p53/TAp63 and AKT regulate mammalian target of rapamycin complex 1 (mTORC1) signaling through two independent parallel pathways in the presence of DNA damage.

Under conditions of DNA damage, the mammalian target of rapamycin complex 1 (mTORC1) is inhibited, preventing cell cycle progression and conserving cellular energy by suppressing translation. We show that suppression of mTORC1 signaling to 4E-BP1 requires the coordinated activity of two tumor suppressors, p53 and p63. In contrast, suppression of S6K1 and ribosomal protein S6 phosphorylation by DNA damage is Akt-dependent. We find that loss of either p53, required for the induction of Sestrin 1/2, or p63, required for the induction of REDD1 and activation of the tuberous sclerosis complex, prevents the DNA damage-induced suppression of mTORC1 signaling. These data indicate that the negative regulation of cap-dependent translation by mTORC1 inhibition subsequent to DNA damage is abrogated in most human cancers.

Detection and biological activities of carboxyethylpyrrole ethanolamine phospholipids (CEP-EPs).

Oxidation of docosahexaenoate phospholipids produces 4-hydroxy-7-oxo-hept-5-eonyl phospholipids (HOHA-PLs) that react with protein lysyl ε-amino residues to generate 2-ω-carboxyethylpyrrole (CEP) derivatives, endogenous factors that induce angiogenesis in the retina and tumors. It seemed likely, but remained unproven, that HOHA-PLs react with ethanolamine phospholipids (EPs) in vivo to generate CEP-EPs. We now show that CEP-EPs are present in human blood at 4.6-fold higher levels in age-related macular degeneration plasma than in normal plasma. We also show that CEP-EPs are pro-angiogenic, inducing tube formation by human umbilical vein endothelial cells by activating Toll-like receptor 2. CEP-EP levels may be a useful biomarker for clinical assessment of AMD risk and CEP-associated tumor progression and a tool for monitoring the efficacy of therapeutic interventions.

Redundant Signaling as the Predominant Mechanism for Resistance to Antibodies Targeting the Type-I Insulin-Like Growth Factor Receptor in Cells Derived from Childhood Sarcoma.

Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R-targeted antibodies. Sensitivity to the IGF-1R-targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1-resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (∼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R-targeted antibodies tested against this model.

Vitamin D receptor as a therapeutic target for benign prostatic hyperplasia.

The bioactive form of vitamin D, 1α, 25-dihydroxyvitamin D3 (1α, 25(OH)2D3), is a secosteroid hormone that binds to the vitamin D receptor (VDR), a member of the nuclear receptor super-family expressed in many cell types, and modulates a variety of biological functions. 1α, 25(OH)2D3 is essential for bone and mineral homeostasis, but also regulates growth and differentiation of multiple cell types, and displays immunoregulatory and anti-inflammatory activities. The antiproliferative, prodifferentiative, antibacterial, immunomodulatory and anti-inflammatory properties of synthetic VDR agonists could be exploited to treat a variety of chronic inflammatory and autoimmune diseases, including benign prostatic hyperplasia (BPH). It has been hypothesized that VDR may influence both the risk of a variety of diseases and their occurrence and prognosis. However, earlier studies investigating the associations between specific VDR polymorphisms and various diseases often show controversial results. We performed a systematic review of the current literature on vitamin D and BPH using the PubMed and Web of Knowledge databases. The aim of this review is to summarize the current knowledge on the utility of the VDR gene regarding prostate growth as well as the pathogenesis and treatment of BPH, a complex syndrome characterized by a static component related to prostate overgrowth, a dynamic component responsible for urinary storage symptoms, and an inflammatory component. Despite the massive advances in recent decades, further research is needed to fully characterize the exact underlying mechanisms of VDR action on BPH and to comprehend how these cellular changes translate into clinical development in physical concert.

Targeting angiogenesis in childhood sarcomas.

Angiogenesis and vasculogenesis constitute two processes in the formation of new blood vessels and are essential for progression of solid tumors. Consequently, targeting angiogenesis, and to a lesser extent vasculogenesis, has become a major focus in cancer drug development. Angiogenesis inhibitors are now being tested in pediatric populations whereas inhibitors of vasculogenesis are in an earlier stage of development. Despite the initial enthusiasm for targeting angiogenesis for treatment of cancer, clinical trials have shown only incremental increases in survival, and agents have been largely cytostatic rather than inducing tumor regressions. Consequently, the role of such therapeutic approaches in the context of curative intent for childhood sarcomas is less clear. Here we review the literature on blood vessel formation in sarcomas with a focus on pediatric sarcomas and developments in targeting angiogenesis for treatment of these rare cancers.

