Eric C. Dietze

Triple-negative breast cancer in African-American women: disparities versus biology

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that disproportionately affects BRCA1 mutation carriers and young women of African origin. There is evidence that African-American women with TNBC have worse clinical outcomes than women of European descent. However, it is unclear whether survival differences persist after adjusting for disparities in access to health-care treatment, co-morbid disease and income. It remains controversial whether TNBC in African-American women is a molecularly distinct disease or whether African-American women have a higher incidence of aggressive biology driven by disparities: there is evidence in support of both. Understanding the relative contributions of biology and disparities is essential for improving the poor survival rate of African-American women with TNBC.

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)

Morphologically normal-appearing mammary epithelial cells obtained from high-risk women exhibit methylation silencing of INK4a/ARF.

Purpose: p16(INK4a) has been appreciated as a key regulator of cell cycle progression and senescence. Cultured human mammary epithelial cells that lack p16(INK4a) activity have been shown to exhibit premalignant phenotypes, such as telomeric dysfunction, centrosomal dysfunction, a sustained stress response, and, most recently, a dysregulation of chromatin remodeling and DNA methylation. These data suggest that cells that lack p16(INK4a) activity would be at high risk for breast cancer development and may exhibit an increased frequency of DNA methylation events in early cancer. Experimental Design: To test this hypothesis, the frequencies of INK4a/ARF promoter hypermethylation, as well as four additional selected loci, were tested in the initial random periareolar fine needle aspiration samples from 86 asymptomatic women at high risk for development of breast cancer, stratified using the Masood cytology index. Results:INK4a/ARF promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia and, importantly, in morphologically normal-appearing mammary epithelial cells; 29 of 86 subjects showed INK4a/ARF promoter hypermethylation in at least one breast. Importantly, INK4a/ARF promoter hypermethylation was not associated with atypia, and the frequency of hypermethylation did not increase with increasing Masood cytology score. The frequency of INK4a/ARF promoter hypermethylation was associated with the combined frequency of promoter hypermethylation of retinoic acid receptor-β2, estrogen receptor-α, and breast cancer-associated 1 genes (P = 0.001). Conclusions: Because INK4a/ARF promoter hypermethylation does not increase with age but increases with the frequency of other methylation events, we predict that INK4a/ARF promoter hypermethylation may serve as a marker of global methylation dysregulation.

Interferon-regulatory factor-1 is critical for tamoxifen-mediated apoptosis in human mammary epithelial cells

Unlike estrogen receptor-positive (ER(+)) breast cancers, normal human mammary epithelial cells (HMECs) typically express low nuclear levels of ER (ER poor). We previously demonstrated that 1.0 μM tamoxifen (Tam) promotes apoptosis in acutely damaged ER-poor HMECs through a rapid, ‘nonclassic’ signaling pathway. Interferon-regulatory factor-1 (IRF-1), a target of signal transducer and activator of transcription-1 transcriptional regulation, has been shown to promote apoptosis following DNA damage. Here we show that 1.0 μM Tam promotes apoptosis in acutely damaged ER-poor HMECs through IRF-1 induction and caspase-1/3 activation. Treatment of acutely damaged HMEC-E6 cells with 1.0 μM Tam resulted in recruitment of CBP to the γ-IFN-activated sequence element of the IRF-1 promoter, induction of IRF-1, and sequential activation of caspase-1 and -3. The effects of Tam were blocked by expression of siRNA directed against IRF-1 and caspase-1 inhibitors. These data indicate that Tam induces apoptosis in HMEC-E6 cells through a novel IRF-1-mediated signaling pathway that results in activated caspase-1 and -3.

