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1.

Background

Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure.

Methods/Findings

To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/β-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118–310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118–310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118–310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118–310 treated mice were unable to seed the growth of secondary tumors after transplant.

Conclusions

These studies demonstrate that inhibitors of Wnt/β-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.  相似文献   

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The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/β-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, β-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/β-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.  相似文献   

5.

Background

While the role of canonical (β-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known.

Methodology/Principal Findings

Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while β-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes.

Conclusions/Significance

Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis through a pathway involving Wnt4, Fz6, Rac1 and Jnk kinases.  相似文献   

6.

Introduction

Canonical and non-canonical Wnt pathways are involved in the genesis of multiple tumors; however, their role in pituitary tumorigenesis is mostly unknown.

Objective

This study evaluated gene and protein expression of Wnt pathways in pituitary tumors and whether these expression correlate to clinical outcome.

Materials and Methods

Genes of the Wnt canonical pathway: activating ligands (WNT11, WNT4, WNT5A), binding inhibitors (DKK3, sFRP1), β-catenin (CTNNB1), β-catenin degradation complex (APC, AXIN1, GSK3β), inhibitor of β-catenin degradation complex (AKT1), sequester of β-catenin (CDH1), pathway effectors (TCF7, MAPK8, NFAT5), pathway mediators (DVL-1, DVL-2, DVL-3, PRICKLE, VANGL1), target genes (MYB, MYC, WISP2, SPRY1, TP53, CCND1); calcium dependent pathway (PLCB1, CAMK2A, PRKCA, CHP); and planar cell polarity pathway (PTK7, DAAM1, RHOA) were evaluated by QPCR, in 19 GH-, 18 ACTH-secreting, 21 non-secreting (NS) pituitary tumors, and 5 normal pituitaries. Also, the main effectors of canonical (β-catenin), planar cell polarity (JNK), and calcium dependent (NFAT5) Wnt pathways were evaluated by immunohistochemistry.

Results

There are no differences in gene expression of canonical and non-canonical Wnt pathways between all studied subtypes of pituitary tumors and normal pituitaries, except for WISP2, which was over-expressed in ACTH-secreting tumors compared to normal pituitaries (4.8x; p = 0.02), NS pituitary tumors (7.7x; p = 0.004) and GH-secreting tumors (5.0x; p = 0.05). β-catenin, NFAT5 and JNK proteins showed no expression in normal pituitaries and in any of the pituitary tumor subtypes. Furthermore, no association of the studied gene or protein expression was observed with tumor size, recurrence, and progressive disease. The hierarchical clustering showed a regular pattern of genes of the canonical and non-canonical Wnt pathways randomly distributed throughout the dendrogram.

Conclusions

Our data reinforce previous reports suggesting no activation of canonical Wnt pathway in pituitary tumorigenesis. Moreover, we describe, for the first time, evidence that non-canonical Wnt pathways are also not mis-expressed in the pituitary tumors.  相似文献   

7.

Background

Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients.

Methods

Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway.

Results

In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration.

Conclusions

We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.  相似文献   

8.

Background

Sonic hedgehog (Shh) signaling in the mouse requires the microtubule-based organelle, the primary cilium. The primary cilium is assembled and maintained through the process of intraflagellar transport (IFT) and the response to Shh is blocked in mouse mutants that lack proteins required for IFT. Although the phenotypes of mouse IFT mutants do not overlap with phenotypes of known Wnt pathway mutants, recent studies report data suggesting that the primary cilium modulates responses to Wnt signals.

Methodology/Principal Findings

We therefore carried out a systematic analysis of canonical Wnt signaling in mutant embryos and cells that lack primary cilia because of loss of the anterograde IFT kinesin-II motor (Kif3a) or IFT complex B proteins (Ift172 or Ift88). We also analyzed mutant embryos with abnormal primary cilia due to defects in retrograde IFT (Dync2h1). The mouse IFT mutants express the canonical Wnt target Axin2 and activate a transgenic canonical Wnt reporter, BAT-gal, in the normal spatial pattern and to the same quantitative level as wild type littermates. Similarly, mouse embryonic fibroblasts (MEFs) derived from IFT mutants respond normally to added Wnt3a. The switch from canonical to non-canonical Wnt also appears normal in IFT mutant MEFs, as both wild-type and mutant cells do not activate the canonical Wnt reporter in the presence of both Wnt3a and Wnt5a.

