PDGFR-induced autocrine SDF-1 signaling in cancer cells promotes metastasis in advanced skin carcinoma.
ABSTRACT: Advanced and undifferentiated skin squamous cell carcinomas (SCCs) exhibit aggressive growth and enhanced metastasis capability, which is associated in mice with an expansion of the cancer stem-like cell (CSC) population and with changes in the regulatory mechanisms that control the proliferation and invasion of these cells. Indeed, autocrine activation of PDGFR? induces CSC invasion and promotes distant metastasis in advanced SCCs. However, the mechanisms involved in this process were unclear. Here, we show that CSCs of mouse advanced SCCs (L-CSCs) express CXCR4 and CXCR7, both receptors of SDF-1. PDGFR? signaling induces SDF-1 expression and secretion, and the autocrine activation of this pathway in L-CSCs. Autocrine SDF-1/CXCR4 signaling induces L-CSC proliferation and survival, and mediates PDGFR?-induced invasion, promoting in vivo lung metastasis. Validation of these findings in patient samples of skin SCCs shows a strong correlation between the expression of SDF1, PDGFRA, and PDGFRB, which is upregulated, along CXCR4 in tumor cells of advanced SCCs. Furthermore, PDGFR regulates SDF-1 expression and inhibition of SDF-1/CXCR4 and PDGFR pathways blocks distant metastasis of human PD/S-SCCs. Our results indicate that functional crosstalk between PDGFR/SDF-1 signaling regulates tumor cell invasion and metastasis in human and mouse advanced SCCs, and suggest that CXCR4 and/or PDGFR inhibitors could be used to block metastasis of these aggressive tumors.
Project description:Cancer stem cells (CSCs) play a key role in tumour growth and metastasis. Although an expansion of CSC population was previously described in advanced cutaneous squamous cell carcinoma (SCC), it remains unknown whether CSC regulatory mechanisms also change through tumour progression to promote malignancy. Here, we generate mouse models of skin SCCs to study alterations in CSC regulation over progression. We found that features of CSCs change at late stage of progression, correlating with a switch from epithelial to mesenchymal tumour shape. beta-catenin and EGFR signalling are down-regulated, whereas autocrine FGFR1 and PDGFR alpha pathways are up-regulated in CSCs of advanced tumours. Inhibition of FGFR and PDGFR signalling repress malignant SCCs growth and metastasis, respectively. An enhanced epithelial-to-mesenchymal transition (EMT) program and over-expression of PDGFR alpha and FGFR1 are observed in malignant human skin SCCs, suggesting that these pathways are also induced over human SCC progression. Therefore, uncover signalling pathways controlling CSCs at specific stage of progression may improve the selection of more accurate targeted therapeutic strategies according to tumour stage, blocking SCC relapse and metastasis. Mouse models of skin SCCs progression were first generated. For that, individual samples of spontaneous or DMBA-TPA induced skin SCCs generated in K14-HPV16 mice were orthotopically engrafted in immunodeficient mice and then serially transplanted, generating lineages in which the progression from WD-SCCs to PD-SCCs was observed. Then, tumour cells expressing CD34 and alpha 6-integrin, previously identified as cutaneous cancer stem cells, were isolated by flow cytometry-sorter from WD-SCCs and PD-SCCs of different tumour lineages. RNA extraction and hybridization on Affymetrix microarrays was carried out to compare whole gene profiling of these populations of CSCs.
Project description:Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4-mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4-mediated phosphoproteome, including construction of kinase-substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, ?-catenin, and PPP1C? in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy.
Project description:Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras(G12D) activation and Smad4 deletion, to mouse keratin 15-expressing (K15+) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified ?-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with ?-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras(G12D) activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs.
Project description:Sarcomas are malignant tumors derived from mesenchymal tissues and may harbor a subset of cells with cancer stem-like cell (CSC) properties. Platelet-derived growth factor receptors ? and ? (PDGFR-?/?) play an important role in the maintenance of mesenchymal stem cells. Here we examine the role of PDGFR-?/? in sarcoma CSCs. PDGFR-?/? activity and the effects of PDGFR-?/? inhibition were examined in 3 human sarcoma cell lines using in vitro assays and mouse xenograft models. In all three cell lines, PDGFR-?/? activity was significantly higher in cells grown as spheroids (to enrich for CSCs) and in cells sorted for CD133 expression (a marker of sarcoma CSCs). Self-renewal transcription factors Nanog, Oct4, and Slug and epithelial-to-mesenchymal transition (EMT) proteins Snail, Slug, and Zeb1 were also significantly higher in spheroids cells and CD133(+) cells. Spheroid cells and CD133(+) cells demonstrated 2.9- to 4.2-fold greater migration and invasion and resistance to doxorubicin chemotherapy. Inhibition of PDGFR-?/? in CSCs using shRNA or pharmacologic inhibitors reduced expression of certain self-renewal and EMT proteins, reduced spheroid formation by 74-82%, reduced migration and invasion by 73-80%, and reversed chemotherapy resistance. In mouse xenograft models, combining PDGFR-?/? inhibition (using shRNA or imatinib) with doxorubicin had a more-than-additive effect in blocking tumor growth, with enhanced apoptosis, especially in CD133(+) cells. These results indicate that PDGFR-?/? activity is upregulated in sarcoma CSCs and promote CSC phenotypes including migration, invasion, and chemotherapy resistance. Thus, the PDGFR-?/? pathway represents a new potential therapeutic target to reduce metastatic potential and increase chemosensitivity.
