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.

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.

Characterizing Breast Cancer in a Population with Increased Prevalence of Triple-Negative Breast Cancer: Androgen Receptor and ALDH1 Expression in Ghanaian Women.

BACKGROUND: The androgen receptor (AR) is a commonly-expressed hormone receptor in breast cancer and may be a marker of response to targeted anti-androgen therapy, a particularly attractive option for triple-negative breast cancer (TNBC). Gene expression studies suggest that ARs may distinguish a luminal/AR TNBC subtype from stem cell-like subtypes. TNBC frequency is two to three times higher in African American and African breast cancers compared with White American and European breast cancers, yet little is known regarding TNBC subtypes in high-frequency African-ancestry populations. We evaluated ARs and the mammary stem cell marker aldehyde dehydrogenase 1 (ALDH1) among breast cancers from Ghana, Africa. METHODS: Overall, 147 formalin-fixed, paraffin-embedded invasive breast cancers from the Komfo Anoyke Teaching Hospital in Ghana were studied at the University of Michigan, and analyzed immunohistochemically for estrogen receptor (ER), progesterone receptor (PR), HER2/neu, ALDH1, and AR expression. RESULTS: The median age of patients was 45 years. Only 31 cases (21 %) were ER-positive, and 14 (10 %) were HER2-positive; 89 (61 %) were TNBCs. For the entire group, 44 % were AR-positive and 45 % were ALDH1-positive. ER/PR-positive tumors were more likely to be AR-positive compared with ER/PR-negative tumors (87 vs. 26 %; p < 0.0001), but there was no association between ALDH1 and AR expression. Among the TNBC cases, 45 % were ALDH1-positive and 24 % were AR-positive. ALDH1 positivity was associated with AR positivity within the subset of TNBC (36 vs. 14 %; p = 0.019). CONCLUSION: We confirmed other studies showing a high frequency of TNBC in Africa. Surprisingly, ALDH1 was found to correlate with AR expression among TNBC, suggesting that novel TNBC subtypes may exist among populations with African ancestry.

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

BACKGROUND: 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. METHODS: Formalin-fixed, paraffin-embedded specimens were transported from the Komfo Anoyke Teaching Hospital in Kumasi, Ghana to the University of Michigan for centralized histopathology study. Expression of ER, PR, HER2, and ALDH1 was assessed by immunohistochemistry. ALDH1 staining was further characterized by its presence in stromal versus epithelial and/or tumor components of tissue. RESULTS: A total of 173 women contributed to this study: 69 with benign breast conditions, mean age 24 years, and 104 with breast cancer, mean age 49 years. The proportion of benign breast conditions expressing stromal ALDH1 (n = 40, 58%) was significantly higher than those with cancer (n = 44, 42.3%) (P = .043). Among the cancers, TNBC had the highest prevalence of ALDH1 expression, either in stroma or in epithelial cells. More than 2-fold higher likelihood of ALDH1 expression was observed in TNBC cases compared with other breast cancer subtypes (odds ratio = 2.38, 95% confidence interval 1.03-5.52, P = .042). CONCLUSIONS: ALDH1 expression was higher in stromal components of benign compared with cancerous lesions. Of the ER-, PR-, and HER2-defined subtypes of breast cancer, expression of ALDH1 was highest in TNBC.

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.

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.

Androgen Receptor and ALDH1 Expression Among Internationally Diverse Patient Populations.

PURPOSE: Population-based incidence rates of breast cancers that are negative for estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2/ neu (triple-negative breast cancer [TNBC]) are higher among African American (AA) compared with white American (WA) women, and TNBC prevalence is elevated among selected populations of African patients. The extent to which TNBC risk is related to East African versus West African ancestry, and whether these associations extend to expression of other biomarkers, is uncertain. METHODS: We used immunohistochemistry to evaluate estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2/ neu, androgen receptor and aldehyde dehydrogenase 1 (ALDH1) expression among WA (n = 153), AA (n = 76), Ethiopian (Eth)/East African (n = 90), and Ghanaian (Gh)/West African (n = 286) patients with breast cancer through an institutional review board-approved international research program. RESULTS: Mean age at diagnosis was 43, 49, 60, and 57 years for the Eth, Gh, AA, and WA patients, respectively. TNBC frequency was higher for AA and Gh patients (41% and 54%, respectively) compared with WA and Eth patients (23% and 15%, respectively; P < .001) Frequency of ALDH1 positivity was higher for AA and Gh patients (32% and 36%, respectively) compared with WA and Eth patients (23% and 17%, respectively; P = .007). Significant differences were observed for distribution of androgen receptor positivity: 71%, 55%, 42%, and 50% for the WA, AA, Gh, and Eth patients, respectively ( P = .008). CONCLUSION: Extent of African ancestry seems to be associated with particular breast cancer phenotypes. West African ancestry correlates with increased risk of TNBC and breast cancers that are positive for ALDH1.

