Tomoko Oh-hara

Cytotoxic Activity of Tivantinib (ARQ 197) Is Not Due Solely to c-MET Inhibition

The receptor tyrosine kinase c-MET is the high-affinity receptor for the hepatocyte growth factor (HGF). The HGF/c-MET axis is often dysregulated in tumors. c-MET activation can be caused by MET gene amplification, activating mutations, and auto- or paracrine mechanisms. Thus, c-MET inhibitors are under development as anticancer drugs. Tivantinib (ARQ 197) was reported as a small-molecule c-MET inhibitor and early clinical studies suggest antitumor activity. To assess whether the antitumor activity of tivantinib was due to inhibition of c-MET, we compared the activity of tivantinib with other c-MET inhibitors in both c-MET-addicted and nonaddicted cancer cells. As expected, other c-MET inhibitors, crizotinib and PHA-665752, suppressed the growth of c-MET-addicted cancers, but not the growth of cancers that are not addicted to c-MET. In contrast, tivantinib inhibited cell viability with similar potency in both c-MET-addicted and nonaddicted cells. These results suggest that tivantinib exhibits its antitumor activity in a manner independent of c-MET status. Tivantinib treatment induced a G(2)-M cell-cycle arrest in EBC1 cells similarly to vincristine treatment, whereas PHA-665752 or crizotinib treatment markedly induced G(0)-G(1) cell-cycle arrest. To identify the additional molecular target of tivantinib, we conducted COMPARE analysis, an in silico screening of a database of drug sensitivities across 39 cancer cell lines (JFCR39), and identified microtubule as a target of tivantinib. Tivantinib-treated cells showed typical microtubule disruption similar to vincristine and inhibited microtubule assembly in vitro. These results suggest that tivantinib inhibits microtubule polymerization in addition to inhibiting c-MET.

Comparative study on reversal efficacy of SDZ PSC 833, cyclosporin A and verapamil on multidrug resistance in vitro and in vivo

A non-immunosuppressive cyclosporin, SDZ PSC 833 (PSC833), shows a reversal effect on multidrug resistance (MDR) by functional modulation of MDR1 gene product, P-glycoprotein. The objective of the present study was to compare the reversal efficacy of three multidrug resistance modulators, PSC833, cyclosporin A (CsA) and verapamil (Vp). PSC833 has approximately 3-10-fold greater potency than CsA and Vp with respect to the restoring effect on reduced accumulation of doxorubicin (ADM) and vincristine (VCR) in ADM-resistant K562 myelogenous leukemia cells (K562/ADM) in vitro and also on the sensitivity of K562/ADM to ADM and VCR in in vitro growth inhibition. The in vivo efficacy of a combination of modifiers (PSC833 and CsA: 50 mg/kg, Vp 100 mg/kg administered p.o. 4 h before the administration of anticancer drugs) with anticancer drugs (ADM 2.5 mg/kg i.p., Q4D days 1, 5 and 9, VCR 0.05 mg/kg i.p., QD days 1-5) was tested in ADM-resistant P388-bearing mice. PSC833 significantly enhanced the increase in life span by more than 80%, whereas CsA and Vp enhanced by less than 50%. This reversal potency, which exceeded that of CsA and Vp, was confirmed by therapeutic experiments using colon adenocarcinoma 26-bearing mice. These results demonstrated that PSC833 has significant potency to reverse MDR in vitro and in vivo, suggesting that PSC833 is a good candidate for reversing multidrug resistance in clinical situations.

Platelets Promote Tumor Growth and Metastasis via Direct Interaction between Aggrus/Podoplanin and CLEC-2

The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus–CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus–CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet–tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.

Transport of cyclosporin A across the brain capillary endothelial cell monolayer by P-glycoprotein.

P-glycoprotein, a multidrug transporter protein, exists in the brain capillary endothelium. To study the function of P-glycoprotein in brain capillary endothelium as a barrier against cyclosporin A, we examined the interaction of cyclosporin A with P-glycoprotein expressed in cultured brain capillary endothelial cells (MBEC4). P-glycoprotein of MBEC4 specifically bound [125I]iodoaryl azidoprazosin, and the binding was inhibited by cyclosporin A and vincristine. Intracellular accumulation of cyclosporin A in MBEC4 was about one-third the amount accumulated in mouse aortic endothelial cells (MAEC3), a cell line that did not express P-glycoprotein. The reduced accumulation of cyclosporin A in MBEC4 was increased by verapamil, a competitive inhibitor of transport function of P-glycoprotein. Cyclosporin A was preferentially transported from basal to apical side when the cell monolayer of MBEC4 was formed; however this transendothelial transport was not observed across cell monolayer of MAEC3. Verapamil inhibited the transendothelial transport of cyclosporin A across the MBEC4 monolayer. Thus P-glycoprotein in brain capillary endothelium could transport cyclosporin A across the endothelium from the basal to the apical side. These observations suggest that P-glycoprotein is involved in the complex function of the blood-brain barrier as a secretory detoxifying transporter of cyclosporin A.

PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has selective antitumor activity. Although TNF-α-induced intracellular signaling pathways have been well studied, TRAIL signaling is not fully understood. Here, we identified a novel TRAIL receptor-binding protein, protein arginine methyltransferase 5 (PRMT5), as a result of proteomic screening. PRMT5 selectively interacted with death receptor 4 and death receptor 5 but not with TNF receptor 1 or Fas. PRMT5 gene silencing sensitized various cancer cells to TRAIL without affecting TRAIL resistance in nontransformed cells. PRMT5 contributed to TRAIL-induced activation of inhibitor of κB kinase (IKK) and nuclear factor-κB (NF-κB), leading to induction of several NF-κB target genes. Although IKK inhibition increased sensitivity to both TRAIL and TNF-α, PRMT5 knockdown potentiated TRAIL-mediated cytotoxicity alone. PRMT5 had no effect on TNF-α-mediated NF-κB signaling. These results show the selectivity of PRMT5 for TRAIL signaling. The PRMT5 small interfering RNA-mediated susceptibility to TRAIL was rescued by ectopic expression of active IKKβ, confirming the involvement of PRMT5 in TRAIL resistance by activating the NF-κB pathway. Collectively, our findings suggest the therapeutic potential of PRMT5 in TRAIL-based cancer treatments.(Mol Cancer Res 2009;7(4):557–69)

Reversal of multidrug resistance by an immunosuppressive agent FK-506

SummaryFK-506, a novel immunosuppressive agent, was examined for its reversing effect on multidrug-resistant tumor cells. FK-506 at 3 μM completely reversed the resistance against vincristine (VCR) in vitro in VCR-resistant mouse leukemia P388 cells (P388/VCR). FK-506 also enhanced the cytotoxicity of VCR in Adriamycin(ADM)-resistant human ovarian cancer A2780 cells (AD10) and ADM-resistant human myelogenous leukemia K562 cells (K562/ADM) in vitro. FK-506 was also effective in modulating sensitivity to ADM in AD10 cells in vitro. FK-506 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. When 20 mg/kg FK-506 was combined with 200 μg/kg VCR, a T/C value of 151% was obtained. Under the protocol used in this study, FK-506 was more potent than cyclosporin A (CsA) and verapamil. FK-506 inhibited [3H]azidopine binding to P-glycoprotein efficiently. The binding of VCR to K562/ADM plasma membrane was inhibited by FK-506 as effectively as by CsA. Moreover, the accumulation of VCR in AD10 cells was increased by FK-506 as efficiently as that of CsA and verapamil. These results indicate that FK-506 directly interacts with P-glycoprotein like CsA and verapamil, inhibits the active efflux of vincristine from resistant cells, increases the vincristine accumulation in resistant cells, and thus overcomes multidrug resistance in vitro and in vivo.

Production of interleukin-11 in bone-derived endothelial cells and its role in the formation of osteolytic bone metastasis

The interactions of the cells in the bone microenvironment play important roles in bone remodeling. Osteoblasts are involved in the bone remodeling through the production of soluble factors that regulate proliferation and differentiation of osteoclasts and through cell–cell interactions. Histological studies have suggested that endothelial cells are also associated with some osteolytic bone diseases. However, it is still unclear how endothelial cells contribute to bone resorption. We established bone-derived endothelial cells (BDECs) to study their roles in bone remodeling. The established BDECs promoted bone resorption in a murine neonatal calvaria organ culture system by secreting a soluble bone resorption-inducing factor(s) when stimulated by several inflammatory cytokines. This bone resorption-inducing factor was identified as interleukin-11 (IL-11). IL-11 is known to enhance bone resorption by promoting osteoclastogenesis and by suppressing the activity of osteoblasts. The production of IL-11 in BDECs was also promoted by conditioned medium of human melanoma A375M cells. Because A375M cells formed osteolytic bone metastasis in vivo, BDECs might be involved in pathological osteolysis by producing IL-11. These results suggest that endothelial cells in bone play important roles in the promotion of bone resorption by secreting IL-11 in physiological and pathological conditions.

