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See detailContribution à l'étude des mécanismes transcriptionnels du facteur de transcription SIP1/ZEB2
Koopmansch, Benjamin ULg

Doctoral thesis (2013)

Lors de la conversion métastatique des tumeurs épithéliales, certaines cellules tumorales acquièrent la capacité d’envahir le tissu sous-jacent et de former des métastases à distance. De nombreuses ... [more ▼]

Lors de la conversion métastatique des tumeurs épithéliales, certaines cellules tumorales acquièrent la capacité d’envahir le tissu sous-jacent et de former des métastases à distance. De nombreuses données de la littérature montrent que l’acquisition de ces propriétés est accompagnée d’un phénomène de transdifférenciation appelé « transition épithéliomésenchymateuse » (TEM), impliquant la perte de caractéristiques de cellules épithéliales au profit de caractéristiques de cellules mésenchymateuses. Parmi les modifications moléculaires caractéristiques de la TEM, on observe une diminution de l’expression de cadhérine E ainsi et l’expression de novo de filaments de vimentine. L’expression accrue de différents facteurs de transcription inducteurs de la TEM est aussi rapportée. SIP1 est un des facteurs de transcription impliqués dans les phénomènes de TEM tumorale. Il a été clairement montré que SIP1 réprime l’expression de la cadhérine E en liant son promoteur. Le mécanisme de répression n’est pas précisément connu, mais il n’implique pas le co-répresseur CtBP. Un modèle de répression suggère que SIP1 empêche l’accès de facteurs activateurs aux promoteurs des gènes réprimés. Les données obtenues au cours de ce travail nous permettent d’appuyer cette hypothèse et impliquent KLF4, un facteur de transcription activateur liant le promoteur de la cadhérine E, dans ce modèle. Nos résultats révèlent en effet une compétition entre SIP1 et KLF4 pour la liaison sur le promoteur de la cadhérine E. Nous montrons aussi que les deux facteurs ont des effets opposés sur l’activité du promoteur de la cadhérine E et que KLF4 n’active celui-ci que lorsque SIP1 ne peut s’y lier. Enfin, nos données nous ont permis de localiser plus précisément la région du promoteur de la cadhérine E liée par KLF4. Cette région chevauche un des sites liés par SIP1. SIP1 est considéré comme répresseur de la transcription, mais les données s’accumulent montrant l’induction rapide de gènes cibles de la TEM, dont la vimentine, suite à une surexpression de SIP1. Nous avons entrepris de mieux comprendre l’activation de la transcription par SIP1 en utilisant le promoteur de la vimentine comme modèle, et par une approche globale à l’aide de la technique de ChIPSeq. Cette partie n’a malheureusement pas atteint ses objectifs. [less ▲]

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See detailInterplay between KLF4 and ZEB2/SIP1 in the regulation of E-cadherin expression.
Koopmansch, Benjamin ULg; Berx, Geert; Foidart, Jean-Michel ULg et al

in Biochemical and Biophysical Research Communications (2013), 431(4), 652

E-cadherin expression is repressed by ZEB2/SIP1 while it is induced by KLF4. Independent data from the literature indicate that these two transcription factors could bind close to each other in the ... [more ▼]

E-cadherin expression is repressed by ZEB2/SIP1 while it is induced by KLF4. Independent data from the literature indicate that these two transcription factors could bind close to each other in the proximal region of the E-cadherin gene promoter. We have here explored a potential competition between ZEB2 and KLF4 for the binding to the E-cadherin promoter. We show an inverse correlation between ZEB2 expression levels and KLF4 recruitment on the E-cadherin promoter in three breast cancer cell lines and in A431/HA.ZEB2 cells in which ZEB2 expression is induced by doxycycline (DOX). We identified a region of the E-cadherin promoter bound by KLF4 which is necessary for the activation of the E-cadherin promoter activity after KLF4 overexpression. This region is localized between positions -28 and -10 and thus overlaps with one of the ZEB2 binding sites. Deleting the bipartite ZEB2 binding site results in increased KLF4 induced E-cadherin promoter activity. Taken together, our results suggest that E-cadherin expression in cancer cells is controlled by a balance between ZEB2 and KLF4 expression levels. [less ▲]

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See detailKu proteins interact with activator protein-2 transcription factors and contribute to ERBB2 overexpression in breast cancer cell lines.
Nolens, Grégory ULg; Pignon, Jean-Christophe ULg; Koopmansch, Benjamin ULg et al

in Breast Cancer Research [=BCR] (2009), 11(6),

INTRODUCTION: Activator protein-2 (AP-2) alpha and AP-2 gamma transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene ... [more ▼]