Conclusions

We conclude that loss of primary cilia or defects in retrograde IFT do not affect the response of the midgestation embryo or embryo-derived fibroblasts to Wnt ligands.  相似文献   

9.

Background  

The Wnt signal transduction pathway is important in a wide variety of developmental processes as well as in the genesis of human cancer. Vertebrate Wnt pathways can be functionally separated into two classes, the canonical Wnt/beta-catenin pathway and the non-canonical Wnt/Ca2+ pathway. Supporting differences in Wnt signaling, gain of function of Wnt-1 in C57mg mouse mammary epithelial cells leads to their morphological transformation while loss of function of Wnt-5a leads to the same transformation. Many downstream target genes of the Wnt/beta-catenin pathway have been identified. In contrast, little is known about the Wnt/Ca2+ pathway and whether it regulates gene expression.  相似文献   

10.

Background

A relationship between the increased density of tumor-associated macrophages (TAMs) and decreased survival was recently reported in thyroid cancer patients. Among these tumors, anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumors in humans. TAMs (type M2) have been recognized as promoting tumor growth. The purpose of our study was to analyze with immunohistochemistry the presence of TAMs in a series of 27 ATC.

Methodology/Principal Findings

Several macrophages markers such as NADPH oxidase complex NOX2-p22phox, CD163 and CD 68 were used. Immunostainings showed that TAMs represent more than 50% of nucleated cells in all ATCs. Moreover, these markers allowed the identification of elongated thin ramified cytoplasmic extensions, bestowing a “microglia-like” appearance on these cells which we termed “Ramified TAMs” (RTAMs). In contrast, cancer cells were totally negative. Cellular stroma was highly simplified since apart from cancer cells and blood vessels, RTAMs were the only other cellular component. RTAMs were evenly distributed and intermingled with cancer cells, and were in direct contact with other RTAMs via their ramifications. Moreover, RTAMs displayed strong immunostaining for connexin Cx43. Long chains of interconnected RTAMs arose from perivascular clusters and were dispersed within the tumor parenchyma. When expressed, the glucose transporter Glut1 was found in RTAMs and blood vessels, but rarely in cancer cells.

Conclusion

ATCs display a very dense network of interconnected RTAMs in direct contact with intermingled cancer cells. To our knowledge this is the first time that such a network is described in a malignant tumor. This network was found in all our studied cases and appeared specific to ATC, since it was not found in differentiated thyroid cancers specimens. Taken together, these results suggest that RTAMs network is directly related to the aggressiveness of the disease via metabolic and trophic functions which remain to be determined.  相似文献   

11.

Introduction

Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures.

Objective

We tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.

Results

We found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.

Conclusions

MSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.  相似文献   

12.

Background

Because of their regenerative and paracrine abilities, cardiac stem cells (CSCs) are the most appropriate, optimal and promising candidates for the development of cardiac regenerative medicine strategies. However, native and exogenous CSCs in ischemic hearts are exposed to various pro-apoptotic or cytotoxic factors preventing their regenerative and paracrine abilities.

Methods and Results

We examined the effects of H2O2 on mouse CSCs (mCSCs), and observed that hydrogen peroxide (H2O2) treatment induces mCSCs apoptosis via the caspase 3 pathway, in a dose-dependent manner. We then examined the effects of Wnt1 over-expression on H2O2-induced apoptosis in mCSCs and observed that Wnt1 significantly decreased H2O2-induced apoptosis in mCSCs. On the other hand, inhibition of the canonical Wnt pathway by the secreted frizzled related protein 2 (SFRP2) or knockdown of β-catenin in mCSCs reduced cells resistance to H2O2-induced apoptosis, suggesting that Wnt1 predominantly prevents H2O2-induced apoptosis through the canonical Wnt pathway.

