Loss of 15-hydroxyprostaglandin dehydrogenase indicates a tumor suppressor role in pituitary adenomas

15-hydroxyprostaglandin dehydrogenase (15-PGDH) may function as a tumor suppressor that antagonizes the action of the cyclooxygenase-2 (COX-2) oncogene in several types of tumors. However, it is unknown if it has a role in the pituitary. Recently, our group found that 15-PGDH expression was low in prolactin (PRL) secreting adenomas (prolactinomas) and growth hormone (GH) secreting adenomas (GHomas) using fiber-optic BeadArray technology. In this study, we examined the relative expression of 15-PGDH and COX-2 mRNA in clinical specimens and examined the effects of 15-PGDH on GH3 rat pituitary tumor cell proliferation, apoptosis and hormone secretion. 15-PGDH expression was lower and COX-2 expression was higher in prolactinomas and GHomas compared with normal controls. Overexpressed 15-PGDH inhibited tumor cell proliferation and induced apoptosis. It had a significant suppressive effect on mRNA levels and on the secretion of PRL and GH in GH3 cells. The inhibition of cell proliferation was accompanied by the decreased expression of cox-2, matrix metalloproteinase-9 (MMP-9) and B cell leukemia/lymphoma-2 (Bcl-2). These data are suggestive of a previously unrecognized pathway in pituitary tumorigenesis, and this novel observation may shed light on therapeutic strategies for pituitary tumors.     

Epidermal growth factor competes with EGF receptor inhibitors to induce cell death in EGFR-overexpressing tumor cells

Epidermal growth factor receptor (EGFR) signaling plays an important role in cell growth and differentiation. Mutations in the EGFR gene and EGFR gene amplifications have been associated with increased responsiveness to selective EGFR tyrosine kinase inhibitors (EGFR-TKIs). By contrast, EGF may also stimulate apoptosis in tumor cells, depending on EGFR and Her2 (erbB-2) expression levels. In the present study, we investigated cellular responses after EGFR activation by EGF, or inhibition by cetuximab and gefitinib. EGF treatment induced a near-immediate increase in p38 MAPK phosphorylation together with inactivation of ERK1/2. In contrast, gefitinib- and cetuximab-induced phosphorylation of p38 MAPK was much delayed, and gefitinib also induced a delayed activation of ERK1/2. EGF induced progressive cell death of A431 cells with prolonged treatment, whereas cetuximab- or gefitinib-treated cells showed temporary growth arrest and subsequent re-growth. Moreover, in combination treatment experiments, cetuximab or gefitinib competitively inhibited EGF-induced cell death. Normal WI38-VA13 cells did not display any noticeable changes in cell proliferation in response to EGF, gefitinib or cetuximab. EGF-induced death signaling is apparently irreversible: EGF induced significant EGFR phosphorylation/internalization and activated caspase-3, -8 and -9, effects that were not observed in cetuximab- or gefitinib-treated cells. Collectively, these results indicate that EGF may be a more potent cytotoxic agent than EGFR blockers in EGFR-overexpressing cancer cells.     

Transforming growth factor alpha expression drives constitutive epidermal growth factor receptor pathway activation and sensitivity to gefitinib (Iressa) in human pancreatic cancer cell lines

The epidermal growth factor receptor (EGFR) is considered an important therapeutic target in pancreatic cancer, but it is currently impossible to identify those patients who are most likely to benefit from EGFR-directed therapy. We examined the biological effects of the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in a panel of nine human pancreatic cancer cell lines. The drug strongly inhibited DNA synthesis and induced low levels of apoptosis at clinically relevant concentrations in a subset of three of the lines (L3.6pl, BxPC3, and Cfpac1). Sensitivity to gefitinib correlated directly with ligand [transforming growth factor-alpha (TGF-alpha)] expression (r(2) = 0.71, P = 0.004) but not with surface EGFR expression. The gefitinib-sensitive cells displayed constitutive baseline EGFR phosphorylation, whereas the gefitinib-resistant cells did not. Exposure to gefitinib or a small interfering RNA construct specific for TGF-alpha reversed the constitutive EGFR phosphorylation and downstream target [extracellular signal-regulated kinases (ERK), AKT] phosphorylation in the gefitinib-sensitive cells but had no effects on ERK or AKT phosphorylation in gefitinib-resistant cells. Baseline EGFR phosphorylation was lower in a subclone of L3.6pl selected for low TGF-alpha expression, and these cells were also resistant to gefitinib-mediated growth inhibition. Gefitinib blocked the growth of tumor xenografts derived from L3.6pl cells but had no effect on the growth of tumors derived from EGFR-independent MiaPaCa-2 cells. Together, our data show that TGF-alpha expression identifies a subset of human pancreatic cancer cells that is dependent on EGFR signaling in vitro and in vivo. Quantification of TGF-alpha expression may therefore represent an effective means of identifying EGFR-responsive primary tumors.     

