Kathy Toy

Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1

Increased levels of enhancer of zeste homolog 2 (EZH2), a critical regulator of cellular memory, are associated with negative estrogen receptor (ER) expression and disease progression in breast cancer. High levels of EZH2 signal the presence of metastasis and poor outcome in breast cancer patients. To test the hypothesis that deregulation of EZH2 contributes to ER-negative breast cancer progression, EZH2 expression was inhibited in ER-negative breast cancer cells MDA-MB-231 and CAL51 using a lentivirus system. EZH2 knockdown decreased proliferation and delayed the G2/M cell-cycle transition, although not affecting apoptosis. In vivo, EZH2 downregulation significantly decreased breast xenograft growth and improved survival. EZH2 knockdown upregulated BRCA1 protein. Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 downregulation on proliferation, G2/M arrest, and on the levels of hyperphosphorylated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis. Invasive ER-negative breast carcinomas show significant overexpression of EZH2 and downregulation of BRCA1 proteins. Taken together, we show that EZH2 is important in ER-negative breast cancer growth in vivo and in vitro, and that BRCA1 is required for the proliferative effects of EZH2. Blockade of EZH2 may provide a prime target to prevent and/or halt ER-negative breast cancer progression.

EZH2 expands breast stem cells through activation of NOTCH1 signaling

Significance Triple-negative breast cancers comprise 10% of invasive breast carcinomas but are responsible for a disproportionate number of deaths and remain poorly understood. Unfortunately, current therapies are only weakly effective, and the median disease-free survival is 4 y among young women. Clinical studies support the relevance of Enhancer of Zeste Homolog 2 (EZH2) overexpression to the progression of triple-negative breast carcinomas. Our study shows that EZH2 acts as an activator of the NOTCH1 promoter and signaling to expand the stem cell pool, leading to accelerated breast cancer initiation and growth. We discovered that this function is independent of EZH2 histone methyltransferase activity and of its Polycomb Repressive Complex 2-binding partners, paving the way for novel therapeutic strategies. Breast cancer is the second-leading cause of cancer-related deaths in women, but the details of how it begins remain elusive. Increasing evidence supports the association of aggressive triple-negative (TN) breast cancer with heightened expression of the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) and increased tumor-initiating cells (TICs). However, mechanistic links between EZH2 and TICs are unclear, and direct demonstration of a tumorigenic function of EZH2 in vivo is lacking. Here, we identify an unrecognized EZH2/NOTCH1 axis that controls breast TICs in TN breast carcinomas. EZH2 overexpression increases NOTCH1 expression and signaling, and inhibition of NOTCH1 activity prevents EZH2-mediated stem cell expansion in nontumorigenic breast cells. We uncover a unique role of EZH2 in activating, rather than repressing, NOTCH1 signaling through binding to the NOTCH1 promoter in TN breast cancer cells. EZH2 binding is independent of its catalytic histone H3 lysine 27 methyltransferase activity and of the Polycomb Repressive Complex 2 but corresponds instead to transcriptional activation marks. In vivo, EZH2 knockdown decreases the onset and volume of xenografts derived from TN breast TICs. Conversely, transgenic EZH2 overexpression accelerates mammary tumor initiation and increases NOTCH1 activation in mouse mammary tumor virus-neu mice. Consonant with these findings, in clinical samples, high levels of EZH2 are significantly associated with activated NOTCH1 protein and increased TICs in TN invasive carcinomas. These data reveal a functional and mechanistic link between EZH2 levels, NOTCH1 signaling activation, and TICs, and provide previously unidentified evidence that EZH2 enhances breast cancer initiation.

EZH2 and ALDH-1 mark breast epithelium at risk for breast cancer development

It is well established that benign proliferative lesions and atypical hyperplasia increase the risk of breast cancer, which can develop in either breast. At present, there is no radiological, pathological, or molecular marker capable of distinguishing which proliferative or atypical lesions will progress to carcinoma. EZH2, a protein involved in stem cell renewal and carcinogenesis is upregulated in the morphologically normal breast epithelium from BRCA1 mutation carriers. Here, we tested the hypothesis that EZH2 expression alone or in combination with the breast stem cell marker aldehyde dehydrogenase-1 (ALDH-1) may identify benign breast biopsies that progress to breast cancer in the future. Benign breast biopsy samples obtained from 59 women who subsequently developed (study group, n=29) or who did not develop (control group, n=30) breast cancer in the same time period were subjected to immunohistochemical analyses of EZH2 and ALDH-1 proteins. When present, EZH2 was expressed in the nuclei of benign epithelial cells, whereas ALDH-1 was expressed in the cytoplasm of epithelial cells and/or in the stroma. EZH2, epithelial ALDH-1, and expanded stromal ALDH-1-positive cells were present in 95, 43, and 69%, respectively, of study group biopsies, compared with 16, 13, and 37%, respectively, of control biopsies (P <0.05 for all). The mean percentage of EZH2-positive cells was higher in the study group than in the control group (34 and 6%, respectively). EZH2 expression was associated with breast cancer development (P=8.2 × 10−6) and with younger age at cancer diagnosis (P=0.0086). Both stromal and epithelial ALDH-1 were associated with development of breast cancer (P=0.001 and P=0.049, respectively). Our study provides first evidence that EZH2 and epithelial and stromal ALDH-1 detection in benign breast biopsies may predict increased risk for breast cancer, with implications for breast cancer prevention.

