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Recruitment of mesenchymal stem cells into prostate tumours promotes metastasis.

Jung Y, Kim JK, Shiozawa Y, Wang J, Mishra A, Joseph J, Berry JE, McGee S, Lee E, Sun H, Wang J, Jin T, Zhang H, Dai J, Krebsbach PH, Keller ET, Pienta KJ, Taichman RS - Nat Commun (2013)

Bottom Line: Here we show that CXCL16, a ligand for CXCR6, facilitates mesenchymal stem cell or very small embryonic-like cells recruitment into prostate tumours.CXCR6 signalling stimulates the conversion of mesenchymal stem cells into cancer-associated fibroblasts, which secrete stromal-derived factor-1, also known as CXCL12.CXCL12 expressed by cancer-associated fibroblasts then binds to CXCR4 on tumour cells and induces an epithelial-to-mesenchymal transition, which ultimately promotes metastasis to secondary tumour sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Tumours recruit mesenchymal stem cells to facilitate healing, which induces their conversion into cancer-associated fibroblasts that facilitate metastasis. However, this process is poorly understood on the molecular level. Here we show that CXCL16, a ligand for CXCR6, facilitates mesenchymal stem cell or very small embryonic-like cells recruitment into prostate tumours. CXCR6 signalling stimulates the conversion of mesenchymal stem cells into cancer-associated fibroblasts, which secrete stromal-derived factor-1, also known as CXCL12. CXCL12 expressed by cancer-associated fibroblasts then binds to CXCR4 on tumour cells and induces an epithelial-to-mesenchymal transition, which ultimately promotes metastasis to secondary tumour sites. Our results provide the molecular basis for mesenchymal stem cell recruitment into tumours and how this process leads to tumour metastasis.

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EMT-mediated CXCR4 is highly involved in prostate cancer metastasis(a) Migration assays were performed in Transwell® plates using 10% serum or CXCL12 as chemoattractants. Migration toward 0.5% serum was used as a negative control. (b) Blockade of CXCR4 by AMD3100 or anti-CXCR4 antibody prevents prostate cancer migration towards CXCL12 or MSCs isolated from CXCR6+/+, but not CXCR6−/− animals. Data in (a,b) are representativedata from two independent studies (mean±s.d., ANOVA). Significance was determined using a Student’s t-test. RFP-labeled RM1WT or RM1EMT cells (Supplementary Fig. S5a) were incubated with vehicle or AMD3100 in vitro, and then inoculated by intra-cardiac (i.c.) injection into CXCR6+/+ or CXCR6−/− (n = 7). Metastasis was assessed by qPCR for RFP in a number of tissues. (c,d) Number of metastatic RM1 cells following i.c. injection. *Significance between RM1WT treated with vehicle and RM1WT treated with AMD3100 (P < 0.05). #Significance between RM1WT treated with vehicle and RM1EMT cells treated with vehicle (P < 0.05). †Significance between RM1EMT treated with vehicle and RM1EMT treated with AMD3100 (P < 0.05). Error bars represents mean±s.d., n = 2 independent experiments, P < 0.05; Student’s t-test. (e-h) RM1 cells expressing RFP were identified in the femur of CXCR6+/+ or CXCR6−/− mice following i.c. injection. Red arrows identify RM1 cells. White arrows identify osteoblast on the bone surface staining positive for CXCL12 expression. Scale bars, 100μm. (f,h) Quantification of Fig. 5e and Fig. 5g, respectively. The numbers of RM1 cells were quantified on the endosteal region of the 7 long bones. Endosteal regions were defined as 12 cell diameters from bone surfaces. ((Mean±s.d. (n = 3)., ANOVA).
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Figure 5: EMT-mediated CXCR4 is highly involved in prostate cancer metastasis(a) Migration assays were performed in Transwell® plates using 10% serum or CXCL12 as chemoattractants. Migration toward 0.5% serum was used as a negative control. (b) Blockade of CXCR4 by AMD3100 or anti-CXCR4 antibody prevents prostate cancer migration towards CXCL12 or MSCs isolated from CXCR6+/+, but not CXCR6−/− animals. Data in (a,b) are representativedata from two independent studies (mean±s.d., ANOVA). Significance was determined using a Student’s t-test. RFP-labeled RM1WT or RM1EMT cells (Supplementary Fig. S5a) were incubated with vehicle or AMD3100 in vitro, and then inoculated by intra-cardiac (i.c.) injection into CXCR6+/+ or CXCR6−/− (n = 7). Metastasis was assessed by qPCR for RFP in a number of tissues. (c,d) Number of metastatic RM1 cells following i.c. injection. *Significance between RM1WT treated with vehicle and RM1WT treated with AMD3100 (P < 0.05). #Significance between RM1WT treated with vehicle and RM1EMT cells treated with vehicle (P < 0.05). †Significance between RM1EMT treated with vehicle and RM1EMT treated with AMD3100 (P < 0.05). Error bars represents mean±s.d., n = 2 independent experiments, P < 0.05; Student’s t-test. (e-h) RM1 cells expressing RFP were identified in the femur of CXCR6+/+ or CXCR6−/− mice following i.c. injection. Red arrows identify RM1 cells. White arrows identify osteoblast on the bone surface staining positive for CXCL12 expression. Scale bars, 100μm. (f,h) Quantification of Fig. 5e and Fig. 5g, respectively. The numbers of RM1 cells were quantified on the endosteal region of the 7 long bones. Endosteal regions were defined as 12 cell diameters from bone surfaces. ((Mean±s.d. (n = 3)., ANOVA).

