Jorge Filmus

Downregulation of the pro-apoptotic protein Bak is required for the ras-induced transformation of intestinal epithelial cells.

Anoikis is a form of programmed cell death induced in normal epithelial cells by detachment from the extracellular matrix [1] [2] [3]. In epithelial cells of the intestine and other organs, activated rasinduces resistance to anoikis [3] [4], but the actual molecular effectors directly involved in the apoptotic machinery that execute or block anoikis have not yet been identified. Bak, a pro-apoptotic member of the Bcl-2 family, is downregulated in a high proportion of colorectal tumours [5]. In addition, Bak is an important regulator of apoptosis in normal intestinal epithelial cells [6] [7]. Here, we show that activated rasinduces the downregulation of Bak in rat and human intestinal epithelial cells. This ras-induced downregulation of Bak expression could be suppressed by an inhibitor of phosphatidylinositol (PI) 3-kinase, an enzyme already implicated in ras-induced resistance to anoikis [8]. Ectopic expression of Bak in ras-transformed rat intestinal epithelial IEC-18 cells inhibited ras-induced resistance to anoikis and significantly reduced their tumorigenicity. We conclude, therefore, that the ability of rasto downregulate Bak, and the consequent resistance to anoikis, are essential components of the transforming capacity of this oncogene in intestinal epithelial cells.

A patient with the Simpson–Golabi–Behmel syndrome displays a loss‐of‐function point mutation in GPC3 that inhibits the attachment of this proteoglycan to the cell surface

The Simpson–Golabi–Behmel syndrome (SGBS) is an X-linked disorder characterized by developmental overgrowth and a broad spectrum of clinical manifestations that vary from a mild phenotype in carrier females to infantile lethal forms in males [Chen et al., 1993; Neri et al., 1998]. The list of developmental abnormalities in males affected by SGBS include a distinct facial appearance, macroglossia, cleft palate, syndactyly, polydactyly, cystic and dysplastic kidneys, congenital heart defects, rib and vertebral abnormalities, and umbilical/inguinal hernias [Neri et al., 1998; DeBaun et al., 2001]. In addition, patients with SGBS display a high risk for the development of pediatric tumors [DeBaun et al., 2001]. Pilia et al. [1996] reported that SGBS is the result of loss-of-function mutations in the Glypican-3 (GPC3) gene. GPC3 is a member of the glypican family, which are heparan sulfate proteoglycans (HSPGs) that are bound to the exocytoplasmic surface of the plasma membrane through a covalent glycosyl-phosphatidylinositol (GPI) linkage [Filmus et al., 2008]. Our laboratory has recently reported that GPC3 acts as an inhibitor of Hedgehog (Hh) signaling during development, and that the overgrowth of patients with SGBS is, at least in part, the consequence of hyperactivation of the Hh signaling pathway [Capurro et al., 2008]. Hh signaling is triggered by the binding of this growth factor to Patched, the transmembrane signaling receptor [Hooper and Scott, 2005; Nieuwenhuis and Hui, 2005]. Our study showed that GPC3 inhibits Hh activity by competing with Patched for Hh binding, and that the binding of GPC3 to Hh induces its endocytosis and degradation [Capurro et al., 2008]. We also showed that cell attachment is essential for the Hh-inhibitory activity of GPC3. The vast majority of GPC3 mutations reported in SGBS patients consist of deletions ranging from less than 0.1 kb to more than 300 kb [Hughes-Benzie et al., 1996; Li et al., 2001]. Veugelers et al. [2000] identified a patient with a p.W296R mutation and reported that this mutant GPC3 protein is poorly expressed and is less substituted with heparan sulfate. A second GPC3 c.1666G>A mutation, which predicts p.G556R in a patient with SGBS was reported by Penisson-Besnier et al. [2008], but the consequences of this mutation are unknown. Proteins anchored to GPI are first generated as precursors that display a C-terminal hydrophobic region preceded by a short hydrophilic spacer region [Hooper, 2001]. These two regions have to be cleaved by the GPI-anchoring machinery before the GPI anchor is added to the corresponding core protein [Hooper, 2001]. The cleavage site (termed w) is located at the N-terminus of the spacer region. Interestingly, glycine 556 in GPC3 is located at the wþ 2 position, which is critical for cleavage [Hooper, 2001]. Sequencing of many GPI-anchored proteins has established that in mammals the wþ 2 position is usually a glycine or a serine [Udenfriend and Kodukula, 1995]. Based on this, we have hypothesized that the p.G556R mutation inhibits the insertion of the GPI anchor to GPC3, and that without the lipid anchor GPC3 is released into the extracellular environment losing in this way the capacity to inhibit Hh signaling. To test this hypothesis we generated a mutant GPC3 cDNA containing the c.1666G>A mutation. The mutation was verified by sequencing the whole cDNA. We also confirmed the

Oncogenes as Regulators of Cell Survival

During the past few years, it has become obvious that resistance to programmed cell death, or apoptosis, plays a major role in the process of malignant transformation and subsequent tumor progression. Molecular mechanisms of such resistance induced by the activation of various proto-oncogenes and the loss of activity of tumor suppressor genes have begun to emerge. In parallel, our knowledge of the principles governing the functioning of the intrinsic cell death machinery has expanded considerably.

Insertion of metal-responsive elements increases the inducibility of the mouse mammary tumor virus promoter

The promoter of the mouse mammary tumor virus (MMTV) is commonly used in inducible mammalian expression vectors. We have modified this promoter by inserting metal-responsive elements (MRE) at different sites. Surprisingly, MMTV promoters containing MRE 5 with respect to the glucocorticoid responsive elements (GRE), were not induced by metal ions. However, in the simultaneous presence of dexamethasone and metal ions, the modified promoters displayed a significantly increased transcriptional strength [up to sevenfold more than wild-type (wt) MMTV] in transiently transfected cells and permanent cell lines. This increment in the inducible expression was achieved without a concomitant increase in the basal level of expression. These modified MMTV promoters will offer the opportunity to develop tightly regulated inducible mammalian expression systems that are significantly more potent than the ones based on the wt MMTV promoter.