Project description:Robust genes were up-regulated in the oral cancer cells with SDF-1/CXCR4 system; however, most of the genes did not exhibit metastasis-related functions. Mock cells and B88-SDF-1 cells, which have an autocrine SDF-1/CXCR4 system and exhibit distant metastatic potential in vivo were used.
Project description:Stromal cell-derived growth factor (SDF)-1? acts as a ligand to C-X-C chemokine receptors 4 (CXCR4) and 7 (CXCR7), which are involved in the formation of choroidal neovascularization. Previous studies have demonstrated crosstalk between the platelet-derived growth factor (PDGF)-BB/PDGF receptor (PDGFR)-? and SDF-1?/CXCR4 axes during tumor neovascularization by increasing the recruitment of pericytes. However, the effects of interactions between these two signaling pathways in retinal microvascular pericytes remain poorly understood. Western blotting and reverse transcription-quantitative PCR were used to measure CXCR4 and CXCR7 expression in PDGF-BB-treated pericytes, whilst Cell Counting Kit-8 and Transwell migration assays were used to investigate cell viability and migration following PDGF-BB pretreatment on SDF-1?-treated pericytes. Exogenous PDGF-BB enhanced CXCR4 and CXCR7 expression through PDGFR-? in a dose- and time-dependent manners. In addition, PDGF-BB increased cell viability and migration in SDF-1?-treated pericytes, which were inhibited by AMD3100 and niclosamide, inhibitors for CXCR4 and STAT3 respectively. Crosstalk between PDGF-BB/PDGFR-? and SDF-1?/CXCR4/CXCR7 were involved in the JAK2/STAT3 signaling pathway. PDGF-BB treatment enhanced CXCR4, CXCR7 and PDGFR-?expression, which may be associated with the phosphorylation of STAT3. siRNA-PDGFR-? transfection reduced CXCR4 and CXCR7 expression in pericytes. Therefore, PDGF-BB directly targets PDGFR-? and serves an important role in regulating CXCR4 and CXCR7 expression, ultimately affecting viability and migration in SDF-1?-treated pericytes. Therefore, targeting CXCR4/CXCR7 may serve as a potential therapeutic strategy for fundus diseases.
Project description:Stromal cell-derived factor 1 (SDF-1) is a chemokine that can be expressed in injured cardiomyocytes after myocardial infarction (MI). By combining with its receptor CXCR4, SDF-1 induced stem and progenitor cells migration. CXCR7, a novel receptor for SDF-1, has been identified recently. We aimed to explore the roles of SDF-1/CXCR4 and SDF-1/CXCR7 pathway and their crosstalk in CSCs migration. In the present study, CXCR4 and CXCR7 expression were identified in CSCs. Transwell assay showed that SDF-1 caused CSCs migration in a dose- and time-dependent manner, which could be significantly suppressed by CXCR4 or CXCR7 siRNA. Phospho-ERK, phospho-Akt and Raf-1 significantly elevated in CSCs with SDF-1 stimulation. Knockdown of CXCR4 or CXCR7 significantly decreased phospho-ERK or phospho-Akt, respectively, and eventually resulted in the inhibition of CSCs migration. Moreover, western blot showed that MK2206 (Akt inhibitor) increased the expression of phospho-ERK and Raf-1, whereas PD98059 (ERK inhibitor) had no effect on phospho-Akt and Raf-1. GW5074 (Raf-1 inhibitor) upregulated the expression of phospho-ERK, but had no effect on phospho-Akt. The present study indicated that SDF-1/CXCR7/Akt and SDF-1/CXCR4/ERK pathway played important roles in CSCs migration. Akt phosphorylation inhibited Raf-1 activity, which in turn dephosphorylated ERK and negatively regulated CSCs migration.
