Expression of secreted frizzled-related protein 2 in a primary canine mammary tumor cell line: a candidate tumor marker for mammary tumor cells

Canine mammary tumors (CMTs) have been proposed to be a good animal model for human breast cancer. To provide a basis for the tumorigenic study of CMTs, cell lines were established using a modified cell culture technique. The epithelial morphology and immunostaining with cytokeratin 18 confirmed the epithelial origin of the cells. In an investigation of possible mammary tumorigenesis-related factors, the expression of Wnt signaling-related proteins was detected in cell lines. Secreted frizzled-related protein 2 (SFRP2) was abundantly expressed in CMT cells but not in normal canine mammary gland (MG) cells. Secreted frizzled-related protein 2 was secreted into the culture medium and was associated with the extracellular matrix. In addition, increased expressions of beta-catenin and cyclin D1 were observed in cells overexpressing SFRP2. The marked differential expression of SFRP2 reveals that this protein may be a potential candidate marker for CMTs. The CMT cell line established in this study provides a useful tool and experimental model for understanding both the tumorigenesis of CMTs and the role of Wnt signaling in cancers.     

Loss of sfrp1 promotes ductal branching in the murine mammary gland

ABSTRACT: BACKGROUND: Secreted frizzled-related proteins (SFRPs) are a family of proteins that block the Wnt signaling pathway and loss of SFRP1 expression is found in breast cancer along with a multitude of other human cancers. Activated Wnt signaling leads to inappropriate mammary gland development and mammary tumorigenesis in mice. When SFRP1 is knocked down in immortalized non-malignant mammary epithelial cells, the cells exhibit a malignant phenotype which resembles the characteristics observed in metastatic breast cancer stem-like cells. However, the effects of SFRP1 loss on mammary gland development in vivo are yet to be elucidated. The work described here was initiated to investigate the role of SFRP1 in mammary gland development and whether SFRP1/ mice exhibit changes in mammary gland morphology and cell signaling pathways shown to be associated with SFRP1 loss in vitro. RESULTS: 10 week old nulliparous SFRP1/ mammary glands exhibited branching with clear lobulo-alveolar development, which normally only occurs in hormonally stimulated mid-pregnant wt mammary glands. Explant cultures of SFRP1/ mammary glands display increased levels of a well known Wnt signaling target gene, Axin2. Histomorphologic evaluation of virgin glands revealed that by 10 weeks of age, the duct profile is markedly altered in SFRP1/ mice showing a significantly higher density of ducts with distinct alveoli present throughout the mammary gland, and with focal ductal epithelial hyperplasia. These findings persist as the mice age and are evident at 23 weeks of age. Changes in gene expression, including c-Myc, TGFbeta-2, Wnt4, RANKL, and Rspo2 early in mammary gland development are consistent with the excessive hyper branching phenotype. Finally, we found that loss of SFRP1 significantly increases the number of mammary epithelial cells capable of mammosphere formation. CONCLUSIONS: Our study indicates that SFRP1 gene is critical for maintaining proper mammary gland development, and that reduced levels of SFRP1 results in hyperplastic lesions and its loss may be a critical event in cancer initiation.     

Non-steroidal anti-inflammatory drugs target the pro-tumorigenic extracellular matrix of the postpartum mammary gland

Breast cancer patients diagnosed postpartum have poor prognosis. The postpartum mammary gland undergoes tissue regression to return to the pre-pregnant state. This involution is characterized by wound healing programs known to be tumor promotional in other contexts. Previous studies have shown that mammary extracellular matrix (ECM) from nulliparous rats has tumor suppressive attributes, while mammary ECM from involuting mammary glands is promotional. In models of pregnancy-associated breast cancer, non-steroidal anti-inflammatory drug (NSAID) treatment targeted to postpartum involution inhibits tumor progression, in part by suppressing COX-2 dependent collagen deposition. Because mammary ECM proteins are coordinately regulated, NSAID treatment is anticipated to result in additional protective changes in the mammary extracellular matrix. Here, systemic NSAID treatment was utilized during postpartum involution to reduce mammary COX-2 activity. ECM was isolated from actively involuting glands of rats treated with NSAIDs and compared to ECM isolated from control-involution and nulliparous rats in 3D cell culture and xenograft assays. Compositional changes in ECM between groups were identified by proteomics. In four distinct 3D culture assays, normal and transformed mammary epithelial cells plated in NSAID-involution ECM, phenocopied cells plated in ECM from nulliparous rats rather than ECM from control-involution rats. Tumor cells mixed with NSAID-involution ECM and injected orthotopically in mice formed smaller tumors than cells mixed with control-involution ECM. Proteomic analyses identified and 3D culture assays implicated the ECM protein tenascin-C as a potential mediator of tumor progression during involution that is decreased by NSAID treatment. In summary, NSAID treatment decreases tumor-promotional attributes of postpartum involution mammary ECM.     

Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice

The cyclooxygenase (COX)-2 gene encodes an inducible prostaglandin synthase enzyme that is overexpressed in adenocarcinomas and other tumors. Deletion of the murine Cox-2 gene in Min mice reduced the incidence of intestinal tumors, suggesting that it is required for tumorigenesis. However, it is not known if overexpression of Cox-2 is sufficient to induce tumorigenic transformation. We have derived transgenic mice that overexpress the human COX-2 gene in the mammary glands using the murine mammary tumor virus promoter. The human Cox-2 mRNA and protein are expressed in mammary glands of female transgenic mice and were strongly induced during pregnancy and lactation. Female virgin Cox-2 transgenic mice showed precocious lobuloalveolar differentiation and enhanced expression of the beta-casein gene, which was inhibited by the Cox inhibitor indomethacin. Mammary gland involution was delayed in Cox-2 transgenic mice with a decrease in apoptotic index of mammary epithelial cells. Multiparous but not virgin females exhibited a greatly exaggerated incidence of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors. Cox-2-induced tumor tissue expressed reduced levels of the proapoptotic proteins Bax and Bcl-x(L) and an increase in the anti-apoptotic protein Bcl-2, suggesting that decreased apoptosis of mammary epithelial cells contributes to tumorigenesis. These data indicate that enhanced Cox-2 expression is sufficient to induce mammary gland tumorigenesis. Therefore, inhibition of Cox-2 may represent a mechanism-based chemopreventive approach for carcinogenesis.     

Pten对奶牛乳腺上皮细胞活力及乳蛋白合成的影响

Pten作为抑癌基因,参与调控细胞生长、粘附、凋亡以及其它细胞活动.目前,国内外关于Pten在奶牛乳腺发育过程中表达及调节的研究鲜有报道.为了揭示Pten的表达与奶牛乳腺发育与泌乳之间的关系,本研究应用qRT-PCR技术检测Pten在不同泌乳时期和不同乳品质的奶牛乳腺组织中的表达差异,进而应用脂质体转染方法,通过siRNA介导的RNA干扰技术改变Pten基因在奶牛乳腺上皮细胞中的表达量,CASY法检测细胞活力,用ELISA试剂盒检测细胞分泌β-酪蛋白的含量,采用qRT-PCR、Western 印迹等技术检测Pten对奶牛乳腺上皮细胞中乳蛋白相关信号通路基因表达的影响.结果显示,泌乳期高乳品质奶牛乳腺组织中Pten表达水平显著低于泌乳期低乳品质及干乳期奶牛;Pten基因沉寂后,细胞活力提高,β-酪蛋白质量浓度增加,CSN2、AKT、MTOR、STAT5表达量增加.研究表明,Pten可通过抑制细胞活力和乳蛋白分泌而影响泌乳.     

Down-regulation of sfrp1 in a mammary epithelial cell line promotes the development of a cd44high/cd24low population which is invasive and resistant to anoikis

Background  

The Wnt family of secreted proteins is implicated in the regulation of cell fate during development, as well as in cell proliferation, morphology, and migration. Aberrant activation of the Wnt/β-catenin signaling pathway leads to the development of several human cancers, including breast cancer. Secreted frizzled-related protein 1 (SFRP1) antagonizes this pathway by competing with the Frizzled receptor for Wnt ligands resulting in an attenuation of the signal transduction cascade. Loss of SFRP1 expression is observed in breast cancer, along with several other cancers, and is associated with poor patient prognosis. However, it is not clear whether the loss of SFRP1 expression predisposes the mammary gland to tumorigenesis.     

The proto-oncogene int-1 encodes a secreted protein associated with the extracellular matrix.

