Non-neoplastic Breast Epithelium Research Articles

Over-expression of miR-183-5p or miR-492 triggers invasion and proliferation and loss of polarity in non-neoplastic breast epithelium

microRNAs (miRNAs) serve as novel noninvasive cancer biomarkers. In an HMT-3522 S1 (S1) breast epithelial risk-progression three-dimensional (3D) culture model, non-neoplastic S1 cells form a fully polarized epithelium. When silenced for the gap junction and tumor suppressor Cx43, Cx43-KO-S1 cells recapitulate pre-neoplastic phenotypes observed in tissues at risk for breast cancer in vivo. To delineate the role of miRNAs in breast tumorigenesis and identify key miRNA players in breast epithelial polarity, the miRNA profile specific to Cx43 loss in Cx43-KO-S1 compared to S1 cells was sequenced, revealing 65 differentially expressed miRNAs. A comparative analysis was conducted between these miRNAs and tumor-associated miRNAs from a young Lebanese patient validation cohort. miR-183-5p, downstream of Cx43 loss, was commonly upregulated in the patient cohort and the 3D culture model. miR-492, not attributed to Cx43 loss, was only specifically up-regulated in the young Lebanese patients. Ectopic expression of either miR-183-5p or miR-492 in S1 cells, through pLenti-III-miR-GPF vectors, resulted in the formation of larger multi-layered acini devoid of lumen, with disrupted epithelial polarity, as shown by an altered localization of Cx43, ß-catenin and Scrib, and decreased nuclear circularity in 3D cultures. Enhanced proliferation and invasion capacity were also observed. Over-expression of miR-183-5p or miR-492, therefore, induces pre-neoplastic phenotypes similar to those reported upon Cx43 loss, and may act as oncomiRs and possible biomarkers of increased breast cancer risk.

Hyaluronic Acid-Targeted Stimuli-Sensitive Nanomicelles Co-Encapsulating Paclitaxel and Ritonavir to Overcome Multi-Drug Resistance in Metastatic Breast Cancer and Triple-Negative Breast Cancer Cells

Active targeting and overcoming multi-drug resistance (MDR) can be some of the important attributes of targeted therapy for metastatic breast cancer (MBC) and triple-negative breast cancer (TNBC) treatment. In this study, we constructed a hyaluronic acid (HA)-decorated mixed nanomicelles-encapsulating chemotherapeutic agent paclitaxel (PTX) and P-glycoprotein inhibitor ritonavir (RTV). HA was conjugated to poly (lactide) co-(glycolide) (PLGA) polymer by disulfide bonds (HA-ss-PLGA). HA is a natural ligand for CD44 receptors overexpressed in breast cancer cells. Disulfide bonds undergo rapid reduction in the presence of glutathione, present in breast cancer cells. The addition of RTV can inhibit the P-gp and CYP3A4-mediated metabolism of PTX, thus aiding in reversing MDR and sensitizing the cells toward PTX. An in vitro uptake and cytotoxicity study in MBC MCF-7 and TNBC MDA-MB-231 cell lines demonstrated the effective uptake of the nanomicelles and drug PTX compared to non-neoplastic breast epithelium MCF-12A cells. Interestingly, in vitro potency determination showed a reduction in mitochondrial membrane potential and reactive oxygen species in breast cancer cell lines, indicating effective apoptosis of cancer cells. Thus, stimuli-sensitive nanomicelles along with HA targeting and RTV addition can effectively serve as a chemotherapeutic drug delivery agent for MBC and TNBC.

Potential biomarkers of ductal carcinoma in situ progression

BackgroundDuctal carcinoma in situ is a non-obligate precursor of invasive breast carcinoma and presents a potential risk of over or undertreatment. Finding molecular biomarkers of disease progression could allow for more adequate patient treatment. We aimed to identify potential biomarkers that can predict invasiveness risk.MethodsIn this epithelial cell-based study archival formalin-fixed paraffin-embedded blocks from six patients diagnosed with invasive lesions (pure invasive ductal carcinoma), six with in-situ lesions (pure ductal carcinoma in situ), six with synchronous lesions (invasive ductal carcinoma with an in-situ component) and three non-neoplastic breast epithelium tissues were analyzed by gene expression profiling of 770 genes, using the nCounter® PanCancer Pathways panel of NanoString Technologies.ResultsThe results showed that in comparison with non-neoplastic tissue the pure ductal carcinoma in situ was one with the most altered gene expression profile. Comparing pure ductal carcinoma in situ and in-situ component six differentially expressed genes were found, three of them (FGF2, GAS1, and SFRP1), play a role in cell invasiveness. Importantly, these genes were also differentially expressed between invasive and noninvasive groups and were negatively regulated in later stages of carcinogenesis.ConclusionsWe propose these three genes (FGF2, GAS1, and SFRP1) as potential biomarkers of ductal carcinoma in situ progression, suggesting that their downregulation may be involved in the transition of stationary to migrating invasive epithelial cells.