Senescence mediates pituitary hypoplasia and restrains pituitary tumor growth

Understanding factors subserving pituitary cell proliferation enables understanding mechanisms underlying uniquely benign pituitary tumors. Pituitary tumor-transforming gene (Pttg) deletion results in pituitary hypoplasia, low pituitary cell proliferation rates, and rescue of pituitary tumor development in Rb(+/-) mice. Pttg(-/-) pituitary glands exhibit ARF/p53/p21-dependent senescence pathway activation evidenced by up-regulated p19, cyclin D1, and Bcl-2 protein levels and p53 stabilization. High pituitary p21 levels in the absence of PTTG were associated with suppressed cyclin-dependent kinase 2 activity, Rb phosphorylation, and cyclin A expression, all required for cell cycle progression. Although senescence-associated beta-galactosidase was enhanced in Pttg-deficient pituitary glands, telomere lengths were increased. DNA damage signaling pathways were activated and aneuploidy was evident in the Pttg-deficient pituitary, triggering senescence-associated genes. To confirm the p21 dependency of decreased proliferation and senescence in the Pttg-null pituitary, mouse embryonic fibroblast (MEF) colony formation was tested in wild-type, Pttg(-/-), Rb(+/-), Rb(+/-)Pttg(-/-), and Rb(+/-)Pttg(-/-)p21(-/-) cells. Rb(+/-)Pttg(-/-) MEFs, unlike Rb(+/-) cells, failed to produce colonies and exhibited high levels of senescence. p21 deletion from Rb(+/-)Pttg(-/-) MEFs enhanced anchorage-independent cell growth, accompanied by a marked decrease in senescence. As cell proliferation assessed by bromodeoxyuridine incorporation was higher in Rb(+/-)Pttg(-/-)p21(-/-) relative to Rb(+/-)Pttg(-/-) pituitary glands, p21-dependent senescence provoked by Pttg deletion may underlie pituitary hypoplasia and decreased tumor development in Rb(+/-)Pttg(-/-) mice.     

Analysis of prolactin and growth hormone production in hyperplastic and neoplastic rat pituitary tissues by the hemolytic plaque assay

The reverse hemolytic plaque assay (RHPA) was used to detect hormone release from cultured normal, hyperplastic, and neoplastic rat pituitary cells. Hyperplastic pituitary cells were produced by s.c. diethylstilbestrol (DES) treatment (10 mg in Silastic tubes) for 3, 6, and 9 weeks. Neoplastic pituitary cells from rats with MtT/W15 transplantable tumors treated with DES for 3 weeks were also analyzed. Aliquots of the same cells were also analyzed by immunocytochemical staining. DES treatment resulted in an increase in prolactin (PRL)-producing cells in hyperplastic pituitaries compared to untreated pituitaries after 9 weeks of treatment by the RHPA [61.2 +/- 5.2 (SE) versus 32 +/- 3.0] and by immunocytochemical staining [70.9 +/- 2.4 versus 36 +/- 1.4]. The percentage of mammosomatotropic cells decreased from 11.3 +/- 3.8 to 4.2 +/- 2.6% in pituitary cells from these same groups of animals. After 3 weeks of DES treatment in rats with MtT/W15 tumor, there was an increase in growth hormone (GH)-producing cells and a decrease in PRL-producing cells when analyzed by the RHPA (control: percentage of GH, 36.3 +/- 6.2; percentage of PRL, 39.0 +/- 1.6 versus DES-treated tumors: percentage of GH of 68.2 +/- 1.9; and percentage of PRL, 3.2 +/- 1.8%). The percentage of mammosomatotropic cells declined from 12.4 +/- 2.3 to 0.77 +/- 2.4%. A combined procedure of RHPA followed by immunocytochemical staining on the same slides also revealed a decline in mammosomatotropic cells after chronic DES treatment in hyperplastic and neoplastic MtT/W15 tumor cells. These results show that DES has different effects on PRL and GH secretion and storage in hyperplastic pituitary and in the MtT/W15 pituitary tumor cells.     