Mentions: To explore the extent to which CXCL16 drives metastasis, we determined whether CAF-derived CXCL12 activates an EMT in prostate cancer cells (Supplementary Fig. S4a). Loss of cell-cell contacts and the emergence of a spindle-shaped morphology was observed following CXCL12 treatments of prostate cancer cells or when they were cocultured with MSCsCXCR6+/+, but not when cocultered with MSCCXCR6−/− (Fig. 4a). In fact, when prostate cancer cells were treated with CXCL12 or cocultured with MSCsCXCR6+/+, but not MSCsCXCR6−/− cells, a near complete loss of the epithelial transcriptome occurred including E-cadherin, reduced cytokeratin, enhanced expression of N-cadherin, vimentin, α-SMA, β-catenin, snail, and slug were observed (Fig. 4a-c). When tumor microarrays were stained for E-cadherin or N-cadherin, more E-cadherin expressing prostate cancer cells were detected in the benign prostate tissues, whereas more N-cadherin expressing prostate cancer cells were detected in the Gleason 4+5 prostate cancers (Fig. 4d,e; Supplementary Fig. S4b)36-38. Enhanced expression of the CXCL12 receptor CXCR4 is known to facilitate migration and metastasis in vivo39,40. We observed that CXCR4 expression by prostate cancer was enhanced following induction of an EMT phenotype in vitro and was associated with enhanced tumor growth in vivo (Fig. 4f,g). Studies with anti-CXCR4 antibody and the CXCR4 inhibitor AMD3100 showed that CXCL12 induces prostate cancer towards an EMT phenotype (Fig. 4h; Supplementary Fig. S4c-e). In fact, prostate cancer cells which had undergone an EMT were significantly more responsive than their parental counterparts to CXCL12 or serum (Fig. 5a). Such that CXCR4 blockade prevented prostate cancer migration in vitro (Fig. 5b).


Recruitment of mesenchymal stem cells into prostate tumours promotes metastasis.

Jung Y, Kim JK, Shiozawa Y, Wang J, Mishra A, Joseph J, Berry JE, McGee S, Lee E, Sun H, Wang J, Jin T, Zhang H, Dai J, Krebsbach PH, Keller ET, Pienta KJ, Taichman RS - Nat Commun (2013)

