RANK induces epithelial-mesenchymal transition and stemness in human mammary epithelial cells and promotes tumorigenesis and metastasis

Paracrine signaling through receptor activator of NF-κB (RANK) pathway mediates the expansion of mammary epithelia that occurs during pregnancy, and activation of RANK pathway promotes mammary tumorigenesis in mice. In this study we extend these previous data to human cells and show that the RANK pathway promotes the development of mammary stem cells and breast cancer. Overexpression of RANK (FL-RANK) in a panel of tumoral and normal human mammary cells induces the expression of breast cancer stem and basal/stem cell markers. High levels of RANK in untransformed MCF10A cells induce changes associated with both stemness and transformation, including mammary gland reconstitution, epithelial-mesenchymal transition (EMT), increased migration, and anchorage-independent growth. In addition, spheroids of RANK overexpressing MCF10A cells display disrupted acinar formation, impair growth arrest and polarization, and luminal filling. RANK overexpression in tumor cells with nonfunctional BRCA1 enhances invasiveness in acinar cultures and increases tumorigenesis and metastasis in immunodeficient mice. High levels of RANK were found in human primary breast adenocarcinomas that lack expression of the hormone receptors, estrogen and progesterone, and in tumors with high pathologic grade and proliferation index; high RANK/RANKL expression was significantly associated with metastatic tumors. Together, our findings show that RANK promotes tumor initiation, progression, and metastasis in human mammary epithelial cells by increasing the population of CD44(+)CD24(-) cells, inducing stemness and EMT. These results suggest that RANK expression in primary breast cancer associates with poor prognosis.     

HOXC8-Dependent Cadherin 11 Expression Facilitates Breast Cancer Cell Migration through Trio and Rac

HOXC8 expression is upregulated in diverse cancer types, and a high level of HOXC8 is often associated with the aggressive/metastatic phenotypes. We previously reported that the presence of HOXC8 is essential for breast cancer cell migration and metastasis. However, the underlying molecular mechanism of HOXC8 regulation of cell migration is unclear. Here, we demonstrate that the presence of HOXC8 is required for cadherin 11 (CDH11) expression in breast cancer cells and that HOXC8 regulation of cell migration is mediated by CDH11. To understand the role of HOXC8-CDH11 axis in cell migration, we show that depleting either HOXC8 or CDH11 diminishes the formation of actin-based membrane ruffles, an event essential for cell migration. The loss of membrane ruffles in HOXC8- or CDH11-knockdown cells is apparently caused by reduced Rac activity because ectopically expressing active Rac1 restores cytoskeleton reorganization. CDH11 physically interacts with Trio, a Rac GEF. We show that Trio is responsible for the majority of endogenous Rac activity in migratory breast cancer cells. Because knockdown of CDH11 prevents the plasma membrane localization of Trio, our study indicates that CDH11 may play a role in recruiting Trio to the plasma membrane where Trio activates Rac, leading to cell migration. This study reveals a novel HOXC8-CDH11-Trio-Rac signaling axis that contributes significantly to breast cancer cell migration.     

Dietary energy balance modulates epithelial-to-mesenchymal transition and tumor progression in murine claudin-low and basal-like mammary tumor models

