ABSTRACT
Many tumors display intracellular heterogeneity with subsets of cancer stem cells (CSC) that sustain tumor growth, recurrence, and therapy resistance. Cancer-associated fibroblasts (CAF) have been shown to support and regulate CSC function. Here, we investigate the interactions between CSCs and CAFs in mammary gland tumors driven by combined activation of Wnt/ß-catenin and Hgf/Met signaling in mouse mammary epithelial cells. In this setting, CSCs secrete the Hedgehog ligand SHH, which regulate CAFs via paracrine activation of Hedgehog signaling. CAFs subsequently secrete factors that promote expansion and self-renewal of CSCs. In vivo treatment of tumors with the Hedgehog inhibitor vismodegib reduce CAF and CSC expansion, resulting in an overall delay of tumor formation. Our results identify a novel intracellular signaling module that synergistically regulates CAFs and CSCs. Targeting CAFs with Hedgehog inhibitors may offer a novel therapeutic strategy against breast cancer. Cancer Res; 77(8); 2134-47. ©2017 AACR.
Subject(s)
Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Hedgehog Proteins/metabolism , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Anilides/pharmacology , Animals , Cell Communication/physiology , Female , Mice , Proto-Oncogene Proteins c-met/metabolism , Pyridines/pharmacology , Signal Transduction , Wnt Proteins/metabolism , Wnt Signaling Pathway , beta Catenin/metabolismABSTRACT
OBJECTIVES: We have previously identified a 115-gene signature that characterises the metastatic potential of human primary colon cancers. The signature included the canonical Wnt target gene BAMBI, which promoted experimental metastasis in mice. Here, we identified three new direct Wnt target genes from the signature, and studied their functions in epithelial-mesenchymal transition (EMT), cell migration and experimental metastasis. DESIGN: We examined experimental liver metastases following injection of selected tumour cells into spleens of NOD/SCID mice. Molecular and cellular techniques were used to identify direct transcription target genes of Wnt/ß-catenin signals. Microarray analyses and experiments that interfered with cell migration through inhibitors were performed to characterise downstream signalling systems. RESULTS: Three new genes from the colorectal cancer (CRC) metastasis signature, BOP1, CKS2 and NFIL3, were identified as direct transcription targets of ß-catenin/TCF4. Overexpression and knocking down of these genes in CRC cells promoted and inhibited, respectively, experimental metastasis in mice, EMT and cell motility in culture. Cell migration was repressed by interfering with distinct signalling systems through inhibitors of PI3K, JNK, p38 mitogen-activated protein kinase and/or mTOR. Gene expression profiling identified a series of migration-promoting genes, which were induced by BOP1, CKS2 and NFIL3, and could be repressed by inhibitors that are specific to these pathways. CONCLUSIONS: We identified new direct Wnt/ß-catenin target genes, BOP1, CKS2 and NFIL3, which induced EMT, cell migration and experimental metastasis of CRC cells. These genes crosstalk with different downstream signalling systems, and activate migration-promoting genes. These pathways and downstream genes may serve as therapeutic targets in the treatment of CRC metastasis.
Subject(s)
Basic-Leucine Zipper Transcription Factors/genetics , CDC28 Protein Kinase, S cerevisiae/genetics , Cell Movement/genetics , Colorectal Neoplasms/genetics , Epithelial-Mesenchymal Transition/genetics , Liver Neoplasms , Nuclear Proteins/genetics , Wnt Signaling Pathway/genetics , Animals , CDC2-CDC28 Kinases , Cell Cycle Proteins , Disease Models, Animal , Humans , Liver Neoplasms/genetics , Liver Neoplasms/secondary , Liver Neoplasms, Experimental , Mice , Neoplasm Metastasis , RNA-Binding Proteins , Tumor Cells, CulturedABSTRACT
In this study, we have used techniques from cell biology, biochemistry, and genetics to investigate the role of the tyrosine phosphatase Shp2 in tumor cells of MMTV-PyMT mouse mammary glands. Genetic ablation or pharmacological inhibition of Shp2 induces senescence, as determined by the activation of senescence-associated ß-gal (SA-ß-gal), cyclin-dependent kinase inhibitor 1B (p27), p53, and histone 3 trimethylated lysine 9 (H3K9me3). Senescence induction leads to the inhibition of self-renewal of tumor cells and blockage of tumor formation and growth. A signaling cascade was identified that acts downstream of Shp2 to counter senescence: Src, focal adhesion kinase, and Map kinase inhibit senescence by activating the expression of S-phase kinase-associated protein 2 (Skp2), Aurora kinase A (Aurka), and the Notch ligand Delta-like 1 (Dll1), which block p27 and p53. Remarkably, the expression of Shp2 and of selected target genes predicts human breast cancer outcome. We conclude that therapies, which rely on senescence induction by inhibiting Shp2 or controlling its target gene products, may be useful in blocking breast cancer.