Predisposition of genetic polymorphism with the risk of urolithiasis.

Urolithiasis is a relevant clinical problem with a subsequent burden for health system. The aim of this review is to provide recent progress made using genetic polymorphisms to define pathophysiology, to identify persons at risk for kidney stone disease and to predict treatment response. Population case-control studies are useful both as an alternative and an adjunct as compared to family studies. These involve either whole genome scanning or candidate gene approaches. While whole genome scanning is likely to be widely used in future, at present, candidate gene studies are more feasible. When performing candidate gene case-control studies factors such as study design, methods for recruitment of case and controls, selection of candidate genes, functional significance of polymorphisms chosen for study and statistical analysis require close attention to ensure that only genuine associations are detected. Some of the significant genes that play role in stone formation include calcitonin receptor gene (CTR), vitamin D receptor (VDR), Urokinase, Interleukin, (IL-1ß, IL-Ra), E-Cadherin, Androgen & oestrogen receptor gene, vascular endothelial growth factor (VEGF) and Arginine p21. In our case-control study we studied CTR, VDR, Urokinase, IL-1ß(-511 and +3954), IL-Ra from north India and predict that VDR, IL-ß (-511) and IL-1Ra gene may be used as a possible genetic marker for earlier detection in patients who are at risk for calcium oxalate stone disease. Further, linkage disequilibrium and haplotype structure of a certain candidate gene is important for association analysis. When a certain polymorphic allele has been found to be associated with disease, it is further explained on basis of LD and haplotype structure by one or more other alleles. Once it is determined which haplotype carries the risk allele, by means of molecular biological functional analyses, the variants on that haplotype allele truly causing the effect can be determined.

IL-6 and CXCL8 mediate osteosarcoma-lung interactions critical to metastasis.

Osteosarcoma (OS), a malignant tumor of bone, kills through aggressive metastatic spread almost exclusively to the lung. Mechanisms driving this tropism for lung tissue remain unknown, though likely invoke specific interactions between tumor cells and other cells within the lung metastatic niche. Aberrant overexpression of ΔNp63 in OS cells directly drives production of IL-6 and CXCL8. All these factors were expressed at higher levels in OS lung metastases than in matched primary tumors from the same patients. Expression in cell lines correlated strongly with lung colonization efficiency in murine xenograft models. Lentivirus-mediated expression endowed poorly metastatic OS cells with increased metastatic capacity. Disruption of IL-6 and CXCL8 signaling using genetic or pharmaceutical inhibitors had minimal effects on tumor cell proliferation in vitro or in vivo, but combination treatment inhibited metastasis across multiple models of metastatic OS. Strong interactions occurred between OS cells and both primary bronchial epithelial cells and bronchial smooth muscle cells that drove feed-forward amplification of IL-6 and CXCL8 production. These results identify IL-6 and CXCL8 as primary mediators of OS lung tropism and suggest pleiotropic, redundant mechanisms by which they might effect metastasis. Combination therapy studies demonstrate proof of concept for targeting these tumor-lung interactions to affect metastatic disease.

Genotype and haplotype determination of IL1B (g. -511C>T and g. +3954C>T) and (IL1RN) in pediatric nephrolithiasis.