Suppression of p53 function in normal human mammary epithelial cells increases sensitivity to extracellular matrix–induced apoptosis

Little is known about the fate of normal human mammary epithelial cells (HMECs) that lose p53 function in the context of extracellular matrix (ECM)–derived growth and polarity signals. Retrovirally mediated expression of human papillomavirus type 16 (HPV-16) E6 and antisense oligodeoxynucleotides (ODNs) were used to suppress p53 function in HMECs as a model of early breast cancer. p53+ HMEC vector controls grew exponentially in reconstituted ECM (rECM) until day 6 and then underwent growth arrest on day 7. Ultrastructural examination of day 7 vector controls revealed acinus-like structures characteristic of normal mammary epithelium. In contrast, early passage p53− HMEC cells proliferated in rECM until day 6 but then underwent apoptosis on day 7. p53− HMEC-E6 passaged in non-rECM culture rapidly (8–10 passages), lost sensitivity to both rECM-induced growth arrest and polarity, and also developed resistance to rECM-induced apoptosis. Resistance was associated with altered expression of α3-integrin. Treatment of early passage p53− HMEC-E6 cells with either α3- or β1-integrin function-blocking antibodies inhibited rECM-mediated growth arrest and induction of apoptosis. Our results indicate that suppression of p53 expression in HMECs by HPV-16 E6 and ODNs may sensitize cells to rECM-induced apoptosis and suggest a role for the α3/β1-heterodimer in mediating apoptosis in HMECs grown in contact with rECM.

CBP/p300 induction is required for retinoic acid sensitivity in human mammary cells.

The coactivators CBP and p300 are recruited by retinoic acid receptors (RARs) during retinoid mediated transcriptional regulation. To assess the role of CBP/p300 in all-trans-retinoic acid (ATRA)-mediated growth arrest in mammary epithelial cells, two systems were tested: (1) ATRA resistant MCF-7 cells were transduced with a functional RAR-beta 2; (2) normal human mammary epithelial cells (HMECs) were transduced with a pan-RAR dominant negative, RAR-alpha 403. Expression of RAR-beta 2 in MCF-7 cells resulted in increased sensitivity to ATRA-induced growth arrest and correlated with induction of CBP/p300 mRNA and protein. Inhibition of RAR function in HMECs resulted in resistance to ATRA-induced growth arrest and loss of CBP/p300 induction. Antisense suppression of CBP/p300 in HMECs resulted in decreased retinoic acid response element reporter trans-activation and decreased ATRA-mediated growth arrest. Thus, in human mammary epithelial cells, CBP/p300 were both modulated by an ATRA signaling pathway and were required for a normal response to ATRA.

Tamoxifen and tamoxifen ethyl bromide induce apoptosis in acutely damaged mammary epithelial cells through modulation of AKT activity

Normal human mammary epithelial cells (HMECs), unlike estrogen receptor-positive (ER+) breast cancers, typically express low nuclear levels of ER (ER-‘poor’). We previously demonstrated that 1.0 μM tamoxifen (Tam) induced apoptosis in ER-‘poor’ HMECs acutely transduced with human papillomavirus-16 E6 (HMEC-E6) through a rapid mitochondrial signaling pathway. Here, we show that plasma membrane-associated E2-binding sites initiate the rapid apoptotic effects of Tam in HMEC-E6 cells through modulation of AKT activity. At equimolar concentrations, Tam and tamoxifen ethyl bromide (QTam), a membrane impermeant analog of Tam, rapidly induced apoptosis in HMEC-E6 cells associated with an even more rapid decrease in phosphorylation of AKT at serine-473. Treatment of HMEC-E6 cells with 1.0 μM QTam resulted in a 50% decrease in mitochondrial transmembrane potential, sequential activation of caspase-9 and -3, and a 90% decrease in AKT Ser-473 phosphorylation. The effects of both Tam and QTam were blocked by expression of constitutively active AKT (myristoylated AKT or AKT-Thr308Asp/Ser473Asp). These data indicate that Tam and QTam induce apoptosis in HMEC-E6 cells through a plasma membrane-activated AKT-signaling pathway that results in (1) decreased AKT phosphorylation at Ser-473, (2) mitochondrial membrane depolarization, and (3) activated caspase-9 and -3.