Subject(s)
Cellular Senescence , Mammary Neoplasms, Animal/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Animals , Aurora Kinase A/genetics , Aurora Kinase A/metabolism , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Calcium-Binding Proteins , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Female , Histones , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/pathology , Methylation , Mice , Mice, Transgenic , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , S-Phase Kinase-Associated Proteins/genetics , S-Phase Kinase-Associated Proteins/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
HER2-positive breast tumors are associated with a high risk of brain relapse. HER3 is thought to be an indispensible signaling substrate for HER2 (encoded by ERBB2) and is induced in breast cancer-brain metastases, though the molecular mechanisms by which this oncogenic dimer promotes the development of brain metastases are still elusive. We studied the effects of the HER3-HER2 ligand, heregulin (neuregulin-1, broadly expressed in the brain), on luminal breast cancer cell lines in vitro. Treatment of SKBr3 (ERBB2-amplified), MDA-MB-361 (ERBB2-amplified, metastatic brain tumor-derived) and MCF7 (HER2-positive, not ERBB2-amplified) cells with exogenous heregulin increased proliferation and adhesive potential, concomitant with induction of cyclin D1 and ICAM-1, and suppression of p27. All three cell lines invaded through matrigel toward a heregulin chemotactic signal in transwell experiments, associated with activation of extracellular cathepsin B and matrix metalloproteinase-9 (MMP-9). Moreover, heregulin induced breast cancer cell transmigration across a tight barrier of primary human brain microvascular endothelia. This was dependent on the activity of HER2, HER3 and MMPs, and was completely abrogated by combination HER2-HER3 blockade using Herceptin® and the humanized HER3 monoclonal antibody, EV20. Collectively these data suggest mechanisms by which the HER3-HER2 dimer promotes development of metastatic tumors in the heregulin-rich brain microenvironment.
Subject(s)
Blood-Brain Barrier/metabolism , Breast Neoplasms/pathology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Neuregulin-1/metabolism , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/metabolism , Blood-Brain Barrier/enzymology , Blood-Brain Barrier/pathology , Brain/blood supply , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/physiology , Endothelial Cells/metabolism , Female , Humans , MCF-7 Cells , Signal TransductionABSTRACT
Prognosis for patients with estrogen-receptor (ER)-negative basal breast cancer is poor, and chemotherapy is currently the best therapeutic option. We have generated a compound-mutant mouse model combining the activation of ß-catenin and HGF (Wnt-Met signaling), which produced rapidly growing basal mammary gland tumors. We identified the chemokine system CXCL12/CXCR4 as a crucial driver of Wnt-Met tumors, given that compound-mutant mice also deficient in the CXCR4 gene were tumor resistant. Wnt-Met activation rapidly expanded a population of cancer-propagating cells, in which the two signaling systems control different functions, self-renewal and differentiation. Molecular therapy targeting Wnt, Met, and CXCR4 in mice significantly delayed tumor development. The expression of a Wnt-Met 322 gene signature was found to be predictive of poor survival of human patients with ER-negative breast cancers. Thus, targeting CXCR4 and its upstream activators, Wnt and Met, might provide an efficient strategy for breast cancer treatment.
Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/diagnosis , Chemokine CXCL12/metabolism , Mammary Neoplasms, Experimental/metabolism , Proto-Oncogene Proteins c-met/metabolism , Receptors, CXCR4/metabolism , Wnt Signaling Pathway , Animals , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Chemokine CXCL12/genetics , Female , Genetic Therapy , Hepatocyte Growth Factor/genetics , Hepatocyte Growth Factor/metabolism , Humans , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/therapy , Mice , Middle Aged , Prognosis , Proto-Oncogene Proteins c-met/genetics , Receptors, CXCR4/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
We show that activation of Wnt/ß-catenin and attenuation of Bmp signals, by combined gain- and loss-of-function mutations of ß-catenin and Bmpr1a, respectively, results in rapidly growing, aggressive squamous cell carcinomas (SCC) in the salivary glands of mice. Tumours contain transplantable and hyperproliferative tumour propagating cells, which can be enriched by fluorescence activated cell sorting (FACS). Single mutations stimulate stem cells, but tumours are not formed. We show that ß-catenin, CBP and Mll promote self-renewal and H3K4 tri-methylation in tumour propagating cells. Blocking ß-catenin-CBP interaction with the small molecule ICG-001 and small-interfering RNAs against ß-catenin, CBP or Mll abrogate hyperproliferation and H3K4 tri-methylation, and induce differentiation of cultured tumour propagating cells into acini-like structures. ICG-001 decreases H3K4me3 at promoters of stem cell-associated genes in vitro and reduces tumour growth in vivo. Remarkably, high Wnt/ß-catenin and low Bmp signalling also characterize human salivary gland SCC and head and neck SCC in general. Our work defines mechanisms by which ß-catenin signals remodel chromatin and control induction and maintenance of tumour propagating cells. Further, it supports new strategies for the therapy of solid tumours.
Subject(s)
Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Salivary Gland Neoplasms/genetics , Salivary Gland Neoplasms/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Bone Morphogenetic Proteins/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , CREB-Binding Protein/antagonists & inhibitors , CREB-Binding Protein/metabolism , Carcinoma, Squamous Cell/pathology , Cell Proliferation/drug effects , Epigenesis, Genetic , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Histone Methyltransferases , Humans , Mice , Mice, Inbred NOD , Mice, Mutant Strains , Mice, SCID , Mice, Transgenic , Mutation , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Pyrimidinones/pharmacology , Salivary Gland Neoplasms/pathology , Transplantation, Heterologous , Wnt Signaling Pathway/drug effects , beta Catenin/antagonists & inhibitorsABSTRACT
The functional versatility of Wnt/ß-catenin signaling can be seen by its ability to act in stem cells of the embryo and of the adult as well as in cancer stem cells. During embryogenesis, stem cells demonstrate a requirement for ß-catenin in mediating the response to Wnt signaling for their maintenance and transition from a pluripotent state. In adult stem cells, Wnt signaling functions at various hierarchical levels to contribute to specification of different tissues. This has raised the possibility that the tightly regulated self-renewal mediated by Wnt signaling in stem and progenitor cells is subverted in cancer cells to allow malignant progression. Intensive work is currently being performed to resolve how intrinsic and extrinsic factors that regulate Wnt/ß-catenin signaling coordinate the stem and cancer stem cell states.
