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Differential roles of Hath1, MUC2 and P27Kip1 in relation with gamma-secretase inhibition in human colonic carcinomas: a translational study.

Souazé F, Bou-Hanna C, Kandel C, Leclair F, Devallière J, Charreau B, Bézieau S, Mosnier JF, Laboisse CL - PLoS ONE (2013)

Bottom Line: Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation.Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ).In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment.

View Article: PubMed Central - PubMed

Affiliation: EA Biometadys, Université de Nantes, Nantes, France. frederique.souaze@inserm.fr

ABSTRACT
Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation. Our aim was fourfold: 1) determine whether Hath1 is able to alter the phenotype of colon cancer cells that are committed to a differentiated phenotype, 2) determine whether the Hath1-dependent alteration of differentiation is coupled to a restriction of anchorage-dependent growth, 3) decipher the respective roles of three putative tumor suppressor genes Hath1, MUC2 and P27kip1 in this coupling and, 4) examine how our findings translate to primary tumors. Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ). Then the cells were detached and their ability to survive/proliferate in the absence of substratum was assessed. γ-secretase inhibition led to a Hath1-mediated preferential induction of MUC2 over MUC5AC, without DPPIV modification, in association with a decrease in anchorage-independent growth. While P27kip1 silencing relieved the cells from the Hath1-induced decrease of anchorage-independent growth, MUC2 silencing did not modify this parameter. Hath1 ectopic expression in the Hath1 negative enterocytic Caco2 cells led to a decreased anchorage-independent growth in a P27kip1-independent manner. In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment. Parallel MUC2 up-regulation occurred in 4 (4/7) and P27kip1 in only 2 (2/7) tumors. Interestingly, the response patterns of primary tumors to DBZ fitted with the hierarchical model of divergent signalling derived from our findings on cell lines.

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Effect of Hath1, MUC2 or P27Kip1 gene silencing on anchorage independent-growth. A,B, C,D,G,H,I-Filter-grown HT29-Cl.27H, treated or not (control) with DBZ for 21 days, were resuspended and plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in the presence of the indicated siRNA target smart pool (NT, non target; Hath1; MUC2 or P27Kip1). A- RT-PCR detection of Hath1 (left) and Muc-2 (right) mRNA in HT29-Cl.27H cells transfected by siRNA (NT, or Hath1): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; **, p<0.01; ***, p<0.001. B- Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM of 3 experiments; ***, p<0.001. C- RT-PCR detection of MUC2 mRNA in HT29-Cl.27H cells transfected by siRNA (NT and MUC2): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; ***, p<0.001. D- Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM of 3 experiments; ***, p<0.001. E- Filter-grown HT29-Cl.16E sox9 cells, treated or not (control) with DBZ for 21 days, were resuspended and plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in 10% FCS containing medium with or without addition of doxycycline. RT-PCR detection of Hath1 and MUC2 mRNA: relative expression to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; **, P<0.01; *, P<0.05. Insert: Flag-Sox9 was detected by immunoblot on cellular extracts after 72-hour doxycycline induction. F- Relative cell viability determined by Trypan blue dye exclusion cell count (in the same culture conditions as Fig. 4E). Mean ± SEM of 3 experiments; ***, p<0.001. G- and H- RT-PCR detection of P27Kip1 mRNA (G and H left) and MUC2 mRNA (H right) in HT29-Cl.27H cells transfected by siRNA (G -NT and Hath1, H- NT and P27): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; ***, p<0.001. Insert: P27Kip1 and β-actin were detected by immunoblot on DBZ pre-treated cells transfected by siRNA (G -NT and Hath1, H left- NT and P27). I- Relative cell viability determined by Trypan blue dye exclusion cell count of HT29-Cl.27H cells transfected by siRNA NT or P27; Mean ± SEM of 3 experiments; ***, p<0.001.
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pone-0055904-g004: Effect of Hath1, MUC2 or P27Kip1 gene silencing on anchorage independent-growth. A,B, C,D,G,H,I-Filter-grown HT29-Cl.27H, treated or not (control) with DBZ for 21 days, were resuspended and plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in the presence of the indicated siRNA target smart pool (NT, non target; Hath1; MUC2 or P27Kip1). A- RT-PCR detection of Hath1 (left) and Muc-2 (right) mRNA in HT29-Cl.27H cells transfected by siRNA (NT, or Hath1): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; **, p<0.01; ***, p<0.001. B- Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM of 3 experiments; ***, p<0.001. C- RT-PCR detection of MUC2 mRNA in HT29-Cl.27H cells transfected by siRNA (NT and MUC2): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; ***, p<0.001. D- Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM of 3 experiments; ***, p<0.001. E- Filter-grown HT29-Cl.16E sox9 cells, treated or not (control) with DBZ for 21 days, were resuspended and plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in 10% FCS containing medium with or without addition of doxycycline. RT-PCR detection of Hath1 and MUC2 mRNA: relative expression to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; **, P<0.01; *, P<0.05. Insert: Flag-Sox9 was detected by immunoblot on cellular extracts after 72-hour doxycycline induction. F- Relative cell viability determined by Trypan blue dye exclusion cell count (in the same culture conditions as Fig. 4E). Mean ± SEM of 3 experiments; ***, p<0.001. G- and H- RT-PCR detection of P27Kip1 mRNA (G and H left) and MUC2 mRNA (H right) in HT29-Cl.27H cells transfected by siRNA (G -NT and Hath1, H- NT and P27): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; ***, p<0.001. Insert: P27Kip1 and β-actin were detected by immunoblot on DBZ pre-treated cells transfected by siRNA (G -NT and Hath1, H left- NT and P27). I- Relative cell viability determined by Trypan blue dye exclusion cell count of HT29-Cl.27H cells transfected by siRNA NT or P27; Mean ± SEM of 3 experiments; ***, p<0.001.

