James O’Kelly

Cyr61 Is Overexpressed in Gliomas and Involved in Integrin-Linked Kinase-Mediated Akt and β-Catenin-TCF/Lef Signaling Pathways

Cyr61 is a member of the CCN family of growth factors; these proteins are secreted and can act as ligands of distinct integrins. We show that Cyr61 can enhance tumorigenicity of glioma cells acting through activated integrin-linked kinase (ILK) to stimulate β-catenin-TCF/Lef and Akt signaling pathways. Overexpression of Cyr61 occurred in highly tumorigenic glioma cell lines and in 68% of the most malignant glioblastoma multiforme brain tumors. Forced expression of Cyr61 in U343 glioma cells accelerated their growth in liquid culture, enhanced their anchorage-independent proliferation in soft agar, and significantly increased their ability to form large, vascularized tumors in nude mice. Overexpression of Cyr61 in the U343 cells led to the up-regulation of distinct integrins, including β1 and ανβ3, which have been shown to interact with Cyr61 and ILK. The activity of ILK was increased dramatically in these cells. Overexpression of Cyr61 also resulted in the phosphorylation of glycogen synthase kinase-3β and accumulation and nuclear translocation of β-catenin, leading to activation of the β-catenin-TCF/Lef-1 signaling pathway. Furthermore, forced expression of Cyr61 in the glioma cells activated phosphatidylinositol 3′-kinase pathway, resulting in prominent phosphorylation of Akt and the antiapoptotic protein Bad. Cyr61 appears to stimulate several signaling pathways in the development of gliomas.

Cucurbitacin B has a potent antiproliferative effect on breast cancer cells in vitro and in vivo

Cucurbitacins are a group of diverse triterpenoid substances isolated from plants with medicinal properties. One particularly potent family member is cucurbitacin B (CuB). The antiproliferative effects of CuB against human breast cancer cells were tested. Six human breast cancer cell lines were examined because they represent a diverse mix of breast cancer subtypes varying in expression of estrogen receptor (ER), Her2/neu, and p53 mutation. The antiproliferative effect of CuB were also studied in vivo. The effective dose inhibiting 50% growth (ED50) was between 10−8 M and 10−7 M for this collection of breast cancer cell lines. These cells underwent rapid morphologic changes after 15–20 min exposure to CuB (5 × 10−7 M), which was associated with disruption of the microtubules and F‐actin, as observed by confocal microscopy. Human MDA‐MB‐231 (ER‐, p53 mutated) breast cancer cells were orthotopically implanted into the breasts of nude mice who intraperitoneally received either CuB 1.0 mg/kg or vehicle. Tumor volume was reduced by 55% in the group that received CuB for 6 weeks compared with vehicle controls. No apparent organ tissue damage was observed by pathological assessment. Interestingly, the experimental mice had lower serum glucose levels, consistent with use of CuB as an antidiabetic drug in China. This drug appears to be a third in a family of drugs targeting the microtubules (taxanes [e.g. taxol], vinca alkaloid [e.g. vincristine], and now CuB). Our in vitro and in vivo results suggest that CuB may be an effective, new approach for the treatment of ER‐, Her2/neu amplified, and p53 mutant breast cancers. (Cancer Sci 2008; 99: 1793–1797)

Regulation of the CAMP gene by 1,25(OH)2D3 in various tissues

The induction of antimicrobial peptides such as the human cathelicidin, CAMP/hCAP18, by 1,25(OH)(2)D(3) provides a very exciting therapeutic approach in boosting immunity against infectious diseases. To explore the range of cell types and expand the number of cell models for studying the regulation of CAMP gene expression by 1,25(OH)(2)D(3), we treated cell lines from various tissue types and determined CAMP gene expression. Also, we tested additional compounds together with 1,25(OH)(2)D(3) to look for possible cooperative activation of the gene. We identified 1,25(OH)(2)D(3)-mediated induction of the CAMP gene in B-cell lymphomas, prostate and endometrial cancer lines and found cooperative activation with the histone deacetylase inhibitor sodium butyrate. The data suggest that regulation of CAMP by 1,25(OH)(2)D(3) is potentially important in a wide range of tissues.

