Claude Sardet

Abrogation of De novo Lipogenesis by Stearoyl-CoA Desaturase 1 Inhibition Interferes with Oncogenic Signaling and Blocks Prostate Cancer Progression in Mice

Increased de novo fatty acid (FA) synthesis is one hallmark of tumor cells, including prostate cancer. We present here our most recent results showing that lipid composition in human prostate cancer is characterized by an increased ratio of monounsaturated FA to saturated FA, compared with normal prostate, and evidence the overexpression of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1) in human prostate cancer. As a new therapeutic strategy, we show that pharmacologic inhibition of SCD1 activity impairs lipid synthesis and results in decreased proliferation of both androgen-sensitive and androgen-resistant prostate cancer cells, abrogates the growth of prostate tumor xenografts in nude mice, and confers therapeutic benefit on animal survival. We show that these changes in lipid synthesis are translated into the inhibition of the AKT pathway and that the decrease in concentration of phosphatidylinositol-3,4,5-trisphosphate might at least partially mediate this effect. Inhibition of SCD1 also promotes the activation of AMP-activated kinase and glycogen synthase kinase 3α/β, the latter on being consistent with a decrease in β-catenin activity and mRNA levels of various β-catenin growth-promoting transcriptional targets. Furthermore, we show that SCD1 activity is required for cell transformation by Ras oncogene. Together, our data support for the first time the concept of targeting the lipogenic enzyme SCD1 as a new promising therapeutic approach to block oncogenesis and prostate cancer progression. Mol Cancer Ther; 9(6); 1740–54. ©2010 AACR.

The CDK4-pRB-E2F1 pathway controls insulin secretion.

CDK4–pRB–E2F1 cell-cycle regulators are robustly expressed in non-proliferating β cells, suggesting that besides the control of β-cell number the CDK4–pRB–E2F1 pathway has a role in β-cell function. We show here that E2F1 directly regulates expression of Kir6.2, which is a key component of the KATP channel involved in the regulation of glucose-induced insulin secretion. We demonstrate, through chromatin immunoprecipitation analysis from tissues, that Kir6.2 expression is regulated at the promoter level by the CDK4–pRB–E2F1 pathway. Consistently, inhibition of CDK4, or genetic inactivation of E2F1, results in decreased expression of Kir6.2, impaired insulin secretion and glucose intolerance in mice. Furthermore we show that rescue of Kir6.2 expression restores insulin secretion in E2f1−/− β cells. Finally, we demonstrate that CDK4 is activated by glucose through the insulin pathway, ultimately resulting in E2F1 activation and, consequently, increased expression of Kir6.2. In summary we provide evidence that the CDK4–pRB–E2F1 regulatory pathway is involved in glucose homeostasis, defining a new link between cell proliferation and metabolism.

Cyclin A is a mediator of p120E4F-dependent cell cycle arrest in G1

ABSTRACT E4F is a ubiquitously expressed GLI-Krüppel-related transcription factor which has been identified for its capacity to regulate transcription of the adenovirus E4 gene in response to E1A. However, cellular genes regulated by E4F are still unknown. Some of these genes are likely to be involved in cell cycle progression since ectopic p120 E4F expression induces cell cycle arrest in G1. Although p21 WAF1 stabilization was proposed to mediate E4F-dependent cell cycle arrest, we found that p120 E4F can induce a G1 block in p21−/− cells, suggesting that other proteins are essential for the p120 E4F -dependent block in G1. We show here that cyclin A promoter activity can be repressed by p120 E4F and that this repression correlates with p120 E4F binding to the cyclic AMP-responsive element site of the cyclin A promoter. In addition, enforced expression of cyclin A releases p120 E4F -arrested cells from the G1block. These data identify the cyclin A gene as a cellular target for p120 E4F and suggest a mechanism for p120 E4F -dependent cell cycle regulation.

The Na+/H+ exchanger is phosphorylated in human platelets in response to activating agents

α‐Thrombin, phorbol esters (PMA) and 1,2‐diacylglycerol (DAG), three activators of the amiloride‐sensitive Na+/H+ exchange in human platelets, rapidly increase the intracellular pH and the level of phosphorylation of the Na+/H+ exchanger protein (NHE1). This stimulatory effect is suppressed by staurosporine, a potent kinase inhibitor, and increased by okadaic acid, a potent inhibitor of phosphatase 1 and 2A. The modulations of NHE1 phosphorylation by these factors correlate well with their effects on platelet pH. Thus, we conclude that in platelets (i) Na+/H+ exchange is mediated by NHE1. and (ii) platelet activating agents stimulate NHE1 via the modulation of the kinase/phosphatase equilibrium.

