Yasemin D. Yazici

Sorafenib inhibits the angiogenesis and growth of orthotopic anaplastic thyroid carcinoma xenografts in nude mice

Anaplastic thyroid carcinoma (ATC) remains one of the most lethal human cancers. We hypothesized that sorafenib, a multikinase inhibitor of the BRaf, vascular endothelial growth factor receptor-2, and platelet-derived growth factor receptor-β kinase, would decrease tumor growth and angiogenesis in an orthotopic model of ATC. The in vitro antiproliferative and proapoptotic effects of sorafenib on ATC cell lines were examined. To study the in vivo effects of sorafenib on orthotopic ATC tumors in nude mice, sorafenib was given p.o. at 40 or 80 mg/kg daily. Intratumoral effects were studied using immunohistochemical analysis. The effect of sorafenib on survival of the mice was also studied. Sorafenib inhibited the in vitro proliferation of ATC cell lines. Sorafenib also significantly inhibited tumor angiogenesis via the induction of endothelial apoptosis in an orthotopic model of thyroid cancer. As result, the growth of orthotopic ATC xenografts was reduced and the survival of the test animals was improved. Sorafenib exerts significant antitumor activity in an orthotopic xenograft model of ATC via a potent antiangiogenic effect. The antiangiogenic effects of sorafenib suggest that its use in clinical setting may not depend on the BRAF mutational status of thyroid tumors. Given the lack of curative options for patients with ATC, sorafenib warrants further study as a therapeutic agent against ATC. [Mol Cancer Ther 2007;6(6):1785–92]

Integrin-linked kinase is a potential therapeutic target for anaplastic thyroid cancer

We investigated integrin-linked kinase (ILK), a focal adhesion serine-threonine protein kinase, as a new molecular target for treating anaplastic thyroid cancer. ILK mediates cell growth and survival signals and is overexpressed in a number of cancers. Therefore, we hypothesized that inhibition of ILK leads to growth arrest and apoptosis of thyroid cancer cells. According to Western blotting, the level of ILK protein was highly expressed in one papillary (NPA187) and four of five (Hth74, DRO, ARO, KAT4, and K4) anaplastic thyroid cancer cell lines. Immunohistochemical analysis of a human tissue microarray revealed that ILK was highly expressed in anaplastic thyroid cancer but not in normal human thyroid tissue. Treating thyroid cancer cell lines with a new ILK inhibitor, QLT0267, inhibited epidermal growth factor–induced phosphorylation of AKT, inhibited cell growth, and induced apoptosis in the NPA187, DRO, and K4 cell lines. QLT0267 also inhibited the kinase activity of immunoprecipitated ILK in four of five cell lines. Tumor volumes in mice treated with QLT0267 were significantly reduced compared with those in untreated mice. In immunohistochemical studies, QLT0267 suppressed phosphorylated p-AKT and angiogenesis (i.e., reduced mean vascular density) and induced apoptosis in both tumor cells and tumor-associated endothelial cells of the thyroid DRO xenografts. In summary, we found that ILK expression and activity were elevated in human anaplastic thyroid cancer and ILK inhibition led to growth arrest and apoptosis in vitro and in vivo. Our results provide preliminary evidence that ILK is a potential therapeutic target for treating anaplastic thyroid cancer.

Cetuximab and Irinotecan Interact Synergistically to Inhibit the Growth of Orthotopic Anaplastic Thyroid Carcinoma Xenografts in Nude Mice

Purpose: Anaplastic thyroid carcinoma (ATC) remains one of the most lethal known human cancers. Targeted molecular therapy with cetuximab, a monoclonal antibody against epidermal growth factor receptor, offers new treatment potentials for patient with ATC. Cetuximab has also been reported to have synergistic effects when combined with irinotecan, a topoisomerase inhibitor. Therefore, we hypothesized that cetuximab and irinotecan would be effective in inhibiting the growth and progression of ATC in a murine orthotopic model. Experimental Design: The in vitro antiproliferative effects of cetuximab and irinotecan on ATC cell line ARO were examined. We also studied the in vivo effects of cetuximab and irinotecan on the growth, invasion, and metastasis of orthotopic ATC tumors in nude mice. The in vivo antitumor efficacy of cetuximab/irinotecan combination was also compared with that of doxorubicin. Results: Cetuximab alone did not show any antiproliferative or proapoptotic effect on this cell line. However, when combined with irinotecan, cetuximab potentiated the in vitro antiproliferative and proapoptotic effect of irinotecan. Cetuximab, irinotecan, and cetuximab/irinotecan combination resulted in 77%, 79%, and 93% in vivo inhibition of tumor growth, respectively. Incidences of lymph node metastasis, laryngeal invasion, and tumor microvessel density were also significantly decreased in these treatment groups. Furthermore, the cetuximab/irinotecan combination was significantly more effective than doxorubicin in inhibiting the growth of orthotopic ATC xenografts. Conclusions: Combination therapy with cetuximab/irinotecan inhibits the growth and progression of orthotopic ATC xenografts in nude mice. Given the lack of curative options for patients with ATC, combination therapy with cetuximab and irinotecan treatment warrants further study.

