SUMMARY The tumor stroma is believed to contribute to some of the most malignant characteristics of epithelial tumors. However, signaling between stromal and tumor cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumors. This was associated with the massive remodeling of the extra-cellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumors ameliorated disruption of the tumor microenvironment and was sufficient to decrease tumor growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumor stroma of breast cancer patients. These findings identify the Pten-Ets2 axis as a critical stroma-specific signaling pathway that suppresses mammary epithelial tumors.

Translational initiation factor 2 (IF2) is a guanine nucleotidebinding protein that can bind guanosine 3 ,5 -(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.fast kinetics ͉ GTP ͉ translation regulation ͉ nutritional stress I nitiation factor 2 (IF2) is the only initiation factor that is ribosome-bound throughout the entire translation initiation pathway, participating initially in the formation of the 30S initiation complex (30SIC) and subsequently in the assembly of the 70S initiation complex (70SIC), a process that ultimately results in formation of the first peptide bond (initiation dipeptide) and generates the first ribosomal pretranslocation complex (for reviews, see refs. 1-5). Thus, it could be predicted that IF2 functions are accompanied͞modulated by conformational changes that could be either consequence or cause of the interactions of IF2 with its various ligands (30S and 50S ribosomal subunits, fMet-tRNA, GTP, GDP⅐Pi, and GDP). Crystallographic (6) and NMR (7) studies have, in fact, shown that, depending upon the nature of their ligand (i.e., GTP or GDP), aIF5B, the archaeal homologue of bacterial IF2, as well as isolated IF2G2, the G domain of IF2, undergo large structural changes. On the other hand, chemical probing (8) and cryo-EM (9, 10) have clearly shown that several conformational changes of the factor occur during the early, middle, and late events of translation initiation.Bacterial cells growing under optimal nutritional conditions contain a high (Ͼ1 mM) concentration of GTP and a vanishingly low level of GDP. Thus, IF2 is expected to exist and to bind the 30S subunit almost exclusively in the GTP form, because it displays similar affinity for GTP and GDP, both K d s being in the 10-to 100-M range (11). The IF2⅐GTP was shown to have a higher affinity for the 30S ribosomal subunit than either IF2⅐GDP or free IF2 (11). The adjustment of fMet-tRNA in the P site (ref. 12 and refs. therein) and the release of IF2 from 70SIC (13-15) have been attributed to the IF2-dependent GTP hydrolysis, which is very rapidly triggered by th...

The interaction between fMet-tRNA f Met and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the speci®city of this interaction are localized within the acceptor hexanucleotide fMet-3¢ACCAAC of the initiator tRNA and a fairly small area at the surface of the b-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2). A weak but speci®c interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA f Met has been mapped using two independent approaches, sitedirected mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA f Met , and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3¢ to 5¢. Keywords: NMR spectroscopy/protein±RNA interaction/ site-directed mutagenesis/translation initiation IntroductionThe speci®c recognition of fMet-tRNA f Met by initiation factor IF2 represents one of the most important interactions occurring during translation initiation in bacteria (for reviews see Spurio et al., 1993; Schmitt et al., 1996;Gualerzi et al., 2000). This interaction determines the accuracy in the selection of the correct initiation site of both leadered (containing a 5¢-UTR) and unleadered mRNAs (Grill et al., 2000 and references therein), in the speed and ef®ciency of both 30S and 70S initiation complex formation (Gualerzi et al., 1986) and in the formation of the ®rst peptide bond (initiation dipeptide), which marks the transition from the initiation to the elongation phase of translation (La Teana et al., 1996;Toms Ïic et al., 2000).Important progress in the elucidation of the IF2 structure was made recently. The molecular dissection of Bacillus stearothermophilus IF2 (82 kDa) allowed the identi®cation of three domains in the molecule (the N-terminal domain, the central G-domain and the carboxyl-terminal C-domain), the site responsible for the recognition and binding of fMet-tRNA f Met being located in the 24.5 kDa C-terminal part of the protein (IF2 C) (Gualerzi et al., 1991). Further studies have shown that IF2 C is constituted by two domains of approximately equal size (IF2 C-1 and IF2 C-2) (Misselwitz et al., 1997). IF2 C-2, of 110 residues, was found to contain all the structural determinants involved in the recognition of fMet-tRNA f Met , and its complex with fMet-tRNA f Met displayed the same stability and properties as those formed by intact IF2 and IF2 C Spurio et al., 2000). The three dimensional (3D) solution structure of B.stearothermophilus IF2 C-2 determined by multinuclear NMR spectroscopy consists of a compact b-barrel, structurally homologous to domains II of elongation factors EF-Tu and EF-G, despite the lack of an...

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