The proto-oncogene int-1 plays an important role in mammary tumorigenesis when activated by proviral insertions of the mouse mammary tumor virus. In normal mouse tissues the gene is expressed in the embryonic neural tube, suggesting a developmental function, while in Drosophila the homolog of int-1 is the segment polarity gene wingless. In order to study the protein products of int-1 we have derived fibroblast cell lines infected with multiple copies of a retroviral vector expressing int-1 cDNA. By Western blot analysis and immunoprecipitation we have identified a 44 kd form of int-1 protein which is secreted from these cells. The 44 kd species is distinct from the major intracellular forms of int-1 protein as judged by its slower mobility in SDS-polyacrylamide gels and by its longer half-life in pulse-chase experiments. Under normal growth conditions, little or none of the 44 kd protein is detectable in the cell culture medium but instead the majority is found associated with the extracellular matrix (ECM). The protein appears to bind heparin in vitro, suggesting that it might bind glycosaminoglycans in the ECM. These data support the view that int-1 protein may play a role in cell-cell communication over short distances.     

SFRP2 and Slug Contribute to Cellular Resistance to Apoptosis in Hypertrophic Scars

Hypertrophic scars (HS) are skin disorders which occur after wounding and thermal injury. Our previous studies have suggested that secreted frizzled-related protein 2 (SFRP2) is involved in HS formation and that the suppression of SFRP2 promotes apoptosis of hypertrophic scar fibroblasts (HSFBs). However, the mechanisms have not been clarified. Previous studies revealed that Slug expression inhibits cell apoptosis, in vitro and in vivo, and SFRP2 regulates the expression of Slug in cervical cancer cells. In the present study, we quantified differential expression levels of expression of SFRP2 and Slug in HS and normal skin tissues by immunohistochemistry, both of which have important anti-apoptosis roles. Furthermore, a short hairpin RNA approach was adopted to investigate the potential function of SFRP2 and Slug in HSFB apoptosis. Cell apoptosis was detected using fluorescence-activated cell sorting and Caspase-3 activity was assayed by spectrophotometry. This study demonstrates that SFRP2 expression, as well as Slug, is dramatically up-regulated in HS relative to normal skin tissues, and the Slug expression is positively correlated with SFRP2. Slug expression was down-regulated in SFRP2-deficient cells, and the down-regulation of Slug expression increased sensitivity to apoptosis which was induced through a caspase-3-dependent pathway. The infected cells with reduced levels of Slug were tested for the expression of apoptosis-related genes (Bcl-2, Bax and PUMA) which were previously identified as Slug targets. Bcl-2 expression was down-regulated in Slug-deficient cells. In conclusion, SFRP2 appears to interact with Slug to affect the apoptosis of hypertrophic scar fibroblasts.     

The estrogen-responsive Agr2 gene regulates mammary epithelial proliferation and facilitates lobuloalveolar development

Agr2 is a putative protein disulfide isomerase (PDI) initially identified as an estrogen-responsive gene in breast cancer cell lines. While Agr2 expression in breast cancer is positively correlated with estrogen receptor (ER) expression, it is upregulated in both hormone dependent and independent carcinomas. Several in vitro and xenograft studies have implicated Agr2 in different oncogenic features of breast cancer; however, the physiological role of Agr2 in normal mammary gland development remains to be defined. Agr2 expression is developmentally regulated in the mammary gland, with maximum expression during late pregnancy and lactation. Using a mammary gland specific knockout mouse model, we show that Agr2 facilitates normal lobuloalveolar development by regulating mammary epithelial cell proliferation; we found no effects on apoptosis in Agr2(-/-) mammary epithelial cells. Consequently, mammary glands of Agr2(-/-) females exhibit reduced expression of milk proteins, and by two weeks post-partum their pups are smaller in size. Utilizing a conditional mouse model, we show that Agr2 constitutive expression drives precocious lobuloalveolar development and increased milk protein expression in the virgin mammary gland. In vitro studies using knock down and overexpression strategies in estrogen receptor positive and negative mammary epithelial cell lines demonstrate a role for Agr2 in estradiol-induced cell proliferation. In conclusion, the estrogen-responsive Agr2, a candidate breast cancer oncogene, regulates epithelial cell proliferation and lobuloalveolar development in the mammary gland. The pro-proliferative effects of Agr2 may explain its actions in early tumorigenesis.     