Subject(s)
Neoplastic Stem Cells/metabolism , Stem Cells/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway , Animals , Cell Transformation, Neoplastic , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Hematopoietic System/cytology , Hematopoietic System/metabolism , Hematopoietic System/pathology , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Intestinal Mucosa/metabolism , Intestines/cytology , Mammary Glands, Human/metabolism , Mammary Glands, Human/pathology , Neoplastic Stem Cells/pathology , Nervous System/cytology , Nervous System/metabolism , Nervous System/pathology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Skin/cytology , Skin/metabolism , Stem Cells/cytology , Sumoylation , Transcription, Genetic , Ubiquitination , beta Catenin/metabolismABSTRACT
Canonical Wnt signaling supports the formation and maintenance of stem and cancer stem cells. Recent studies have elucidated epigenetic mechanisms that control pluripotency and stemness, and allow a first assessment how embryonic and tissue stem cells are generated and maintained, and how Wnt signaling might be involved. The core of this review highlights the roles of Wnt signaling in stem and cancer stem cells of tissues such as skin, intestine and mammary gland. Lastly, we refer to the characterization of novel and powerful inhibitors of canonical Wnt signaling and describe attempts to bring these compounds into preclinical and clinical studies.
Subject(s)
Neoplastic Stem Cells/metabolism , Signal Transduction , Stem Cells/metabolism , Wnt Proteins/metabolism , Animals , Histones/metabolism , HumansABSTRACT
CXCR4 is a G protein-coupled receptor of considerable biological significance, and among its numerous functions, it is suggested to play a critical role in cancer metastasis. We have investigated the expression and function of CXCR4 in a range of breast cancer cell lines covering a spectrum of invasive phenotypes and found that, while surface levels of CXCR4 were uniform across the entire panel, only highly invasive cells that are metastatic in immunocompromised mice expressed functional receptors. CXCL12/SDF-1 induced cellular responses such as calcium mobilization, actin polymerization, and chemotaxis in metastatic cells, whereas noninvasive cells were unresponsive. Moreover, CXCL12 activated multiple signaling pathways downstream of G proteins in highly invasive cells but failed to activate any of the examined kinase cascades in noninvasive cell lines. This blockade in nonmetastatic cell lines seems to be due to the inability of G protein alpha and beta subunits to form a heterotrimeric complex with CXCR4. Galpha and Gbeta were able to bind to CXCR4 independently in all cell lines, but the association of G protein alphabetagamma heterotrimers with the receptor, a prerequisite for signal transduction downstream from G protein-coupled receptors, was only observed in the highly invasive cell lines. Our findings show, for the first time, that CXCR4 function is subject to complex and potentially tightly controlled regulation in breast cancer cells via differential G protein-receptor complex formation, and this regulation may play a role in the transition from nonmetastatic to malignant tumors.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , GTP-Binding Proteins/metabolism , Receptors, CXCR4/metabolism , Cell Line, Tumor , Cell Movement/physiology , Chemokine CXCL12 , Chemokines, CXC/metabolism , Humans , Ligands , Neoplasm Metastasis , Receptors, CXCR4/biosynthesis , Signal TransductionABSTRACT
In the multimolecular environment in tissues and organs, cross-talk between growth factor and G protein-coupled receptors is likely to play an important role in both normal and pathological responses. In this report, we demonstrate transactivation of the chemokine receptor CXCR4 by the growth factor insulin-like growth factor (IGF)-1 is required for IGF-1-induced cell migration in metastatic MDA-MB-231 cells. The induction of chemotaxis in MDA-MB-231 cells by IGF-1 was inhibited by pretreatment of the cells with pertussis toxin (PTX) and by RNAi-mediated knockdown of CXCR4. Transactivation of the CXCR4 pathway by IGF-1 occurred independently of CXCL12, the chemokine ligand of CXCR4. Neither CXCR4 knockdown nor PTX had any effect on the ability of IGF-1 to activate IGF-1R, suggesting that CXCR4 and G proteins are activated subsequent to, or independently of, phosphorylation of IGF-1R by IGF-1. Coprecipitation studies revealed the presence of a constitutive complex containing IGF-1R, CXCR4, and the G protein subunits, G(i)alpha2 and Gbeta, and stimulation of MDA-MB-231 cells with IGF-1 led to the release of G(i)alpha2 and Gbeta from CXCR4. Based on our findings, we propose that CXCR4 constitutively forms a complex with IGF-1R in MDA-MB-231 cells, and that this interaction allows IGF-1 to activate migrational signaling pathways through CXCR4, G(i)alpha2 and Gbeta.