Connexin 43 Loss Triggers Cell Cycle Entry and Invasion in Non-Neoplastic Breast Epithelium: A Role for Noncanonical Wnt Signaling.

(1) Background: The expression of connexin 43 (Cx43) is disrupted in breast cancer, and re-expression of this protein in human breast cancer cell lines leads to decreased proliferation and invasiveness, suggesting a tumor suppressive role. This study aims to investigate the role of Cx43 in proliferation and invasion starting from non-neoplastic breast epithelium. (2) Methods: Nontumorigenic human mammary epithelial HMT-3522 S1 cells and Cx43 shRNA-transfected counterparts were cultured under 2-dimensional (2-D) and 3-D conditions. (3) Results: Silencing Cx43 induced mislocalization of β-catenin and Scrib from apicolateral membrane domains in glandular structures or acini formed in 3-D culture, suggesting the loss of apical polarity. Cell cycle entry and proliferation were enhanced, concomitantly with c-Myc and cyclin D1 upregulation, while no detectable activation of Wnt/β-catenin signaling was observed. Motility and invasion were also triggered and were associated with altered acinar morphology and activation of ERK1/2 and Rho GTPase signaling, which acts downstream of the noncanonical Wnt pathway. The invasion of Cx43-shRNA S1 cells was observed only under permissive stiffness of the extracellular matrix (ECM). (4) Conclusion: Our results suggest that Cx43 controls proliferation and invasion in the normal mammary epithelium in part by regulating noncanonical Wnt signaling.

Abstract 4505: Silencing connexin 43 induces cell cycle entry and invasion in non-neoplastic mammary epithelial cells

Abstract Studies demonstrated loss of connexin 43 (Cx43) expression in breast cancer cells in-vitro and in-vivo. Our previous studies have shown that silencing Cx43 in HMT-3522 S1 cells, non-neoplastic human mammary epithelial cells (S1 cells), altered apical polarity and mitotic spindle orientation under 3-dimensional (3-D) culture conditions. The purpose of this study is to investigate the effects of Cx43 loss on proliferation and invasion of non-neoplastic breast epithelium. Cx43 shRNA-transfected and control S1 cells were cultured under 2-D and 3-D conditions. Cx43-knockdown S1 cells exhibited enhanced proliferation both in 2-D and 3-D throughout the culture period, as shown by the increased cell counts and acinar size, respectively. In addition, cell cycle entry was enhanced, as indicated by the increased percentages of cells in S and G2/M phases and the reduced percentage of cells in G0/G1 phase. Cx43-silenced S1 cells upregulated the expression of proliferation genes, c-Myc and cyclin D1, in 2-D and 3-D, but maintained β-catenin levels. Furthermore, immunoprecipitation studies showed that Cx43 associated with β-catenin and ZO-2 in 2-D and 3-D cultures of S1 cells, and co-localized with β-catenin in 3-D, suggesting assembly of gap junction complexes in S1 cells. The loss of Cx43 caused mis-localization of β-catenin in 3-D cultures of S1 cells, suggesting involvement of the Wnt/β-catenin pathway downstream of Cx43 signaling. β-Catenin primarily exhibited apico-lateral distribution in S1 cells and re-localized to basolateral membrane domains in Cx43 knockdown cells. Silencing Cx43 enhanced migration of S1 cells by 40%, as shown by time-lapse imaging. In addition, Cx43-silenced S1 cells exhibited loss of the characteristic spheroid morphology and acquired a migratory phenotype in acini. Matrigel-invasion and MMP-9 activity were also enhanced. Cx43-silenced S1 cells were capable of invading the matrigel by two-fold compared to control cells. This was concomitant with enhanced expression and activity levels of Rho GTPases (RhoA, Rac1 and Cdc42), suggesting that Cx43 regulates non-canonical Wnt signaling, as a mechanism to induce migration and invasion. We propose a role for Cx43 loss in triggering proliferation and invasion events in non-neoplastic breast epithelium downstream of disrupted Wnt signaling. Citation Format: Sabreen F. Fostok, Dana Bazzoun, Sophie A. Lelievre, Mirvat El-Sibai, Rabih S. Talhouk. Silencing connexin 43 induces cell cycle entry and invasion in non-neoplastic mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4505.

