Inhibition Reduces Tumor Growth Research Articles

Abstract 1646: Combining molecular target drugs to inhibit cancer-stromal cell interaction in gastric cancer.

Abstract Recent studies in molecular and cellular biology have shown that tumor growth and metastasis are not determined by cancer cells alone but also by various stromal cells. Many human tumors secrete platelet-derived growth factor (PDGF) ligands, and PDGF receptors (PDGF-Rs) are expressed by various stromal cell populations, including carcinoma-associated fibroblasts (CAFs). Tumor cells in surgical specimens and orthotopic tumors have been shown to express PDGF-B, whereas PDGF-Rβ has been shown to be expressed predominantly by stromal cells, including CAFs, pericytes, and lymphatic endothelial cells. mTOR (mammalian target of rapamycin) is a central regulatory kinase that increases the production of proteins that stimulate key cellular processes such as cell growth and proliferation, cell metabolism, and angiogenesis. mTOR also increases the translation of hypoxia-inducible factor-1a (HIF-1a), which drives the expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF), resulting in new vasculature. In the present study, we examined the effects of PDGF-R tyrosine kinase inhibitors (imatinib, nilotinib) and mTOR inhibitor (everolimus) on tumor stroma in an orthotopic nude mouse model of human gastric cancer. Treatment with PDGF-R tyrosine kinase inhibitors did not inhibit tumor growth but did significantly decrease the stromal reaction, lymphatic invasion, lymphatic vessel area, and pericyte coverage of tumor microvessels. In contrast, treatment with mTOR inhibitor reduced tumor growth and microvessel density but did not decrease the stromal reaction. PDGF-R inhibitor and mTOR inhibitor used in combination reduced both the growth rate and stromal reaction. (control (5/5, 0.3±0.1g), nilotinib (6/6, 0.3±0.1g), everolimus (4/6, 0.08±0.02g: p<0.05), combination (1/6, 0.04g)) Target molecule-based inhibition of cancer-stromal cell interaction may hold promise as an effective anti-tumor therapy. Citation Format: Mieko Onoyama, Yasuhiko Kitadai, Kei Shinagawa, Ryo Yuge, Mayu Ohnishi, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Combining molecular target drugs to inhibit cancer-stromal cell interaction in gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1646. doi:10.1158/1538-7445.AM2013-1646

Inhibition of GTP cyclohydrolase attenuates tumor growth by reducing angiogenesis and M2‐like polarization of tumor associated macrophages

GTP cyclohydrolase (GCH1) is the key-enzyme to produce the essential enzyme cofactor, tetrahydrobiopterin. The byproduct, neopterin is increased in advanced human cancer and used as cancer-biomarker, suggesting that pathologically increased GCH1 activity may promote tumor growth. We found that inhibition or silencing of GCH1 reduced tumor cell proliferation and survival and the tube formation of human umbilical vein endothelial cells, which upon hypoxia increased GCH1 and endothelial NOS expression, the latter prevented by inhibition of GCH1. In nude mice xenografted with HT29-Luc colon cancer cells GCH1 inhibition reduced tumor growth and angiogenesis, determined by in vivo luciferase and near-infrared imaging of newly formed blood vessels. The treatment with the GCH1 inhibitor shifted the phenotype of tumor associated macrophages from the proangiogenic M2 towards M1, accompanied with a shift of plasma chemokine profiles towards tumor-attacking chemokines including CXCL10 and RANTES. GCH1 expression was increased in mouse AOM/DSS-induced colon tumors and in high grade human colon and skin cancer and oppositely, the growth of GCH1-deficient HT29-Luc tumor cells in mice was strongly reduced. The data suggest that GCH1 inhibition reduces tumor growth by (i) direct killing of tumor cells, (ii) by inhibiting angiogenesis, and (iii) by enhancing the antitumoral immune response.

