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

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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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Design and validation of lineage tracing reporters of META. Conceptual design of E-cadCreIIIcI2. In mesenchymal cells, the E-cadherin promoter is inactive and the IIIc exon is included; no Cre is produced. In epithelial cells, Cre is actively transcribed from the E-cadherin promoter and exon IIIc is efficiently skipped, leading to expression of Cre recombinase. E-cadCreIIIcI2 acts on RG, which contains a DsRed ORF and stop codon (red octagons) flanked by Loxp sites (triangles) followed by the EGFP ORF and a stop codon. MET leads to activation of Cre recombinase and a switch from DsRed to EGFP expression. B. DT and AT3 cells were stably transfected with RG. Cells subsequently transfected with pcDNA6 exclusively expressed DsRed, and cells harboring the Cre ORF activated EGFP expression. When transfected with the E-cadCreIIIcI2 reporter, only epithelial DT cells switched from DsRed to EGFP expression while AT3 cells maintained DsRed expression. Scale bar = 50 μm. C. Flow cytometry analysis shows that a small sub-population of AT3 cells undergo MET. D. RT-qPCR of DT and AT3 cells transfected with RG and E-cadCreIIIcI2 reveals approximately 10-fold higher expression of Cre in epithelial DT cells than mesenchymal AT3 cells.
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Figure 2: Design and validation of lineage tracing reporters of META. Conceptual design of E-cadCreIIIcI2. In mesenchymal cells, the E-cadherin promoter is inactive and the IIIc exon is included; no Cre is produced. In epithelial cells, Cre is actively transcribed from the E-cadherin promoter and exon IIIc is efficiently skipped, leading to expression of Cre recombinase. E-cadCreIIIcI2 acts on RG, which contains a DsRed ORF and stop codon (red octagons) flanked by Loxp sites (triangles) followed by the EGFP ORF and a stop codon. MET leads to activation of Cre recombinase and a switch from DsRed to EGFP expression. B. DT and AT3 cells were stably transfected with RG. Cells subsequently transfected with pcDNA6 exclusively expressed DsRed, and cells harboring the Cre ORF activated EGFP expression. When transfected with the E-cadCreIIIcI2 reporter, only epithelial DT cells switched from DsRed to EGFP expression while AT3 cells maintained DsRed expression. Scale bar = 50 μm. C. Flow cytometry analysis shows that a small sub-population of AT3 cells undergo MET. D. RT-qPCR of DT and AT3 cells transfected with RG and E-cadCreIIIcI2 reveals approximately 10-fold higher expression of Cre in epithelial DT cells than mesenchymal AT3 cells.

Mentions: Validation of the combinatorial reporters indicated that multiple regulatory elements provided enhanced specificity, which is critical for faithful reporter readout. As a result, the lineage tracing MET reporters were generated based on the design of the E-cadFFIIIcI2, in which the Firefly ORF was replaced by the Cre recombinase ORF to give E-cadCreIIIcI2. Low E-cadherin promoter activation and exon IIIc inclusion in mesenchymal cells should lead to very low, if any, Cre expression (Figure 2A). Conversely, epithelial cells should express Cre via activation of the E-cadherin promoter and exon IIIc skipping (Figure 2A). The E-cadCreIIIcI2 reporter acts on a second plasmid, RG (21), in which the DsRed ORF containing a stop codon is flanked by LoxP sites followed by the EGFP ORF containing a stop codon (Figure 2A). Expression of Cre during MET should lead to permanent removal of DsRed by recombination at LoxP sites and consistent expression of EGFP.


Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways
Design and validation of lineage tracing reporters of META. Conceptual design of E-cadCreIIIcI2. In mesenchymal cells, the E-cadherin promoter is inactive and the IIIc exon is included; no Cre is produced. In epithelial cells, Cre is actively transcribed from the E-cadherin promoter and exon IIIc is efficiently skipped, leading to expression of Cre recombinase. E-cadCreIIIcI2 acts on RG, which contains a DsRed ORF and stop codon (red octagons) flanked by Loxp sites (triangles) followed by the EGFP ORF and a stop codon. MET leads to activation of Cre recombinase and a switch from DsRed to EGFP expression. B. DT and AT3 cells were stably transfected with RG. Cells subsequently transfected with pcDNA6 exclusively expressed DsRed, and cells harboring the Cre ORF activated EGFP expression. When transfected with the E-cadCreIIIcI2 reporter, only epithelial DT cells switched from DsRed to EGFP expression while AT3 cells maintained DsRed expression. Scale bar = 50 μm. C. Flow cytometry analysis shows that a small sub-population of AT3 cells undergo MET. D. RT-qPCR of DT and AT3 cells transfected with RG and E-cadCreIIIcI2 reveals approximately 10-fold higher expression of Cre in epithelial DT cells than mesenchymal AT3 cells.
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Figure 2: Design and validation of lineage tracing reporters of META. Conceptual design of E-cadCreIIIcI2. In mesenchymal cells, the E-cadherin promoter is inactive and the IIIc exon is included; no Cre is produced. In epithelial cells, Cre is actively transcribed from the E-cadherin promoter and exon IIIc is efficiently skipped, leading to expression of Cre recombinase. E-cadCreIIIcI2 acts on RG, which contains a DsRed ORF and stop codon (red octagons) flanked by Loxp sites (triangles) followed by the EGFP ORF and a stop codon. MET leads to activation of Cre recombinase and a switch from DsRed to EGFP expression. B. DT and AT3 cells were stably transfected with RG. Cells subsequently transfected with pcDNA6 exclusively expressed DsRed, and cells harboring the Cre ORF activated EGFP expression. When transfected with the E-cadCreIIIcI2 reporter, only epithelial DT cells switched from DsRed to EGFP expression while AT3 cells maintained DsRed expression. Scale bar = 50 μm. C. Flow cytometry analysis shows that a small sub-population of AT3 cells undergo MET. D. RT-qPCR of DT and AT3 cells transfected with RG and E-cadCreIIIcI2 reveals approximately 10-fold higher expression of Cre in epithelial DT cells than mesenchymal AT3 cells.
Mentions: Validation of the combinatorial reporters indicated that multiple regulatory elements provided enhanced specificity, which is critical for faithful reporter readout. As a result, the lineage tracing MET reporters were generated based on the design of the E-cadFFIIIcI2, in which the Firefly ORF was replaced by the Cre recombinase ORF to give E-cadCreIIIcI2. Low E-cadherin promoter activation and exon IIIc inclusion in mesenchymal cells should lead to very low, if any, Cre expression (Figure 2A). Conversely, epithelial cells should express Cre via activation of the E-cadherin promoter and exon IIIc skipping (Figure 2A). The E-cadCreIIIcI2 reporter acts on a second plasmid, RG (21), in which the DsRed ORF containing a stop codon is flanked by LoxP sites followed by the EGFP ORF containing a stop codon (Figure 2A). Expression of Cre during MET should lead to permanent removal of DsRed by recombination at LoxP sites and consistent expression of EGFP.

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.