Using novel murine models of claudin-low and basal-like breast cancer, we tested the hypothesis that diet-induced obesity (DIO) and calorie restriction (CR) differentially modulate progression of these aggressive breast cancer subtypes. For model development, we characterized two cell lines, "mesenchymal (M)-Wnt" and "epithelial (E)-Wnt," derived from MMTV-Wnt-1 transgenic mouse mammary tumors. M-Wnt, relative to E-Wnt, cells were tumor-initiating cell (TIC)-enriched (62% vs. 2.4% CD44(high)/CD24(low)) and displayed enhanced ALDEFLUOR positivity, epithelial-to-mesenchymal transition (EMT) marker expression, mammosphere-forming ability, migration, invasion, and tumorigenicity (P < 0.001; each parameter). M-Wnt and E-Wnt cells clustered with claudin-low and basal-like breast tumors, respectively, in gene expression profiles and recapitulated these tumors when orthotopically transplanted into ovariectomized C57BL/6 mice. To assess the effects of energy balance interventions on tumor progression and EMT, mice were administered DIO, control, or CR diets for 8 weeks before orthotopic transplantation of M-Wnt or E-Wnt cells (for each cell line, n = 20 mice per diet) and continued on their diets for 6 weeks while tumor growth was monitored. Relative to control, DIO enhanced M-Wnt (P = 0.01), but not E-Wnt, tumor progression; upregulated EMT- and TIC-associated markers including N-cadherin,fibronectin, TGFβ, Snail, FOXC2, and Oct4 (P < 0.05, each); and increased intratumoral adipocytes. Conversely, CR suppressed M-Wnt and E-Wnt tumor progression (P < 0.02, each) and inhibited EMT and intratumoral adipocyte accumulation. Thus, dietary energy balance interventions differentially modulate EMT and progression of claudin-low and basal-like tumors. EMT pathway components may represent targets for breaking the obesity-breast cancer link, particularly for preventing and/or controlling TIC-enriched subtypes such as claudin-low breast cancer.     

Alterations of EGFR,p53 and PTEN that mimic changes found in basal-like breast cancer promote transformation of human mammary epithelial cells

Breast cancer can be classified into different molecular subtypes with varying clinical and pathological characteristics. The basal-like breast cancer subtype represents one of the most aggressive and lethal types of breast cancer, and due to poor mechanistic understanding, it lacks targeted therapy. Many basal-like breast cancer patient samples display alterations of established drivers of cancer development, including elevated expression of EGFR, p53 inactivating mutations and loss of expression of the tumor suppressor PTEN; however, their contribution to human basal-like breast cancer pathogenesis remains ill-defined. Using non-transformed human mammary epithelial cells, we set out to determine whether altering EGFR, p53 and PTEN in different combinations could contribute to basal-like breast cancer progression through transformation of cells. Altering PTEN in combination with either p53 or EGFR in contrast to any of the single alterations caused increased growth of transformed colonies in soft agar. Concomitantly modifying all three genes led to the highest rate of cellular proliferation and the greatest degree of anchorage-independent colony formation. Results from our effort to engineer a model of BBC expressing alterations of EGFR, p53 and PTEN suggest that these changes are cooperative and likely play a causal role in basal-like breast cancer pathogenesis. Consideration should be given to targeting EGFR and restoring p53 and PTEN signaling simultaneously as a strategy for treatment of this subtype of breast cancer.     

MAGE-D4B is a novel marker of poor prognosis and potential therapeutic target involved in breast cancer tumorigenesis

Melanoma-associated antigen (MAGE) family members are generally described as tumor-specific antigens. An association between MAGE-D4B and breast cancer has yet to be reported and the functional role of the encoded protein has never been established. We performed microarray analysis of 104 invasive breast tumors and matched non-cancerous breast biopsies. qPCR was used for validation in an independent biobank. To investigate the biological relevance of MAGE-D4B in breast tumorigenesis, its phenotypic effects were assessed in vitro. Overall, MAGE-D4B was detected in 43% of tumors while undetected in normal breast tissue. MAGE-D4B was found to correlate with tumor progression and to be an independent prognostic marker for poor outcome in term of relapse-free and overall survival, with potential predictive relevance in relation to response to chemotherapy. RNA interference-mediated knockdown of MAGE-D4B significantly hampered the invasive properties of Hs578T cells by affecting anchorage-independent growth, adhesion, migration and invasion affecting anchorage-independent growth, adhesion, migration and invasion and by modulating expression of invasion-suppressor gene E-cadherin.     