Histone Methyltransferase EZH2 Induces Akt-Dependent Genomic Instability and BRCA1 Inhibition in Breast Cancer

Increased levels of EZH2, a critical regulator of cellular memory, signal the presence of metastasis and poor outcome in breast cancer patients. High levels of EZH2 are associated with nuclear pleomorphism, lack of estrogen receptor expression, and decreased nuclear levels of BRCA1 tumor suppressor protein in invasive breast carcinomas. The mechanism by which EZH2 overexpression promotes the growth of poorly differentiated invasive carcinomas remains to be defined. Here, we show that EZH2 controls the intracellular localization of BRCA1 protein. Conditional doxycycline-induced upregulation of EZH2 in benign mammary epithelial cells results in nuclear export of BRCA1 protein, aberrant mitoses with extra centrosomes, and genomic instability. EZH2 inhibition in CAL51 breast cancer cells induces BRCA1 nuclear localization and rescues defects in ploidy and mitosis. Mechanistically, EZH2 overexpression is sufficient for activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway specifically through activation of Akt isoform 1. EZH2-induced BRCA1 nuclear export, aneuploidy, and mitotic defects were prevented by treatment with the PI3K inhibitors LY294002 or wortmannin. Targeted inhibition of Akt-1, Akt-2, and Akt-3 isoforms revealed that the EZH2-induced phenotype requires specific activation of Akt-1. The relevance of our studies to human breast cancer is highlighted by the finding that high EZH2 protein levels are associated with upregulated expression of phospho-Akt-1 (Ser473) and decreased nuclear expression of phospho-BRCA1 (Ser1423) in 39% of invasive breast carcinomas. These results enable us to pinpoint one mechanism by which EZH2 regulates BRCA1 expression and genomic stability mediated by the PI3K/Akt-1 pathway.

Metaplastic breast carcinomas are enriched in markers of tumor-initiating cells and epithelial to mesenchymal transition

Metaplastic breast carcinomas constitute a distinct aggressive form of invasive breast cancer with histological evidence of epithelial to mesenchymal transition toward spindle, chondroid, or osseous cell types. During tumorigenesis, epithelial to mesenchymal transition promotes invasion and metastasis and has been linked to the presence of stem cells. We hypothesized that metaplastic carcinomas may express epithelial to mesenchymal transition markers and may be enriched in tumor-initiating cells specifically in the non-glandular metaplastic elements. In 27 primary metaplastic carcinomas of the breast we tested the expression of epithelial to mesenchymal transition inducers ZEB1 and E-cadherin and the presence of tumor-initiating cells by using aldehyde dehydrogenase-1 (ALDH-1) and CD44+/CD24−/low immunohistochemistry. Of the 27 metaplastic carcinomas, 20 (74%) had squamous and/or spindle areas and 7 (26%) had heterologous elements (6 chondroid and 1 osseous). ALDH-1-positive and CD44+/CD24−/low-expressing cells were detected in the non-glandular metaplastic components (Fisher’s exact, P=0.0017). E-cadherin expression was reduced or absent (aberrant) in all metaplastic components whereas it was normal in the glandular areas. On the contrary, overexpression of ZEB1 was detected in 41% (11 of 27) of the non-glandular, metaplastic components, and in none of the glandular areas. The presence of tumor-initiating cells, aberrant E-cadherin, and ZEB1 upregulation was associated in over 90% of the spindle areas and heterologous elements (χ2 test, P<0.05). We provide first in situ evidence that epithelial to mesenchymal transition inducers and tumor-initiating cells are present specifically in the non-glandular components of metaplastic carcinomas.

CCN6 modulates BMP signaling via the Smad-independent TAK1/p38 pathway, acting to suppress metastasis of breast cancer

CCN6 (WISP3) is an extracellular matrix protein that exerts tumor suppressive functions in breast cancer, where its decreased expression is a feature of advanced disease. However, neither its role nor mechanism of action in breast cancer metastasis has been established. Bone morphogenetic proteins (BMPs), which constitute ligands of the TGF-β superfamily, are multifunctional cytokines that induce epithelial-mesenchymal transition, cell invasion, and metastasis. In this study, we identify a CCN6-BMP4-TAK1 kinase signaling pathway that controls the ability of the p38 MAP kinase to regulate acinar morphogenesis and invasion of breast cells. ShRNA-mediated attenuation of CCN6 in human mammary epithelial cells led to BMP4 upregulation as a major response to exposure to the TGF-β superfamily. CCN6 attenuation also induced BMP4-mediated activation of the Smad-independent TAK1 and p38 kinases. Conversely, ectopic expression of CCN6 in breast cancer cells antagonized BMP4-mediated TAK1/p38 activation and invasive capacity, both by binding BMP4 protein as well as decreasing BMP4 protein levels. Effects on BMP4 and p38 were confirmed in vivo where they correlated with decreased metastasis. In clinical specimens, we found that CCN6 expression was inversely associated with BMP4 and phospho-p38 levels in 69% of invasive breast carcinomas examined, consistent with the functional results. Together our findings identify a novel modifier pathway through which CCN6 acts to limit breast cancer invasion and metastasis.