Abstract P1-07-01: Attainment of extremely high concordance rates between fluorescence in situ hybridization and immunohistochemistry in testing for human epidermal growth factor receptor 2 (HER2) in breast cancer using a normalized scoring system for immunohistochemistry: a four year experience with over 9000 cases

Abstract Background: The 2007 ASCO-CAP guidelines mandate a high level (95%) of concordance between different testing modalities such as fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) for demonstration of the human epidermal growth factor receptor 2 (HER2) status in breast cancer. As few laboratories had been able to attain this level of concordance, the ASCO-CAP guidelines mandated regulation of pre-analytical variables such as fixation time in an attempt to improve concordance. We wished to test our hypothesis that the major reason for discordance between IHC and FISH results is false positive IHC, i.e., IHC 3+ immunostaining in cases which are non amplified by FISH, and that the vast majority of these false positives can be eliminated by using a normalized IHC scoring system. Materials and Methods: A total of 9,022 breast cancer cases, representing all those submitted to PhenoPath Laboratories for FISH testing from pathology laboratories around the United States between September 2008 and May 2012, had parallel sections immunostained with the rabbit monoclonal antibody SP3 to the HER2 gene product, with localization via a polymer based detection system. Tumor was scored according to ASCO-CAP criteria, as 0, 1+, 2+ or 3+; however, scoring was tabulated in two ways. The first was an absolute HER2 score based solely on analysis of the carcinoma, and the second was a normalized HER2 score, obtained by subtracting the score of the non-neoplastic breast epithelium from that of the infiltrating cancer. FISH was performed using the Vysis PathVysion combined HER2 and CEP17 probe kit with morphometric analysis performed using a MetaSystems image analysis system that incorporated the Metafer software. A score of > 2.2 was required for classification as amplified, and <1.8 for classification as non amplified. Results: Using the standard scoring system, negative concordance (0 or 1+ IHC, non-amplified by FISH) was obtained in 3448/3496 (98.6%) of cases, and positive concordance (3+ IHC, amplified by FISH) was obtained in 502/666 (75.3%) of cases, indicating the presence of significant numbers of falsely IHC positive cases. However, using the normalization method, the negative concordance rate remained unchanged at 3903/3960 (98.6%) but the positive concordance rate improved to 450/463 (97.2%). The absolute number of false IHC positive cases, using the normalization method, was reduced by over 90%, from 164 to 13. Discussion: Extremely high concordance rates, with elimination of the vast majority of false IHC positive cases, can be attained in a clinical laboratory examining tissues from divergent sources, even in the absence of controlling pre-analytical variables, by employing a normalized scoring system for IHC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-01.

Evaluation of estrogen and progesterone receptors in non-neoplastic breast tissue of women of reproductive age exposed to tamoxifen and raloxifene: a randomized, double-blind study

The objective of the present study was to compare the effects of tamoxifen and raloxifene in non-neoplastic breast epithelium. A randomized, double-blind study was carried out in 57 ovulatory, premenopausal women of 18-40 years of age, who had been diagnosed with fibroadenoma of the breast. The patients were divided into three groups: Group A: placebo, n=20; Group B: tamoxifen 20 mg/day, n = 21; and Group C: raloxifene 60mg/day, n=16. The study medication was given for 22 days starting on the first day of the menstrual cycle. On the 23rd day, the fibroadenoma was removed and a sample of non-neoplastic breast tissue was collected for immunohistochemical evaluation of estrogen and progesterone receptors. Comparison between the mean percentages of stained nuclei in the three groups was performed by analysis of variance and multiple comparisons, using Tukey's method to compare pairwise means, with significance established at P < 0.05. Exposition to tamoxifen or raloxifene resulted in a significant and similar reduction in the mean percentage of stained nuclei for estrogen and progesterone receptors (P<0.0001). Tamoxifen and raloxifene reduce progesterone and estrogen receptor alpha expression significantly and to a similar extent in the non-neoplastic breast tissue of women of reproductive age.

