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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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Proposed divergent signalling for gamma-secretase-dependent pathways of differentiation and anchorage-independent growth in colon cancer cells.Biological effects induced by γ-secretase inhibition in colonic cancer cells were dependent on Hath1 expression. In Hath1 non-expressing cells, γ-secretase inhibition had no effect on MUC2, P27Kip1 and anchorage independent growth. In Hath1 positive cells, γ-secretase inhibition led to an increase of Hath1 expression leading to two separate effects on 1- increase in mucin producing cells with MUC2 expression and 2- reduction in anchorage independent growth mediated by the activation of the cyclin dependent kinase inhibitor P27Kip1.
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pone-0055904-g006: Proposed divergent signalling for gamma-secretase-dependent pathways of differentiation and anchorage-independent growth in colon cancer cells.Biological effects induced by γ-secretase inhibition in colonic cancer cells were dependent on Hath1 expression. In Hath1 non-expressing cells, γ-secretase inhibition had no effect on MUC2, P27Kip1 and anchorage independent growth. In Hath1 positive cells, γ-secretase inhibition led to an increase of Hath1 expression leading to two separate effects on 1- increase in mucin producing cells with MUC2 expression and 2- reduction in anchorage independent growth mediated by the activation of the cyclin dependent kinase inhibitor P27Kip1.

Mentions: Whether the primary colonic cancer cells are able to respond to γ-secretase inhibition is an important issue. Due to the known difficulties for maintaining primary colon carcinomas in culture, we optimized a short term culture method. Interestingly, while all tumors displayed a Hes1 mRNA down-regulation upon DBZ treatment, only a minor fraction of the primary tumors displayed a “full” response to DBZ, i.e. an up-regulation of Hath1, MUC2 and P27Kip1. Interestingly Peignon et al [41] recently reported that the hyperactivation of β-catenin signalling overrides the forced differentiation induced by Notch inhibition. However in our experiments we did not find a correlation between β-catenin activation in the primary tumors, as shown by counting the percentage of β-catenin positive nuclei, and the level of Hath1, MUC2 and P27Kip1 expression in DBZ-treated tumor cells. In addition, we did not find any Hath1 mutation that could explain in 2 cases the uncoupling of Hath1 over-expression from MUC2 and P27Kip1 regulation. The response to DBZ fits with our proposed scheme of signalling pathways (Figure 6), (i) MUC2 and-or P27Kip1 augmentation appears to be associated with Hath1 up-regulation, and (ii) P27Kip1 and MUC2 can be separately up-regulated, a finding that is in line with a Hath1-dependent divergent signalling. In addition, P27Kip1 up-regulation upon DBZ treatment occurred only in a minor proportion of primary colon carcinomas. It comes then that a screening strategy based on primary culture of tumor cells is advisable for selecting patients that could response to γ-secretase inhibitors.


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)

Proposed divergent signalling for gamma-secretase-dependent pathways of differentiation and anchorage-independent growth in colon cancer cells.Biological effects induced by γ-secretase inhibition in colonic cancer cells were dependent on Hath1 expression. In Hath1 non-expressing cells, γ-secretase inhibition had no effect on MUC2, P27Kip1 and anchorage independent growth. In Hath1 positive cells, γ-secretase inhibition led to an increase of Hath1 expression leading to two separate effects on 1- increase in mucin producing cells with MUC2 expression and 2- reduction in anchorage independent growth mediated by the activation of the cyclin dependent kinase inhibitor P27Kip1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569436&req=5

pone-0055904-g006: Proposed divergent signalling for gamma-secretase-dependent pathways of differentiation and anchorage-independent growth in colon cancer cells.Biological effects induced by γ-secretase inhibition in colonic cancer cells were dependent on Hath1 expression. In Hath1 non-expressing cells, γ-secretase inhibition had no effect on MUC2, P27Kip1 and anchorage independent growth. In Hath1 positive cells, γ-secretase inhibition led to an increase of Hath1 expression leading to two separate effects on 1- increase in mucin producing cells with MUC2 expression and 2- reduction in anchorage independent growth mediated by the activation of the cyclin dependent kinase inhibitor P27Kip1.
Mentions: Whether the primary colonic cancer cells are able to respond to γ-secretase inhibition is an important issue. Due to the known difficulties for maintaining primary colon carcinomas in culture, we optimized a short term culture method. Interestingly, while all tumors displayed a Hes1 mRNA down-regulation upon DBZ treatment, only a minor fraction of the primary tumors displayed a “full” response to DBZ, i.e. an up-regulation of Hath1, MUC2 and P27Kip1. Interestingly Peignon et al [41] recently reported that the hyperactivation of β-catenin signalling overrides the forced differentiation induced by Notch inhibition. However in our experiments we did not find a correlation between β-catenin activation in the primary tumors, as shown by counting the percentage of β-catenin positive nuclei, and the level of Hath1, MUC2 and P27Kip1 expression in DBZ-treated tumor cells. In addition, we did not find any Hath1 mutation that could explain in 2 cases the uncoupling of Hath1 over-expression from MUC2 and P27Kip1 regulation. The response to DBZ fits with our proposed scheme of signalling pathways (Figure 6), (i) MUC2 and-or P27Kip1 augmentation appears to be associated with Hath1 up-regulation, and (ii) P27Kip1 and MUC2 can be separately up-regulated, a finding that is in line with a Hath1-dependent divergent signalling. In addition, P27Kip1 up-regulation upon DBZ treatment occurred only in a minor proportion of primary colon carcinomas. It comes then that a screening strategy based on primary culture of tumor cells is advisable for selecting patients that could response to γ-secretase inhibitors.

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