Siya Lem

Optical Redox Ratio Differentiates Breast Cancer Cell Lines Based on Estrogen Receptor Status

Autofluorescence spectroscopy is a powerful imaging technique that exploits endogenous fluorophores. The endogenous fluorophores NADH and flavin adenine dinucleotide (FAD) are two of the principal electron donors and acceptors in cellular metabolism, respectively. The optical oxidation-reduction (redox) ratio is a measure of cellular metabolism and can be determined by the ratio of NADH/FAD. We hypothesized that there would be a significant difference in the optical redox ratio of normal mammary epithelial cells compared with breast tumor cell lines and that estrogen receptor (ER)-positive cells would have a higher redox ratio than ER-negative cells. To test our hypothesis, the optical redox ratio was determined by collecting the fluorescence emission for NADH and FAD via confocal microscopy. We observed a statistically significant increase in the optical redox ratio of cancer compared with normal cell lines (P < 0.05). Additionally, we observed a statistically significant increase in the optical redox ratio of ER(+) breast cancer cell lines. The level of ESR1 expression, determined by real-time PCR, directly correlated with the optical redox ratio (Pearsons correlation coefficient = 0.8122, P = 0.0024). Furthermore, treatment with tamoxifen and ICI 182,870 statistically decreased the optical redox ratio of only ER(+) breast cancer cell lines. The results of this study raise the important possibility that fluorescence spectroscopy can be used to identify subtypes of breast cancer based on receptor status, monitor response to therapy, or potentially predict response to therapy. This source of optical contrast could be a potentially useful tool for drug screening in preclinical models.

CpG Island Tumor Suppressor Promoter Methylation in Non-BRCA-Associated Early Mammary Carcinogenesis

Background: Only 5% of all breast cancers are the result of BRCA1/2 mutations. Methylation silencing of tumor suppressor genes is well described in sporadic breast cancer; however, its role in familial breast cancer is not known. Methods: CpG island promoter methylation was tested in the initial random periareolar fine-needle aspiration sample from 109 asymptomatic women at high risk for breast cancer. Promoter methylation targets included RARB (M3 and M4), ESR1, INK4a/ARF, BRCA1, PRA, PRB, RASSF1A, HIN-1, and CRBP1. Results: Although the overall frequency of CpG island promoter methylation events increased with age (P < 0.0001), no specific methylation event was associated with age. In contrast, CpG island methylation of RARB M4 (P = 0.051), INK4a/ARF (P = 0.042), HIN-1 (P = 0.044), and PRA (P = 0.032), as well as the overall frequency of methylation events (P = 0.004), was associated with abnormal Masood cytology. The association between promoter methylation and familial breast cancer was tested in 40 unaffected premenopausal women in our cohort who underwent BRCA1/2 mutation testing. Women with BRCA1/2 mutations had a low frequency of CpG island promoter methylation (15 of 15 women had ≤4 methylation events), whereas women without a mutation showed a high frequency of promoter methylation events (24 of 25 women had 5-8 methylation events; P < 0.0001). Of women with a BRCA1/2 mutation, none showed methylation of HIN-1 and only 1 of 15 women showed CpG island methylation of RARB M4, INK4a/ARF, or PRB promoters. Conclusions: This is the first evidence of CpG island methylation of tumor suppressor gene promoters in non-BRCA1/2 familial breast cancer. (Cancer Epidemiol Biomarkers Prev 2009;18(3):901–14)

ESR1 Promoter Hypermethylation Does Not Predict Atypia in RPFNA nor Persistent Atypia after 12 Months Tamoxifen Chemoprevention