Mentions: To examine whether there was a common control of anchorage independent cellular growth and MUC2 expression upon γ-secretase inhibition, we performed Hath1 silencing by RNA interference in suspension culture. As shown in figure 4, transiently transfected Hath1 siRNA was able to significantly reverse the effect of DBZ on Hath1 expression (Figure 4A, Left) and concomitantly reduced MUC2 expression (Figure 4A, Right). In parallel, Hath1 siRNA reversed the inhibitory effect of DBZ on anchorage-independent growth (Figure 4B).


Differential roles of Hath1, MUC2 and P27Kip1 in relation with gamma-secretase inhibition in human colonic carcinomas: a translational study.

Souazé F, Bou-Hanna C, Kandel C, Leclair F, Devallière J, Charreau B, Bézieau S, Mosnier JF, Laboisse CL - PLoS ONE (2013)

Effect of Hath1, MUC2 or P27Kip1 gene silencing on anchorage independent-growth. A,B, C,D,G,H,I-Filter-grown HT29-Cl.27H, treated or not (control) with DBZ for 21 days, were resuspended and plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in the presence of the indicated siRNA target smart pool (NT, non target; Hath1; MUC2 or P27Kip1). A- RT-PCR detection of Hath1 (left) and Muc-2 (right) mRNA in HT29-Cl.27H cells transfected by siRNA (NT, or Hath1): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; **, p<0.01; ***, p<0.001. B- Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM of 3 experiments; ***, p<0.001. C- RT-PCR detection of MUC2 mRNA in HT29-Cl.27H cells transfected by siRNA (NT and MUC2): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; ***, p<0.001. D- Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM of 3 experiments; ***, p<0.001. E- Filter-grown HT29-Cl.16E sox9 cells, treated or not (control) with DBZ for 21 days, were resuspended and plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in 10% FCS containing medium with or without addition of doxycycline. RT-PCR detection of Hath1 and MUC2 mRNA: relative expression to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; **, P<0.01; *, P<0.05. Insert: Flag-Sox9 was detected by immunoblot on cellular extracts after 72-hour doxycycline induction. F- Relative cell viability determined by Trypan blue dye exclusion cell count (in the same culture conditions as Fig. 4E). Mean ± SEM of 3 experiments; ***, p<0.001. G- and H- RT-PCR detection of P27Kip1 mRNA (G and H left) and MUC2 mRNA (H right) in HT29-Cl.27H cells transfected by siRNA (G -NT and Hath1, H- NT and P27): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; ***, p<0.001. Insert: P27Kip1 and β-actin were detected by immunoblot on DBZ pre-treated cells transfected by siRNA (G -NT and Hath1, H left- NT and P27). I- Relative cell viability determined by Trypan blue dye exclusion cell count of HT29-Cl.27H cells transfected by siRNA NT or P27; Mean ± SEM of 3 experiments; ***, p<0.001.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569436&req=5