Concomitant inhibition of epidermal growth factor and vascular endothelial growth factor receptor tyrosine kinases reduces growth and metastasis of human salivary adenoid cystic carcinoma in an orthotopic nude mouse model

We hypothesized that epidermal growth factor (EGF) receptor (EGFR) activation and vascular endothelial growth factor (VEGF)–induced angiogenic signals are important for the progression and metastasis of human salivary adenoid cystic carcinoma (ACC). To test this hypothesis, we evaluated the therapeutic effect of AEE788, a dual inhibitor of EGF and VEGF receptor (VEGFR) tyrosine kinases, on human salivary ACC. In clinical specimens of salivary ACC, EGF and VEGF signaling proteins were expressed at markedly higher levels than in adjacent normal glandular tissues. We examined the effects of AEE788 on salivary ACC cell growth and apoptosis and on the phosphorylation of EGFR and VEGFR-2 in salivary ACC cells. Treatment of salivary ACC cells with AEE788, alone or in combination with chemotherapy, led to growth inhibition, induction of apoptosis, and dose-dependent inhibition of EGFR and VEGFR-2 phosphorylation. To determine the in vivo antitumor effects of AEE788, nude mice with orthotopic parotid tumors were randomized to receive oral AEE788 alone, paclitaxel alone, cisplatin alone, a combination of AEE788 plus paclitaxel, a combination of AEE788 plus cisplatin, or a placebo. AEE788 inhibited tumor growth and prevented lung metastasis in nude mice. To study the mechanism of interaction between AEE788 and chemotherapy, AEE788 was found to potentiate growth inhibition and apoptosis of ACC tumor cells mediated by chemotherapy. Tumors of mice treated with AEE788 and AEE788 plus chemotherapy exhibited down-regulation of activated EGFR and VEGFR-2, increased tumor and endothelial cell apoptosis, and decreased microvessel density, which correlated with a decrease in the level of matrix metalloproteinase-9 and matrix metalloproteinase-2 expression and a decrease in the incidence of vascular metastasis. These data show that EGFR and VEGFR can be molecular targets for therapy of salivary ACC. [Mol Cancer Ther 2006;5(11):2696–705]

Antivascular Therapy of Human Follicular Thyroid Cancer Experimental Bone Metastasis by Blockade of Epidermal Growth Factor Receptor and Vascular Growth Factor Receptor Phosphorylation

Patients suffering from bone metastases of follicular thyroid carcinoma (FTC) have a poor prognosis because of the lack of effective treatment strategies. The overexpression of epidermal growth factor receptor (EGFR) associated with increased vascularity has been implicated in the pathogenesis of FTC and subsequent bone metastases. We hypothesized that inhibiting the phosphorylation of the EGFR and vascular endothelial growth factor receptor (VEGFR) by AEE788, a dual tyrosine kinase inhibitor of EGFR and VEGFR, in combination with paclitaxel would inhibit experimental FTC bone lesions and preserve bone structure. We tested this hypothesis using the human WRO FTC cell line. In culture, AEE788 inhibited the EGF-mediated phosphorylation of EGFR, VEGFR2, mitogen-activated protein kinase, and Akt in culture. AEE788, alone and in combination with paclitaxel, inhibited cell growth and induced apoptosis. When WRO cells were injected into the tibia of nude mice, tumor and endothelial cells within the lesions expressed phosphorylated EGFR, VEGFR, Akt, and mitogen-activated protein kinase that were inhibited by the oral administration of AEE788. Therapy consisting of orally given AEE788 and i.p. injected paclitaxel induced a high level of apoptosis in tumor-associated endothelial cells and tumor cells with the inhibition of tumor growth in the bone and the preservation of bone structure. Collectively, these data show that blocking the phosphorylation of EGFR and VEGFR with AEE788 combined with paclitaxel can significantly inhibit experimental human FTC in the bone of nude mice.

An orthotopic model of anaplastic thyroid carcinoma in athymic nude mice

Purpose: To develop an orthotopic model of anaplastic thyroid carcinoma (ATC) in athymic nude mice. Experimental Design: Various thyroid carcinoma cell lines were injected into the thyroid gland of athymic nude mice to determine whether such injection was technically feasible. ATC cells were then injected into the thyroid gland or the subcutis of nude mice at various concentrations, and the mice were then followed for tumor development. The tumors were examined histopathologically for local invasion or regional or distant metastasis. Results: Injection of tumor cells into the thyroid glands of nude mice was technically feasible and resulted in the formation of thyroid tumors. The ATC cell line DRO showed significantly higher tumorigenicity in the thyroid gland than in the subcutis. In contrast, oral squamous cell carcinoma cell line TU167 shows no significantly higher tumorigenicity in the thyroid gland than in the subcutis. ATC tumors established in the thyroid gland also produced symptomatic compression of the esophagus and the trachea. Local invasion of the larynx and trachea was as well as high rates of pulmonary metastasis were also observed. Immunohistochemical staining showed higher microvessel density as well as higher expression of vascular endothelial growth factor and interleukin-8 in the orthotopic thyroid tumors than in ectopic tumors. Conclusion: An orthotopic model of ATC in athymic nude mice was developed that closely recapitulates the clinical findings of human ATC. This model should facilitate the understanding of the pathogenesis of ATC and aid in the development of novel therapies against ATC.

