Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of γ-secretase inhibition on cellular viability in anchorage-independent culture conditions in relation with Hath1 and MUC2 expression.A and B- Filter-grown HT29-Cl.27H or Caco2 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 without DBZ. A- RT-PCR detection of Hath1 and MUC2 mRNA: mean expression relative to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; **, p<0.01. B- Relative cell viability versus control determined by trypan blue dye exclusion cell count. C- Filter-grown HT29-Cl.27H or Caco2 cells, pre-treated or not (control) with DBZ for 21 days were detached and seeded at 10, 000 cells per 60 mm dish in 0.35% agarose. The cells were maintained in culture for additional 15 days without supplementation of DBZ. Clusters of minimum 10 cells were counted. Mean relative expression to the control DMSO; Mean ± SEM of 3 experiments (3–4 dishes per experiment); ***, p<0.001. D- RT-PCR detection of Hes1 in Caco2 cells, treated or not (control) with DBZ for 21 days on filters: mean expression relative to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3569436&req=5

pone-0055904-g003: Effect of γ-secretase inhibition on cellular viability in anchorage-independent culture conditions in relation with Hath1 and MUC2 expression.A and B- Filter-grown HT29-Cl.27H or Caco2 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 without DBZ. A- RT-PCR detection of Hath1 and MUC2 mRNA: mean expression relative to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; **, p<0.01. B- Relative cell viability versus control determined by trypan blue dye exclusion cell count. C- Filter-grown HT29-Cl.27H or Caco2 cells, pre-treated or not (control) with DBZ for 21 days were detached and seeded at 10, 000 cells per 60 mm dish in 0.35% agarose. The cells were maintained in culture for additional 15 days without supplementation of DBZ. Clusters of minimum 10 cells were counted. Mean relative expression to the control DMSO; Mean ± SEM of 3 experiments (3–4 dishes per experiment); ***, p<0.001. D- RT-PCR detection of Hes1 in Caco2 cells, treated or not (control) with DBZ for 21 days on filters: mean expression relative to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments.

Mentions: To examine the impact of differentiation on the anchorage-independent growth, HT29-Cl27H cells were first induced to differentiate by DBZ and then the cells were detached and maintained without DBZ in suspension culture in polyhema coated wells. After 72 hours culture, MUC2 and Hath1 mRNA levels remained elevated in DBZ-pretreated cells (Figure 3A). Parallel to this maintenance of differentiation, we observed a 40% decrease in anchorage independent cell growth (Figure 3B). This effect resulted from a reduced proliferation and a 2 fold increase in cell apoptosis determined by the percentage of M30 positive cells (DBZ: 4.5% ±0.29 versus control: 1.9% ±0.38, ** p<0.01). DBZ-pretreated cells displayed a 50% decrease in soft agarose clonogenicity (Figure 3C). Interestingly, DBZ-pretreated Caco2 cells showed no modification of anchorage independent growth in suspension culture or in a soft agarose clonogenicity assay (Figure 3B and C). Although in Caco2 cells DBZ treatment did not alter Hath1 mRNA level, it was able to act upstream of Hath1 by decreasing Hes1 mRNA expression (Figure 3D).


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 γ-secretase inhibition on cellular viability in anchorage-independent culture conditions in relation with Hath1 and MUC2 expression.A and B- Filter-grown HT29-Cl.27H or Caco2 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 without DBZ. A- RT-PCR detection of Hath1 and MUC2 mRNA: mean expression relative to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; **, p<0.01. B- Relative cell viability versus control determined by trypan blue dye exclusion cell count. C- Filter-grown HT29-Cl.27H or Caco2 cells, pre-treated or not (control) with DBZ for 21 days were detached and seeded at 10, 000 cells per 60 mm dish in 0.35% agarose. The cells were maintained in culture for additional 15 days without supplementation of DBZ. Clusters of minimum 10 cells were counted. Mean relative expression to the control DMSO; Mean ± SEM of 3 experiments (3–4 dishes per experiment); ***, p<0.001. D- RT-PCR detection of Hes1 in Caco2 cells, treated or not (control) with DBZ for 21 days on filters: mean expression relative to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0055904-g003: Effect of γ-secretase inhibition on cellular viability in anchorage-independent culture conditions in relation with Hath1 and MUC2 expression.A and B- Filter-grown HT29-Cl.27H or Caco2 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 without DBZ. A- RT-PCR detection of Hath1 and MUC2 mRNA: mean expression relative to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments; *, P<0.05; **, p<0.01. B- Relative cell viability versus control determined by trypan blue dye exclusion cell count. C- Filter-grown HT29-Cl.27H or Caco2 cells, pre-treated or not (control) with DBZ for 21 days were detached and seeded at 10, 000 cells per 60 mm dish in 0.35% agarose. The cells were maintained in culture for additional 15 days without supplementation of DBZ. Clusters of minimum 10 cells were counted. Mean relative expression to the control DMSO; Mean ± SEM of 3 experiments (3–4 dishes per experiment); ***, p<0.001. D- RT-PCR detection of Hes1 in Caco2 cells, treated or not (control) with DBZ for 21 days on filters: mean expression relative to control DMSO after normalization to actin gene expression; Mean ± SEM of 3 experiments.
Mentions: To examine the impact of differentiation on the anchorage-independent growth, HT29-Cl27H cells were first induced to differentiate by DBZ and then the cells were detached and maintained without DBZ in suspension culture in polyhema coated wells. After 72 hours culture, MUC2 and Hath1 mRNA levels remained elevated in DBZ-pretreated cells (Figure 3A). Parallel to this maintenance of differentiation, we observed a 40% decrease in anchorage independent cell growth (Figure 3B). This effect resulted from a reduced proliferation and a 2 fold increase in cell apoptosis determined by the percentage of M30 positive cells (DBZ: 4.5% ±0.29 versus control: 1.9% ±0.38, ** p<0.01). DBZ-pretreated cells displayed a 50% decrease in soft agarose clonogenicity (Figure 3C). Interestingly, DBZ-pretreated Caco2 cells showed no modification of anchorage independent growth in suspension culture or in a soft agarose clonogenicity assay (Figure 3B and C). Although in Caco2 cells DBZ treatment did not alter Hath1 mRNA level, it was able to act upstream of Hath1 by decreasing Hes1 mRNA expression (Figure 3D).

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