Involvement of Rad51 in cytotoxicity induced by epidermal growth factor receptor inhibitor (gefitinib, IressaR) and chemotherapeutic agents in human lung cancer cells

Gefitinib (Iressa(R), ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling activation. Rad51 is an essential component of the homologous recombination repair pathway. High level of Rad51 expression has been reported in chemo- or radioresistant carcinomas. We hypothesized that gefitinib may enhance the effects of the alkylating agent cisplatin- or the antitumor antibiotic mitomycin C (MMC)-mediated cytotoxicity by decreasing ERK1/2 activation and Rad51 expression. Exposure of human non-small lung cancer cells to gefitinib decreased cisplatin- or MMC-elicited ERK1/2 activation and Rad51 protein induction. Neither cisplatin nor MMC treatment affected Rad51 messenger RNA (mRNA). However, gefitinib cotreatment with cisplatin or MMC significantly decreased Rad51 mRNA levels. In addition, gefitinib decreased cisplatin- or MMC-elicited Rad51 protein levels by increasing Rad51 protein instability. Enhancement of ERK1/2 signaling by constitutively active mitogen-activated protein kinase kinase 1/2 (MKK1/2-CA) increased Rad51 protein levels and protein stability in gefitinib and cisplatin or MMC cotreated cells. Moreover, the synergistic cytotoxic effects induced by gefitinib cotreatment with cisplatin or MMC were remarkably decreased by MKK1-CA-mediated enhancement of ERK1/2 activation. Depletion of endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced lung cancer cell death upon treatment with cisplatin or MMC. We conclude that Rad51 protein protects lung cancer cells from synergistic cytotoxic effects induced by gefitinib and chemotherapeutic agents. Suppression of Rad51 expression may be a novel lung cancer therapeutic modality to overcome drug resistance to EGFR inhibitors and chemotherapeutic agents.     

Combined targeting of endothelin A receptor and epidermal growth factor receptor in ovarian cancer shows enhanced antitumor activity

Ovarian carcinomas overexpress endothelin A receptors (ET(A)R) and epidermal growth factor (EGF) receptor (EGFR). In these cells, endothelin-1 (ET-1) triggers mitogenic and invasive signaling pathways that are in part mediated by EGFR transactivation. Combined targeting of ET(A)R, by the specific ET(A)R antagonist ZD4054, and of EGFR by the EGFR inhibitor gefitinib (IRESSA), may offer improvements in ovarian carcinoma treatment. In HEY and OVCA 433 ovarian carcinoma cells, ET-1 or EGF induced rapid activation of EGFR, p42/44 mitogen-activated protein kinase (MAPK), and AKT. ZD4054 was able to reduce the ET-1-induced EGFR transactivation. Gefitinib significantly inhibited EGF- and ET-1-induced EGFR phosphorylation, but incompletely reduced the ET-1-induced activation of downstream targets. ZD4054 plus gefitinib resulted in a greater inhibition of EGFR, MAPK, and AKT phosphorylation, indicating the critical role of these interconnected signaling proteins. ZD4054 effectively inhibited cell proliferation, invasiveness, and vascular endothelial growth factor (VEGF) secretion. Concomitantly, ZD4054 enhanced apoptosis and E-cadherin promoter activity and expression. In both cell lines, the drug combination resulted in a significant decrease in cell proliferation (65%), invasion (52%), and VEGF production (50%), accompanied by a 2-fold increase in apoptosis. The coadministration of ZD4054 enhanced the efficacy of gefitinib leading to partial (82%) or complete tumor regression on HEY ovarian carcinoma xenografts. Antitumor effects were paralleled by biochemical and immunohistologic evidence of decreased vascularization, Ki-67, matrix metalloproteinase-2 (MMP-2), VEGF, MAPK and EGFR, and enhanced E-cadherin expression. The cross-signaling between the EGFR/ET(A)R pathways provides a rationale to combine EGFR inhibitors with ET(A)R antagonists, identifying new effective therapeutic opportunities for ovarian cancer.     

