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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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Fluorescence-based MET reporters reveal epithelial plasticity in mesenchymal rat prostate tumorsA. Schematic of the Gint and RIIIcI2 reporters. B. Skipping of exon IIIc leads to production of DsRed in epithelial DT cells while inclusion of exon IIIc in mesenchymal AT3 cells prevents expression of DsRed. Epithelial DT cells express high levels of E-cadherin with low vimentin while mesenchymal AT3 cells have almost no E-cadherin, but robustly express vimentin. C. AT3+Gint+RIIIcI2 tumors reveal foci of DsRed expressing cells near the tumor edge. A dotted line indicates the border between the inner tumor mass and the unlabeled cells of the tumor capsule. Arrows highlight tumor cells that reside within the tumor capsule that have undergone MET. Scale bars = 50 μm unless otherwise specified.
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Figure 1: Fluorescence-based MET reporters reveal epithelial plasticity in mesenchymal rat prostate tumorsA. Schematic of the Gint and RIIIcI2 reporters. B. Skipping of exon IIIc leads to production of DsRed in epithelial DT cells while inclusion of exon IIIc in mesenchymal AT3 cells prevents expression of DsRed. Epithelial DT cells express high levels of E-cadherin with low vimentin while mesenchymal AT3 cells have almost no E-cadherin, but robustly express vimentin. C. AT3+Gint+RIIIcI2 tumors reveal foci of DsRed expressing cells near the tumor edge. A dotted line indicates the border between the inner tumor mass and the unlabeled cells of the tumor capsule. Arrows highlight tumor cells that reside within the tumor capsule that have undergone MET. Scale bars = 50 μm unless otherwise specified.

Mentions: To monitor MET in vivo, we developed a fluorescence-based reporter of MET, which was driven by epithelial-specific skipping of exon IIIc from fibroblast growth factor receptor 2 (FGFR2) (16–18, 20) (Figure 1A). Epithelial cells skip exon IIIc while mesenchymal cells include exon IIIc. The RIIIcI2 reporter contains the DsRed open reading frame (ORF) interrupted by the IIIc exon and flanking introns (20). Skipping of exon IIIc in epithelial Dunning rat prostate cancer DT cells leads to an in-frame DsRed mRNA and expression of DsRed (Figure 1B), while exon IIIc inclusion in mesenchymal AT3 cells interrupts the DsRed ORF, with little to no DsRed expression (Figure 1B). DsRed expression correlated with high E-cadherin staining and low levels of vimentin in the epithelial DT cells. Conversely, the mesenchymal AT3 cells expressed little to no DsRed, low E-cadherin, and high levels of vimentin (Figure 1B).


Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways
Fluorescence-based MET reporters reveal epithelial plasticity in mesenchymal rat prostate tumorsA. Schematic of the Gint and RIIIcI2 reporters. B. Skipping of exon IIIc leads to production of DsRed in epithelial DT cells while inclusion of exon IIIc in mesenchymal AT3 cells prevents expression of DsRed. Epithelial DT cells express high levels of E-cadherin with low vimentin while mesenchymal AT3 cells have almost no E-cadherin, but robustly express vimentin. C. AT3+Gint+RIIIcI2 tumors reveal foci of DsRed expressing cells near the tumor edge. A dotted line indicates the border between the inner tumor mass and the unlabeled cells of the tumor capsule. Arrows highlight tumor cells that reside within the tumor capsule that have undergone MET. Scale bars = 50 μm unless otherwise specified.
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Related In: Results  -  Collection

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Figure 1: Fluorescence-based MET reporters reveal epithelial plasticity in mesenchymal rat prostate tumorsA. Schematic of the Gint and RIIIcI2 reporters. B. Skipping of exon IIIc leads to production of DsRed in epithelial DT cells while inclusion of exon IIIc in mesenchymal AT3 cells prevents expression of DsRed. Epithelial DT cells express high levels of E-cadherin with low vimentin while mesenchymal AT3 cells have almost no E-cadherin, but robustly express vimentin. C. AT3+Gint+RIIIcI2 tumors reveal foci of DsRed expressing cells near the tumor edge. A dotted line indicates the border between the inner tumor mass and the unlabeled cells of the tumor capsule. Arrows highlight tumor cells that reside within the tumor capsule that have undergone MET. Scale bars = 50 μm unless otherwise specified.
Mentions: To monitor MET in vivo, we developed a fluorescence-based reporter of MET, which was driven by epithelial-specific skipping of exon IIIc from fibroblast growth factor receptor 2 (FGFR2) (16–18, 20) (Figure 1A). Epithelial cells skip exon IIIc while mesenchymal cells include exon IIIc. The RIIIcI2 reporter contains the DsRed open reading frame (ORF) interrupted by the IIIc exon and flanking introns (20). Skipping of exon IIIc in epithelial Dunning rat prostate cancer DT cells leads to an in-frame DsRed mRNA and expression of DsRed (Figure 1B), while exon IIIc inclusion in mesenchymal AT3 cells interrupts the DsRed ORF, with little to no DsRed expression (Figure 1B). DsRed expression correlated with high E-cadherin staining and low levels of vimentin in the epithelial DT cells. Conversely, the mesenchymal AT3 cells expressed little to no DsRed, low E-cadherin, and high levels of vimentin (Figure 1B).

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