Conclusions

Our results provide the first evidences that Wnt1 plays an important role in CSCs’ defenses against H2O2-induced apoptosis through the canonical Wnt1/GSK3β/β-catenin signaling pathway.  相似文献   

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Background

Morphogenesis results from the coordination of distinct cell signaling pathways controlling migration, differentiation, apoptosis, and proliferation, along stem/progenitor cell dynamics. To decipher this puzzle, we focused on epithelial-mesenchymal transition (EMT) “master genes”. EMT has emerged as a unifying concept, involving cell-cell adhesion, migration and apoptotic pathways. EMT also appears to mingle with stemness. However, very little is known on the physiological role and relevance of EMT master-genes. We addressed this question during mammary morphogenesis. Recently, a link between Slug/Snai2 and stemness has been described in mammary epithelial cells, but EMT master genes actual localization, role and targets during mammary gland morphogenesis are not known and we focused on this basic question.

Methodology/Principal Findings

Using a Slug–lacZ transgenic model and immunolocalization, we located Slug in a distinct subpopulation covering about 10–20% basal cap and duct cells, mostly cycling cells, coexpressed with basal markers P-cadherin, CK5 and CD49f. During puberty, Slug-deficient mammary epithelium exhibited a delayed development after transplantation, contained less cycling cells, and overexpressed CK8/18, ER, GATA3 and BMI1 genes, linked to luminal lineage. Other EMT master genes were overexpressed, suggesting compensation mechanisms. Gain/loss-of-function in vitro experiments confirmed Slug control of mammary epithelial cell luminal differentiation and proliferation. In addition, they showed that Slug enhances specifically clonal mammosphere emergence and growth, cell motility, and represses apoptosis. Strikingly, Slug-deprived mammary epithelial cells lost their potential to generate secondary clonal mammospheres.

Conclusions/Significance

We conclude that Slug pathway controls the growth dynamics of a subpopulation of cycling progenitor basal cells during mammary morphogenesis. Overall, our data better define a key mechanism coordinating cell lineage dynamics and morphogenesis, and provide physiological relevance to broadening EMT pathways.  相似文献   

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16.

Introduction

Wnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear.

Methods

We used a panel of normal and breast cancer cell lines to assess Wnt pathway gene and protein expression, and for the investigation of Wnt signalling within stem cell-enriched populations, mRNA and protein expression was analysed after the selection of anoikis-resistant cells. Finally, cell lines and patient-derived samples were used to investigate Wnt pathway effects on stem cell activity in vitro.

Results

Wnt pathway signalling increased in cancer compared to normal breast and in both cell lines and patient samples, expression of Wnt pathway genes correlated with estrogen receptor (ER) expression. Furthermore, specific Wnt pathway genes were predictive for recurrence within subtypes of breast cancer. Canonical Wnt pathway genes were increased in breast cancer stem cell-enriched populations in comparison to normal breast stem cell-enriched populations. Furthermore in cell lines, the ligand Wnt3a increased whilst the inhibitor DKK1 reduced mammosphere formation with the greatest inhibitory effects observed in ER+ve breast cancer cell lines. In patient-derived metastatic breast cancer samples, only ER-ve mammospheres were responsive to the ligand Wnt3a. However, the inhibitor DKK1 efficiently inhibited both ER+ve and ER-ve breast cancer but not normal mammosphere formation, suggesting that the Wnt pathway is aberrantly activated in breast cancer mammospheres.

Conclusions

Collectively, these data highlight differential Wnt signalling in breast cancer subtypes and activity in patient-derived metastatic cancer stem-like cells indicating a potential for Wnt-targeted treatment in breast cancers.  相似文献   

17.

Background

Targeted therapies, associated with standard chemotherapies, have improved breast cancer care. However, primary and acquired resistances are frequently observed and the development of new concepts is needed. High-throughput approaches to identify new active and safe molecules with or without an “a priori” are currently developed. Also, repositioning already-approved drugs in cancer therapy is of growing interest. The thiomorpholine hydroxamate compound TMI-1 has been previously designed to inhibit metalloproteinase activity for the treatment of rheumatoid arthritis. We present here the repositioning of TMI-1 drug in breast cancer.