Combination with bortezomib enhances the antitumor effects of nanoparticle-encapsulated thiostrepton

Bortezomib is well-known for inducing cell death in cancer cells, specifically through the mechanism of proteasome inhibition. Thiostrepton, a thiazole antibiotic, has also been described for its proteasome inhibitory action, although differing slightly to bortezomib in the proteasomal site to which it is active. Previously we had shown the synergic effect of bortezomib and thiostrepton in breast cancer cells in vitro, where sub-apoptotic concentrations of both proteasome inhibitors resulted in synergic increase in cell death when combined as a treatment. Here, we administered such a combination to MDA-MB-231 xenograft tumors in vivo, and found that the effect of complementary proteasome inhibitors reduced tumor growth rates more efficiently than compared with when administered alone. Increased induction of apoptotic activity in tumors was found be associated with the growth inhibitory activity of combination treatment. Further examination additionally revealed that combination-treated tumors exhibited reduced proteasome activity, compared with non-treated and single drug-treated tumors. These data suggest that this drug combination may be useful as a therapy for solid tumors.

Efficacy of Tie2 Receptor Antagonism in Angiosarcoma

Angiosarcomas are malignant endothelial cell tumors with few effective systemic treatments. Despite a unique endothelial origin, molecular candidates for targeted therapeutic intervention have been elusive. In this study, we explored the tunica internal endothelial cell kinase 2 (Tie2) receptor as a potential therapeutic target in angiosarcoma. Human angiosarcomas from diverse sites were shown to be universally immunoreactive for Tie2. Tie2 and vascular endothelial growth factor receptor (VEGFR) antagonists inhibited SVR and MS1-VEGF angiosarcoma cell survival in vitro. In the high-grade SVR cell line, Tie2 and VEGF antagonists inhibited cell survival synergistically, whereas effects were largely additive in the low-grade MS1-VEGF cell line. Xenograft modeling using these cell lines closely recapitulated the human disease. In vivo, Tie2 and VEGFR inhibition resulted in significant angiosarcoma growth delay. The combination proved more effective than either agent alone. Tie2 inhibition seemed to elicit tumor growth delay through increased tumor cell apoptosis, whereas VEGFR inhibition reduced tumor growth by lowering tumor cell proliferation. These data identify Tie2 antagonism as a potential novel, targeted therapy for angiosarcomas and provide a foundation for further investigation of Tie2 inhibition, alone and in combinations, in the management of this disease.

Covering the Cover

Covering the Cover

Fatty acid synthase as a novel target for meningioma therapy

High levels of fatty acid synthase (FAS) expression have been reported in hormone receptor-positive tumors, including prostate, breast, and ovarian cancers, and its inhibition reduces tumor growth in vitro and in vivo. Similar to other hormone receptor-positive tumor types, meningiomas are progesterone receptor- and estrogen receptor-immunoreactive brain tumors. To define the role of FAS in human meningioma growth control, we first analyzed the FAS expression using a tissue microarray containing 38 meningiomas and showed increased FAS expression in 70% of atypical WHO grade II and anaplastic WHO grade III meningiomas compared with 10% of benign WHO grade I tumors. We next confirmed this finding by real-time PCR and Western blotting. Second, we demonstrated that treatment with the FAS inhibitor, cerulenin (Cer), significantly decreased meningioma cell survival in vitro. Third, we showed that Cer treatment reduced FAS expression by modulating Akt phosphorylation (activation). Fourth, we demonstrated that Cer treatment of mice bearing meningioma xenografts resulted in significantly reduced tumor volumes associated with increased meningioma cell death. Collectively, our data suggest that the increased FAS expression in human meningiomas represents a novel therapeutic target for the treatment of unresectable or malignant meningioma.

Targeting EGFR activity in blood vessels is sufficient to inhibit tumor growth and is accompanied by an increase in VEGFR-2 dependence in tumor endothelial cells

Epidermal growth factor receptor (EGFR) targeting agents such as kinase inhibitors reduce tumor growth and progression. We have previously reported that EGFR is not only expressed by the tumor cells but by the tumor endothelial cells (EC) as well (Amin, D. N., Hida, K., Bielenberg, D. R., Klagsbrun, M., 2006. Tumor endothelial cells express epidermal growth factor receptor (EGFR) but not ErbB3 and are responsive to EGF and to EGFR kinase inhibitors. Cancer Res. 66, 2173–80). Thus, targeting tumor blood vessel EGFR may be a viable strategy for tumor growth inhibition. We describe here a melanoma xenograft model where the tumor cells express very little or no EGFR but the tumor blood vessels express activated EGFR. The EGFR kinase inhibitor, gefitinib (Iressa), retarded tumor growth with a size decrease of 38% compared to control mice, ostensibly due to targeting of the blood vessels. EC were isolated from tumors of gefitinib-treated mice. These EC were unable to proliferate in response to EGF and displayed relatively weaker activation of MAPK and AKT signaling in response to EGF compared to tumor EC isolated from vehicle-treated mice. In contrast, the tumor EC from gefitinib-treated mice expressed higher levels of VEGFR-2 both at the mRNA and protein level. In addition, these cells were less sensitive to EGFR kinase inhibitors in vitro but more sensitive to a VEGFR-2 kinase inhibitor. These results suggest that in tumor EC from gefitinib-treated mice there is a switch from dependence on EGFR activity to signaling via VEGFR-2. Our data provide a molecular rationale for combination therapies targeting both EGF and VEGF signaling on the tumor vasculature.

CCAAT/Enhancer Binding Protein β Regulates Aromatase Expression via Multiple and Novel Cis-Regulatory Sequences in Uterine Leiomyoma

Control of aromatase expression in uterine leiomyoma has significant clinical implications because aromatase inhibitors reduce tumor growth and associated irregular uterine bleeding. The mechanisms that regulate aromatase expression in leiomyoma are unknown. We previously demonstrated that the cAMP-responsive proximal promoters I.3 and II regulate aromatase expression in vivo in uterine leiomyoma tissue. Here, we investigated the cellular and molecular mechanisms responsible for promoter I.3/II usage. In smooth muscle cells isolated from leiomyoma (LSMCs), dibutyryl cAMP significantly induced aromatase mRNA and enzyme activity. Reporter constructs of promoter I.3/II deletion and site-directed mutants with selective disruption of cis-regulatory elements in the -517/-16 bp region revealed that five out of seven elements, including three CCAAT/enhancer binding protein (C/EBP) binding sites and two cAMP response elements, were essential for cAMP-induced promoter activity. EMSAs demonstrated that nuclear extracts from LSMCs contain complexes assembled on four of the five cis-elements, with C/EBP binding sites, including a novel -245/-231 bp sequence, clearly associating with C/EBPbeta. Chromatin immunoprecipitation assays revealed that C/EBPbeta binds specifically to the promoter I.3/II region in intact cells. Dibutyryl cAMP significantly induced nuclear C/EBPbeta protein levels in LSMCs in a time-dependent manner. Conversely, knockdown of C/EBPbeta dramatically suppressed cAMP-induced aromatase mRNA and enzyme activity. C/EBPbeta, which binds to multiple cis-regulatory elements in promoter I.3/II, is a key factor in the transcriptional complex controlling aromatase expression in uterine leiomyoma cells. Definition of this mechanism further may assist in designing inhibitors of aromatase specific for leiomyoma tissue.