pone-0055904-g004: Effect of Hath1, MUC2 or P27Kip1 gene silencing on anchorage independent-growth. A,B, C,D,G,H,I-Filter-grown HT29-Cl.27H, treated or not (control) with DBZ for 21 days, were resuspended and plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in the presence of the indicated siRNA target smart pool (NT, non target; Hath1; MUC2 or P27Kip1). A- RT-PCR detection of Hath1 (left) and Muc-2 (right) mRNA in HT29-Cl.27H cells transfected by siRNA (NT, or Hath1): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; **, p<0.01; ***, p<0.001. B- Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM of 3 experiments; ***, p<0.001. C- RT-PCR detection of MUC2 mRNA in HT29-Cl.27H cells transfected by siRNA (NT and MUC2): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; ***, p<0.001. D- Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM of 3 experiments; ***, p<0.001. E- Filter-grown HT29-Cl.16E sox9 cells, treated or not (control) with DBZ for 21 days, were resuspended and plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in 10% FCS containing medium with or without addition of doxycycline. RT-PCR detection of Hath1 and MUC2 mRNA: relative expression to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; **, P<0.01; *, P<0.05. Insert: Flag-Sox9 was detected by immunoblot on cellular extracts after 72-hour doxycycline induction. F- Relative cell viability determined by Trypan blue dye exclusion cell count (in the same culture conditions as Fig. 4E). Mean ± SEM of 3 experiments; ***, p<0.001. G- and H- RT-PCR detection of P27Kip1 mRNA (G and H left) and MUC2 mRNA (H right) in HT29-Cl.27H cells transfected by siRNA (G -NT and Hath1, H- NT and P27): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; ***, p<0.001. Insert: P27Kip1 and β-actin were detected by immunoblot on DBZ pre-treated cells transfected by siRNA (G -NT and Hath1, H left- NT and P27). I- Relative cell viability determined by Trypan blue dye exclusion cell count of HT29-Cl.27H cells transfected by siRNA NT or P27; Mean ± SEM of 3 experiments; ***, p<0.001.
Mentions: To examine whether there was a common control of anchorage independent cellular growth and MUC2 expression upon γ-secretase inhibition, we performed Hath1 silencing by RNA interference in suspension culture. As shown in figure 4, transiently transfected Hath1 siRNA was able to significantly reverse the effect of DBZ on Hath1 expression (Figure 4A, Left) and concomitantly reduced MUC2 expression (Figure 4A, Right). In parallel, Hath1 siRNA reversed the inhibitory effect of DBZ on anchorage-independent growth (Figure 4B).

Bottom Line: Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation.Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ).In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment.

View Article: PubMed Central - PubMed

Affiliation: EA Biometadys, Université de Nantes, Nantes, France. frederique.souaze@inserm.fr

ABSTRACT
Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation. Our aim was fourfold: 1) determine whether Hath1 is able to alter the phenotype of colon cancer cells that are committed to a differentiated phenotype, 2) determine whether the Hath1-dependent alteration of differentiation is coupled to a restriction of anchorage-dependent growth, 3) decipher the respective roles of three putative tumor suppressor genes Hath1, MUC2 and P27kip1 in this coupling and, 4) examine how our findings translate to primary tumors. Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ). Then the cells were detached and their ability to survive/proliferate in the absence of substratum was assessed. γ-secretase inhibition led to a Hath1-mediated preferential induction of MUC2 over MUC5AC, without DPPIV modification, in association with a decrease in anchorage-independent growth. While P27kip1 silencing relieved the cells from the Hath1-induced decrease of anchorage-independent growth, MUC2 silencing did not modify this parameter. Hath1 ectopic expression in the Hath1 negative enterocytic Caco2 cells led to a decreased anchorage-independent growth in a P27kip1-independent manner. In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment. Parallel MUC2 up-regulation occurred in 4 (4/7) and P27kip1 in only 2 (2/7) tumors. Interestingly, the response patterns of primary tumors to DBZ fitted with the hierarchical model of divergent signalling derived from our findings on cell lines.

Show MeSH
Related in: MedlinePlus