Epithelial-to-mesenchymal transition confers resistance to apoptosis in three murine mammary epithelial cell lines

Epithelial-to-mesenchymal transition (EMT) is an essential embryogenic and developmental process, characterized by altered cellular morphology, loss of cell adhesion, and gain of migratory ability. Dysregulation of this process has been implicated in tumorigenesis, mediating the acquisition of migratory and invasive phenotypes by tumor cells. Mammary epithelial cells provide an excellent model in which to study the process, being derived from mammary gland tissue that utilizes EMT to facilitate branching morphogenesis through which the developing gland migrates into and invades the fat pad. Inappropriate EMT has been heavily implicated in the progression of ductal hyperplasia and mammary tumor metastasis. We examined the morphological and molecular changes of three murine mammary epithelial cell lines following EMT induction. EMT was induced in the EpH-4 and NMuMG cell lines by transforming growth factor (TGF)-beta1 but not by ethanol, while the KIM-2 cell line was partially resistant to TGF-beta1 but responded fully to ethanol. The response to EMT-inducing reagent was shown to be critically dependent on the time of treatment, with confluent cells failing to respond. Timelapse photography identified increased motility during wound healing in cells pre-treated with EMT-inducing reagent compared with untreated controls. Furthermore, EMT conferred resistance to UV-induced apoptosis. Our data indicate that evaluation of characteristics other than loss and gain of phenotypic markers may be of benefit when assessing EMT, and contribute to the evidence suggesting that inappropriate EMT facilitates the acquisition of resistance to apoptosis, a key characteristic required for tumor survival.     

Cyr61 suppresses growth of human endometrial cancer cells

Cyr61 (CCN1) is a member of the CCN protein family; these secreted proteins are involved in diverse biological processes such as cell adhesion, angiogenesis, apoptosis, and either growth arrest or growth stimulation depending on the cellular context. We studied the role of Cyr61 in endometrial tumorigenesis. Levels of Cyr61 were decreased in endometrial tumors compared with normal endometrium. Knockdown of Cyr61 expression by RNA interference in a well differentiated endometrial adenocarcinoma cell line (Ishikawa) stimulated its cellular growth. Conversely, overexpression of the protein in the undifferentiated AN3CA endometrial cancer cell line decreased their growth concurrently with increased apoptosis in liquid culture. These same cells had decreased clonogenic capacity and a nearly complete loss of tumorigenicity in vivo. Furthermore, partially purified Cyr61 suppressed growth of endometrial cancer cells. The increased apoptosis in these endometrial cancer cells with forced overexpression of Cyr61 was associated with elevated expression of the pro-apoptotic proteins Bax, Bad, and TRAIL (tumor necrosis factor receptor-associated ligand). Cyr61-induced caspase-3 activation and depolarization of mitochondrial membrane. In summary, endometrial cancer cells have decreased expression of Cyr61 compared with normal endometrium, and this lowered expression may provide the transformed cells a growth advantage over their normal counterpart.     

The Role of the Microenvironment in Mammary Gland Development and Cancer

The mammary gland is composed of a diverse array of cell types that form intricate interaction networks essential for its normal development and physiologic function. Abnormalities in these interactions play an important role throughout different stages of tumorigenesis. Branching ducts and alveoli are lined by an inner layer of secretory luminal epithelial cells that produce milk during lactation and are surrounded by contractile myoepithelial cells and basement membrane. The surrounding stroma comprised of extracellular matrix and various cell types including fibroblasts, endothelial cells, and infiltrating leukocytes not only provides a scaffold for the organ, but also regulates mammary epithelial cell function via paracrine, physical, and hormonal interactions. With rare exceptions breast tumors initiate in the epithelial compartment and in their initial phases are confined to the ducts but this barrier brakes down with invasive progression because of a combination of signals emitted by tumor epithelial and various stromal cells. In this article, we overview the importance of cellular interactions and microenvironmental signals in mammary gland development and cancer.The mammary gland is composed of a combination of multiple cell types that together form complex interaction networks required for the proper development and functioning of the organ. The branching milk ducts are formed by an outer myoepithelial cell layer producing the basement membrane (BM) and an inner luminal epithelial cell layer producing milk during lactation. The ducts are surrounded by the microenvironment composed of extracellular matrix (ECM) and various stromal cell types (e.g., endothelial cells, fibroblasts, myofibroblasts, and leukocytes). Large amount of data suggest that cell-cell and cell-microenvironment interactions modify the proliferation, survival, polarity, differentiation, and invasive capacity of mammary epithelial cells. However, the molecular mechanisms underlying these effects are poorly understood. The purification and comprehensive characterization of each cell type comprising normal and neoplastic human breast tissue combined with hypothesis testing in cell culture and animal models are likely to improve our understanding of the role these cells play in the normal functioning of the mammary gland and in breast tumorigenesis. In this article, we overview cellular and microenvironmental interactions that play important roles in the normal functioning of the mammary gland and their abnormalities in breast cancer.     