Prevention of hematogenous metastasis by neutralizing mice and its chimeric anti-Aggrus/podoplanin antibodies

The platelet aggregation‐inducing factor, Aggrus (also known as podoplanin), is reported to contribute to cancer metastasis by mediating cancer cell–platelet interaction. Aggrus has been shown to be upregulated in many different types of cancers. Thus, not only the functional inhibition of Aggrus, but also its application as a cancer‐specific antigen has therapeutic potential. Among a series of anti‐Aggrus mAb established previously, no mouse anti‐human Aggrus mAb exists that possesses the ability to neutralize platelet aggregation. For precise preclinical examinations of mouse and monkey models, the establishment of Aggrus‐neutralizing mouse mAb and their chimeric Abs is needed. In this study, we established two mouse anti‐human Aggrus mAb, P2‐0 and HAG‐3. A precise analysis of their epitopes revealed that P2‐0 recognized the conformation near the bioactive O‐glycosylation site at the Thr52 residue. In contrast, HAG‐3 recognized the amino‐terminus side at a short distance from the conformation recognized by P2‐0. We observed that only P2‐0 attenuated Aggrus‐induced platelet aggregation and Aggrus binding to its platelet receptor, that is, the C‐type lectin‐like receptor‐2. Consistent with these data, only P2‐0 prevented the experimental metastasis of human Aggrus‐overexpressing CHO cells. Subsequently, we cloned the complementary determining region of P2‐0 and produced the murine/human chimeric P2‐0 antibody. This chimeric antibody maintained its inhibitory activity of Aggrus‐induced platelet aggregation and experimental metastasis. Thus, P2‐0 and its chimeric antibody are expected to aid the development of preclinical and clinical examinations of Aggrus‐targeted cancer therapy. (Cancer Sci 2011; 102: 2051–2057)

Casein Kinase 2–Interacting Protein-1, a Novel Akt Pleckstrin Homology Domain-Interacting Protein, Down-regulates PI3K/Akt Signaling and Suppresses Tumor Growth In vivo

The serine/threonine kinase Akt plays a central role in cell survival and proliferation. Its activation is linked to tumorigenesis in several human cancers. Although many Akt substrates have been elucidated, the Akt-binding proteins that regulate Akt function remain unclear. We report herein having identified casein kinase 2-interacting protein-1 (CKIP-1) as an Akt pleckstrin homology (PH) domain-binding protein with Akt inhibitory function. CKIP-1 formed a complex with each Akt isoform (Akt1, Akt2, and Akt3) via its NH2 terminus. Dimerization of CKIP-1 via its leucine zipper (LZ) motif at the COOH terminus was found to be associated with Akt inactivation because deletion of the LZ motif eliminated Akt inhibitory function, although it could still bind to Akt. Expression of the NH2 terminus-deleted CKIP-1 mutant containing the LZ motif, but lacking Akt-binding ability, induced Akt phosphorylation and activation by sequestering the ability of endogenous CKIP-1 to bind to Akt. Stable CKIP-1 expression caused Akt inactivation and cell growth inhibition in vitro. In addition, the growth of stable CKIP-1 transfectants xenografted into nude mice was slower than that of mock transfectants. These results indicate that CKIP-1, a novel Akt PH domain-interacting protein, would be a candidate of tumor suppressor with an Akt inhibitory function.

Potentiation of the reversal activity of SDZ PSC833 on multi‐drug resistance by an anti‐p‐glycoprotein monoclonal antibody MRK‐16

SDZ PSC833 (PSC833), an analogue of cyclosporines, is one of the most potent modulators of multi‐drug resistance (MDR). We previously reported that MRK‐16, an anti‐P‐glycoprotein MAb, enhanced MDR reversal activity of cyclosporin A (CsA) through inhibition of P‐glycoprotein‐mediated CsA transport. We have examined here whether MRK‐16 can enhance MDR reversal activity of PSC833. We found that MRK‐16 potentiated the MDR reversal activity of PSC833, and of CsA, in MDR sublines of human myelocytic leukemia K562 and human ovarian cancer A2780 cells. Like MRK‐16 combined with CsA, MRK‐16 enhanced the effect of a sub‐optimum dose of PSC833 on vincristine accumulation in MDR cells. However, MRK‐16 could not increase cellular accumulation of PSC833 in MDR tumor cells, yet it could increase cellular accumulation of CsA. P‐glycoprotein could not transport PSC833 but could transport CsA. Our results indicate that MRK‐16 potentiates the MDR reversal activity of both PSC833 and CsA, yet also suggest that the molecular mechanism of the potentiation differs between the two substances.