ESR1及其亚型△5-Del-ESR1 mRNA在泌乳素腺瘤中的表达

目的：研究ESR及其亚型mRNA在泌乳素腺瘤中的表达.方法：应用RT-PCR测定20例泌乳素腺瘤标本ESRI、ESR2及△5-Del-ESR1 mRNA的表达,研究其表达水平与患者性别、肿瘤体积、侵袭性及PRL水平的关系.结果：男性和绝经后女性患者肿瘤ESR1 mRNA表达高于育龄女性患者（P=0.08）;侵袭性泌乳素腺瘤高于非侵袭性肿瘤（P=0.01）;表达水平与PRL的对数值呈正相关,R2=0.665（P=0.00）.PRL≥1000ng/ml的患者△5-Del-ESR1 mRNA表达水平较PRL〈1000ng/ml的患者明显增高（P=0.031）.结论：ESR1及其亚型A5-Del-ESRI mRNA表达与泌乳素腺瘤PRL分泌及肿瘤侵袭有关.     

Effects of estradiol on prolactin and growth hormone messenger RNAs in cultured normal and neoplastic (MtT/W15 and GH3) rat pituitary cells

The effects of 17 beta-estradiol (E17 beta) on prolactin (PRL) cell proliferation and on the expression of PRL and growth hormone (GH) proteins and mRNAs were analyzed in cultured pituitary cells by immunocytochemistry, in situ hybridization, and Northern blot hybridization studies. Three different cell cultures were used: (a) normal pituitary cells; (b) GH3 tumor cell line; and (c) MtT/W15, a transplantable PRL and GH-producing pituitary tumor. E17 beta (10(-7) M) caused a significant increase in PRL cell proliferation in normal pituitary [3.9 +/- 0.4 versus 7.7 +/- 0.9% (SEM) of immunostained PRL cells with thymidine incorporation] [P less than 0.01] but produced a significant decrease in PRL cell proliferation in MtT/W15 primary cell cultures [6.7 +/- 1.0 versus 3.7 +/- 0.8%] [P less than 0.05]. PRL mRNA was significantly increased in normal pituitary and in GH3 tumor cells by E17 beta treatment. There was a significant decrease in PRL mRNA and an increase in GH mRNA expression in cultured MtT/W15 tumor cells by immunocytochemistry and in situ hybridization analyses. The percentage of cells producing both PRL and GH or mammosomatotropic cells analyzed by two different techniques declined after one week in culture in normal pituitary cells and in cultured MtT/W15 tumor cells after E17 beta treatment. These results show that E17 beta has a direct stimulatory effect on normal pituitary and GH3 cells and a direct inhibitory effect on MtT/W15 tumor cells with respect to cell proliferation and PRL hormone and mRNA expression.     

EXPRESSION OF EPIDERMAL GROWTH FACTOR, TRANSFORMINGGROWTH FACTOR-α AND THEIR RECEPTOR IN HUMAN PITUITARY TUMORS

The development of pituitary tumor was regulated by multiple-step signal transduction and their complicated process. A lot of growth factors participated in the cell's proliferation and their function activity, so the abnormal in growth factor and/or its receptor may involve in the pituitary tumorigenesis.[1] To investigate the role of epidermal growth factor receptor (EGFR) and its protein tyrosine kinases signaling conduction in the pathogenesis of pituitary tumors, we examined the expressi…     

Suppression of proliferation of two independent NF1 malignant peripheral nerve sheath tumor cell lines by the pan-ErbB inhibitor CI-1033

