Simian virus 40 (SV40) large T antigen (LT) is a multifunctional protein that is important for viral replication and oncogenic transformation. Previously, infection of monkey or human cells with SV40 was shownto lead to the induction of DNA damage response signaling, which is required for efficient viral replication. However, it was not clear if LT is sufficient to induce the damage response and, if so, what the genetic requirements and functional consequences might be. Here, we show that the expression of LT alone, without a replication origin, can induce key DNA damage response markers including the accumulation of ␥-H2AX and 53BP1 in nuclear foci. Other DNA damage-signaling components downstream of ATM/ATR kinases were induced, including chk1 and chk2. LT also bound the Claspin mediator protein, which normally facilitates the ATR activation of chk1 and monitors cellular replication origins. Stimulation of the damage response by LT depends mainly on binding to Bub1 rather than to the retinoblastoma protein. LT has long been known to stabilize p53 despite functionally inactivating it. We show that the activation of a DNA damage response by LT via Bub1 appears to play a major role in p53 stabilization by promoting the phosphorylation of p53 at Ser15. Accompanying the DNA damage response, LT induces tetraploidy, which is also dependent on Bub1 binding. Taken together, our data suggest that LT, via Bub1 binding, breaches genome integrity mechanisms, leading to DNA damage responses, p53 stabilization, and tetraploidy.Simian virus 40 (SV40) is a small DNA tumor virus, belonging to the polyomavirus family, that induces a productive infection in its natural host, the rhesus macaque, but yields oncogenic transformation in nonpermissive hosts such as rodent cells. The highly multifunctional large T antigen (LT) is the key early protein essential for both driving viral replication as well as inducing cellular transformation. Two other early proteins, small t antigen and 17k T antigen (17k) may perform auxiliary functions during the viral life cycle (39, 69). LT has served as a powerful model system for understanding fundamental cellular processes such as nuclear translocation, transcriptional regulation, eukaryotic DNA replication, immortalization, and malignant transformation (reviewed in references 26 and 37).LT overrides cellular control mechanisms and reprograms the host cell to create a permissive environment for viral replication. The deregulation of cellular proliferation is dependent on LT's interaction with specific host proteins, among which the tumor suppressors p53 and the retinoblastoma protein (pRB) are the best characterized (reviewed in reference 37). Transformation in vitro and tumor induction in vivo frequently depend on LT binding and functionally inactivating these key tumor suppressors (37).

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP 3 ). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP 3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. [Cancer Res 2009;69(15):6299-306]

The therapeutic index for chemotherapeutic drugs is determined in part by systemic toxicity, so strategies for dose intensification to improve efficacy must also address tolerability. In addressing this issue, we have investigated a novel combinatorial strategy of reconstructing a drug molecule and using sequential drug-induced nanoassembly to fabricate supramolecular nanomedicines (SNM). Using cabazitaxel as a target agent, we established that individual synthetic prodrugs tethered with polyunsaturated fatty acids were capable of recapitulating self-assembly behavior independent of exogenous excipients. The resulting SNM could be further refined by PEGylation with amphiphilic copolymers suitable for preclinical studies. Among these cabazitaxel derivatives, docosahexaenoic acid-derived compound 1 retained high antiproliferative activity. SNM assembled with compound 1 displayed an unexpected enhancement of tolerability in animals along with effective therapeutic efficacy in a mouse xenograft model of human cancer, compared with free drug administered in its clinical formulation. Overall, our studies showed how attaching flexible lipid chains to a hydrophobic and highly toxic anticancer drug can convert it to a systemic self-deliverable nanotherapy, preserving its pharmacologic efficacy while improving its safety profile. Cancer Res; 77(24); 6963-74. Ó2017 AACR.

The availability of precisely modulated chemical modifi cations dramatically affects the physicochemical properties of pristine drugs and should facilitate the amphiphilic self-assembly of prodrugs into supramolecular nanoprodrugs (SNPs). However, rationally designing such prodrugs to achieve favorable clinical outcomes still remains a challenge. Here, a library of prodrugs through site-specifi c attachment of a variety of lipophilic moieties to the antitumor agent SN-38 (7-ethyl-10-hydroxycamptothecin) is constructed. Taking advantage of the role of hydroxyl groups as solvophilic moieties, these prodrugs exhibit self-assembly in aqueous environments, allowing for the identifi cation of fi ve prodrugs capable of self-assembling into SNPs at high drug concentrations. Importantly, in vivo studies demonstrate that the antitumor activity of the SNPs correlates well with their stability and long-term circulation. In addition, the modular feature of this SNP design strategy offers the opportunity to readily incorporate additional valuable functionalities (e.g., tumor-specifi c targeting ligands) to the particle surface, which is further exploited to improve antitumor effi cacy in mouse xenograft models. Thus, this structure-based reconstruction of SN-38 molecules signifi cantly improves the potency of SNPs for clinical use. These results also provide novel mechanistic insights into the rational design of prodrugs.

