The ubiquitin-like SUMO proteins covalently modify protein substrates and regulate their functional properties. In a broad spectrum of cancers, the tumor suppressor PML undergoes ubiquitin-mediated degradation primed by CK2 phosphorylation. Here we report that the SUMO E3-ligase inhibitor PIAS1 regulates oncogenic signaling through its ability to sumoylate PML and the PML-RARA oncoprotein of acute promyelocytic leukemia (APL). PIAS1-mediated SUMOylation of PML promoted CK2 interaction and ubiquitin/proteasome-mediated degradation of PML, attenuating its tumor suppressor functions. In addition, PIAS1-mediated SUMOylation of PML-RARA was essential for induction of its degradation by arsenic trioxide, an effective APL treatment. Moreover, PIAS1 suppression abrogated the ability of arsenic trioxide to trigger apoptosis in APL cells. Lastly, PIAS1 was also essential for PML degradation in non-small cell lung cancer cells, and PML and PIAS1 were inversely correlated in NSCLC cell lines and primary specimens. Together, our findings reveal novel roles for PIAS1 and the SUMOylation machinery in regulating oncogenic networks and the response to leukemia therapy.

Membrane microdomains, the so-called lipid rafts, function as platforms to concentrate receptors and assemble the signal transduction machinery. Internalization, in most cases, is carried out by different specialized structures, the clathrincoated pits. Here, we show that several endocytic proteins are efficiently recruited to morphologically identified plasma membrane lipid rafts, upon activation of the epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase. Analysis of detergent-resistant membrane fractions revealed that the EGF-dependent association of endocytic proteins with rafts is as efficient as that of signaling effector molecules, such as Grb2 or Shc. Finally, the EGFR, but not the nonsignaling transferrin receptor, could be localized in nascent coated pits that almost invariably contained raft membranes. Thus, specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization. INTRODUCTIONOn engagement of receptor tyrosine kinases (RTKs) by their cognate ligands, their intrinsic kinase activity is stimulated with ensuing receptor autophosphorylation and recruitment/activation of the signal transduction machinery, in turn responsible for several effector functions. Concurrently, activated receptors trigger their own endocytosis, whose ultimate goal is to extinguish signaling through removal of receptors from the cell surface (Carpenter, 2000). In the case of the epidermal growth factor (EGF) receptor (EGFR) the localization of the two processes is well characterized (Jorissen et al., 2003). Signaling occurs within specialized membrane microdomains, lipid rafts (Simons and Toomre, 2000;Maxfield, 2002), whereas endocytosis occurs mostly through the clathrin-coated pits (CCPs; Conner and Schmid, 2003).Membrane rafts are cholesterol-and sphingolipid-rich membrane regions characterized by higher order and lower buoyant density than bulk plasma membrane (Simons and Toomre, 2000;Sprong et al., 2001;Kusumi et al., 2004). These structures are also characterized by their insolubility in some detergents at 4°C (DRM, detergent resistant membranes; Brown and Rose, 1992). Several transmembrane receptors have been reported to associate with membrane rafts (Cheng et al., 1999;Krauss and Altevogt, 1999;Mineo et al., 1999;Lamaze et al., 2001;Giurisato et al., 2003), including the EGFR . The association of receptors with lipid rafts is thought to be functional to the activation of signaling cascades (Cheng et al., 1999;Waugh et al., 1999;Drevot et al., 2002;Matveev and Smart, 2002;Pierce, 2002;Stoddart et al., 2002;del Pozo et al., 2004). Accordingly, specific signaling (Gingras et al., 1998;Iwabuchi et al., 1998;Michaely et al., 1999;Kindzelskii et al., 2004) and adaptor proteins (e.g., shc and grb2; Biedi et al., 2003;Ridyard and Robbins, 2003;Yang et al., 2004) have been found associated to rafts. However, lipid rafts are rather small, possibly containing only few molecules (P...

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC often harbors oncogenic K-RAS mutations that lead to the aberrant activation of several intracellular networks including the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. Oncogenic K-RAS predicts poor prognosis and resistance to treatment with ionizing radiation (IR). Oncogenic K-Ras expression in the respiratory epithelium is sufficient to initiate NSCLC tumorigenesis, which requires the catalytic subunit of PI3K. Thus, effective inhibition of the PI3K signaling should lead to significant antitumor effects. However, therapy with rapamycin analogues has yielded disappointing results due in part to compensatory up-regulation of AKT. We hypothesized that dual PI3K/mTOR blockade would overcome these limitations. We tested this hypothesis with BEZ235, a novel dual PI3K/mTOR inhibitor that has recently entered clinical development. We found that BEZ235 induces a striking antiproliferative effect both in transgenic mice with oncogenic K-RAS-induced NSCLC and in NSCLC cell lines expressing oncogenic K-RAS. We determined that treatment with BEZ235 was not sufficient to induce apoptosis. However, we found that dual PI3K/mTOR blockade effectively sensitizes NSCLC expressing oncogenic K-RAS to the proapoptotic effects of IR both in vitro and in vivo. We conclude that dual PI3K/mTOR blockade in combination with IR may benefit patients with NSCLC expressing oncogenic K-RAS. These findings may have general applicability in cancer therapy, because aberrant activation of PI3K occurs frequently in human cancer.