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.

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.

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.

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.

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.

Clinical profile, BRCA2 expression, and the androgen receptor CAG repeat region in Egyptian and Moroccan male breast cancer patients.

INTRODUCTION: Male breast cancer (MBC) is a rare disease. Rates of MBC in Northern Africa vary by region. The age-standardized incidence for MBC is higher in Morocco than in Egypt, and the Egyptian rate is similar to the U.S of approximately 1/10(5)This study aimed at investigating the clinical and molecular characteristics of MBC in Egypt and Morocco. METHODS: This case-case study included 211 cases from Egypt and 132 from Morocco. Tumor tissues were available for 47 Egyptian and 18 Moroccan patients. Medical record information was abstracted for patients' demographics, medical history, and treatment. BRCA2 protein expression status was examined in Egyptian and Moroccan tumors. Androgen receptor CAG repeat length was analyzed using the tissue samples in Egyptian MBC tumors and controls. Limited amount of tissues from Morocco did not allow for the analysis of CAG repeats. RESULTS: Egyptian MBC patients had a significantly lower age at diagnosis (Egypt: 57.5 ± 15.1, Morocco: 63.9 ± 14.4, P=0.0002) and a higher prevalence of liver cirrhosis (Egypt: 28.0%, Morocco: 0.8%, P=< 0.0001). MBC patients also had higher tumor grades [I (0.9%), II (81.0%), III (18.1%)] in Egypt vs. [I (10.7%), II (81.0%), III (8.3%)] in Morocco (P=0.0017). The clinical and molecular characteristics of the groups from the 2 countries did not significantly differ. There was no significant difference with respect to BRCA2 expression amongst countries (Egypt: 28.9% non-wild type, Morocco: 27.8% non-wild type, P=0.9297) or CAG lengths amongst BRCA2 expression types in Egyptians (Wild type: 54.6% with CAG repeat lengths of 20+, Non-wild type: 50% with CAG repeat lengths of 20+, P=0.7947). CONCLUSIONS: Differences in MBC between Egypt and Morocco are more likely due to differences in other risk factors such as consanguinity and use of xenoestrogenic pesticides.

Inflammatory breast cancer in north Africa: comparison of clinical and molecular epidemiologic characteristics of patients from Egypt, Tunisia, and Morocco.

Understanding molecular characteristics that distinguish inflammatory breast cancer (IBC) from non-IBC is crucial for elucidating breast cancer etiology and management. We included 3 sets of patients from Egypt (48 IBC and 64 non-IBC), Tunisia (24 IBC and 40 non-IBC), and Morocco (42 IBC and 41 non-IBC). Egyptian IBC patients had the highest combined erythema, edema, peau d'orange, and metastasis among the 3 IBC groups. Egyptian IBC tumors had the highest RhoC expression than Tunisians and Moroccan IBCs (87% vs. 50%, vs. 38.1, for the 3 countries, respectively). Tumor emboli were more frequent in Egyptian IBC than non-IBC (Mean ± SD: 14.1 ± 14.0 vs. 7.0 ± 12.9, respectively) (P < 0.001) and Tunisians (Mean ± SD: 3.4 ± 2.5 vs. 1.9 ± 2.0, respectively) (P < 0.01). There was no difference of emboli in Moroccan tumors (1.7 ± 1.2 vs. 1.8 ± 1.2 for IBC and non-IBC, respectively (P=0.66). This study illustrates that RhoC overexpression and tumor emboli are more frequent in IBC relative to non-IBC from Egypt and Tunisia. Tumors of Moroccans were significantly different from Egyptian and Tunisian tumors for RhoC expression and emboli. Future studies should focus on relating epidemiologic factors and clinical pictures to molecular features of IBC in these and other populations.

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.