E2Fs and the Retinoblastoma Protein Family

The orderly progression through the cell cycle is mediated by the sequential activation of several cyclin/cyclin-dependent kinase (cdk) complexes. These kinases phosphorylate a number of cellular substrates, among which are the product of the retinoblastoma gene, pRB, and the pRB-related proteins p107 and p130. Phosphorylation of these proteins in late G1 causes their release from the heterodimeric transcription factors E2F/DP. This results in the transcriptional activation of E2F-responsive genes which encode proteins that either directly control cell-cycle progression or function in metabolic processes linked to the cell cycle. Thus E2F/DP complexes are key signal transducers connecting the cell cycle machinery with the transcriptional control of sets of genes that mediate the passage through the G1 and S phases of the cell cycle. This critical regulatory pathway, which gates cell cycle progression, is often disrupted during the pathogenesis of many mammalian tumors. It also plays a pivotal role in animal development and in promoting cellular differentiation. We review the current state of knowledge regarding this pRB/E2F pathway. (Figure 1.1)

The human amiloride-sensitive Na+/H+ antiporter: localization to chromosome 1 by in situ hybridization

The Na+/H+ antiporter is a ubiquitous membrane-bound enzyme involved in pH regulation of vertebrate cells. We cloned the human gene capable of complementing antiporter-deficient mouse fibroblasts and isolated an exon-containing genomic DNA fragment. Using this genomic probe, we mapped the putative structural gene of the amiloride-sensitive Na+/H+ antiporter to the human chromosome region 1p35----p36.1 by in situ hybridization.

The periodic down regulation of Cyclin E gene expression from exit of mitosis to end of G(1) is controlled by a deacetylase- and E2F-associated bipartite repressor element.

The expression of cyclin E and that of a few other bona fide cell cycle regulatory genes periodically oscillates every cycle in proliferating cells. Although numerous experiments have documented the role of E2F sites and E2F activities in the control of these genes as cells exit from G0 to move through the initial G1/S phase transition, almost nothing is known on the role of E2Fs during the subsequent cell cycles. Here we show that a variant E2F-site that is part of the Cyclin E Repressor Module (CERM) (Le Cam et al., 1999b) accounts for the periodic down regulation of the cyclin E promoter observed between the exit from mitosis until the mid/late G1 phase in exponentially cycling cells. This cell cycle-dependent repression correlates with the periodic binding of an atypical G1-specific high molecular weight p107-E2F complex (Cyclin E Repressor Complex: CERC2) that differs in both size and DNA binding behaviors from known p107-E2F complexes. Notably, affinity purified CERC2 displays a TSA-sensitive histone deacetylase activity and, consistent with this, derepression of the cyclin E promoter by trichostatin A depends on the CERM element. Altogether, this shows that the cell cycle-dependent control of cyclin E promoter in cycling cells is embroiled in acetylation pathways via the CERM-like E2F element.

Erythroid-specific Inhibition of the tal-1 Intragenic Promoter Is Due to Binding of a Repressor to a Novel Silencer

The basic helix-loop-helix tal-1 gene plays a key role in hematopoiesis, and its expression is tightly controlled through alternative promoters and complex interactions of cis-acting regulatory elements. tal-1 is not expressed in normal T cells, but its transcription is constitutive in a large proportion of human T cell leukemias. We have previously described a downstream initiation of tal-1 transcription specifically associated with a subset of T cell leukemias that leads to the production of NH2-truncated TAL-1 proteins. In this study, we characterize the human promoter (promoter IV), embedded within a GC-rich region in exon IV, responsible for this transcriptional activity. The restriction of promoter IV usage is assured by a novel silencer element in the 3′-unstranslated region of the human gene that represses its activity in erythroid but not in T cells. The silencer activity is mediated through binding of a tissue-specific nuclear factor to a novel protein recognition motif (designated tal-RE) in the silencer. Mutation of a single residue within the tal-RE abolishes both specific protein binding and silencing activity. Altogether, our results demonstrate that the tal-1 promoter IV is actively repressed in cells of the erythro-megakaryocytic lineage and that this repression is released in leukemic T cells, resulting in the expression of the tal-1 truncated transcript.

A functional genetic screen identifies TFE3 as a gene that confers resistance to the anti-proliferative effects of the retinoblastoma protein and transforming growth factor-β

The helix-loop-helix transcription factor TFE3 has been suggested to play a role in the control of cell growth by acting as a binding partner of transcriptional regulators such as E2F3, SMAD3, and LEF-1 (1–4). Furthermore, translocations/TFE3 fusions have been directly implicated in tumorigenesis (5–7). Surprisingly, however, a direct functional role for TFE3 in the regulation of proliferation has not been reported. By screening retroviral cDNA expression libraries to identify cDNAs that confer resistance to a pRB-induced proliferation arrest, we have found that TFE3 overrides a growth arrest in Rat1 cells induced by pRB and its upstream regulator p16INK4A. In addition, TFE3 expression blocks the anti-mitogenic effects of TGF-β in rodent and human cells. We provide data supporting a role for endogenous TFE3 in the direct regulation of CYCLIN E expression in an E2F3-dependent manner. These observations establish TFE3 as a functional regulator of proliferation and offer a potential mechanism for its involvement in cancer.

E4F1 connects the Bmi1-ARF-p53 pathway to epidermal stem cell-dependent skin homeostasis.

Comment on: Lacroix M, et al. Proc Natl Acad Sci USA 2010; 107:21076-81.