Secondary Amides of Sulfonylated 3-Amidinophenylalanine. New Potent and Selective Inhibitors of Matriptase

Matriptase is an epithelium-derived type II transmembrane serine protease and has been implicated in the activation of substrates such as pro-HGF/SF and pro-uPA, which are likely involved in tumor progression and metastasis. Through screening, we have identified bis-basic secondary amides of sulfonylated 3-amidinophenylalanine as matriptase inhibitors. X-ray analyses of analogues 8 and 31 in complex with matriptase revealed that these inhibitors occupy, in addition to part of the previously described S4-binding site, the cleft formed by the molecular surface and the unique 60 loop of matriptase. Therefore, optimization of the inhibitors included the incorporation of appropriate sulfonyl substituents that could improve binding of these inhibitors into both characteristic matriptase subsites. The most potent derivatives inhibit matriptase highly selective with K(i) values below 5 nM. Molecular modeling revealed that their improved affinity results from interaction with the S4 site of matriptase. Analogues 8 and 59 were studied in an orthotopic xenograft mouse model of prostate cancer. Compared to control, both inhibitors reduced tumor growth, as well as tumor dissemination.

Infiltration of COX-2–expressing macrophages is a prerequisite for IL-1β–induced neovascularization and tumor growth

Inflammatory angiogenesis is a critical process in tumor progression and other diseases. The inflammatory cytokine IL-1beta promotes angiogenesis, tumor growth, and metastasis, but its mechanisms remain unclear. We examined the association between IL-1beta-induced angiogenesis and cell inflammation. IL-1beta induced neovascularization in the mouse cornea at rates comparable to those of VEGF. Neutrophil infiltration occurred on day 2. Macrophage infiltration occurred on days 4 and 6. The anti-Gr-1 Ab-induced depletion of infiltrating neutrophils did not affect IL-1beta- or VEGF-induced angiogenesis. The former was reduced in monocyte chemoattractant protein-1-deficient (MCP-1(-/-)) mice compared with wild-type mice. After day 4, clodronate liposomes, which kill macrophages, reduced IL-1beta-induced angiogenesis and partially inhibited VEGF-induced angiogenesis. Infiltrating macrophages near the IL-1beta-induced neovasculature were COX-2 positive. Lewis lung carcinoma cells expressing IL-1beta (LLC/IL-1beta) developed neovasculature with macrophage infiltration and enhanced tumor growth in wild-type but not MCP-1(-/-) mice. A COX-2 inhibitor reduced tumor growth, angiogenesis, and macrophage infiltration in LLC/IL-1beta. Thus, macrophage involvement might be a prerequisite for IL-1beta-induced neovascularization and tumor progression.

Reverse transcriptase inhibitors reduce tumor growth in vivo and induce cell differentiation

3134 Background: Endogenous, nontelomeric reverse transcriptase (RT) is encoded by two classes of genomic repeated elements: retrotransposons and endogenous retroviruses. Expression of RT-coding genes is generally repressed in differentiated non pathological tissues, yet is active in the germ-line, embryonic tissue and tumor cells. We reported that nevirapine, a non-nucleosidic RT inhibitor, reduces cell growth and induces differentiation in murine and human cell lines. Furthermore, nevirapine treatment causes a reprogramming of gene expression in murine transformed cells: particularly significant changes occur in genes involved in cell cycle control, such as down-regulation of cyclin D1 and up-regulation of p16, (Mangiacasale, Oncogene 2003). Finally, RT inhibitors are currently used in the treatment of HIV patients and epidemiological studies suggested that these drugs induce a dramatic decrease in the incidence of HIV-related neoplasms. Results: We report that treatment with two RT inhibitors, nevirapine and efavirenz, effectively induces a reversible inhibition of cell growth in human prostate (PC3), colon (HT29), small-cell lung (H69) cancer cell lines as well as in human melanoma (A-375) and glioma (U343), in vitro. Neither drug displays significant cytotoxic effects, producing minimal induction of necrosis and apoptosis. The inhibition of cell growth correlates with the accumulation of cells in the G0/G1 phase of cell cycle, concomitant with decrease in S-phase cells. Moreover, treated cells acquire a differentiated phenotype. Tumor xenografts from human prostate, colon, melanoma and small-cell lung cancer cells in nude mice treated with efavirenz show a strong reversible inhibition of growth. Maximal inhibition of cancer growth is obtained with a dose of 20 mg/kg b.w., which is within the therapeutical dose range used to achieve RT inhibition in patients with retrovirus infections such as HIV. Conclusions: These findings suggest that endogenous RT may represent a novel target in human cancer therapy and support the proposal that efavirenz could be tested as a new safe drug in association with standard anti-cancer treatments in humans. No significant financial relationships to disclose.