A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells

TAZ (WWTR1), identified as a 14-3-3 binding protein with a PDZ binding motif, modulates mesenchymal stem cell differentiation. We now show that TAZ plays a critical role in the migration, invasion, and tumorigenesis of breast cancer cells. TAZ is conspicuously expressed in human breast cancer cell lines in which its expression levels generally correlate with the invasiveness of cancer cells. Overexpression of TAZ in low-expressing MCF10A cells causes morphologic changes characteristic of cell transformation and promotes cell migration and invasion. Conversely, RNA interference-mediated knockdown of TAZ expression in MCF7 and Hs578T cells reduces cell migration and invasion. TAZ knockdown in MCF7 cells also retards anchorage-independent growth in soft agar and tumorigenesis in nude mice. Significantly, TAZ is overexpressed in approximately 20% of breast cancer samples. These results indicate that TAZ plays a role in the migration, invasion, and tumorigenesis of breast cancer cells and thus presents a novel target for the detection and treatment of breast cancer.     

自发Basal-like乳腺癌小鼠模型研究

探讨TA2小鼠自发乳腺癌生物学行为、形态及免疫表型特征,比较其与人Basal-like乳腺癌的相似程度,研究TA2小鼠乳腺癌发生与妊娠及小鼠乳腺癌病毒（mouse mammary tumor virus,MMTV）的关系。方法:收集TA2自发乳腺癌小鼠84只,采用H&E染色和免疫组织化学染色检测TA2自发乳腺癌组织形态、免疫表型与转移情况,比较与人Basal-like乳腺癌的相似程度。电化学法检测TA2自发乳腺癌小鼠（n=10）、妊娠15天小鼠（n=6）与正常小鼠（n=6）血清雌二醇和孕酮水平。电镜观察肿瘤细胞内是否存在MMTV病毒颗粒,使用PCR和Real-time PCR分别检测MMTV LTR在TA2正常小鼠乳腺、妊娠小鼠乳腺和乳腺癌中基因组与mRNA表达变化。结果:TA2自发乳腺癌小鼠平均见瘤年龄（329.81±95.32）天,平均分娩次数（2.70±1.82）次,且肿瘤在妊娠期内迅速增长。肿瘤多发生内脏转移,淋巴结转移未见,80.0%（64/80）发生肺转移。TA2自发乳腺癌形态一致,癌巢由胞浆稀少的小圆细胞构成,周围被发育良好的基质包绕。实性癌巢中央为未分化或分化差的癌细胞,且核分裂像多见,中央可见坏死;癌巢边缘的癌细胞多发生分化,呈腺泡状,腺管状或乳头状;腺泡状癌巢周围有单层或多层肌上皮和致密基质包绕;腺管状分化癌巢中央可见粉色分泌物积聚;实性癌巢中央可见坏死。免疫组化染色显示TA2自发乳腺癌不表达ER、PR和HER2,高表达Cyclin D1、PCNA、p53和CK5/6。电镜观察显示肿瘤细胞内存在MMTV病毒颗粒。TA2自发乳腺癌小鼠血清雌二醇和孕酮浓度及瘤组织内MMTV LTR mRNA表达均升高,其改变与妊娠小鼠相似。结论:TA2小鼠自发乳腺癌形态、免疫表型和生物学特征与人Basal-like乳腺癌极为相似,能够反映Basal-like乳腺癌发生发展的全过程,是研究Basal-like乳腺癌的良好模型。妊娠激素水平升高导致的MMTV激活在Basal-like乳腺癌发生过程中起重要作用。      

Enhanced invasion and tumor growth of fibroblast growth factor 8b-overexpressing MCF-7 human breast cancer cells