Vitamin D and bone physiology: demonstration of vitamin D deficiency in an implant osseointegration rat model.

PURPOSEnThe patient population varies in nutritional deficiencies, which may confound the host response to biomaterials. The objective of this study was to evaluate the effect of a common deficiency of vitamin D on implant osseointegration in the rat model.nnnMATERIALS AND METHODSnMale Sprague-Dawley rats were maintained under the cessation of vitamin D intake and UV exposure. The serum levels of 1,25(OH)(2)D(3), 25 OHD(3), Ca, and P were determined. Miniature cylindrical Ti6Al4V implants (2-mm long, 1-mm diameter) were fabricated with double acid-etched (DAE) surface or modified DAE with discrete crystalline deposition (DCD) of hydroxyapatite nanoparticles. DAE and DCD implants were placed in the femurs of vitamin D-insufficient and control rats. After 14 days of healing, the femur-implant samples were subjected to implant push-in test and nondecalcified histology. The surfaces of recovered implant specimens after the push-in test were further evaluated by scanning electron microscopy (SEM).nnnRESULTSnThe decreased serum level of 25 OHD(3) demonstrated the establishment of vitamin D insufficiency in this model. The implant push-in test revealed that DAE and DCD implants in the vitamin D-insufficient group (15.94 +/- 8.20 N, n = 7; 15.63 +/- 3.96 N, n = 7, respectively) were significantly lower than those of the control group (24.99 +/- 7.92 N, n = 7, p < 0.05; 37.48 +/- 17.58 N, n = 7, p < 0.01, respectively). The transcortical bone-to-implant contact ratio (BIC) was also significantly decreased in the vitamin D-insufficient group. SEM analyses further suggested that the calcified tissues remaining next to the implant surface after push-in test appeared unusually fragmented.nnnCONCLUSIONSnThe effect of vitamin D insufficiency significantly impairing the establishment of Ti6Al4V implant osseointegration in vivo was unexpectedly profound. The outcome of Ti-based endosseous implants may be confounded by the increasing prevalence of vitamin D insufficiency in our patient population.

Aberrant expression of neutrophil and macrophage-related genes in a murine model for human neutrophil-specific granule deficiency

Neutrophil‐specific granule deficiency involves inheritance of germline mutations in the CCAAT/enhancer‐binding protein ε (C/EBPE) gene. Humans and mice lacking active C/EBPε suffer frequent bacterial infections as a result of functionally defective neutrophils and macrophages. We hypothesized that these defects reflected dysregulation of important immune response genes. To test this, gene expression differences of peritoneally derived neutrophils and macrophages from C/EBPε−/− and wild‐type mice were determined with DNA microarrays. Of 283 genes, 146 known genes and 21 expressed sequence tags (ESTs) were down‐regulated, and 85 known genes and 31 ESTs were up‐regulated in the C/EBP−/− mice. These included genes involved in cell adhesion/chemotaxis, cytoskeletal organization, signal transduction, and immune/inflammatory responses. The cytokines CC chemokine ligand 4, CXC chemokine ligand 2, and interleukin (IL)‐6, as well as cytokine receptors IL‐8RB and granulocyte‐colony stimulating factor, were down‐regulated. Chromatin immunoprecipitation analysis identified binding of C/EBPε to their promoter regions. Increased expression for lipid metabolism genes apolipoprotein E (APOE), scavenger receptor class B‐1, sorting protein‐related receptor containing low‐density lipoprotein receptor class A repeat 1, and APOC2 in the C/EBPε−/− mice correlated with reduced total cholesterol levels in these mice before and after maintenance on a high‐fat diet. Also, C/EBPε‐deficient macrophages showed a reduced capacity to accumulate lipids. In summary, dysregulation of numerous, novel C/EBPε target genes impairs innate immune response and possibly other important biological processes mediated by neutrophils and macrophages.