INTRODUCTION: Activator protein-2 (AP-2) alpha and AP-2 gamma transcription factors contribute to ERBB2 gene overexpression in breast cancer. In order to understand the mechanism by which the ERBB2 gene is overexpressed we searched for novel AP-2 interacting factors that contribute to its activity. METHODS: Ku proteins were identified as AP-2 alpha interacting proteins by glutathione serine transferase (GST)-pull down followed by mass spectrometry. Transfection of the cells with siRNA, expression vectors and reporter vectors as well as chromatin immunoprecipitation (ChIP) assay were used to ascertain the implication of Ku proteins on ERBB2 expression. RESULTS: Nuclear proteins from BT-474 cells overexpressing AP-2 alpha and AP-2 gamma were incubated with GST-AP2 or GST coated beads. Among the proteins retained specifically on GST-AP2 coated beads Ku70 and Ku80 proteins were identified by mass spectrometry. The contribution of Ku proteins to ERBB2 gene expression in BT-474 and SKBR3 cell lines was investigated by downregulating Ku proteins through the use of specific siRNAs. Depletion of Ku proteins led to downregulation of ERBB2 mRNA and protein levels. Furthermore, reduction of Ku80 in HCT116 cell line decreased the AP-2 alpha activity on a reporter vector containing an AP-2 binding site linked to the ERBB2 core promoter, and transfection of Ku80 increased the activity of AP-2 alpha on this promoter. Ku siRNAs also inhibited the activity of this reporter vector in BT-474 and SKBR3 cell lines and the activity of the ERBB2 promoter was further reduced by combining Ku siRNAs with AP-2 alpha and AP-2 gamma siRNAs. ChIP experiments with chromatin extracted from wild type or AP-2 alpha and AP-2 gamma or Ku70 siRNA transfected BT-474 cells demonstrated Ku70 recruitment to the ERBB2 proximal promoter in association with AP-2 alpha and AP-2 gamma. Moreover, Ku70 siRNA like AP-2 siRNAs, greatly reduced PolII recruitment to the ERBB2 proximal promoter. CONCLUSIONS: Ku proteins in interaction with AP-2 (alpha and gamma) contribute to increased ERBB2 mRNA and protein levels in breast cancer cells. [less ▲]

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See detailAndrogen receptor controls EGFR and ERBB2 gene expression at different levels in prostate cancer cell lines.
Pignon, Jean-Christophe ULg; Koopmansch, Benjamin ULg; Nolens, Grégory ULg et al

in Cancer Research (2009), 69(7), 2941-2949

EGFR or ERBB2 contributes to prostate cancer (PCa) progression by activating the androgen receptor (AR) in hormone-poor conditions. Here, we investigated the mechanisms by which androgens regulate EGFR ... [more ▼]

EGFR or ERBB2 contributes to prostate cancer (PCa) progression by activating the androgen receptor (AR) in hormone-poor conditions. Here, we investigated the mechanisms by which androgens regulate EGFR and ERBB2 expression in PCa cells. In steroid-depleted medium (SDM), EGFR protein was less abundant in androgen-sensitive LNCaP than in androgen ablation-resistant 22Rv1 cells, whereas transcript levels were similar. Dihydrotestosterone (DHT) treatment increased both EGFR mRNA and protein levels and stimulated RNA polymerase II recruitment to the EGFR gene promoter, whereas it decreased ERBB2 transcript and protein levels in LNCaP cells. DHT altered neither EGFR or ERBB2 levels nor the abundance of prostate-specific antigen (PSA), TMEPA1, or TMPRSS2 mRNAs in 22Rv1 cells, which express the full-length and a shorter AR isoform deleted from the COOH-terminal domain (ARDeltaCTD). The contribution of both AR isoforms to the expression of these genes was assessed by small interfering RNAs targeting only the full-length or both AR isoforms. Silencing of both isoforms strongly reduced PSA, TMEPA1, and TMPRSS2 transcript levels. Inhibition of both AR isoforms did not affect EGFR and ERBB2 transcript levels but decreased EGFR and increased ERBB2 protein levels. Proliferation of 22Rv1 cells in SDM was inhibited in the absence of AR and ARDeltaCTD. A further decrease was obtained with PKI166, an EGFR/ERBB2 kinase inhibitor. Overall, we showed that ARDeltaCTD is responsible for constitutive EGFR expression and ERBB2 repression in 22Rv1 cells and that ARDeltaCTD and tyrosine kinase receptors are necessary for sustained 22Rv1 cell growth. [less ▲]

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