Attenuated Podoplanin Staining in Breast Myoepithelial Cells

Although podoplanin expression in breast myoepithelial (ME) cells has been reported to be readily distinguishable from that of lymphatic endothelium (LE), we recently encountered a case in which attenuated expression led to an incorrect interpretation of lymphatic invasion. This prompted us to further analyze the pattern and intensity of podoplanin expression in LE and in ME cells surrounding both non-neoplastic and neoplastic breast epithelium in 42 cases of breast carcinoma. In addition, cases with presumed lymphatic invasion (tumor cells within podoplanin-positive lymphatic-like structures) were further characterized on the basis of histologic review and results of additional endothelial (CD31 and/or CD34) and ME (p63+/-calponin) immunostains. Normal LE always displayed a strong (3+) linear podoplanin expression pattern, whereas ME cells surrounding non-neoplastic breast epithelium had a granular, branching membranous staining pattern that was either moderate (2+) or strong (3+) in 13 (34%) and 25 (66%) foci, respectively. ME cells surrounding ductal carcinoma in situ (DCIS) displayed weaker (1+ to 2+) podoplanin expression with 26 (72%) foci showing only a residual thin/discontinuous pattern of expression, whereas the other 10 (28%) foci showed a pattern similar to that around non-neoplastic epithelium. Further evaluation of 10 foci of presumed lymphatic invasion confirmed the presence of lymphatic invasion in 6 (60%) cases, whereas 4 displayed surrounding ME cells and were thus interpreted as DCIS. One case, in which both DCIS and lymphatic invasion were unequivocally present, displayed a few nests of tumor cells surrounded by both podoplanin-positive and p63-positive cells in different areas of the periphery and could not be readily classified. These findings represent an important caveat in the diagnosis of lymphatic invasion and, accordingly, one should always interpret results of podoplanin staining in the context of the histologic appearance and/or consider evaluation of additional endothelial or ME immunostains, especially when the characteristically strong linear pattern of expression of LE is not evident.

High concordance between immunohistochemistry and fluorescence in situ hybridization testing for HER2 status in breast cancer requires a normalized IHC scoring system

The American Society of Clinical Oncologists and College of American Pathologists have recently released new guidelines for laboratory testing of HER2 status in breast cancer, which require high levels (95%) of concordance between immunohistochemistry positive (3+) and fluorescence in situ hybridization-amplified cases, and between immunohistochemistry negative (0/1+) and fluorescence in situ hybridization-nonamplified cases; these required levels of concordance are significantly higher than those found in most published studies. We tested the hypothesis that a modification of the HER2 immunohistochemistry scoring system could significantly improve immunohistochemistry and fluorescence in situ hybridization concordance. A total of 6604 breast cancer specimens were evaluated for HER2 status by both immunohistochemistry and fluorescence in situ hybridization using standard methodologies. Results were compared when the standard immunohistochemistry scoring system was replaced by a normalized scoring system in which the HER2 score was derived by subtracting the score on the non-neoplastic breast epithelium from that on the tumor cells. Among the 6604 tumors, using a non-normalized immunohistochemistry scoring system, 267/872 (30.6%) of the immunohistochemistry 3+ cases proved to be fluorescence in situ hybridization nonamplified, whereas using the normalized scoring system only 30/562 (5.3%) of immunohistochemistry 3+ cases proved to be ‘false positive’. The concordance rate between immunohistochemistry 3+ and fluorescence in situ hybridization-amplified cases using the normalized scoring method was 94.7%, whereas the concordance using the non-normalized method was only 69.4%. Extremely high concordance between immunohistochemistry and fluorescence in situ hybridization assessment of HER2 status in breast cancer is achievable, but to attain this high level of concordance, modification of the FDA-approved immunohistochemistry scoring system is required.