RhoC Impacts the Metastatic Potential and Abundance of Breast Cancer Stem Cells

Cancer stem cells (CSCs) have been shown to promote tumorigenesis of many tumor types, including breast, although their relevance to cancer metastasis remains unclear. While subpopulations of CSCs required for metastasis have been identified, to date there are no known molecular regulators of breast CSC (BCSC) metastasis. Here we identify RhoC GTPase as an important regulator of BCSC metastasis, and present evidence suggesting that RhoC also modulates the frequency of BCSCs within a population. Using an orthotopic xenograft model of spontaneous metastasis we discover that RhoC is both necessary and sufficient to promote SUM149 and MCF-10A BCSC metastasis–often independent from primary tumor formation–and can even induce metastasis of non-BCSCs within these cell lines. The relationship between RhoC and BCSCs persists in breast cancer patients, as expression of RhoC and the BCSC marker ALDH1 are highly correlated in clinical specimens. These results suggest new avenues to combating the deadliest cells driving the most lethal stage of breast cancer progression.

Expression of aldehyde dehydrogenase 1 as a marker of mammary stem cells in benign and malignant breast lesions of Ghanaian women

Breast cancers that are negative for the estrogen receptor (ER), the progesterone receptor (PR), and the HER2 (human epidermal growth factor receptor 2) marker are more prevalent among African women, and the biologically aggressive nature of these triple‐negative breast cancers (TNBCs) may be attributed to their mammary stem cell features. Little is known about expression of the mammary stem cell marker aldehyde dehydrogenase 1 (ALDH1) in African women. Novel data are reported regarding ALDH1 expression in benign and cancerous breast tissue of Ghanaian women.

Blockade of CCN6 (WISP3) activates growth factor-independent survival and resistance to anoikis in human mammary epithelial cells.

CCN6 is a secreted cysteine-rich matricellular protein (36.9 kDa) that exerts growth-inhibitory functions in breast cancer. Reduction or loss of CCN6 protein has been reported in invasive carcinomas of the breast with lymph node metastasis and in inflammatory breast cancer. However, the mechanism by which CCN6 loss promotes breast cancer growth remains to be defined. In the present study, we developed lentiviral-mediated short hairpin RNA CCN6 knockdown (KD) in nontumorigenic mammary epithelial cells MCF10A and HME. We discovered that CCN6 KD protects mammary epithelial cells from apoptosis and activates growth factor-independent survival. In the absence of exogenous growth factors, CCN6 KD was able to promote growth under anchorage-independent conditions and triggered resistance to detachment-induced cell death (anoikis). On serum starvation, CCN6 KD was sufficient for activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Growth factor-independent cell survival was stunted in CCN6 KD cells when treated with either human recombinant CCN6 protein or the PI3K inhibitor LY294002. Targeted inhibition of Akt isoforms revealed that the survival advantage rendered by CCN6 KD requires specific activation of Akt-1. The relevance of our studies to human breast cancer is highlighted by the finding that low CCN6 protein levels are associated with upregulated expression of phospho-Akt-1 (Ser(473)) in 21% of invasive breast carcinomas. These results enable us to pinpoint one mechanism by which CCN6 controls survival of breast cells mediated by the PI3K/Akt-1 pathway.

Breast Cancer and African Ancestry: Lessons Learned at the 10-Year Anniversary of the Ghana-Michigan Research Partnership and International Breast Registry

Women with African ancestry in western, sub-Saharan Africa and in the United States represent a population subset facing an increased risk of being diagnosed with biologically aggressive phenotypes of breast cancer that are negative for the estrogen receptor, the progesterone receptor, and the HER2/neu marker. These tumors are commonly referred to as triple-negative breast cancer. Disparities in breast cancer incidence and outcome related to racial or ethnic identity motivated the establishment of the International Breast Registry, on the basis of partnerships between the Komfo Anokye Teaching Hospital in Kumasi, Ghana, the University of Michigan Comprehensive Cancer Center in Ann Arbor, Michigan, and the Henry Ford Health System in Detroit, Michigan. This research collaborative has featured educational training programs as well as scientific investigations related to the comparative biology of breast cancer in Ghanaian African, African American, and white/European American patients. Currently, the International Breast Registry has expanded to include African American patients throughout the United States by partnering with the Sisters Network (a national African American breast cancer survivors’ organization) and additional sites in Ghana (representing West Africa) as well as Ethiopia (representing East Africa). Its activities are now coordinated through the Henry Ford Health System International Center for the Study of Breast Cancer Subtypes. Herein, we review the history and results of this international program at its 10-year anniversary.