Purpose: Currently, we lack biomarkers to predict whether high-risk women with mammary atypia will respond to tamoxifen chemoprevention. Experimental Design: Thirty-four women with cytologic mammary atypia from the Duke University High-Risk clinic were offered tamoxifen chemoprevention. We tested whether ESR1 promoter hypermethylation and/or estrogen receptor (ER) protein expression by immunohistochemistry predicted persistent atypia in 18 women who were treated with tamoxifen for 12 months and in 16 untreated controls. Results: We observed a statistically significant decrease in the Masood score of women on tamoxifen chemoprevention for 12 months compared with control women. This was a significant interaction effect of time (0, 6, and 12 months) and treatment group (tamoxifen versus control) P = 0.0007. However, neither ESR1 promoter hypermethylation nor low ER expression predicted persistent atypia in Random Periareolar Fine Needle Aspiration after 12 months tamoxifen prevention. Conclusions: Results from this single institution pilot study provide evidence that, unlike for invasive breast cancer, ESR1 promoter hypermethylation and/or low ER expression is not a reliable marker of tamoxifen-resistant atypia. (Cancer Epidemiol Biomarkers Prev 2008;17(8):1884–90)

Protein Microarray Analysis of Mammary Epithelial Cells from Obese and Nonobese Women at High Risk for Breast Cancer: Feasibility Data

Background: Obesity is a well-established risk factor for cancer, accounting for up to 20% of cancer deaths in women. Studies of women with breast cancer have shown obesity to be associated with an increased risk of dying from breast cancer and increased risk of developing distant metastasis. While previous studies have focused on differences in circulating hormone levels as a cause for increased breast cancer incidence in postmenopausal women, few studies have focused on potential differences in the protein expression patterns of mammary epithelial cells obtained from obese versus nonobese women. Methods: Protein expression was assessed by reverse-phase protein microarray in mammary epithelial cells from 31 random periareolar fine needle aspirations performed on 26 high-risk women. Results: In this pilot and exploratory study, vimentin (unadjusted P = 0.028) expression was significantly different between obese and nonobese women. Conclusions: Vimentin is integral both to adipocyte structure and function and to the epithelial-to-mesenchymal transition needed for cancer cell metastasis. Further research is needed to confirm this finding and determine the possible effects of the adipocyte microenvironment on the initiation and progression of breast cancer in high-risk women. Impact: Differential protein expression patterns obtained from a future expanded study may serve to elaborate the underlying pathology of breast cancer initiation and progression in obese women and identify potential biomarkers of response to preventative interventions such as dietary changes and exercise. Cancer Epidemiol Biomarkers Prev; 20(3); 476–82. ©2011 AACR.

Fluoxetine induces cytotoxic endoplasmic reticulum stress and autophagy in triple negative breast cancer

AIM To investigate the mechanism of action of lipophilic antidepressant fluoxetine (FLX) in representative molecular subtypes of breast cancer. METHODS The anti-proliferative effects and mechanistic action of FLX in triple-negative (SUM149PT) and luminal (T47D and Au565) cancer cells and non-transformed MCF10A were investigated. Reverse phase protein microarray (RPPM) was performed with and without 10 μmol/L FLX for 24 and 48 h to determine which proteins are significantly changed. Viability and cell cycle analysis were also performed to determine drug effects on cell growth. Western blotting was used to confirm the change in protein expression examined by RPPM or pursue other signaling proteins. RESULTS The FLX-induced cell growth inhibition in all cell lines was concentration- and time-dependent but less pronounced in early passage MCF10A. In comparison to the other lines, cell growth reduction in SUM149PT coincided with significant induction of endoplasmic reticulum (ER) stress and autophagy after 24 and 48 h of 10 μmol/L FLX, resulting in decreased translation of proteins along the receptor tyrosine kinase/Akt/mammalian target of rapamycin pathways. The increase in autophagy marker, cleaved microtubule-associated protein 1 light chain 3, in SUM149PT after 24 h of FLX was likely due to increased metabolic demands of rapidly dividing cells and ER stress. Consequently, the unfolded protein response mediated by double-stranded RNA-dependent protein kinase-like ER kinase resulted in inhibition of protein synthesis, growth arrest at the G1 phase, autophagy, and caspase-7-mediated cell death. CONCLUSION Our study suggests a new role for FLX as an inducer of ER stress and autophagy, resulting in death of aggressive triple negative breast cancer SUM149PT.