Neurofibromatosis Type 1 (NF1) is characterized by the abnormal proliferation of neuroectodermal tissues and the development of certain tumors, particularly neurofibromas, which may progress into malignant peripheral nerve sheath tumors (MPNSTs). Effective pharmacological therapy for the treatment of NF1 tumors is currently unavailable and the prognosis for patients with MPNSTs is poor. Loss of neurofibromin correlates with increased expression of the epidermal growth factor receptor (EGFR) and ErbB2 tyrosine kinases and these kinases have been shown to promote NF1 tumor-associated pathologies in vivo. We show here that while NF1 MPNST cells have higher EGFR expression levels and are more sensitive to EGF when compared to a non-NF1 MPNST cell line, the ability of the EGFR inhibitor gefitinib to selectively inhibit NF1 MPNST cell proliferation is marginal. We also show that NF1 MPNST proliferation correlates with activated ErbB2 and can be suppressed by nanomolar concentrations of the pan-ErbB inhibitor CI-1033 (canertinib). Consequently, targeting both EGFR and ErbB2 may prove an effective strategy for suppressing NF1 MPNST tumor growth in vivo.     

Cytocidal effects of bromocriptine, somatostatin analog, and heat on growth hormone-secreting pituitary adenoma in vitro.

The effects of bromocriptine (BC), a somatostatin analog (SMS), and heat on the secretion of growth hormone (GH) and prolactin (PRL), and on the morphologic features of human GH-secreting pituitary adenoma were studied in vitro. The treatment with BC, SMS, or heat (41.5 degrees C and 42.5 degrees C) markedly suppressed the secretion of GH and PRL from the adenoma cells and reduced the number of cells immunoreactive with GH or PRL. The combined treatment with BC and heat induced a marked reduction in the number of GH and PRL cells consistent with the effect on the secretion of GH and PRL. These results suggest that BC, SMS, and heat treatments produced the cytotoxic effects on pituitary adenoma cells, and that the simultaneous treatment of BC and heat enhanced this effect.     

Activation of MEK/ERK and PI3K/Akt pathways by fibronectin requires integrin alphav-mediated ADAM activity in hepatocellular carcinoma: a novel functional target for gefitinib

We have shown that the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839) inhibits the development of intrahepatic metastases of hepatocellular carcinoma CBO140C12, and EGFR transactivation by tumor necrosis factor-alpha is a possible target of gefitinib. In the present study, we focused on the fibronectin (FN)-dependent signaling pathway to further elucidate the antimetastatic activity of gefitinib in CBO140C12 cells. We initially observed that FN induced activation of extracellular signal-regulated kinase (ERK), p38 and Akt, as well as cell proliferation and CBO140C12 cell invasion. These responses were mediated by EGFR tyrosine kinase, because gefitinib inhibited these effects of FN. FN-induced ERK, p38 and Akt activation was partly blocked by the Arg-Gly-Asp (RGD)-pseudo-peptide FC-336, anti-alphav integrin antibody RMV-7, the broad-spectrum matrix metalloprotease inhibitor GM6001 and the broad spectrum a disintegrin and metalloprotease (ADAM) inhibitor TAPI-1. But these inhibitors had no effect on EGF-induced signaling pathways, suggesting that integrins and ADAM may be upstream components of EGFR in these responses. These results suggest that FN-induced activation of ERK, p38, Akt, cell proliferation and invasion was mediated, at least in part, via integrins, ADAM and EGFR, and that this FN-induced signaling pathway might be involved in the antimetastatic activity of gefitinib.     

Effects of epidermal growth factor receptor blockade on ependymoma stem cells in vitro and in orthotopic mouse models

Some lines of evidence suggest that tumors, including ependymoma, might arise from a subpopulation of cells, termed cancer stem cells (CSCs), with self-renewal and tumor-initiation properties. Given the strict dependence of CSCs on epidermal growth factor (EGF) through EGF receptor (EGFR), we investigated the effects of EGFR inhibitors in ependymoma-stem cells (SCs) in vitro and in orthotopic mouse models. We established two ependymoma-SC lines from two recurrent pediatric ependymoma. Both lines expressed markers of radial glia--the candidate SCs of ependymoma--and showed renewal ability, multipotency, and tumorigenicity after orthotopic implantation, despite markedly different expression of CD133 (94 vs. 6%). High phosphorylated-EGFR/EGFR ratio was detected, which decreased after differentiation. EGFR inhibitors (gefitinib and AEE788) reduced clonogenicity, proliferation and survival of ependymoma-SC lines dose-dependently, and blocked EGF-induced activation of EGFR, Akt and extracellular signal-regulated kinase 1/2. Overall, AEE788 was more effective than gefitinib. EGFR blockade as well as differentiation strongly reduced CD133 expression. However, ex vivo treatment with AEE788 did not impair orthotopic tumor engraftment, whereas ex vivo differentiation did, suggesting that CD133 does not absolutely segregate for tumorigenicity in ependymoma-SCs. Orally administered AEE788 prolonged survival of mice bearing ependymoma-SC-driven orthotopic xenografts from 56 to 63 days, close to statistical significance (log-rank p=0.06). Our study describes for the first time EGFR signaling in ependymoma-SCs and the effects of EGFR blockade in complementary in vitro and in vivo systems. The experimental models we developed can be used to further investigate the activity of EGFR inhibitors or other antineoplastic agents in this tumor.     

