Systemic administration of a recombinant adenovirus encoding the human interferon-beta gene (H5.110CMVhIFN-beta) results in transduction of hepatocytes and detectable circulating levels of IFN-beta protein. In preclinical studies in mice, we noticed a distinctly nonlinear dose response, with low levels of virus (1-3 x 10(10) viral particles) yielding barely detectable levels of IFN-beta but with a higher viral dose (1 x 10(11) particles) resulting in disproportionately high IFN-beta levels. Further studies showed that transgene expression levels from low viral doses could be dramatically enhanced by coadministering an unrelated recombinant adenovirus (H5.110CMVlacZ), suggesting that there was a viral dose threshold effect for efficient viral transduction and/or IFN-beta expression. This enhancement of reporter expression by a nonreporter adenovirus, effective upon coadministration, was further enhanced by preadministration of H5.110CMVlacZ (up to 8 h), but was ineffective if the helper virus was administered as little as 5 min after the H5.110CMVhIFN-beta reporter virus. Our data suggest that the reticuloendothelial system plays a role in this threshold effect, such that low doses of virus are efficiently taken up by the RES/Kupffer cells without leading to appreciable transgene expression, whereas high doses saturate these cells and are able to productively transduce hepatocytes. A better understanding of this phenomenon could have an impact on gene therapy clinical trial safety and efficacy.

Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti-IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti-IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone. Mol Cancer Ther; 9(10); 2652-64. ©2010 AACR.

BackgroundCancer-related weight loss is associated with increased inflammation and decreased survival. The novel inflammatory mediator growth differentiation factor (GDF)15 is associated with poor prognosis in cancer but its role in cancer-related weight loss (C-WL) remains unclear. Our objective was to measure GDF15 in plasma samples of cancer subjects and controls and establish its association with other inflammatory markers and clinical outcomes.MethodsWe measured body weight, appetite, plasma GDF15, and other inflammatory markers in men with cancer-related weight loss (C-WL, n = 58), weight stable patients with cancer (C-WS, n = 72), and non-cancer controls (Co, n = 59) matched by age and pre-illness body mass index. In a subset of patients we also measured handgrip strength, appendicular lean body mass (aLBM), Eastern Cooperative Oncology Group (ECOG), and Karnofsky performance scores.ResultsGDF15, interleukin (IL)-6 and IL-8 were increased in C-WL versus other groups. IL-1 receptor antagonist, IL-4, interferon–gamma, tumour necrosis factor alpha, and vascular endothelial growth factor A were increased in C-WL versus C-WS, and Activin A was significantly downregulated in Co versus other groups. C-WL patients had lower handgrip strength, aLBM, and fat mass, and Eastern Cooperative Oncology Group and Karnofsky performance scores were lower in both cancer groups.GDF15, IL-6, and IL-8 significantly correlated with weight loss; GDF15 negatively correlated with aLBM, handgrip strength, and fat mass. IL-8 and Activin A negatively correlated with aLBM and fat mass. GDF15 and IL-8 predicted survival adjusting for stage and weight change (Cox regression P < 0.001 for both).ConclusionGDF15 and other inflammatory markers are associated with weight loss, decreased aLBM and strength, and poor survival in patients with cancer. GDF15 may serve as a prognostic indicator in cancer patients and is being evaluated as a potential therapeutic target for cancer-related weight loss.

The A v B 6 integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of A v B 6 expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of A v B 6 in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent A v B 6 expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate A v B 6 expression. Detroit 562 cells showed A v B 6 -dependent adhesion and activation of transforming growth factor-B (TGF-B) that was inhibited >90% with an A v B 6 blocking antibody, 6.3G9. Although both recombinant soluble TGF-B receptor type-II (rsTGF-BRII-Fc) and 6.3G9 inhibited TGF-B-mediated Smad2/ 3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-BRII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by A v B 6 antibodies, our findings support a role for A v B 6 in human cancer and underscore the therapeutic potential of function blocking A v B 6 antibodies. [Cancer Res 2008;68(2):561-70]

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.

Purpose: Estrogen receptor-positive (ER þ ) breast cancers are typically treated with endocrine agents, and dependence on the ER pathway is often retained even after multiple rounds of antiestrogen therapy. Selective estrogen receptor degraders (SERD) are being developed as a strategy to more effectively target ER and exploit ER dependence in these cancers, which includes inhibiting both wild-type and mutant forms of ER. The purpose of this study was to evaluate the efficacy of a novel orally bioavailable SERD, elacestrant (RAD1901), in preclinical models of ER þ breast cancer. Experimental Design: Elacestrant was evaluated as a single agent and in combination with palbociclib or everolimus in multiple ER þ breast cancer models, including several patientderived xenograft models.

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-␤ has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-␤ concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-␤ gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-␤ gene delivery into established tumors generated high local concentrations of IFN-␤, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-␤ gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

As the culmination of several years of experiments, we propose a complete amino-acid sequence for porcine pepsin, an enzyme containing 327 amino-acid residues in a single polypeptide chain. In the sequence determination, the enzyme was treated with cyanogen bromide. Five resulting fragments were purified. The amino-acid sequence of four of the fragments accounted for 290 residues. Because the structure of a 37-residue carboxyl-terminal fragment was already known, it wag not studied. The alignment of these fragments was determined from the sequence of methionyl-peptides we had previously reported. We also discovered the locations of activesite aspartyl residues, as well as the pairing of the three disulfide bridges. A minor component of commercial crystalline pepsin was found to contain two extra aminoacid residues, Ala-Leu-, at the amino-terminus of the molecule. This minor component was apparently derived from a different site of cleavage during the activation of porcine pepsinogen.Although pepsin (EC 4.3.3.1) was one of the first enzymes to be discovered and purified in crystalline form, its structurefunction relationship is understood only superficially. In recent years, workers have realized that the complete elucidation of the mechanism of action of an enzyme depends upon the detailed knowledge of the chemical and three-dimensional structure of its molecule. For this reason, we undertook a long-term study of the complete amino-acid sequence of porcine pepsin.A number of studies have contributed to our knowledge of the partial sequence of porcine pepsin. They include investigations of the amino-acid sequences at the regions near the carboxyl-terminus (1-4), the amino-terminus (5, 6), the three disulfide bonds (7), the tryptophanyl residues (8, 9), the phosphoseryl residue (7, 10), two separate active-site aspartyl residues (11,12), and a number of small peptides (13,14). We have confirmed most of these previous findings and now present the complete amino-acid sequence of this enzyme. METHODSPorcine pepsin (three-times crystallized) was obtained from Worthington. The enzyme was either alkali-denatured (7) or reduced and aminoethylated (15) and subjected to cyanogenbromide cleavage (16). Preliminary experiments indicated that at room temperature one of the methionyl bonds (MetThr at residues 80-81) was cleaved less than 5% by cyanogen * Present address: