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Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways

View Article: PubMed Central - PubMed

ABSTRACT

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). While there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET-independent. Indeed, the MET-independent pathway was supported by reanalysis of pre-clinical and clinical data. These results provide evidence for both MET-dependent and MET-independent metastatic pathways.

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AT3 and DU145 cells metastasize via MET-independent pathwaysA. DTs and AT3 cells were left untransfected or transfected with an empty vector (pcDNA6) or E-cadDipIIIcI2 and subsequently selected with blasticidin. All untransfected DT and AT3 cells are killed by blasticidin, while DTs and AT3 cells transfected with an empty vector containing a blasticidin resistance marker survive blasticidin selection. Importantly, only mesenchymal AT3 cells transfected with the E-cadDipIIIcI2 suicide reporter grow out during blasticidin selection while all epithelial DT cells with the reporter die. B. Quantification of DT and AT3 growth during blasticidin selection by WST-1 cell growth assay. C. Representative lung metastases from AT3 cells harboring RIIIcI2+Gint or E-cadDipIIIcI2+Gint. D. No difference was observed in the number of macrometastases in each group. E. Injection of DU145 E-cadCreIIIcI2 (as a control) or E-cadDipIIIcI2 cells led to formation of lung metastases with 100% penetrance. There was no difference in the number of metastases between control cells or cells with the suicide reporter. F. DU145 E-cadDipIIIcI2 metastases lack E-cadherin expression and stain positive for vimentin.
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Figure 4: AT3 and DU145 cells metastasize via MET-independent pathwaysA. DTs and AT3 cells were left untransfected or transfected with an empty vector (pcDNA6) or E-cadDipIIIcI2 and subsequently selected with blasticidin. All untransfected DT and AT3 cells are killed by blasticidin, while DTs and AT3 cells transfected with an empty vector containing a blasticidin resistance marker survive blasticidin selection. Importantly, only mesenchymal AT3 cells transfected with the E-cadDipIIIcI2 suicide reporter grow out during blasticidin selection while all epithelial DT cells with the reporter die. B. Quantification of DT and AT3 growth during blasticidin selection by WST-1 cell growth assay. C. Representative lung metastases from AT3 cells harboring RIIIcI2+Gint or E-cadDipIIIcI2+Gint. D. No difference was observed in the number of macrometastases in each group. E. Injection of DU145 E-cadCreIIIcI2 (as a control) or E-cadDipIIIcI2 cells led to formation of lung metastases with 100% penetrance. There was no difference in the number of metastases between control cells or cells with the suicide reporter. F. DU145 E-cadDipIIIcI2 metastases lack E-cadherin expression and stain positive for vimentin.

Mentions: To test the suicide reporters DT and AT3 cells were transfected with either an empty vector control (pcDNA6) containing a blasticidin resistance gene or pcDNA6+E-cadDipIIIcI2. Two days after transfection, cells were treated with blasticidin. After six days of blasticidin selection, all mock transfected DT and AT3 cells were killed, while both cell types transfected with an empty vector that conferred blasticidin resistance grew out efficiently (Figure 4A). There was, however, a remarkable difference between DT and AT3 cells transfected with E-cadDipIIIcI2. While there were very few, if any, DT colonies, many AT3 colonies were detected (Figure 4A). Notably, AT3 cells containing the suicide reporter grew out at a slower rate than AT3 cells harboring an empty vector, likely due to the toxicity of even minute levels of DipA expression.


Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways
AT3 and DU145 cells metastasize via MET-independent pathwaysA. DTs and AT3 cells were left untransfected or transfected with an empty vector (pcDNA6) or E-cadDipIIIcI2 and subsequently selected with blasticidin. All untransfected DT and AT3 cells are killed by blasticidin, while DTs and AT3 cells transfected with an empty vector containing a blasticidin resistance marker survive blasticidin selection. Importantly, only mesenchymal AT3 cells transfected with the E-cadDipIIIcI2 suicide reporter grow out during blasticidin selection while all epithelial DT cells with the reporter die. B. Quantification of DT and AT3 growth during blasticidin selection by WST-1 cell growth assay. C. Representative lung metastases from AT3 cells harboring RIIIcI2+Gint or E-cadDipIIIcI2+Gint. D. No difference was observed in the number of macrometastases in each group. E. Injection of DU145 E-cadCreIIIcI2 (as a control) or E-cadDipIIIcI2 cells led to formation of lung metastases with 100% penetrance. There was no difference in the number of metastases between control cells or cells with the suicide reporter. F. DU145 E-cadDipIIIcI2 metastases lack E-cadherin expression and stain positive for vimentin.
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Related In: Results  -  Collection

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Figure 4: AT3 and DU145 cells metastasize via MET-independent pathwaysA. DTs and AT3 cells were left untransfected or transfected with an empty vector (pcDNA6) or E-cadDipIIIcI2 and subsequently selected with blasticidin. All untransfected DT and AT3 cells are killed by blasticidin, while DTs and AT3 cells transfected with an empty vector containing a blasticidin resistance marker survive blasticidin selection. Importantly, only mesenchymal AT3 cells transfected with the E-cadDipIIIcI2 suicide reporter grow out during blasticidin selection while all epithelial DT cells with the reporter die. B. Quantification of DT and AT3 growth during blasticidin selection by WST-1 cell growth assay. C. Representative lung metastases from AT3 cells harboring RIIIcI2+Gint or E-cadDipIIIcI2+Gint. D. No difference was observed in the number of macrometastases in each group. E. Injection of DU145 E-cadCreIIIcI2 (as a control) or E-cadDipIIIcI2 cells led to formation of lung metastases with 100% penetrance. There was no difference in the number of metastases between control cells or cells with the suicide reporter. F. DU145 E-cadDipIIIcI2 metastases lack E-cadherin expression and stain positive for vimentin.
Mentions: To test the suicide reporters DT and AT3 cells were transfected with either an empty vector control (pcDNA6) containing a blasticidin resistance gene or pcDNA6+E-cadDipIIIcI2. Two days after transfection, cells were treated with blasticidin. After six days of blasticidin selection, all mock transfected DT and AT3 cells were killed, while both cell types transfected with an empty vector that conferred blasticidin resistance grew out efficiently (Figure 4A). There was, however, a remarkable difference between DT and AT3 cells transfected with E-cadDipIIIcI2. While there were very few, if any, DT colonies, many AT3 colonies were detected (Figure 4A). Notably, AT3 cells containing the suicide reporter grew out at a slower rate than AT3 cells harboring an empty vector, likely due to the toxicity of even minute levels of DipA expression.

View Article: PubMed Central - PubMed

ABSTRACT

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). While there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET-independent. Indeed, the MET-independent pathway was supported by reanalysis of pre-clinical and clinical data. These results provide evidence for both MET-dependent and MET-independent metastatic pathways.

No MeSH data available.


Related in: MedlinePlus