Growth-inhibitory effects of human anti-insulin-like growth factor-I receptor antibody (A12) in an orthotopic nude mouse model of anaplastic thyroid carcinoma.

Purpose: The insulin-like growth factor-I receptor (IGF-IR) and its ligands have been implicated in the pathogenesis and progression of various cancers, including those arising in the thyroid gland. We therefore evaluated whether the IGF-IR could serve as a potential target for therapy of anaplastic thyroid carcinoma (ATC). Experimental Design: The expression and activation of the IGF-IR and some of its downstream signaling pathway components were evaluated in both human thyroid cancer specimens and thyroid cancer cell lines. The therapeutic potential of a humanized monoclonal antibody (A12) directed against IGF-IR was assessed in vitro and in vivo in an orthotopic model of ATC. Tumor volume and overall survival time were analyzed to evaluate the efficacy of A12 in vivo. Results: IGF-IR was overexpressed in 94% of the thyroid cancers. Blockade of IGF-IR with A12 was effective in attenuating IGF-IR signaling both in vitro and in vivo. However, the inhibitory effects of A12 on cell proliferation were cell line dependent, as those ATC cell lines that had detectable levels of pIGF-IR were more sensitive to A12 treatment. A12 was equally effective in vivo, where it brought ∼57% (P = 0.041) inhibition in tumor volume. The concomitant use of A12 and irinotecan produced additive effects and resulted in a 93% (P < 0.001) reduction in tumor volume. Blocking IGF-IR blocked Akt phosphorylation and decreased proliferation and microvessel density but increased apoptosis within the tumor xenografts. Our results also highlighted a previously undefined IGF-IR-mediated antiangiogenic effect on tumor-associated endothelium in thyroid cancers. Conclusion: Blocking the IGF-IR with A12 seems to be a potential avenue for treating patients with ATC by its direct antitumor effects and its effects on the tumor vasculature.

Dual Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Inhibition with NVP-AEE788 for the Treatment of Aggressive Follicular Thyroid Cancer

Purpose: Patients with radioiodine-resistant follicular thyroid cancer (FTC) have a poor prognosis, if metastasized, with currently available treatment modalities. Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) and their receptors (EGFR and VEGFR) have been reported to be overexpressed in FTC and have been implicated in FTC development. We hypothesized that inhibiting the phosphorylation of EGFR and VEGFR by treatment with NVP-AEE788 (AEE788), a novel dual specific EGFR and VEGFR inhibitor, either alone or in combination with paclitaxel, would inhibit the growth of FTC xenografts in an orthotopic nude mouse model. Experimental Design: To confirm previous reports, EGF and EGFR expression and vascularity were analyzed in human samples of FTC, Hürthle cell carcinoma, and normal thyroid tissues. EGFR expression in four FTC cell lines was measured using Western blotting. The antitumor effect of AEE788 on FTC cells in vitro was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and Western blotting. The effect of AEE788, alone and in combination with paclitaxel, on FTC tumor growth in an orthotopic nude mouse model was also investigated. Immunohistochemical analysis of EGFR and VEGFR signaling status, cell proliferation, apoptosis, and microvessel density was done. Results: EGF, EGFR, and vascularity were increased in human thyroid tumor samples and EGFR was increased in FTC cells. AEE788 inhibited FTC cell growth in vitro and reduced the phosphorylation status of EGFR, VEGFR, and two downstream targets, AKT and mitogen-activated protein kinase, in FTC cells. AEE788 alone and, to a greater extent, AEE788 plus paclitaxel suppressed FTC tumor growth in the thyroids of nude mice. Conclusion: Dual inhibition of EGFR and VEGFR by AEE788 could represent a novel approach to the treatment of radioiodine-resistant FTC.

Antivascular therapy of oral tongue squamous cell carcinoma with PTK787

Objectives/Hypothesis: Vascular endothelial growth factor (VEGF) is an important mediator in tumor vascularization, growth, and metastasis. We investigated whether blockade of the VEGF receptor (VEGF‐R) signaling pathway by the tyrosine kinase inhibitor PTK787 combined with CPT‐11, a semisynthetic camptothecin analogue, can inhibit the tumor growth and angiogenesis of squamous cell carcinoma of the oral tongue in an orthotopic nude mouse model.

Growth inhibition of orthotopic anaplastic thyroid carcinoma xenografts in nude mice by PTK787/ ZK222584 and CPT-11

A preclinical evaluation of CPT‐1 (Camptosar, irinotecan) and PTK787/ZK222584, a vascular endothelial growth factor receptor (VEGFR‐2) tyrosine kinase inhibitor, as therapeutic agents against anaplastic thyroid carcinoma (ATC) was performed in vitro and in an orthotopic model of ATC in nude mice.