BACKGROUND: Nephrolithiasis is a multifactorial and polygenic disorder characterized by presence of stones in urinary tract. Interleukin1 (IL1) plays role in process of bone loss/hypercalciuria and is involved in formation of kidney stones. We investigated the association between IL1B promoter region and exon-5 (g.-511C>T and g. +3954C>T) polymorphism and variable number of tandem repeats in IL1 receptor antagonist, IL1RN (IVS2) with risk of stone formation in childhood nephrolithiasis in north Indian population. METHODS: Control group of 60 healthy pediatric individuals (age range =4-16 y) and 50 pediatric nephrolithiasis patients (age range =2-14 y) were studied. Polymorphism was detected by PCR based restriction analysis. Haplotypes for IL1B and IL1RN were constructed using Arlequin v2.0 software. RESULTS: Distribution of IL1RN gene polymorphism demonstrated significant difference (p=0.023). Pediatric patients had significantly higher frequency of allele I in IL1RN (16% vs. 1.7%). The distribution of IL1B (g. -511C>T and g. +3954C>T) genotypes in patients and controls were similar (p=0.263 and 0.694 respectively). There was a significant difference in haplotype frequencies between pediatric patients and control group (p<0.05). Haplotype T-E1-I showed>7-folds risk for nephrolithiasis (p=0.033; OR=7.07, 95% CI=1.16-42.84). CONCLUSIONS: Significant association was observed for allele I(*) of IL1RN however, no association was observed for IL1B. Haplotype T-E1-I was significantly associated with higher risk of pediatric nephrolithiasis. These findings suggest that the IL1RN and haplotyping may be an influential marker for susceptibility to pediatric nephrolithiasis.

Association of interleukin-1beta gene and receptor antagonist polymorphisms with calcium oxalate urolithiasis.

BACKGROUND AND PURPOSE: Genetic polymorphisms of the interleukin-1beta (IL-1beta) promoter region (-511) and exon 5 +3954 and a variable number of tandem repeats in the IL receptor antagonist (IL-1RA) gene have been proposed as markers for calcium oxalate urolithiasis. Because the prevalence of these polymorphisms could be influenced by racial variation/ethnicity, we explored the association of IL-1 gene-cluster polymorphisms with stone formation in a north India population. PATIENTS AND METHODS: The case-control study involved 150 stone-free control subjects (mean age 46.5 +/- 10.5 years) and 130 patients (mean age 40.0 +/- 11.5 years) with calcium oxalate urolithiasis. Biallelic polymorphisms of two loci, IL-1beta (-511) and IL-1beta (+3954), as well as the penta-allelic variable number of tandem repeats of IL-1RA, were genotyped by polymerase chain reaction-based restriction analysis. Haplotypes were constructed for the IL-1 gene cluster using SNP Analyzer software. RESULTS: We observed a significant association between stone disease and IL-1beta -511 and IL-1RA polymorphisms (P < 0.001 and P = 0.039, respectively), whereas no association was observed for IL-1beta +3954 (P = 0.408). The frequency of the TT (-511) and I/II (410/240; IL-1RA) genotypes was higher in patients than in control subjects (50/130 v 16/150 and 55/130 v 38/150, respectively), whereas the frequencies of the haplotypes were similar (P = 0.485). Significant linkage disequilibrium showed that three genes were strongly linked (P < 0.0001). Patients with a combination of high IL-1beta (-511 and +3954) and low IL-1RA genotypes were at significantly higher risk for urolithiasis (P < 0.001; odds ratio = 5.448, 0.013, and 2.560, respectively). CONCLUSION: Our study demonstrated a strong association of IL-1RA and IL-1beta-511 and suggested that differences in the IL-1 gene cluster could be linked to the risk of urolithiasis. A combination of IL-1beta and IL-1RA associations exhibiting gene-gene interaction further substantiates the finding of risk.

Association of urokinase gene 3'-UTR polymorphism with calcium oxalate nephrolithiasis.

PURPOSE: Urokinase might play a role in the formation of kidney stones. The aim of this study was to investigate the role of the urokinase gene in calcium oxalate nephrolithiasis. SUBJECTS AND METHODS: A control group of 150 healthy individuals having no history of stone formation (mean age 40 +/- 11.5 years) and a group of 130 patients (mean age 40.5 +/- 10.5 years) with recurrent calcium oxalate stones were examined. The C/T polymorphism of the urokinase gene was detected using polymerase chain reaction (PCR)-based restriction analysis. RESULTS: A marginally significant difference (P = 0.035) was found in the distribution of the urokinase gene 3' untranslated region (UTR) C/T polymorphism between patients with stones and controls. The odds ratio for the risk of the T allele in stone patients was 1.006 (95% CI 0.63-1.62). CONCLUSION: The T allele of 3' UTR of the urokinase gene may not be associated with a higher risk of stone formation.

The Bromodomain BET Inhibitor JQ1 Suppresses Tumor Angiogenesis in Models of Childhood Sarcoma.