Project description:We have demonstrated that blocking CXCR4 may be a potent anti-metastatic therapy for CXCR4-related oral cancer. However, as CXCR4 antagonists are currently in clinical use to induce the mobilization of hematopoietic stem cells, continuous administration as an inhibitor for the metastasis may lead to persistent leukocytosis. In this study, we investigated the novel therapeutic downstream target(s) of the SDF-1/CXCR4 system, using B88-SDF-1 cells, which have an autocrine SDF-1/CXCR4 system and exhibit distant metastatic potential in vivo. Microarray analysis revealed that 418 genes were upregulated in B88-SDF-1 cells. We identified a gene that is highly upregulated in B88-SDF-1 cells, metabotropic glutamate receptor 5 (mGluR5), which was downregulated following treatment with 1,1' -[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist. The upregulation of mGluR5 mRNA in the SDF-1/CXCR4 system was predominately regulated by the Ras-extracellular signal-regulated kinase (ERK)1/2 pathway. Additionally, the growth of B88-SDF-1 cells was not affected by the mGluR5 agonist (S)-3,5-DHPG (DHPG) or the mGluR5 antagonists 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP). However, we observed that DHPG promoted B88-SDF-1 cell migration, whereas both MPEP and MTEP inhibited B88-SDF-1 cell migration. To assess drug toxicity, the antagonists were intraperitoneally injected into immunocompetent mice for 4 weeks. Mice injected with MPEP (5 mg/kg) and MTEP (5 mg/kg) did not exhibit any side effects, such as hematotoxicity, allergic reactions or weight loss. The administration of antagonists significantly inhibited the metastasis of B88-SDF-1 cells to the lungs of nude mice. These results suggest that blocking mGluR5 with antagonists such as MPEP and MTEP could prevent metastasis in CXCR4-related oral cancer without causing side effects.
Project description:CXCL12/stromal cell-derived factor-1alpha (SDF-1alpha), a chemokine ligand for the G protein-coupled receptor CXCR4, plays an important role in the directed movement of cells. Many studies have documented the importance of CXCR4 in tumor progression and organ-specific metastasis. Recently, several studies have implicated a role for SDF-1alpha in head and neck squamous cell carcinoma (HNSCC) metastasis, but currently there is little information about how SDF-1alpha promotes HNSCC metastasis. In this report we show that the NF-kappaB signaling pathway is activated in response to SDF-1alpha in HNSCC while primary and immortalized keratinocytes show no SDF-1alpha-mediated NF-kappaB activity. We found that SDF-1alpha-mediated NF-kappaB signaling is independent of phosphoinositide 3-kinase/Akt and ERK/MAPK pathways. We observed that SDF-1alpha induces IkappaBalpha phosphorylation and degradation and the nuclear translocation of NF-kappaB in HNSCC cell lines, suggesting that SDF-1alpha activates the classical NF-kappaB signaling pathway. Contrary to previous reports, SDF-1alpha-induced NF-kappaB activation is not mediated by tumor necrosis factor alpha. Furthermore, blocking the NF-kappaB signaling pathway with an IKKbeta inhibitor significantly reduces SDF-1alpha-mediated HNSCC invasion. Taken together, our data suggest SDF-1alpha/CXCR4 may promote HNSCC invasion and metastasis by activating NF-kappaB and that targeting NF-kappaB may provide therapeutic opportunities in preventing HNSCC metastasis mediated by SDF-1alpha.
Project description:In the network of chemokine signaling pathways, recent reports have described the SDF-1?/CXCR4 axis and its role in cancer progression and metastasis. Interestingly, we found downregulation of CXCR4 at both transcript and protein level in cervical cancer cell lines and primary tumors. We also found CXCR4 promoter hypermethylation in cervical cancer cell lines and primary biopsy samples. DNA hypomethylating drug 5-AZA-2'-deoxycytidine and histone deacetylase inhibitor Trichostatin A treatments in cell lines reactivate both CXCR4 transcription and protein expression. Cell adhesion assay demonstrated that autocrine SDF-1? promotes the loss of cell adhesion while paracrine SDF-1? predominantly protects the normal cervical cells from loss of cell adhesion. Cervical cancer cell line C-33A having increased expression of CXCR4 after TSA treatment showed increased cell adhesion by paracrine source of SDF-1? in comparison to untreated C-33A. These findings demonstrate the first evidence that epigenetic silencing of CXCR4 makes the cells inefficient to respond to the paracrine source of SDF-1? leading to loss of cell adhesion, one of the key events in metastases and progression of the disease. Our results provide novel insight of SDF-1?/CXCR4 signaling in tumor microenvironment which may be promising to further delineate molecular mechanism of cervical carcinogenesis.