Methodology/Principal Findings

We tested the effect of TMI-1 on luminal, basal and ERBB2-overexpressing breast tumor cell lines and on MMTV-ERBB2/neu tumor evolution. We measured the effects on i) cell survival, ii) cell cycle, iii) extrinsic and intrinsic apoptotic pathways, iv) association with doxorubicin, docetaxel and lapatinib, v) cancer stem cells compartment. In contrast with conventional cytotoxic drugs, TMI-1 was highly selective for tumor cells and cancer stem cells at submicromolar range. All non-malignant cells tested were resistant even at high concentration. TMI-1 was active on triple negative (TN) and ERBB2-overexpressing breast tumor cell lines, and was also highly efficient on human and murine “primary” ERBB2-overexpressing cells. Treatment of transgenic MMTV-ERBB2/neu mice with 100 mg/kg/day TMI-1 alone induced tumor apoptosis, inhibiting mammary gland tumor occurrence and development. No adverse effects were noticed during the treatment. This compound had a strong synergistic effect in association with docetaxel, doxorubicin and lapatinib. We showed that TMI-1 mediates its selective effects by caspase-dependent apoptosis. TMI-1 was efficient in 34/40 tumor cell lines of various origins (ED50: 0.6 µM to 12.5 µM).

Conclusions/Significance

This is the first demonstration of the tumor selective cytotoxic action of a thiomorpholin hydroxamate compound. TMI-1 is a novel repositionable drug not only for the treatment of adverse prognosis breast cancers but also for other neoplasms.  相似文献   

18.

Background

The pathways regulating the transition of mammalian cells from quiescence to proliferation are mediated by multiple miRNAs. Despite significant improvements in our understanding of miRNA targeting, the majority of miRNA regulatory networks are still largely unknown and require experimental validation.

Results

Here we identified miR-503, miR-103, and miR-494 as negative regulators of proliferation in primary human cells. We experimentally determined their genome wide target profiles using RNA-induced silencing complex (RISC) immunoprecipitations and gene expression profiling. Analysis of the genome wide target profiles revealed evidence of extensive regulation of gene expression through non-canonical target pairing by miR-503. We identified the proto-oncogene DDHD2 as a target of miR-503 that requires pairing outside of the canonical 5′ seed region of miR-503, representing a novel mode of miRNA-target pairing. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis.

Conclusions

Our results provide an extensive genome wide set of targets for miR-503, miR-103, and miR-494, and suggest that miR-503 may act as a tumor suppressor in breast cancer by its direct non-canonical targeting of DDHD2.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1279-9) contains supplementary material, which is available to authorized users.  相似文献   

19.
20.

Background

Our previous studies suggested that aberrant activation of Wnt/ß-catenin signaling might be involved in the pathophysiology of endometriosis. We hypothesized that inhibition of Wnt/ß-catenin signaling might result in inhibition of cell proliferation, migration, and/or invasion of endometrial and endometriotic epithelial and stromal cells of patients with endometriosis.

Objectives

The aim of the present study was to evaluate the effects of a small-molecule antagonist of the Tcf/ß-catenin complex (PKF 115–584) on cell proliferation, migration, and invasion of endometrial and endometriotic epithelial and stromal cells.

Methods

One hundred twenty-six patients (78 with and 48 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of PKF 115–584 on cell proliferation, migration, and invasion and on the Tcf/ß-catenin target genes were evaluated in endometrial epithelial and stromal cells of patients with and without endometriosis, and in endometrial and endometriotic epithelial and stromal cells of the same patients.

Results

The inhibitory effects of PKF 115–584 on cell migration and invasion in endometrial epithelial and stromal cells of patients with endometriosis prepared from the menstrual phase were significantly higher than those of patients without endometriosis. Levels of total and active forms of MMP-9 were significantly higher in epithelial and stromal cells prepared from menstrual endometrium in patients with endometriosis compared to patients without endometriosis. Treatment with PKF 115–584 inhibited MMP-9 activity to undetectable levels in both menstrual endometrial epithelial and stromal cells of patients with endometriosis. The number of invasive cells was significantly higher in epithelial and stromal cells of endometriotic tissue compared with matched eutopic endometrium of the same patients. Treatment with PKF 115–584 decreased the number of invasive endometriotic epithelial cells by 73% and stromal cells by 75%.

Conclusions

The present findings demonstrated that cellular mechanisms known to be involved in endometriotic lesion development are inhibited by targeting the Wnt/β-catenin pathway.  相似文献   

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