The bromodomain and extra-terminal domain inhibitor JQ1 has marked antitumor activity against several hematologic malignancies as well as solid tumor models. Here, we investigated its activity in vitro and in vivo against models of childhood rhabdomyosarcoma and Ewing sarcoma. In vitro, JQ1 (but not the inactive enantiomer JQ1R) inhibited cell proliferation and increased G1 fraction of cells, although there was no correlation between cell line sensitivity and suppression of c-MYC or MYCN. In vivo, xenografts showed significant inhibition of growth during the period of treatment, and rapid regrowth after treatment was stopped, activity typical of antiangiogenic agents. Furthermore, xenografts derived from cell lines intrinsically resistant or sensitive to JQ1 in vitro had similar sensitivity in vivo as xenografts. Further investigation showed that JQ1 reduced tumor vascularization. This was secondary to both drug-induced downregulation of tumor-derived growth factors and direct effects of JQ1 on vascular elements. JQ1 suppressed VEGF-stimulated vascularization of Matrigel plugs in mice, and in vitro suppressed differentiation, proliferation, and invasion of human umbilical cord vascular endothelial cells (HUVEC). In HUVECs, JQ1 partially suppressed c-MYC levels, but dramatically reduced AP-1 levels and activity through suppression of the AP-1-associated protein FOSL1. Our data suggest that the antitumor activity of JQ1 in these sarcoma models is largely a consequence of its antiangiogenic activity. Mol Cancer Ther; 15(5); 1018-28. ©2016 AACR.

Vitamin-D receptor (VDR) gene (Fok-I, Taq-I and Apa-I) polymorphisms in healthy individuals from north Indian population.

The vitamin-D endocrine system is involved in a wide variety of biological processes including bone metabolism, modulation of immune responses, and regulation of cell proliferation and differentiation. Variation in this endocrine system have, thus, been linked to several common diseases, including osteoarthritis (OA), diabetes, cancer, cardiovascular ailments, urolithiasis and tuberculosis. Activity of Vit-D is mediated by the vitamin D receptor (VDR), a ligand dependent receptor. VDR gene polymorphisms thus represent strong positional candidates for different diseases like prostate cancer, urolithiasis, inflammatory bowl disease and osteoporosis. Genetic studies provide excellent opportunities to link molecular insights with epidemiological data and can reveal modest and subtle but true biological effects. The abundance of polymorphisms in the human genome as well as high frequencies in human populations have made them targets to explain variation in risk of common diseases. The present study was carried out to determine the distribution of VDR gene (Fok-I, Taq-I and Apa-I) polymorphisms using a PCR-based restriction analysis in unrelated normal healthy individuals from a north Indian population. We obtained allelic frequencies of (68.5% vs 31.5%), (66% vs 34%) and (58% vs 42%) for (F vs f), (T vs t) and (A vs a) alleles, with 44%, 49% and 7%, respectively, for genotypes FF, Ff and ff , 49%, 40% and 11% for TT, Tt and tt and 36%, 44% and 20% for AA, Aa and aa. Our results suggest that the frequency and distribution of the polymorphisms in India are substantially different from in other populations and ethnic groups. Thus the data signify an impact of ethnicity and provide a basis for future epidemiological and clinical studies.

Ethnicity greatly influences the interleukin-1 gene cluster(IL-1b promoter, exon-5 and IL-1Ra) polymorphisms: a pilot study of a north Indian population.

There is considerable evidence that polymorphisms in the regulatory regions of cytokine genes are highly influenced by ethnicity. Polymorphisms in interleukin 1-beta (IL-1beta) and IL-1 receptor antagonist (IL-1Ra) genes, respectively encoding a potent inflammatory agent and an antagonist, which combines with IL-1 receptors competitively, have been associated with a number of diseases like systemic lupus erythematosus, rheumatoid arthritis, sepsis, kidney diseases, and cancer. In this study, we therefore evaluated the distribution of interleukin-1 gene cluster (IL-1beta promoter region, exon-5 and IL-1Ra) gene polymorphisms in 206 healthy north Indian subjects, using PCR-based restriction analysis. We also constructed various haplotypes and estimated the linkage disequilibrium (LD). We found that genotype and allelic frequencies for these cytokines were conspicuously different when compared among different ethnic populations. The haplotype 'T-E1-1' predominated (41.7%) while the least common was 'C-E2-2' (2%) in our population. Genetic linkage between three loci of IL-1 gene showed strong association among the variants in controls (D'=0.42, p<0.001). Our results suggest that the frequency and distribution of the polymorphisms in India are substantially different from other populations and ethnic groups. Thus they signify an impact of ethnicity and provide a basis for future epidemiological and clinical studies.

