Cell, Vol. 75, 1107-1117,
December
17, 1993, Copyright
0 1993 by Cell Press
Tumor Suppression by RNA from the 3’ Untranslated Region of a=Tropomyosin Fanan Rastinejad, Michael J. Conboy, Thomas A. Rando, and Helen M. Blau Department of Molecular Pharmacology Stanford University School of Medicine Stanford, California 94305-5332
Summary NMUP, a nondifferentiating mutant myogenic cell line, gives rise to rhabdomyosarcomas in mice. We show that constitutive expression of RNA from 0.2 kb of the a-tropomyosin (Tm) 3’ untranslated region (UTR), but not control YllTRs, suppresses anchorage-independent growth and tumor formation by NMU2 cells. When fl-galactosidase (f.I-gal)-labeled cells were implanted into muscles of adult mouse hindllmbs, Tm 3’UTR expression suppressed the proliferation, invasion, and destruction of muscle tissues characteristic of NMUS. In the rare tumors that developed from Tm 3lJTR transfectants, RNA expression was extinguished. These results suggest that suppression of tumorlgenicity is dependent on the continued expression of Tm transcripts lacking a coding region. We conclude that untranslated RNAs can function as regulators (rlboregulators) that suppress tumor formation. Introduction A well defined genetic alteration that bypasses normal growth control and gives rise to cancer is the inactivation of tumor suppressor genes (Knudson, 1971). Cell fusion experiments first showed that loss of tumor suppressor gene activity could lead to neoplastic transformation. In hybrids formed between normal and transformed cells, the normal state is dominant (Harris et al., 1969). The prediction that tumor suppressor genes are inactivated in malignant cells was borne out by the discovery that in naturally occurring human tumors a defective allele is inherited, and the remaining allele is inactivated (Cavenee et al., 1963). Definitive evidence for tumor suppressor genes was obtained first by the introduction into tumorigenie cells of entire chromosomes bearing presumed tumor suppressor genes (Weissman et al., 1967) and more recently by expression of the cloned genes themselves (Huang et al., 1966; Finlay et al., 1989). The protein products of tumor suppressor genes inhibit transformation by a multiplicity of mechanisms including blocking angiogenesis (Rastinejad et al., 1989), enhancing responsiveness to growth inhibitors such as TGF-8 (Kimchi et al., 1968; Gerwin et al., 1992) disrupting the cell cycle (Baker et al., 1990; Hinds et al., 1992; Nigro et al., 1992) or inducing cell differentiation (Mechler et al., 1985; Rosengard et al., 1989). All tumor suppressor genes described to date are thought to act through their encoded proteins. Transformation is often associated with a change in cell shape and cytoskeletal architecture due to altered expres-
sion and organization of microfilament and cell adhesion proteins (Cooper et al., 1987; Chan et al., 1989). Actins, a-actinin, vimentin, and tropomyosins are among the structural proteins that are expressed at diminished levels in tumor-derived or virally transformed cells (Leavitt et al., 1982; Matsumura et al., 1983; Cooper et al., 1985; Leavitt et al., 1985; Lin et al., 1985). Especially well documented are changes in tropomyosins. Certain Tm isoforms decrease in neoplastic cells and increase when the cells revert to normal (Cooper et al., 1985). In addition, upon constitutive expression of single cDNAs encoding adhesion or cytoskeletal proteins including tropomyosin, tumorigenicity of cell lines is suppressed (Eiden et al., 1991; Rodriguez Fernandez et al., 1992; Gliick et al., 1993; Prasad et al., 1993). It should be noted that in each of these cases theexpressed cDNAconstruct included both coding and noncoding sequences. We showed previously that noncoding regions of messenger RNAs can act in trans to control growth and differentiation (Rastinejad and Blau, 1993). When a cDNA expression library was introduced into the nondifferentiating myogenic cell line, NMU2, threeof the cDNAs that partially complemented the mutant phenotype derived from genes encoding cytoskeletal proteins: a-actin, tropomyosin, and troponin I. The activity of these cDNAs mapped to the 3’UTR.s. The Tm 3’UTR was particularly effective in promoting expression of differentiation-specific genes in myogenie cell lines. In fibroblasts, expression of the Tm 3’UTR markedly inhibited growth without inducing muscle-specific gene expression (Rastinejad and Blau, 1993). Given these findings and the evidence for tumor suppressor activity by full-length cytoskeletal cDNAs cited above, we examined whether the noncoding region of the a-Tm mRNA could play a role independent of the protein coding region in suppressing transformation. Here we show that an untranslated region of messenger RNA can function as a tumor suppressor. Specifically, we demonstrate that a 0.2 kb RNA transcribed from the a-Tm 3’UTR in the absence of protein coding sequence can inhibit anchorage-independent growth of neoplastic NMUP cells. Moreover, upon continuous expression of the Tm 3’UTR, P-gal-labeled NMU2 cells implanted in the muscles of mice no longer invade and destroy the tissue or form tumors. These novel findings provide evidence that an RNA from an untranslated region of a transcript can act as a tumor suppressor. Results Stable Transfectants Assayed for Transformation Suppression We used the differentiation-defective myogenic mutant, NMU2, to test the potential of the Tm 3’UTR to suppress transformation and promote differentiation in vivo. NMU2 was isolated following low dose mutagenesis of myoblasts of the C2C12 line (subclone C2F3) and selection for growth in soft agar (Rastinejad and Blau, 1993). Unlike its C2F3