Fibroblast growth factor 8 (FGF-8) is a secreted heparin-binding protein, which has mitogenic and transforming activity. Increased expression of FGF-8 has been found in human breast cancer, and it has a potential autocrine role in its progression. Human FGF-8 is alternatively spliced to generate four protein isoforms (a, b, e, and f). Isoform b has been shown to be the most transforming. In this work, we studied the role of FGF-8b in the growth (in vitro and in vivo) of MCF-7 human breast cancer cells, which proliferate in an estrogen-dependent manner. Constitutive overexpression of FGF-8b in MCF-7 cells down-regulated FGF-8b-binding receptors FGF receptor (FGFR) 1IIIc, FGFR2IIIc, and FGFR4 found to be expressed in these cells. FGF-8b overexpression led to an increase in the anchorage-independent proliferation rate in suspension culture and colony formation in soft agar, when MCF-7 cells were cultured with or without estradiol. FGF-8b also provided an additional growth advantage for cells stimulated with estradiol. In addition, FGF-8b-transfected cells invaded more actively through Matrigel than did control cells. This was possibly due to the increased secretion of matrix metalloproteinase 9. In vivo, FGF-8b-transfected MCF-7 cells formed faster growing tumors than vector-only-transfected cells when xenografted into nude mice. The tumors formed by FGF-8b-transfected cells were more vascular than the tumors formed by vector-only-transfected cells. In conclusion, FGF-8b expression confers a growth advantage to MCF-7 breast carcinoma cells, both in vitro and in vivo. In addition to stimulation of proliferation, this growth advantage probably arises from increased invasion and tumor vascularization induced by FGF-8b. The results suggest that FGF-8b signaling may be an important factor in the regulation of tumorigenesis and progression of human breast cancer.     

PC cell-derived growth factor (PCDGF/GP88, progranulin) stimulates migration, invasiveness and VEGF expression in breast cancer cells

Metastasis is a multi-step process involved in the progression of breast cancer to a disease with poor prognosis. Growth factor and/or growth factor receptor over- expression have been reported to play an important role in this process. The 88 kDa glycoprotein PC cell-derived growth factor (PCDGF/GP88), also known as progranulin, has been shown to play a major role in breast tumorigenesis by stimulating proliferation, mediating survival and conferring resistance to tamoxifen. In the present paper, the metastatic potential of PCDGF/GP88 was examined in breast cancer. Using MCF-7 cells, we showed that PCDGF/GP88 over-expression stimulated anchorage-independent cell growth and accelerated cell migration through matrigel. Similar results were obtained with MCF-7 cells treated exogenously with PCDGF/GP88. Furthermore, gelatin zymograph and immunoblot revealed that matrix metalloprotease-9 was up-regulated by PCDGF/GP88. PCDGF/GP88 stimulated VEGF expression in MCF-7 cells. These results suggest that PCDGF/GP88 could act to promote metastasis and angiogenesis in human breast cancer cells in addition to stimulating their proliferation and survival.     

Oncogenic potential of the nuclear receptor coregulator proline-, glutamic acid-, leucine-rich protein 1/modulator of the nongenomic actions of the estrogen receptor

Proline-, glutamic acid-, leucine-rich protein 1 (PELP1), a novel nuclear receptor coactivator, and its expression is deregulated in hormone-dependent cancers, including those of the breast, endometrium, and ovary. PELP1 interacts with estrogen receptor and modulates its genomic and nongenomic functions. In this study, we examined whether PELP1 functions as an oncogene. The overexpression of PELP1 in fibroblasts and epithelial model cells resulted in cellular transformation. PELP1 also enhanced the transformation potential of c-Src kinase in focus formation assays, and PELP1 overexpression potentiated estradiol-mediated cell migratory potential and anchorage-independent growth. Using PELP1-small interfering RNA, we provided evidence that endogenous PELP1 plays an essential role in E2-mediated anchorage-independent growth, cell migration, and cytoskeletal changes. When compared with control vector transfectants, breast cancer cells stably overexpressing PELP1 showed a rapid tumor growth in xenograft studies. Immunohistochemical analysis of PELP1 expression using a tumor progression array of 252 breast carcinomas and normal breast tissue specimens revealed that PELP1 expression is deregulated to a greater degree in higher grade node-positive invasive tumors than in normal breast tissue or ductal carcinoma in situ. Our data suggest that PELP1 is a potential oncogene, that its expression is deregulated during cancer progression, and that PELP1 may play a role in oncogenesis.     

