clinical challenge and therefore prompts the discovery of novel biomarkers. Transient receptor potential melastatin 7 (TRPM7), a channel protein that also contains a regulatory kinase domain, is overexpressed in IDC and regulates migration. However, the molecular mechanism remains poorly defined. Here, we examined whether TRPM7 regulates migration by its channel function or by its kinase domain. A Magnesium Inhibited Cation current was recorded in two ER− highly metastatic breast cancer cell lines. Down-regulation of TRPM7 neither affected Ca2+-, nor Mg2+-homoeostasis but significantly reduced cell migration via a Ca2+-independent pathway. Notably, the overexpression of the truncated kinase domain form of TRPM7 decreased cell migration, while the overexpression of the wild-type form strongly increased it. Concomitantly, TRPM7 silencing reduced the myosin IIA heavy chain phosphorylation. Furthermore, we found higher TRPM7 expression in ER− IDC tissues and lymph nodes than in the non-invasive tumoural samples. In conclusion, TRPM7 plays a critical role in breast cancer cell migration through its kinase domain, and our data support the consideration of using TRPM7 as a novel biomarker and a potential therapeutic target in the treatment of human ER− IDC.

MO‐D‐141‐08: Multi‐Scale Analysis of Collagen Architecture for Classifying Tumor and Healthy Breast Tissue Images

Purpose: To quantify differences in collagen microarchitecture between healthy human breast tissue and breast cancer images using extended field of view second harmonic generation (EFOV SHG) microscopy coupled with analysis by the curvelet transform. Methods: Two frozen samples of normal associated human breast tissue (NAT) and invasive ductal carcinoma (IDC) were obtained through the University of Wisconsin BioBank. Samples were thawed, fixed, embedded in agar, cut into 300 micron thick sections using a vibratome and imaged using a custom inverted SHG microscope designed to allow for EFOV imaging. Individual SHG Images were captured at 0.79 micron resolution and a FOV of approximately 200 microns. The FOV was extended by stage scanning and image stitching to produce submicron resolution fields of view up to 2 cm2. NAT and IDC tissues were imaged, stitched using the Grid/Collection Stitching ImageJ plugin, and analyzed using features derived from the curvelet transform including variance in orientation and prevalence at various scales. These novel features were tested using support vector machine (SVM) cross validation. Results: IDC tissue contained linearly aligned, crisscrossing collagen while NAT tissue contained wavy, dense collagen. The mean curvelet alignment value for the normal images was approximately 2X higher than that for IDC. In addition, normal images had approximately 20% fewer closely correlated curvelets compared to IDC. Three‐fold cross validation of a linear SVM classifier revealed average sensitivity and specificity of 0.994 and 0.969 respectively. Conclusion: These results demonstrate the excellent potential for collagen architecture quantification based on features derived from the curvelet transform. We have also shown the value of EFOV SHG imaging to capture both large and small scale features necessary for quantifying collagen morphology. Future work will improve EFOV SHG imaging throughput and test classification capabilities of the curvelet based feature set on a large sample population. NIH R01 grants CA114462 and CA136590 to P.J.K. and K.W.E., T32 CA009206 to J.S.B and the Morgridge Institute for Research