Tyrosine kinase discoidin domain receptors DDR1 and DDR2 are coordinately deregulated in triple-negative breast cancer.

Receptor kinases Discoidin Domain Receptors (DDRs) 1 and 2 are emerging as new therapeutic targets in breast cancer (BC). However, the expression of DDR proteins during BC progression and their association with BC subtypes remain poorly defined. Herein we report the first comprehensive immunohistochemical analyses of DDR protein expression in a wide range of breast tissues. DDR1 and DDR2 expression was investigated by immunohistochemistry in 218 samples of normal breast (n = 10), ductal carcinoma in situ (DCIS, n = 10), and invasive carcinomas (n = 198), arrayed in tissue microarrays with comprehensive clinical and follow-up information. Staining was evaluated for cell type, subcellular localization, percentage and intensity (scores 1-4), and association with disease subtype and outcome. In normal epithelium and DCIS, DDR1 was highly expressed, while DDR2 was negative in normal epithelium, and in DCIS it localized to cells at the epithelial-stromal interface. Of the 198 invasive carcinomas, DDR1 was high in 87 (44 %) and low in 103 (52 %), and DDR2 was high in 110 (56 %) and low in 87 (44 %). High DDR2 was associated with high tumor grade (P = 0.002), triple-negative subtype (TNBC) (P < 0.0001), and worse survival (P = 0.037). We discovered a novel concordant deregulation of DDR expression, with a DDR1(Low)/DDR2(High) profile significantly associated with TNBC, compared to luminal tumors (P = 0.012), and with worse overall survival. In conclusion, DDR2 upregulation occurs in DCIS, before stromal invasion, and may reflect epithelial-stromal cross-talk. A DDR1(Low)/DDR2(High) protein profile is associated with TNBC and may identify invasive carcinomas with worse prognosis.

EZH2 expands breast stem cells through activation of NOTCH1 signaling.

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 inhibition decreases p38 signaling and suppresses breast cancer motility and metastasis.

EZH2 is a Polycomb group protein that exerts oncogenic functions in breast cancer, where its overexpression is associated with metastatic disease. While it reportedly acts a transcriptional repressor through trimethylation of histone H3 at lysine 27, EZH2 may exhibit context-dependent activating functions. Despite associations with worse outcome and metastasis in breast cancer, a functional role of EZH2 in breast cancer metastasis in vivo has not been demonstrated. Furthermore, whether EZH2 regulates cancer cell phenotype and motility are unknown. In this study, we discovered that knockdown of EZH2 induces a phenotypic reprogramming from mesenchymal to epithelial, reduces motility, and blocks invasion in breast cancer cell lines. In vivo, EZH2 downregulation in MDA-MB-231 cells decreases spontaneous metastasis to the lungs. We uncover an unexpected role of EZH2 in inducing the p38 mitogen-activated protein kinase signaling pathway, an important regulator of breast cancer invasion and metastasis. In breast cancer cells, EZH2 binds to phosphorylated p38 (p-p38) in association with other core members of the Polycomb repressive complex 2, EED, and SUZ12, and EZH2 overexpression leads to increased levels of p-p38 and of activated, downstream pathway proteins. The effect on p-p38 was confirmed in vivo, where it correlated with decreased spontaneous metastasis. In clinical specimens of matched primary and invasive breast carcinomas, we found that EZH2 expression was upregulated in 100 % of the metastases, and that EZH2 and p-p38 were coexpressed in 63 % of cases, consistent with the functional results. Together our findings reveal a new mechanism by which EZH2 functions in breast cancer, and provide direct evidence that EZH2 inhibition reduces breast cancer metastasis in vivo.

CCN6 knockdown disrupts acinar organization of breast cells in three-dimensional cultures through up-regulation of type III TGF-ß receptor.

While normal cells in the human breast are organized into acinar structures, disruption of the acinar architecture is a hallmark of cancer. In a three-dimensional model of morphogenesis, we show that down-regulation of the matrix-associated tumor suppressor protein CCN6 (WNT1-inducible-signaling pathway protein 3) disrupts breast epithelial cell polarity and organization into acini through up-regulation of the type III transforming growth factor-ß receptor (TßRIII or betaglycan). Down-regulation of CCN6 in benign breast cells led to loss of tissue polarity and resulted in cellular disorganization with loss of α6 integrin-rich basement membrane and the basolateral polarity protein E-cadherin. Silencing of TßRIII with shRNA and siRNA rescued the ability of breast epithelial cells to form polarized acinar structures with reduced matrix invasion and restored the correct expression of α6 integrin and E-cadherin. Conversely, CCN6 overexpression in aggressive breast cancer cells reduced TßRIII in vitro and in a xenograft model of CCN6 overexpression. The relevance of our studies to human breast cancer is highlighted by the finding that CCN6 protein levels are inversely associated with TßRIII protein in 64%of invasive breast carcinomas. These results reveal a novel function of the matricellular protein CCN6 and establish a mechanistic link between CCN6 and TßRIII in maintaining acinar organization in the breast.

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-ß 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-ß 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.

Invasive breast carcinomas in Ghana: high frequency of high grade, basal-like histology and high EZH2 expression.

Breast cancer in African-American women has a worse outcome than in Caucasian women. The ancestors of most African-American women come from West Africa, including Ghana. The Polycomb group protein EZH2 is a marker of poor outcome in breast cancers from Caucasian women. The histopathological features and biomarker expression of African breast cancers remain obscure. Here, we investigated a cohort of Ghanaian breast cancers to better define the prevalent tumor types and to test if EZH2 protein may identify aggressive tumors. A group of 169 breast tissues (100 invasive carcinomas and 69 benign) from women treated at Komfo Anoyke Teaching Hospital between 2006 and 2011 were histologically classified and investigated for EZH2 expression. EZH2 nuclear expression we defined as high or low following previously published criteria. Of the 100 invasive carcinomas, 89 % were ductal, 2 % were lobular, and 9 % were metaplastic. Basal-like pathological features were present in 30 % of the tumors. Of the invasive carcinomas, 7 % were grade 1, 41 % grade 2, and 52 % grade 3. EZH2 protein was overexpressed in invasive carcinomas compared to benign breast (p < 0.0001). In invasive carcinomas nuclear EZH2 overexpression was significantly associated with basal-like subtype (p = 0.03) and high histologic grade (p < 0.05). Cytoplasmic EZH2, which has not been previously reported, was present in 16 % of invasive carcinomas and it was associated with triple negative status (p = 0.02). Our results provide the first comprehensive histopathological study of this patient population and uncover the association of EZH2 with high grade and basal-like tumors. We provide the basis for further detailed investigations on this cohort to advance diagnosis and treatment of African and African-American women.

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 (χ(2) 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.

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.

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.