MicroRNA-432 Acts as a Prognostic Biomarker and an Inhibitor of Cell Proliferation,Migration, and Invasion in Breast Cancer

BackgroundAccumulating studies have demonstrated that microRNAs (miRNAs) are involved in the progression of various cancers. This study aimed to investigate the potential clinical and functional role of miR-432 in breast cancer.Materials and MethodsWe evaluated the expression of miR-432 in 117 breast cancer samples and paired nontumor tissue samples, as well as 4 breast cancer cell lines using RT-qPCR analysis. Kaplan-Meier survival curve and multivariate Cox regression analysis were used to evaluate the prognostic significance of miR-432 in breast cancer patients. CCK-8 assay and Transwell assays were used to evaluate the biological function of miR-432 in the progression of breast cancer.ResultsmiR-432 was downregulated in breast cancer tissues and cell lines, and its exotic expression was associated with tumor size, lymph node metastasis, and TNM stage. In addition, breast cancer patients with low miR-432 expression exhibited a shorter overall survival outcome. Further experiments revealed that overexpression of miR-432 inhibited the cell proliferation, migration, and invasion of breast cancer cells, while knockdown of miR-432 promoted these cellular activities. AXL was a direct target of miR-432 in breast cancer cells.ConclusionThe present study suggested that miR-432 may be a tumor suppressor in the progression of breast cancer through inhibiting cell proliferation, migration, and invasion by targeting AXL. And miR-432 might be a prognostic biomarker and therapeutic target for the treatment of breast cancer. This study provided a novel insight into breast cancer prognosis and treatment.     

促进乳腺癌增殖转移和肿瘤干细胞特征的研究

目的 研究Gankyrin在乳腺肿瘤中的表达及其促进肿瘤进展的分子机制。方法 利用数据库研究Gankyrin在乳腺癌中的表达情况,并分析其表达与患者生存的关系。在BT549和MDA-MB-231乳腺癌细胞系过表达和敲减Gankyrin基因后,利用CCK-8、Transwell和流式细胞实验分析细胞增殖、转移和肿瘤干细胞的比例。结果 通过数据库分析显示Gankyrin在乳腺癌组织表达较高(P＜0.01),其高表达与患者的不良预后有关(P＜0.01)。与正常乳腺组织相比,乳腺癌组织中Gankyrin启动子甲基化水平较低(P＜0.01)。通过在乳腺癌细胞中过表达和敲减Gankyrin基因后,表明Gankyrin具有促进乳腺肿瘤细胞增殖和转移的能力,可以维持乳腺癌细胞的肿瘤干细胞特征(P＜0.01)。结论 Gankyrin在乳腺癌高表达与患者的不良预后相关,其可能作为乳腺癌肿瘤标记物。     

Wwox inactivation enhances mammary tumorigenesis

Breast cancer is the leading cause of cancer-related death in women worldwide. Expression of the WWOX tumor suppressor is absent or reduced in a large proportion of breast tumors suggesting that loss of WWOX may contribute to breast tumorigenesis. Wwox-deficient mice die by 3-4 weeks of age precluding adult tumor analysis. To evaluate the effect of WWOX-altered expression on mammary tumor formation, the Wwox-heterozygous allele was back crossed onto the C3H mammary tumor-susceptible genetic background (Wwox(C3H)+/-) and incidence of mammary tumor formation was evaluated. Although 50% of the female Wwox(C3H)+/- mice developed mammary carcinomas, only 7% of Wwox(C3H)+/+ mice did. Intriguingly, mammary tumors in Wwox(C3H)+/- mice frequently lost WWOX protein expression suggesting a genetic predisposition toward mammary tumorigenesis. Immunohistochemical staining of hormone receptors revealed loss of estrogen receptor-α (ER) and progesterone receptor in the majority of these tumors. In vitro, depletion of WWOX in MCF7 ER-positive cells led to reduced ER expression and reduced sensitivity to tamoxifen and estrogen treatment and was associated with enhanced survival and anchorage-independent growth. Finally, cDNA array analyses of murine normal mammary epithelial cells and mammary tumors identified 163 significantly downreguated and 129 upregulated genes in the tumors. The majority of differentially expressed genes were part of pathways involved in cellular movement, cell-to-cell signaling and interaction, cellular development, cellular growth and proliferation and cell death. These changes in gene expression of mouse mammary tumors in Wwox(C3H)+/- mice resemble, at least in part, human breast cancer development. Our findings demonstrate the critical role that the WWOX tumor suppressor gene has in preventing tumorigenesis in breast cancer.