Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers, in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibiting the † To whom correspondence should be addressed. david.tuveson@cancer.org.uk.

To define the genetic requirements for pancreatic ductal adenocarcinoma (PDA), we have targeted concomitant endogenous expression of Trp53(R172H) and Kras(G12D) to the mouse pancreas, revealing the cooperative development of invasive and widely metastatic carcinoma that recapitulates the human disease. The primary carcinomas and metastases demonstrate a high degree of genomic instability manifested by nonreciprocal translocations without obvious telomere erosion-hallmarks of human carcinomas not typically observed in mice. No mutations were discovered in other cardinal tumor suppressor gene pathways, which, together with previous results, suggests that there are distinct genetic pathways to PDA with different biological behaviors. These findings have clear implications for understanding mechanisms of disease pathogenesis, and for the development of detection and targeted treatment strategies.

Activating mutations in the ras oncogene are not considered sufficient to induce abnormal cellular proliferation in the absence of cooperating oncogenes. We demonstrate that the conditional expression of an endogenous K-ras(G12D) allele in murine embryonic fibroblasts causes enhanced proliferation and partial transformation in the absence of further genetic abnormalities. Interestingly, K-ras(G12D)-expressing fibroblasts demonstrate attenuation and altered regulation of canonical Ras effector signaling pathways. Widespread expression of endogenous K-ras(G12D) is not tolerated during embryonic development, and directed expression in the lung and GI tract induces preneoplastic epithelial hyperplasias. Our results suggest that endogenous oncogenic ras is sufficient to initiate transformation by stimulating proliferation, while further genetic lesions may be necessary for progression to frank malignancy.

Mammalian target of rapamycin (mTOR) functions in two distinct signaling complexes, mTORC1 and mTORC2. In response to insulin and nutrients, mTORC1, consisting of mTOR, raptor (regulatory-associated protein of mTOR), and mLST8, is activated and phosphorylates eukaryotic initiation factor 4E-binding protein (4EBP) and p70 S6 kinase to promote protein synthesis and cell size. Previously we found that activation of mTOR kinase in response to insulin was associated with increased 4EBP1 binding to raptor. Here we identify prolinerich Akt substrate 40 (PRAS40) as a binding partner for mTORC1. A putative TOR signaling motif, FVMDE, is identified in PRAS40 and shown to be required for interaction with raptor. Insulin stimulation markedly decreases the level of PRAS40 bound by mTORC1. Recombinant PRAS40 inhibits mTORC1 kinase activity in vivo and in vitro, and this inhibition depends on PRAS40 association with raptor. Furthermore, decreasing PRAS40 expression by short hairpin RNA enhances 4E-BP1 binding to raptor, and recombinant PRAS40 competes with 4E-BP1 binding to raptor. We, therefore, propose that PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.Mammalian target of rapamycin (mTOR), 2 a highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase, is involved in the control of cell growth, survival, proliferation, and metabolism (1, 2). It is present in two structurally and functionally separate complexes, mTORC1 and mTORC2. mTORC1 is rapamycin-sensitive and contains mTOR, raptor, and mLST8 (also known as G␤L), whereas mTORC2 is rapamycin-insensitive and contains mTOR, rictor, mLST8, and hSIN (2). mTORC1 catalyzes the phosphorylation of eIF4E binding protein-1 (4EBP1, also known as PHAS-I) and p70 S6 kinase 1 (S6K1), whereas mTORC2 phosphorylates Ser-473 in the hydrophobic motif of Akt/PKB (3). Raptor associates with mTOR in the mTORC1 complex by multiple binding regions and has a positive role in mTOR kinase activity as evidenced by experiments that deletion or knockdown of raptor abolished mTORC1 activity (4 -6). Raptor is thought to act as a scaffold to bind and present substrates to mTORC1, mediated by a putative TOR signaling motif (TOS motif) (4, 7-9). mLST8, a 36-kDa protein with 7 WD40 repeats, associates with the mTOR kinase domain and is present in both mTORC1 and -2 (10, 11). The TORC1 and TORC2 form multimeric complexes in yeast, flies, and mammalian cells (12)(13)(14).Because of the critical role of the TOR kinase activity in controlling cell size and growth, many inputs, such as growth factors, amino acids, energy sufficiency, and environmental stress such as hypoxia, can regulate its activity (2). Extensive studies have been performed to understand the mechanisms whereby these inputs control mTOR activity. For example, regulation of the mTOR pathway by growth factors such as insulin appears to be mediated via the phosphatidylinositol-3ЈOH kinase-Akt pathway. The stimulation of 4EBP1 phosphorylation by insulin depends on activation of A...

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Oncogenic BRAF alleles are both necessary and sufficient for cellular transformation, suggesting that chemical inhibition of the activated mutant protein kinase may reverse the tumor phenotype. Here, we report the characterization of SB-590885, a novel triarylimidazole that selectively inhibits Raf kinases with more potency towards B-Raf than c-Raf. Crystallographic analysis revealed that SB-590885 stabilizes the oncogenic B-Raf kinase domain in an active configuration, which is distinct from the previously reported mechanism of action of the multi-kinase inhibitor, BAY43-9006. Malignant cells expressing oncogenic B-Raf show selective inhibition of mitogen-activated protein kinase activation, proliferation, transformation, and tumorigenicity when exposed to SB-590885, whereas other cancer cell lines and normal cells display variable sensitivities or resistance to similar treatment. These studies support the validation of oncogenic B-Raf as a target for cancer therapy and provide the first evidence of a correlation between the expression of oncogenic BRAF alleles and a positive response to a selective B-Raf inhibitor. (Cancer Res 2006; 66(23): 11100-5)

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [ 32 P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/ protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition. Mammalian target of rapamycin (mTOR)2 has been demonstrated as a key element in signaling pathways controlling cell size, proliferation, and metabolism (1, 2). Two mTOR signaling complexes, mTORC1 and mTORC2, have been discovered. The rapamycin-sensitive mTORC1 consists of the catalytic subunit mTOR, the substrate-binding subunit raptor (regulatory associated protein of mTOR), mLST8 (also known as G␤L), and PRAS40 (1, 2) and controls protein translation (1, 2). mTORC2, the rapamycin-insensitive form, contains mTOR, rictor, SIN1, mLST8, and PRR5 (1, 2) and functions as a PDK2 (phosphoinositide-dependent protein kinase 2) to phosphorylate Akt/protein kinase B at Ser-473 and regulates the actin cytoskeleton (1, 2). The best characterized downstream effectors of mTORC1 are S6K1 and 4E-BP1 (also known as PHAS-I), both of which are phosphorylated by mTORC1 at multiple sites and are involved in the control of mRNA translation (1, 2). The nature of the phosphorylation sites in these two mTORC1 substrates is surprisingly different: either (S/T)P (3) or h(S/T)h (where h represents hydrophobic) (4). Thus, the surrounding amino acids appear not to be the major determinant for phosphorylation. A critical motif called the TOR signaling (TOS) motif has been discovered in the NH 2 terminus of S6K1 (FDIDL) and COOH terminus of 4E-BP1 (FEMDI) (5, 6). Mutation of the TOS motif not only decreases the rate of phosphorylation of S6K1 and 4E-BP1 by mTOR but also disrupts interaction between these substrates and raptor (5-8).PRAS40 has been recently identified as a protein associated with mTORC1 (9, 10). PRAS40 predominantly interacts with raptor, althoug...

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