RANK overexpression in transgenic mice with mouse mammary tumor virus promoter-controlled RANK increases proliferation and impairs alveolar differentiation in the mammary epithelia and disrupts lumen formation in cultured epithelial acini

RANK and RANKL, the key regulators of osteoclast differentiation and activation, also play an important role in the control of proliferation and differentiation of mammary epithelial cells during pregnancy. Here, we show that RANK protein expression is strictly regulated in a spatial and temporal manner during mammary gland development. RANK overexpression under the control of the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model results in increased mammary epithelial cell proliferation during pregnancy, impaired differentiation of lobulo-alveolar structures, decreased expression of the milk proteins beta-casein and whey acidic protein, and deficient lactation. We also show that treatment of three-dimensional in vitro cultures of primary mammary cells from MMTV-RANK mice with RANKL results in increased proliferation and decreased apoptosis in the luminal area, resulting in bigger acini with filled lumens. Taken together, these results suggest that signaling through RANK not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells. Moreover, the increased proliferation and survival observed in a three-dimensional culture system suggests a role for aberrant RANK signaling during breast tumorigenesis.     

Collagen XVI expression is upregulated in glioblastomas and promotes tumor cell adhesion

The poor prognosis of glioblastoma patients is related to diffuse brain invasion and interaction of tumor cells with extracellular matrices (ECM). We describe expression and function of the FACIT-collagen XVI in glioblastomas. We found upregulation of collagen XVI mRNA as well as protein in glioblastomas as compared to normal cortex. SiRNA knockdown resulted in decreased cell adhesion whereas increased adhesion was observed on surfaces coated with collagen XVI. The migration of glioblastoma cells on this substrate remained unchanged. Our results demonstrate de-novo expression of collagen XVI in glioblastomas as part of the tumor specific remodeling of the ECM.     

Myofibroblast-Derived SFRP1 as Potential Inhibitor of Colorectal Carcinoma Field Effect

Epigenetic changes of stromal-epithelial interactions are of key importance in the regulation of colorectal carcinoma (CRC) cells and morphologically normal, but genetically and epigenetically altered epithelium in normal adjacent tumor (NAT) areas. Here we demonstrated retained protein expression of well-known Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) in stromal myofibroblasts and decreasing epithelial expression from NAT tissues towards the tumor. SFRP1 was unmethylated in laser microdissected myofibroblasts and partially hypermethylated in epithelial cells in these areas. In contrast, we found epigenetically silenced myofibroblast-derived SFRP1 in CRC stroma. Our results suggest that the myofibroblast-derived SFRP1 protein might be a paracrine inhibitor of epithelial proliferation in NAT areas and loss of this signal may support tumor proliferation in CRC.     

Mouse Mammary Tumor Virus Suppresses Apoptosis of Mammary Epithelial Cells through ITAM-Mediated Signaling

Many receptors in hematopoietic cells use a common signaling pathway that relies on a highly conserved immunoreceptor tyrosine-based activation motif (ITAM), which signals through Src family tyrosine kinases. ITAM-bearing proteins are also found in many oncogenic viruses, including the mouse mammary tumor virus (MMTV) envelope (Env). We previously showed that MMTV Env expression transformed normal mammary epithelial cells and that Src kinases were important mediators in this transformation. To study how ITAM signaling affects mammary cell transformation, we utilized mammary cell lines expressing two different ITAM-containing proteins, one encoding a MMTV provirus and the other a B cell receptor fusion protein. ITAM-expressing cells were resistant to both serum starvation- and chemotherapeutic drug-induced apoptosis, whereas cells transduced with these molecules bearing ITAM mutations were indistinguishable from untransduced cells in their sensitivity to these treatments. We also found that Src kinase was activated in the MMTV-expressing cells and that MMTV-induced apoptosis resistance was completely restored by the Src inhibitor PP2. In vivo, MMTV infection delayed involution-induced apoptosis in the mouse mammary gland. Our results show that MMTV suppresses apoptosis through ITAM-mediated Src tyrosine kinase signaling. These studies could lead to the development of effective treatment of nonhematopoietic cell cancers in which ITAM-mediated signaling plays a role.