Reverse transcriptase inhibitors reduce tumor growth in vivo and induce cell differentiation

3134 Background: Endogenous, nontelomeric reverse transcriptase (RT) is encoded by two classes of genomic repeated elements: retrotransposons and endogenous retroviruses. Expression of RT-coding genes is generally repressed in differentiated non pathological tissues, yet is active in the germ-line, embryonic tissue and tumor cells. We reported that nevirapine, a non-nucleosidic RT inhibitor, reduces cell growth and induces differentiation in murine and human cell lines. Furthermore, nevirapine treatment causes a reprogramming of gene expression in murine transformed cells: particularly significant changes occur in genes involved in cell cycle control, such as down-regulation of cyclin D1 and up-regulation of p16, (Mangiacasale, Oncogene 2003). Finally, RT inhibitors are currently used in the treatment of HIV patients and epidemiological studies suggested that these drugs induce a dramatic decrease in the incidence of HIV-related neoplasms. Results: We report that treatment with two RT inhibitors, nevirapine and efavirenz, effectively induces a reversible inhibition of cell growth in human prostate (PC3), colon (HT29), small-cell lung (H69) cancer cell lines as well as in human melanoma (A-375) and glioma (U343), in vitro. Neither drug displays significant cytotoxic effects, producing minimal induction of necrosis and apoptosis. The inhibition of cell growth correlates with the accumulation of cells in the G0/G1 phase of cell cycle, concomitant with decrease in S-phase cells. Moreover, treated cells acquire a differentiated phenotype. Tumor xenografts from human prostate, colon, melanoma and small-cell lung cancer cells in nude mice treated with efavirenz show a strong reversible inhibition of growth. Maximal inhibition of cancer growth is obtained with a dose of 20 mg/kg b.w., which is within the therapeutical dose range used to achieve RT inhibition in patients with retrovirus infections such as HIV. Conclusions: These findings suggest that endogenous RT may represent a novel target in human cancer therapy and support the proposal that efavirenz could be tested as a new safe drug in association with standard anti-cancer treatments in humans. No significant financial relationships to disclose.

Selective inhibition of cyclooxygenase (COX)-2 inhibits endothelial cell proliferation by induction of cell cycle arrest.

High-level expression of cyclooxygenase (COX)-2 is reported in 80-90% of colorectal adenocarcinomas. Selective inhibition of COX-2 was shown to reduce colorectal tumorigenesis in different models of carcinogenesis and to prevent metastasis in xenograft tumor models, as well as to suppress in vitro induced angiogenesis. Recently, COX-2 was reported to be expressed not only in malignant epithelial cells, but also in the neovasculature that feeds the tumor in a variety of solid human cancers. Thus, one of the possible mechanisms by which selective COX-2 inhibitor reduces tumor growth and metastasis is through inhibition of tumor angiogenesis. Although a report suggested a possible role of endothelial COX-1 in the process of angiogenesis, in a recent study, the selective inhibition of COX-2 was shown to strongly inhibit angiogenesis by inducing endothelial cell (EC) apoptosis. In the present study, using human umbilical vein endothelial cells (HUVECs) as a model of angiogenesis, we investigated the potential antiangiogenic effect of the selective COX-2 inhibitor and its mechanism of action, and clearly demonstrated that selective inhibition of COX-2 caused a dose-dependent decrease in the proliferative activity of ECs, as well as an inhibition of capillary-like tube formation. The inhibitory effect on EC proliferation was dependent on the cell cycle arrest to the G1 phase and not on cell apoptosis.