CREB-binding protein regulates apoptosis and growth of HMECs grown in reconstituted ECM via laminin-5

Interactions between normal mammary epithelial cells and extracellular matrix (ECM) are important for mammary gland homeostasis. Loss of interactions between ECM and normal mammary epithelial cells are thought to be an early event in mammary carcinogenesis. CREB-binding protein (CBP) is an important regulator of proliferation and apoptosis but the role of CBP in ECM signaling is poorly characterized. CBP was suppressed in basal-cytokeratin-positive HMECs (CK5/6+, CK14+, CK8–, CK18–, CK19–). Suppression of CBP resulted in loss of reconstituted ECM-mediated growth control and apoptosis and loss of laminin-5 α3-chain expression. Suppression of CBP in normal human mammary epithelial cells (HMECs) resulted in loss of CBP occupancy of the LAMA3A promoter and decreased LAMA3A promoter activity and laminin-5 α-3 chain expression. Exogenous expression of CBP in CBP-negative HMECs that have lost reconstituted ECM-mediated growth regulation and apoptosis resulted in (1) CBP occupancy of the LAMA3A promoter, (2) increased LAMA3A activity and laminin-5 α3-chain expression, and (3) enhancement of reconstituted ECM-mediated growth regulation and apoptosis. Similarly, suppression of laminin-5 α3-chain expression in HMECs resulted in loss of reconstituted ECM-mediated growth control and apoptosis. These observations suggest that loss of CBP in basal-cytokeratin-positive HMECs results in loss of reconstituted ECM-mediated growth control and apoptosis through loss of LAMA3A activity and laminin-5 α3-chain expression. Results in these studies may provide insight into early events in basal-type mammary carcinogenesis.

Metabolic Syndrome and Breast Cancer Risk: Is There a Role for Metformin?

Obesity is one of the most important known preventable causes of cancer, accounting for up to 20% of cancer deaths in women. Obese women have increased risk of dying from breast cancer as well as an increased risk of distant metastasis. Metabolic Syndrome (MetSyn) is a group of metabolic conditions that include 1) abdominal obesity, 2) atherogenic dyslipidemia, 3) elevated blood pressure, and 4) insulin resistance. MetSyn is known to promote the development of cardiovascular disease and diabetes and may be associated with increased breast cancer risk. Emerging evidence supports an association between mammary adipocytes and their secreted adipocytokines and breast cancer initiation and progression. Metformin (1,1-dimethylbiguanide hydrochloride) is a drug used to treat type 2 diabetes and MetSyn. We review the potential association between MetSyn in promoting breast cancer and emerging evidence for the use of metformin in cancer prevention.

Interferon regulatory factor-1 regulates reconstituted extracellular matrix (rECM)-mediated apoptosis in human mammary epithelial cells.

Interactions between extracellular matrix (ECM) and mammary epithelial cells are critical for mammary gland homeostasis and apoptotic signaling. Interferon regulatory factor-1 (IRF-1) is a transcriptional regulator that promotes apoptosis during mammary gland involution and p53-independent apoptosis. We have recently shown that rapid cell surface tamoxifen (Tam) signaling promotes apoptosis in normal human mammary epithelial cells that were acutely damaged by expression of human papillomavirus type-16 E6 protein (*HMEC-E6). Apoptosis was mediated by recruitment of CREB-binding protein (CBP) to the γ-activating sequence (GAS) element of the IRF-1 promoter, induction of IRF-1 and caspase-1/-3 activation. Here, we show that growth factor-depleted, reconstituted ECM (rECM), similar to Tam, promotes apoptosis in *HMEC-E6 cells through induction of IRF-1. Apoptosis was temporally associated with recruitment of CBP to the GAS element of the IRF-1 promoter, induction of IRF-1 expression and caspase-1/-3 activation. Small interfering RNA-mediated suppression of IRF-1 protein expression in *HMEC-E6 cells blocked (1) induction of IRF-1, (2) caspase-1/-3 activation and (3) apoptosis. These observations demonstrate that IRF-1 promotes rECM-mediated apoptosis and provide evidence that both rECM and rapid Tam signaling transcriptionally activate IRF-1 through recruitment of CBP to the IRF-1 GAS promoter complex.