Synthesis of ingenious nanoassemblies is pursued in materials science. Herein, the in situ synthesis of the self-assembled blends of AB/BAB block copolymers of poly(ethylene glycol)-block-polystyrene/polystyrene-blockpoly(ethylene glycol)-block-polystyrene (PEG-b-PS/PS-b-PEG-b-PS) via two-macro-RAFT agent comediated dispersion polymerization is reported. The synthesis strategy combines the advantages of polymer blending and polymerizationinduced self-assembly. Following this strategy, various nanoassemblies of PEG-b-PS/PS-b-PEG-b-PS blends such as high-genus compartmentalized vesicles, multilayer and bicontinuous nanoassemblies, and porous nanospheres are prepared. The parameters, such as PEG-b-PS/PS-b-PEG-b-PS molar ratio, polymerization degree of the PS block, and fed monomer concentration, affecting morphology/structure of PEG-b-PS/PS-b-PEG-b-PS self-assembled blends are revealed.Computer simulations of self-assembly of the AB/BAB blends are performed, and nanoassemblies similar to those observed in our experiments are obtained, indicating that these morphologies are close to thermodynamical equilibrium. The formation mechanism of compartmentalized vesicles is investigated. The proposed strategy of two-macro-RAFT agent comediated dispersion polymerization is considered to be an efficient approach to construct selfassembled blends of block copolymers.

There is increasing evidence that cyclooxygenase (COX)-2 possess both angiogenic and cardioprotective properties. We examined the effects of hypoxic cardiac myocytes (H9c2 cells) on COX-2 expression in human umbilical vein endothelial cells (HUVECs) to determine the pathway involved in COX-2 regulation. The medium from hypoxic (<1% O2) cardiac myocytes (HMCM) or normoxic cardiac myocytes (21% O2) was added to HUVEC cultures. HMCM induced a transient increase of COX-2 mRNA expression at 1 and 3 h without affecting the COX-1 mRNA level. A similar effect also observed in HMCM from cultured primary cardiac myocytes (rat neonatal cardiac myocytes). The increased COX-2 mRNA was associated with a time-dependent increase in COX-2 protein expression. COX-2 was significantly induced by VEGF (4.86 ± 1.03-fold) and IL-1β (3.93 ± 0.89-fold) and slightly increased by TNF-α but not by FGF2, IGF-1, or PDGFs. Analysis of proteins secreted in HMCM showed increased levels of VEGF but not IL-1β or TNF-α. The HMCM-induced COX-2 expression was inhibited by the addition of an anti-VEGF neutralizing antibody. VEGF induced endothelial cell COX-2 expression by both increasing COX-2 transcription and prolonging the COX-2 mRNA half-life. Furthermore, staurosporine, a nonselective PKC inhibitor, prevented the induction of VEGF by hypoxia. Both a selective PKC-α and -β inhibitor and an inducible nitric oxide synthase (NOS) inhibitor decreased the induction of COX-2 by HMCM and VEGF. Finally, HMCM-induced upregulation of COX-2 was accompanied by upregulation of PGI2 and PGE2. These results suggest that VEGF is one of the principal factors produced by hypoxic myocytes that is responsible for the induction of endothelial cell COX-2 expression. This process likely involves both PKC and NOS pathways. Our findings have important implications regarding the cardiac protection of COX-2 in ischemic heart disease.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor known to be up-regulated in ischemic heart and hypoxic endothelial cells. However, the transcriptional regulation of VEGF in hypoxia-induced angiogenesis is not fully understood. Transcriptional enhancer factor-1 (TEF-1) is a transcriptional factor family that can regulate many genes expressed in cardiac and skeletal muscle cells by binding to myocytespecific chloramphenicol acetyltransferase heptamer elements in the promoters of these genes. In this study, we demonstrated that related TEF-1 (RTEF-1), a member of the TEF-1 family, is up-regulated in hypoxic endothelial cells. Overexpression of RTEF-1 increases VEGF promoter activity and VEGF expression. Sequential deletion and site-directed mutation analyses of the VEGF promoter demonstrated that a GC-rich region containing four Sp1 response elements, located between ؊114 and ؊50, was essential for RTEF-1 function. This region is beyond the hypoxia-inducible factor-1␣ binding site and does not consist of M-CAT-related elements. By electrophoretic mobility shift assay, RTEF-1 was found to interact with the first Sp1 residue (؊97 to ؊87) of the four consecutive Sp1 elements. Binding activity of RTEF-1 to VEGF promoter is also confirmed by chromatin immunoprecipitation. In addition, induction of VEGF promoter activity by RTEF-1 results in an increase of angiogenic processes including endothelial cells proliferation and vascular structure formation. These results indicate that RTEF-1 acts as a transcriptional stimulator of VEGF by regulating VEGF promoter activity through binding to Sp1 site. In addition, RTEF-1-induced VEGF promoter activity was enhanced in a hypoxic condition, indicating that RTEF-1 may play an important role in the regulation of VEGF under hypoxia.

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