Reproducibility of Random Periareolar Fine Needle Aspiration in a Multi-Institutional Cancer and Leukemia Group B (CALGB) Cross-Sectional Study

Background: Random periareolar fine needle aspiration (RPFNA) is a research technique developed to assess short-term breast cancer risk in women at increased risk of breast cancer. Although there is increasing acceptance of RPFNA, neither the reproducibility nor the inter–institutional compatibility of RPFNA has been established. To address these key limitations, the Cancer and Leukemia Group B (CALGB) Prevention Group tested the reproducibility of RPFNA in a multi-institutional cross-sectional study. Methods: Sixty-three high-risk women from five CALGB institutions (Duke, Ohio State, Roswell Park, Dana Farber, and Vermont) underwent RPFNA from July 1, 2007 to June 30, 2008. Duplicate bilateral RPFNA was performed on each woman by a single investigator on a single day. Masood Cytology Index score was assessed by a single blinded cytopathologist. Results: There was a high degree of statistical agreement in the Masood Cytology Index scores of duplicate RPFNA samples from the same breast, with a Spearman correlation coefficient of 0.8312 (P < 0.0001). Importantly, although there was agreement in duplicate samples from the same breast, there was lack of agreement between duplicate samples from the opposite breast. Conclusions: This multi-institutional study shows that RPFNA is a highly reproducible measure of breast cytology in a cooperative group cross-sectional trial. RPFNA did not show a high degree of agreement between breasts, suggesting that breast cancer risk and progression may occur at different rates in individual breasts from a single woman. These studies provide proof-of-principle for future RPFNA-based cooperative group prevention studies. (Cancer Epidemiol Biomarkers Prev 2009;18(5):1379–85)

Abstract 4880: PELP1 localization as a biomarker for early mammary carcinogenesis and response to tamoxifen chemoprevention.

Progress in breast cancer prevention is currently limited by our lack of biological markers to identify which high‐risk women will progress to develop breast cancer and which women will respond to chemoprevention therapies, such as tamoxifen (Tam). While Tam has proven to be effective for breast cancer chemoprevention, Tam therapy only reduces the risk of developing breast cancer by approximately 50%. Therefore, identification of biomarkers is essential to determine which women will benefit from Tam chemoprevention. We have been studying the role of PELP1 in early mammary carcinogenesis and response to Tam chemoprevention. PELP1 subcellular localization is primarily nuclear in normal mammary epithelium, but it has been shown to be localized to the cytoplasm of The objective of our current research is to determine if cytoplasmic PELP1 expression promotes expression of a novel gene signature that predicts response to Tam chemoprevention. We are currently testing models of early mammary carcinogenesis (pre-stasis, post-stasis, and immortal) for their response to Tam in the presence and absence of cytoplasmic PELP1. Microarray analysis will be performed on RNA from cell line that represents the earliest point in mammary carcinogenesis in which we see an effect of PELP1 expression. We expect that these studies will provide us with gene signatures that can predict cytoplasmic PELP1 expression and Tam resistance in early mammary carcinogenesis. Gene signatures will then be validated using existing gene array databases. These studies have the potential to lead to additional targets for chemoprevention and benefit women who are resistant to Tam chemoprevention. Citation Format: Julie Hanson Ostrander, Brian Girard, Tarah M. Regan-Anderson, Siya Lem, Victoria L. Seewaldt. PELP1 localization as a biomarker for early mammary carcinogenesis and response to tamoxifen chemoprevention. [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 4880. doi:10.1158/1538-7445.AM2013-4880

Erratum: Pilot and feasibility study: Prospective proteomic profiling of mammary epithelial cells from high-risk women provides evidence of activation of pro-survival pathways (Breast Cancer Research and Treatment (2012) 132 (487-498) DOI: 10.1007/s10549-011-1609-9)

Catherine Ibarra-Drendall • Michelle M. Troch • William T. Barry • Gloria Broadwater • Emanuel F. Petricoin III • Julia Wulfkuhle • Lance A. Liotta • Siya Lem • Joseph C. Baker Jr. • Anne C. Ford • Lee G. Wilke • Carola Zalles • Nicole M. Kuderer • Abigail W. Hoffman • Melanie Shivraj • Priya Mehta • Jamila Williams • Nora Tolbert • Laurie W. Lee • Patrick G. Pilie • Dihua Yu • Victoria L. Seewaldt