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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 ectopic expression and P27Kip1 silencing on anchorage-independent cell growth of Caco2 cells.A- Polyclonal populations of Caco2 cells stably transfected by empty vector (Caco2 control) or Hath1 expression vector (Caco2 Hath1) were plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in 10% FCS containing medium. Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM; **, p<0.01. B-C- Caco2 cells (B) or Hath1 over-expressing Caco2 cells (C) plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture with siRNA target smart pool (NT or P27). Relative cell viability determined by Trypan blue dye exclusion cell count. (B) Insert: P27Kip1 and β-actin were detected by immunoblot on cells transfected by siRNA.
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pone-0055904-g005: Effect of Hath1 ectopic expression and P27Kip1 silencing on anchorage-independent cell growth of Caco2 cells.A- Polyclonal populations of Caco2 cells stably transfected by empty vector (Caco2 control) or Hath1 expression vector (Caco2 Hath1) were plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in 10% FCS containing medium. Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM; **, p<0.01. B-C- Caco2 cells (B) or Hath1 over-expressing Caco2 cells (C) plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture with siRNA target smart pool (NT or P27). Relative cell viability determined by Trypan blue dye exclusion cell count. (B) Insert: P27Kip1 and β-actin were detected by immunoblot on cells transfected by siRNA.

Mentions: However, Hath1 ectopic expression in Caco2 cells resulted in a reduced anchorage-independent growth of the cells (Figure 5A). Remarkably, P27Kip1 silencing by siRNA did not modify the anchorage-independent growth of parental Caco2 cells or Hath1-overexpressing Caco2 cells (Figure 5B and 5C). Thus the forced expression of Hath1 via genetic manipulation of a Hath1 negative colonic cancer cell line, resulting in very high Hath1 expression, negatively regulates cell proliferation in a manner that is clearly independent from that depending on the pharmacological inhibition of γ-secretase in Hath1 positive colonic cancer cells.


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 ectopic expression and P27Kip1 silencing on anchorage-independent cell growth of Caco2 cells.A- Polyclonal populations of Caco2 cells stably transfected by empty vector (Caco2 control) or Hath1 expression vector (Caco2 Hath1) were plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in 10% FCS containing medium. Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM; **, p<0.01. B-C- Caco2 cells (B) or Hath1 over-expressing Caco2 cells (C) plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture with siRNA target smart pool (NT or P27). Relative cell viability determined by Trypan blue dye exclusion cell count. (B) Insert: P27Kip1 and β-actin were detected by immunoblot on cells transfected by siRNA.
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Related In: Results  -  Collection

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

pone-0055904-g005: Effect of Hath1 ectopic expression and P27Kip1 silencing on anchorage-independent cell growth of Caco2 cells.A- Polyclonal populations of Caco2 cells stably transfected by empty vector (Caco2 control) or Hath1 expression vector (Caco2 Hath1) were plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in 10% FCS containing medium. Relative cell viability determined by Trypan blue dye exclusion cell count. Mean ± SEM; **, p<0.01. B-C- Caco2 cells (B) or Hath1 over-expressing Caco2 cells (C) plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture with siRNA target smart pool (NT or P27). Relative cell viability determined by Trypan blue dye exclusion cell count. (B) Insert: P27Kip1 and β-actin were detected by immunoblot on cells transfected by siRNA.
Mentions: However, Hath1 ectopic expression in Caco2 cells resulted in a reduced anchorage-independent growth of the cells (Figure 5A). Remarkably, P27Kip1 silencing by siRNA did not modify the anchorage-independent growth of parental Caco2 cells or Hath1-overexpressing Caco2 cells (Figure 5B and 5C). Thus the forced expression of Hath1 via genetic manipulation of a Hath1 negative colonic cancer cell line, resulting in very high Hath1 expression, negatively regulates cell proliferation in a manner that is clearly independent from that depending on the pharmacological inhibition of γ-secretase in Hath1 positive colonic cancer cells.

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