ΔNp63 promotes pediatric neuroblastoma and osteosarcoma by regulating tumor angiogenesis.

The tumor suppressor gene p53 and its family members p63/p73 are critical determinants of tumorigenesis. ΔNp63 is a splice variant of p63, which lacks the N-terminal transactivation domain. It is thought to antagonize p53-, p63-, and p73-dependent translation, thus blocking their tumor suppressor activity. In our studies of the pediatric solid tumors neuroblastoma and osteosarcoma, we find overexpression of ΔNp63; however, there is no correlation of ΔNp63 expression with p53 mutation status. Our data suggest that ΔNp63 itself endows cells with a gain-of-function that leads to malignant transformation, a function independent of any p53 antagonism. Here, we demonstrate that ΔNp63 overexpression, independent of p53, increases secretion of interleukin (IL)-6 and IL-8, leading to elevated phosphorylation of STAT3 (Tyr-705). We show that elevated phosphorylation of STAT3 leads to stabilization of hypoxia-inducible factor 1α (HIF-1α) protein, resulting in VEGF secretion. We also show human clinical data, which suggest a mechanistic role for ΔNp63 in osteosarcoma metastasis. In summary, our studies reveal the mechanism by which ΔNp63, as a master transcription factor, modulates tumor angiogenesis.

Targeting constitutively-activated STAT3 in hypoxic ovarian cancer, using a novel STAT3 inhibitor.

Tumor hypoxia, a feature of many solid tumors including ovarian cancer, is associated with resistance to therapies. We previously demonstrated that hypoxic exposure results in increased expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). We hypothesized the activation of STAT3 could lead to chemotherapeutic resistance in ovarian cancer cells in hypoxic conditions. In this study, we demonstrate the level of pSTAT3 Tyr705 is increased in the hypoxic regions of human epithelial ovarian cancer (EOC) specimens, as determined by HIF-1α and CD-31 staining. In vitro mutagenesis studies proved that pSTAT3 Tyr705 is necessary for cell survival and proliferation under hypoxic conditions. In addition, we show that S1PR1, a regulator of STAT3 transcription via the JAK/STAT pathway, is highly expressed in hypoxic ovarian cancer cells (HOCCs). Knock down of S1PR1 in HOCCs reduced pSTAT3 Tyr705 levels and was associated with decreased cell survival. Treatment of HOCCs with the STAT3 inhibitor HO-3867 resulted in a rapid and dramatic decrease in pSTAT3 Tyr705 levels as a result of ubiquitin proteasome degradation. STAT3-target proteins Bcl-xL, cyclin D2 and VEGF showed similar decreases in HO-3867 treated cells. Taken together, these findings suggest that activation of STAT3 Tyr705 promotes cell survival and proliferation in HOCCs, and that S1PR1 is involved in the initiation of STAT3 activation. Targeting hypoxia-mediated STAT3 activation represents a therapeutic option for ovarian cancer and other solid tumors.

HO-3867, a safe STAT3 inhibitor, is selectively cytotoxic to ovarian cancer.

STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (-NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867-mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated.

RAC1: an emerging therapeutic option for targeting cancer angiogenesis and metastasis.

Angiogenesis and metastasis are well recognized as processes fundamental to the development of malignancy. Both processes involve the coordination of multiple cellular and chemical activities through myriad signaling networks, providing a mass of potential targets for therapeutic intervention. This review will focus on one master regulator of cell motility, RAC1, and the existing data with regard to its role in cell motility, including particular roles for tumor angiogenesis and invasion/metastasis. We also emphasize the preclinical investigations carried out with RAC1 inhibitors to evaluate the therapeutic potential of this target. Herein, we explore potential future directions as well as the challenges of targeting RAC1 in the treatment of cancer. Recent insights at the molecular and cellular levels are paving the way for a more directed and detailed approach to target mechanisms of RAC1 regulating angiogenesis and metastasis. Understanding these mechanisms may provide insight into RAC1 signaling components as alternative therapeutic targets for tumor angiogenesis and metastasis.

