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A novel genome-wide in vivo screen for metastatic suppressors in human colon cancer identifies the positive WNT-TCF pathway modulators TMED3 and SOX12.

Duquet A, Melotti A, Mishra S, Malerba M, Seth C, Conod A, Ruiz i Altaba A - EMBO Mol Med (2014)

Bottom Line: Finding these, however, is difficult as in vitro assays do not fully predict metastatic behavior, and the majority of studies have used cloned cell lines, which do not reflect primary tumor heterogeneity.Importantly, whereas TMED3 and SOX12 belong to different families involved in protein secretion and transcriptional regulation, both promote endogenous WNT-TCF activity.Treatments for advanced or metastatic colon cancer may thus not benefit from WNT blockers, and these may promote a worse outcome.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland.

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In vivo Red/Green competition assaysA Scheme of the in vivo red/green competition assay used to test the effect of individual shRNAs (see Varnat et al, 2009; Zbinden et al, 2010). Here, lentivector-transduced experimental GFP+ green and control transduced RFP+ red cells are mixed in equal amounts and co-injected in vivo.B–F Fluorescent images of two (top and bottom rows in each case) representative subcutaneous tumors for each condition after one (E) or two (B) in vivo passages, with shRNAs as noted in the figure. Green color indicates a predominance of GFP signal, whereas red color indicates a predominance of RFP signal. In control conditions with an empty GFP+ lentivector, the tumors retain similar amounts of RFP+ and GFP+ cells and the merged signals give a yellow color. (C) Quantification of the GFP+/RFP+ cell ratio, as determined by FACS analyses after cell dissociation, of control, shTMED3 and shSOX12 tumors over 2 passages in vivo as noted. Representative FACS plots of red and green populations for the given conditions. Note the opposite behavior of shSOX12 and shTMED3 (D) as compared with shYAP1 (F; Supplementary Fig S4). Scale bar = 3.5 mm.
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fig04: In vivo Red/Green competition assaysA Scheme of the in vivo red/green competition assay used to test the effect of individual shRNAs (see Varnat et al, 2009; Zbinden et al, 2010). Here, lentivector-transduced experimental GFP+ green and control transduced RFP+ red cells are mixed in equal amounts and co-injected in vivo.B–F Fluorescent images of two (top and bottom rows in each case) representative subcutaneous tumors for each condition after one (E) or two (B) in vivo passages, with shRNAs as noted in the figure. Green color indicates a predominance of GFP signal, whereas red color indicates a predominance of RFP signal. In control conditions with an empty GFP+ lentivector, the tumors retain similar amounts of RFP+ and GFP+ cells and the merged signals give a yellow color. (C) Quantification of the GFP+/RFP+ cell ratio, as determined by FACS analyses after cell dissociation, of control, shTMED3 and shSOX12 tumors over 2 passages in vivo as noted. Representative FACS plots of red and green populations for the given conditions. Note the opposite behavior of shSOX12 and shTMED3 (D) as compared with shYAP1 (F; Supplementary Fig S4). Scale bar = 3.5 mm.

Mentions: However, in vivo red/green competition assays (Fig 4A; Varnat et al, 2009; Zbinden et al, 2010) showed that both shTMED3 and shSOX12 endowed GFP+-expressing cells with an advantage as compared with sibling CC14 cells transduced with a lentivector expressing RFP in subcutaneous grafts (Fig 4B). This advantage was enhanced upon in vivo passage and could be seen in whole tumor imaging (where overall tumor size did not vary; Fig 4B) and in FACS analyses of dissociated tumor cells (Fig 4C and D). Red/green assays with shYAP1-GFP+ CC14 cells injected subcutaneously into nude mice revealed that YAP1 is cell autonomously essential for tumor growth as green cells disappeared from the tumor (Fig 4E and F, Supplementary Fig S4), again showing the opposite phenotype of shTMED3- and shSOX12-expressing cells (Fig 4B and D).


A novel genome-wide in vivo screen for metastatic suppressors in human colon cancer identifies the positive WNT-TCF pathway modulators TMED3 and SOX12.

Duquet A, Melotti A, Mishra S, Malerba M, Seth C, Conod A, Ruiz i Altaba A - EMBO Mol Med (2014)

In vivo Red/Green competition assaysA Scheme of the in vivo red/green competition assay used to test the effect of individual shRNAs (see Varnat et al, 2009; Zbinden et al, 2010). Here, lentivector-transduced experimental GFP+ green and control transduced RFP+ red cells are mixed in equal amounts and co-injected in vivo.B–F Fluorescent images of two (top and bottom rows in each case) representative subcutaneous tumors for each condition after one (E) or two (B) in vivo passages, with shRNAs as noted in the figure. Green color indicates a predominance of GFP signal, whereas red color indicates a predominance of RFP signal. In control conditions with an empty GFP+ lentivector, the tumors retain similar amounts of RFP+ and GFP+ cells and the merged signals give a yellow color. (C) Quantification of the GFP+/RFP+ cell ratio, as determined by FACS analyses after cell dissociation, of control, shTMED3 and shSOX12 tumors over 2 passages in vivo as noted. Representative FACS plots of red and green populations for the given conditions. Note the opposite behavior of shSOX12 and shTMED3 (D) as compared with shYAP1 (F; Supplementary Fig S4). Scale bar = 3.5 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4119353&req=5

fig04: In vivo Red/Green competition assaysA Scheme of the in vivo red/green competition assay used to test the effect of individual shRNAs (see Varnat et al, 2009; Zbinden et al, 2010). Here, lentivector-transduced experimental GFP+ green and control transduced RFP+ red cells are mixed in equal amounts and co-injected in vivo.B–F Fluorescent images of two (top and bottom rows in each case) representative subcutaneous tumors for each condition after one (E) or two (B) in vivo passages, with shRNAs as noted in the figure. Green color indicates a predominance of GFP signal, whereas red color indicates a predominance of RFP signal. In control conditions with an empty GFP+ lentivector, the tumors retain similar amounts of RFP+ and GFP+ cells and the merged signals give a yellow color. (C) Quantification of the GFP+/RFP+ cell ratio, as determined by FACS analyses after cell dissociation, of control, shTMED3 and shSOX12 tumors over 2 passages in vivo as noted. Representative FACS plots of red and green populations for the given conditions. Note the opposite behavior of shSOX12 and shTMED3 (D) as compared with shYAP1 (F; Supplementary Fig S4). Scale bar = 3.5 mm.
Mentions: However, in vivo red/green competition assays (Fig 4A; Varnat et al, 2009; Zbinden et al, 2010) showed that both shTMED3 and shSOX12 endowed GFP+-expressing cells with an advantage as compared with sibling CC14 cells transduced with a lentivector expressing RFP in subcutaneous grafts (Fig 4B). This advantage was enhanced upon in vivo passage and could be seen in whole tumor imaging (where overall tumor size did not vary; Fig 4B) and in FACS analyses of dissociated tumor cells (Fig 4C and D). Red/green assays with shYAP1-GFP+ CC14 cells injected subcutaneously into nude mice revealed that YAP1 is cell autonomously essential for tumor growth as green cells disappeared from the tumor (Fig 4E and F, Supplementary Fig S4), again showing the opposite phenotype of shTMED3- and shSOX12-expressing cells (Fig 4B and D).

Bottom Line: Finding these, however, is difficult as in vitro assays do not fully predict metastatic behavior, and the majority of studies have used cloned cell lines, which do not reflect primary tumor heterogeneity.Importantly, whereas TMED3 and SOX12 belong to different families involved in protein secretion and transcriptional regulation, both promote endogenous WNT-TCF activity.Treatments for advanced or metastatic colon cancer may thus not benefit from WNT blockers, and these may promote a worse outcome.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland.

Show MeSH
Related in: MedlinePlus