Vitamin D analogs and breast cancer.

Breast cancer is the most frequent malignancy of women in the Western world. Vitamin D compounds constitute a novel alternative to the conventional use of antiestrogens for chemoprevention and chemotherapy. The biologically active form of vitamin D, 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], not only plays an essential role in the control of calcium homeostasis, but also acts on cells of a variety of tissues to promote inhibition of cellular proliferation and induction of differentiation. The potential use of 1,25(OH)2D3 in the treatment of cancer is limited by its propensity to cause hypercalcemia at pharmacologically active doses. This has led to the synthesis of analogs of vitamin D that exhibit potent anticancer effects, but have low calcemic activity. Evidence from both in vitro and in vivo studies has demonstrated that vitamin D compounds can inhibit the growth of breast cancer cells, suggesting their therapeutic value in the treatment or prevention of this disease.

Abstract 5581: Development of 2nd generation indoleamine 2,3-dioxygenase 1 (IDO1) selective inhibitors

Harnessing the immune system via immune checkpoint blockade (e.g. anti PD-1, anti PD-L1, anti CTLA4) has led to fast and long lived responses in cancer patients. Response rates however are low and new treatments that enhance these rates are needed. Recent studies have shown that the administration of immune checkpoint blockers is associated with the overexpression of indoleamine 2,3-dioxygenase 1 (IDO1). The resulting immunoregulatory phenotype counteracts immune checkpoint blockade and allows for cancer progression. The discovery of IDO1 inhibitors, and the potential to combine them with immune checkpoint blockers, therefore represents an attractive strategy to fight cancer. We carried out a ligand-based virtual screen with > 1,000,000 commercially available small molecules. In vitro screening of the resulting 610 virtual hits provided us with 2 IDO1-selective, 2 TDO2-selective and 2 IDO1/TDO2-dual confirmed hits. (TDO2 is a protein with similar biochemical activity to IDO1 that is essential to tryptophan homeostasis.) A subsequent Hit to Lead campaign led to the identification of novel chemotypes that display potency similar or superior to IDO1 inhibitors currently under clinical investigation in IFN-γ stimulated (i.e. IDO1+) HeLa cells, with no sign of cytotoxicity. We have demonstrated that these compounds are > 1000-fold selective for IDO1 over TDO2 using cellular assays. IDO1 upregulation by cancer cells is known to be one of the mechanisms by which cancer cells evade the immune system. In an in vitro co-culture assay of cancer cells and T cells we have demonstrated our compounds can rescue T cell proliferation with EC50 values between 10 and 50 nM. We have also demonstrated that our compounds inhibit IDO1 in monocyte derived human dendritic cells. Interestingly, despite this potent cellular activity demonstrated in multiple disease relevant cellular assays, this chemotype failed to inhibit recombinant IDO1 in an isolated biochemical assay performed under reducing conditions, whereas the reference compound epacadostat provided activity comparable to literature values. In order to confirm our cellular effects were due to direct inhibition of IDO1 we set up thermal shift assays. Thermal shift assays using purified IDO1 protein have demonstrated that our compounds directly bind IDO1, and cellular thermal shift assays have confirmed direct target engagement in intact cells (stimulated for IDO1 expression). These compounds have physicochemical properties that would support oral dosing and display low in vitro CYP450 and hERG inhibition, thus reducing the risk of toxicity in the clinic. Our IDO1 inhibitors show a novel differentiated mode of action at the cellular level, and the consequences of this profile in terms of in vivo characterisation is ongoing. Citation Format: Thomas Pesnot, Sachin Mahale, Philip MacFaul, John Maclean, Caroline Phillips, Matilda Bingham, Catherine Eagle, James Kelly, Abhijith Thippeswamy, Simon Armitage, Aleksandr Grisin, Sheenagh Aiken, Lucy Cartwright, Richard Armer. Development of 2nd generation indoleamine 2,3-dioxygenase 1 (IDO1) selective inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5581. doi:10.1158/1538-7445.AM2017-5581