Microarray comparative genomic hybridization analysis of tubular breast carcinoma shows recurrent loss of the CDH13 locus on 16q

Tubular breast carcinoma is a highly differentiated carcinoma with an excellent prognosis. Distinct genetic alterations in tubular breast carcinoma cells have been described, especially broad genetic losses on the q-arm of chromosome 16. These are more common in lobular breast carcinoma and low-grade ductal carcinoma in situ than in ductal breast carcinoma and high-grade ductal carcinoma in situ. To further delineate the molecular changes involved in tubular breast carcinoma more precisely, we examined 23 formalin-fixed and paraffin wax-embedded tissue samples (21 of tubular breast carcinoma and 2 of nonneoplastic breast epithelium) by microarray-based comparative genomic hybridization focusing on 287 genomic target clones of oncogenes and tumor suppressor genes. The results obtained from all nonneoplastic tissue samples of breast epithelium indicate no DNA copy number changes. In the tubular breast carcinoma samples, the highest frequencies for DNA sequence copy number losses were detected for CDH13 (in 86% of the samples) and MSH2, KCNK12 (in 52% of the samples). The highest frequencies of DNA sequence copy number gains were detected for HRAS and D13S319XYZ (each in 62% of the samples). Using principal component analysis, 3 subgroups of tubular breast carcinomas showing relative genetic changes were identified. For validation, the most frequent DNA copy number loss for CDH13 (18/21) was confirmed using fluorescence in situ hybridization in 4 of 5 tubular breast carcinomas analyzed. The newly identified genes with considerable copy number changes may include so far unknown candidate genes for the development and progression of tubular breast carcinoma, such as CDH13. The study provides the starting point for further delineating their detailed influence on the pathogenesis of tubular breast carcinoma.

Estrogen and progesterone receptor levels in nonneoplastic breast epithelium of breast cancer cases versus benign breast biopsy controls

BackgroundPrevious studies and biological mechanisms of carcinogenesis suggest that the steroid receptor content of benign breast epithelium may be related to breast cancer risk. The objective in this study was to compare the levels of estrogen receptor-α (ER) and progesterone receptor (PR) in nonneoplastic breast epithelium between breast cancer cases and biopsy controls.MethodsBetween 1995 and 1997 at two sites (Women's College Hospital in Toronto and Kingston General Hospital), 667 women who were scheduled for diagnostic excisional breast biopsies completed a questionnaire providing personal information and agreed to allow analysis of routinely resected tissue. Histological slides with nonneoplastic epithelium were available for 101 cancer cases and 200 biopsy controls in Toronto and for 105 cancer cases and 119 controls in Kingston. Nonneoplastic epithelium was examined with immunohistochemical assays to determine the percent of epithelial cells staining for ER and PR. Unconditional logistic regression was used to calculate odds ratios (OR) stratified by study site.ResultsThe ER content of nonneoplastic tissue was higher in cases than biopsy controls in unadjusted analyses; after adjustment for age, however, a weak association remained in only one of the study sites. After adjustment for age, the PR content of nonneoplastic tissue was slightly lower in breast cancer cases than controls in one study site. Furthermore, this inverse association was confined to women with PR negative breast cancer in comparison to the controls. No interaction between ER and PR content of nonneoplastic tissue was observed in relation to the odds of having breast cancer.ConclusionThe results of this study are consistent with only a slight indication of increased ER levels in nonneoplastic tissue in breast cancer cases relative to controls. This study contributes to the understanding of breast cancer by examining both ER and PR in nonneoplastic tissue. Limitations remain, however, such as the necessity of using as controls women with benign breast changes, difficulties in selecting the appropriate tissue for analysis, and tissue sampling concurrent to diagnosis.

Mucin histochemistry of mucous carcinomas of breast and colon and non-neoplastic breast epithelium.

Although epithelial mucin has been demonstrated in ordinary mammary duct carcinomas, it is formed profusely in the uncommon mucous carcinomas. A histochemical study of mucous carcinomas of the breast and colon showed that both types produce similar mucins, viz, acid and neutral, although the mammary carcinomas produce more neutral mucin and small amounts of sulphated mucins are produced by colonic tumours. Similar studies on sections of normal breast and cystic mammary dysplasia have shown that intracellular neutral mucin was present in the ducts and lobules, and that intracellular acid mucin was present within cells of the terminal ducts and lobules. It was demonstrated enzymatically that the acid mucins produced by the normal and neoplastic tissues is sialic acid. Hence it can be shown that the mucin produced by mucous carcinomas is of epithelial rather than connective tissue type. It is contended that mucous carcinoma of breast arises by metaplasia of duct cells that have already undergone malignant change and not from the lobules of the breast.