EMT-mediated CXCR4 is highly involved in prostate cancer metastasis(a) Migration assays were performed in Transwell® plates using 10% serum or CXCL12 as chemoattractants. Migration toward 0.5% serum was used as a negative control. (b) Blockade of CXCR4 by AMD3100 or anti-CXCR4 antibody prevents prostate cancer migration towards CXCL12 or MSCs isolated from CXCR6+/+, but not CXCR6−/− animals. Data in (a,b) are representativedata from two independent studies (mean±s.d., ANOVA). Significance was determined using a Student’s t-test. RFP-labeled RM1WT or RM1EMT cells (Supplementary Fig. S5a) were incubated with vehicle or AMD3100 in vitro, and then inoculated by intra-cardiac (i.c.) injection into CXCR6+/+ or CXCR6−/− (n = 7). Metastasis was assessed by qPCR for RFP in a number of tissues. (c,d) Number of metastatic RM1 cells following i.c. injection. *Significance between RM1WT treated with vehicle and RM1WT treated with AMD3100 (P < 0.05). #Significance between RM1WT treated with vehicle and RM1EMT cells treated with vehicle (P < 0.05). †Significance between RM1EMT treated with vehicle and RM1EMT treated with AMD3100 (P < 0.05). Error bars represents mean±s.d., n = 2 independent experiments, P < 0.05; Student’s t-test. (e-h) RM1 cells expressing RFP were identified in the femur of CXCR6+/+ or CXCR6−/− mice following i.c. injection. Red arrows identify RM1 cells. White arrows identify osteoblast on the bone surface staining positive for CXCL12 expression. Scale bars, 100μm. (f,h) Quantification of Fig. 5e and Fig. 5g, respectively. The numbers of RM1 cells were quantified on the endosteal region of the 7 long bones. Endosteal regions were defined as 12 cell diameters from bone surfaces. ((Mean±s.d. (n = 3)., ANOVA).
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Figure 5: EMT-mediated CXCR4 is highly involved in prostate cancer metastasis(a) Migration assays were performed in Transwell® plates using 10% serum or CXCL12 as chemoattractants. Migration toward 0.5% serum was used as a negative control. (b) Blockade of CXCR4 by AMD3100 or anti-CXCR4 antibody prevents prostate cancer migration towards CXCL12 or MSCs isolated from CXCR6+/+, but not CXCR6−/− animals. Data in (a,b) are representativedata from two independent studies (mean±s.d., ANOVA). Significance was determined using a Student’s t-test. RFP-labeled RM1WT or RM1EMT cells (Supplementary Fig. S5a) were incubated with vehicle or AMD3100 in vitro, and then inoculated by intra-cardiac (i.c.) injection into CXCR6+/+ or CXCR6−/− (n = 7). Metastasis was assessed by qPCR for RFP in a number of tissues. (c,d) Number of metastatic RM1 cells following i.c. injection. *Significance between RM1WT treated with vehicle and RM1WT treated with AMD3100 (P < 0.05). #Significance between RM1WT treated with vehicle and RM1EMT cells treated with vehicle (P < 0.05). †Significance between RM1EMT treated with vehicle and RM1EMT treated with AMD3100 (P < 0.05). Error bars represents mean±s.d., n = 2 independent experiments, P < 0.05; Student’s t-test. (e-h) RM1 cells expressing RFP were identified in the femur of CXCR6+/+ or CXCR6−/− mice following i.c. injection. Red arrows identify RM1 cells. White arrows identify osteoblast on the bone surface staining positive for CXCL12 expression. Scale bars, 100μm. (f,h) Quantification of Fig. 5e and Fig. 5g, respectively. The numbers of RM1 cells were quantified on the endosteal region of the 7 long bones. Endosteal regions were defined as 12 cell diameters from bone surfaces. ((Mean±s.d. (n = 3)., ANOVA).
Mentions: To explore the extent to which CXCL16 drives metastasis, we determined whether CAF-derived CXCL12 activates an EMT in prostate cancer cells (Supplementary Fig. S4a). Loss of cell-cell contacts and the emergence of a spindle-shaped morphology was observed following CXCL12 treatments of prostate cancer cells or when they were cocultured with MSCsCXCR6+/+, but not when cocultered with MSCCXCR6−/− (Fig. 4a). In fact, when prostate cancer cells were treated with CXCL12 or cocultured with MSCsCXCR6+/+, but not MSCsCXCR6−/− cells, a near complete loss of the epithelial transcriptome occurred including E-cadherin, reduced cytokeratin, enhanced expression of N-cadherin, vimentin, α-SMA, β-catenin, snail, and slug were observed (Fig. 4a-c). When tumor microarrays were stained for E-cadherin or N-cadherin, more E-cadherin expressing prostate cancer cells were detected in the benign prostate tissues, whereas more N-cadherin expressing prostate cancer cells were detected in the Gleason 4+5 prostate cancers (Fig. 4d,e; Supplementary Fig. S4b)36-38. Enhanced expression of the CXCL12 receptor CXCR4 is known to facilitate migration and metastasis in vivo39,40. We observed that CXCR4 expression by prostate cancer was enhanced following induction of an EMT phenotype in vitro and was associated with enhanced tumor growth in vivo (Fig. 4f,g). Studies with anti-CXCR4 antibody and the CXCR4 inhibitor AMD3100 showed that CXCL12 induces prostate cancer towards an EMT phenotype (Fig. 4h; Supplementary Fig. S4c-e). In fact, prostate cancer cells which had undergone an EMT were significantly more responsive than their parental counterparts to CXCL12 or serum (Fig. 5a). Such that CXCR4 blockade prevented prostate cancer migration in vitro (Fig. 5b).

Bottom Line: Here we show that CXCL16, a ligand for CXCR6, facilitates mesenchymal stem cell or very small embryonic-like cells recruitment into prostate tumours.CXCR6 signalling stimulates the conversion of mesenchymal stem cells into cancer-associated fibroblasts, which secrete stromal-derived factor-1, also known as CXCL12.CXCL12 expressed by cancer-associated fibroblasts then binds to CXCR4 on tumour cells and induces an epithelial-to-mesenchymal transition, which ultimately promotes metastasis to secondary tumour sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Tumours recruit mesenchymal stem cells to facilitate healing, which induces their conversion into cancer-associated fibroblasts that facilitate metastasis. However, this process is poorly understood on the molecular level. Here we show that CXCL16, a ligand for CXCR6, facilitates mesenchymal stem cell or very small embryonic-like cells recruitment into prostate tumours. CXCR6 signalling stimulates the conversion of mesenchymal stem cells into cancer-associated fibroblasts, which secrete stromal-derived factor-1, also known as CXCL12. CXCL12 expressed by cancer-associated fibroblasts then binds to CXCR4 on tumour cells and induces an epithelial-to-mesenchymal transition, which ultimately promotes metastasis to secondary tumour sites. Our results provide the molecular basis for mesenchymal stem cell recruitment into tumours and how this process leads to tumour metastasis.

Show MeSH
Related in: MedlinePlus