Dual targeting of the type 1 insulin-like growth factor receptor and its ligands as an effective antiangiogenic strategy.

BACKGROUND: In pediatric tumor xenograft models, tumor-derived insulin growth factor (IGF-2) results in intrinsic resistance to IGF-IR-targeted antibodies, maintaining continued tumor angiogenesis. We evaluated the antiangiogenic activity of a ligand-binding antibody (MEDI-573) alone or in combination with IGF-I receptor binding antibodies (MAB391, CP01-B02). METHODS: IGF-stimulated signaling was monitored by increased Akt phosphorylation in sarcoma and human umbilical cord vascular endothelial cells (HUVEC). Angiogenesis was determined in vitro using capillary tube formation in HUVECs and in vivo using a VEGF-stimulated Matrigel assay. Tumor growth delay was examined in 4 sarcoma xenograft models. RESULTS: The IGF ligand-binding antibody MEDI-573 suppressed Akt phosphorylation induced by exogenous IGF-I and IGF-2 in sarcoma cells. Receptor-binding antibodies suppressed IGF-I stimulation of Akt phosphorylation, but IGF-2 circumvented this effect and maintained HUVEC tube formation. MEDI-573 inhibited HUVEC proliferation and tube formation in vitro, but did not inhibit angiogenesis in vivo, probably because MEDI-573 binds murine IGF-I with low affinity. However, in vitro antiangiogenic activity of MEDI-573 was also circumvented by human recombinant IGF-I. The combination of receptor- and ligand-binding antibodies completely suppressed VEGF-stimulated proliferation of HUVECs in the presence of IGF-I and IGF-2, prevented ligand-induced phosphorylation of IGF-IR/IR receptors, and suppressed VEGF/IGF-2-driven angiogenesis in vivo. The combination of CP1-BO2 plus MEDI-573 was significantly superior to therapy with either antibody alone against IGF-I and IGF-2 secreting pediatric sarcoma xenograft models. CONCLUSIONS: These results suggest that combination of antibodies targeting IGF receptor and ligands may be an effective therapeutic strategy to block angiogenesis for IGF-driven tumors.

Development, characterization, and reversal of acquired resistance to the MEK1 inhibitor selumetinib (AZD6244) in an in vivo model of childhood astrocytoma.

PURPOSE: The BT-40 low-grade childhood astrocytoma xenograft model expresses mutated BRAF(V600E) and is highly sensitive to the MEK inhibitor selumetinib (AZD6244). In this study, we developed and characterized selumetinib resistance and explored approaches to circumventing the mechanisms of acquired resistance. EXPERIMENTAL DESIGN: BT-40 xenografts were selected in vivo for selumetinib resistance. Resistant tumors were obtained and characterized, as were tumors that reverted to sensitivity. Characterization included expression profiling, assessment of MEK signature and compensatory pathways, MEK inhibition, BRAF expression, and cytokine levels. Combination treatment of BT-40/AZD-resistant tumors with the MEK inhibitor and a STAT3 inhibitor (LLL12) was assessed. RESULTS: Resistance was unstable, tumors reverting to selumetinib sensitivity when passaged in untreated mice, and MEK was equally inhibited in sensitive and resistant tumors by selumetinib. Drug resistance was associated with an enhanced MEK signature and increased interleukin (IL)-6 and IL-8 expression. Selumetinib treatment induced phosphorylation of STAT3 (Y705) only in resistant xenografts, and similar results were observed in BRAF(V600E) astrocytic cell lines intrinsically resistant to selumetinib. Treatment of BT-40-resistant tumors with selumetinib or LLL12 had no significant effect, whereas combined treatment induced complete regressions of BT-40/AZD-resistant xenografts. CONCLUSIONS: Resistance to selumetinib selected in vivo in BT-40 tumor xenografts was unstable. In resistant tumors, selumetinib activated STAT3, and combined treatment with selumetinib and LLL12 induced complete responses in resistant BT-40 tumors. These results suggest dual targeting BRAF (V600E) signaling and STAT3 signaling may be effective in selumetinib-resistant tumors or may retard or prevent onset of resistance.