Reversible Tumorigenesis Induced by Deficiency of Vasodilator-Stimulated Phosphoprotein

Abstract

Random homozygous knockout (RHKO) is an antisense RNA strategy capable of identifying genes whose homozygous functional inactivation yields a selectable phenotype in cells growing in culture. Using this approach, we isolated NIH 3T3 fibroblast clones that showed the ability to form colonies on 0.5% agar and tumors in nude mice. The gene inactivated in one of these clones was found to encode VASP (vasodilatorstimulated phosphoprotein), a previously identified protein that binds to components of the cadherin-catenin junctional complex and has been implicated in cell-cell interactions, the formation of actin filaments, and the transmission of signals at the cytoskeleton-membrane interface. Fibroblasts made deficient in VASP by RHKO showed loss of contact inhibition, and consequently, continued cell division past confluence. Restoration of VASP function by reversal of RHKO yielded cells that had lost the neoplastic capabilities acquired during RHKO. Overproduction of VASP mRNA in the sense or antisense orientation from expression constructs introduced by transfection into naive NIH 3T3 fibroblasts also resulted in neoplastic transformation, implying that normal cell growth may require the maintenance of VASP expression within a narrow range. Our results implicate VASP in tumorigenesis and/or cancer progression.Vasodilator-stimulated phosphoprotein (VASP), a prolinerich founding member of the Ena-VASP protein family, was discovered both as a substrate of cyclic AMP-and cyclic GMPdependent protein kinases and a component of the actin-based cytoskeleton (10,(25)(26)(27)65). VASP is associated with focal adhesions, actin filaments, and highly dynamic membrane regions (50) and has been shown to be a multiligand protein that binds to actin, profilin, ActA, zyxin, and vinculin (9, 12, 51, 52). Vinculin in turn associates with tensin, ␣-actinin, ␣-catenin, and talin (4,31,63,66,68), and zyxin and profilin are involved in actin filament assembly and organization (3,14,43,56,60). In vivo evidence indicates that VASP is a crucial factor in the formation of actin filaments and the integration of signals transmitted between the cytoskeleton, the cytoskeleton-membrane interface, and two cyclic nucleotide signal transduction pathways (2,25,65). Additionally, recent data indicate that VASP is functionally homologous to the Drosophila melanogaster Enabled (Ena) locus, which was identified initially as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase (1). However, the biological role of VASP has remained largely unknown.Recently, a novel gene discovery strategy termed random homozygous knockout (RHKO) was shown to be capable of identifying genes whose functional inactivation in murine fibroblasts leads to reversible cellular transformation (40). This approach uses a promoter within a randomly inserted, chromosomally integrated gene search vector (GSV) to produce antisense transcripts complementary to those originating in the chromosomal gene containing the GSV, and consequently, also complementary to transcri...

Vasodilator-Stimulated Phosphoprotein Regulates Proliferation and Growth Inhibition by Nitric Oxide in Vascular Smooth Muscle Cells

“…As has been shown in NIH3T3 cells, however, VASP-deficiency and VASP overexpression induce neoplastic transformation and promote tumor growth. 17 Whether similar mechanisms underlie these observations needs to be investigated.…”

“…14 -16 The observation that increased or decreased VASP expression is associated with increased colony formation of NIH3T3 cells in soft agar and tumor formation in nude mice suggests that VASP may play a role in tumorigenesis. 17 Recent studies show that the overexpression of VASP potentiates the activity of serumresponse factor (SRF) in NIH3T3 fibroblasts, possibly by increasing F-actin assembly and depleting the cellular pool of G-actin. 18 -20 Although VASP phosphorylation is used as a biochemical marker for activation of PKG and PKA, its precise cellular and molecular functions remain to be determined.…”

Antisense RNA-mediated Deficiency of the Calpain Protease, nCL-4, in NIH3T3 Cells Is Associated with Neoplastic Transformation and Tumorigenesis

“…Although an anchorage-independent growth phenotype commonly accompanies tumorigenic ability, the capacity to form tumors in nude mice but not colonies on soft agar has been observed previously in a cell line that overexpresses the oncogene MDM2 (52). We showed earlier that reversal of antisense inactivation of genes inactivated by RHKO can result in the loss of tumorigenic capability by some cells, but not by others (40,45,48), consistent with the occurrence of secondary genetic changes common to tumor progression. The inability of Rb, p53, and other tumor suppressor genes to reverse neoplasia associated with mutation of these genes (49 -51) is also believed to result from such secondary changes occurring during the progression of cancer.…”

“…Identification and Characterization of Genomic Clones Containing the GSV-Whereas 5Ј-rapid amplification of cDNA ends preparation of a cDNA library and cDNA capture methods previously have been used to identify other genes inactivated by RHKO (40,45), our initial efforts at using these procedures to identify sequences fused to the GSV reporter in CK cells or their derivatives were not successful. Accordingly, we constructed a cosmid DNA library using genomic DNA isolated in CK cells.…”

Regulation of Cancer Cell Behavior by the Small GTPase Rab13

“…For example, PKA activity has been reported to both stimulate and inhibit cancer cell growth (67). Similarly, both down-regulation and overexpression of VASP result in the loss of contact inhibition and enhanced tumor growth (68). The contribution of VASP to tumorigenicity may depend on which of its residues are phosphorylated.…”