曲格列酮抑制垂体腺瘤细胞GH3的生长及其机制

目的：观察过氧化物酶体增殖物激活受体γ(peroxisome proliferator activated receptorγ , PPARγ)激动剂曲格列酮(troglitazone, TGZ)对垂体腺瘤细胞生长和生长激素（growth hormone,GH）分泌的影响,并探讨其可能的作用机制。方法：采用MTT和ELISA法检测TGZ对大鼠垂体腺瘤细胞系GH3细胞增殖和GH分泌的抑制效应,进一步应用电镜、FCM及Western blotting分别检测细胞凋亡、细胞周期以及Caspase3、Bcl2和Bax蛋白的表达。结果：TGZ呈剂量和时间依赖性方式抑制GH3细胞的增殖和GH的分泌;TGZ干预后的GH3细胞出现典型的凋亡形态特征; TGZ干预GH3细胞后,G2、S期的细胞比例下降,而G1期的细胞比例明显增加;Bax和Caspase3蛋白表达水平明显增加,而Bcl2蛋白表达水平下调,且表现出剂量依赖效应。结论：PPARγ激动剂TGZ可能通过诱导细胞凋亡和阻滞细胞周期来抑制垂体腺瘤细胞生长及其生长激素分泌。     

Enhancement of tumor radioresponse by combined treatment with gefitinib (Iressa, ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, is accompanied by inhibition of DNA damage repair and cell growth in oral cancer

Molecular blockade of EGFR with either an EGFR MAb or an EGFR TKI enhances the radiosensitivity of human SCCs. In the present study, we investigated whether treatment with the EGFR TKI gefitinib (Iressa, ZD1839) improves the response to radiotherapy in the OSCC cell lines HSC2 and HSC3. We examined potential mechanisms that may contribute to the enhanced radiation response induced by gefitinib. Growth inhibition was observed in vitro with radiation or gefitinib. A cooperative antiproliferative effect was obtained when cancer cells were treated with radiation followed by gefitinib. Cells treated with a combination of radiation and gefitinib arrested in G(1) and G(2)-M phases, with a decrease in the S-phase population. While radiation alone did not significantly affect MEK1/2 and p38 MAPK autophosphorylation, the combination of gefitinib and radiation completely inhibited the downstream signaling of EGFR. Results from DNA damage repair analysis in cultured OSCC cells demonstrated that gefitinib had a strong inhibitory effect on DNA-PKc pathways after radiation. Tumor xenograft studies demonstrated that the combination of gefitinib and radiation caused growth inhibition and tumor regression of well-established OSCC tumors in athymic mice; tumor volume was reduced from 1,008.2 to 231.4 mm(3) in HSC2 cells (p < 0.01) and from 284.2 to 12.4 mm(3) in HSC3 cells (p < 0.01). Immunohistochemical analysis of OSCC xenografts revealed that gefitinib caused a striking decrease in tumor cell proliferation when combined with radiotherapy. Overall, we conclude that gefitinib enhances tumor radioresponse by multiple mechanisms that may involve antiproliferative growth inhibition and effects on DNA repair after exposure to radiation.     

Uncoupling of the epidermal growth factor receptor from downstream signal transduction molecules guides the acquired resistance to gefitinib in prostate cancer cells

The clinical efficacy of ErbB1 kinase inhibitors is limited by the development of acquired autoresistance. The activation of alternative signaling pathways can contribute to gefitinib resistance. In this study, we demonstrate that the continuous in vitro exposure of the phosphatase and tensin homologue (deleted from chromosome 10)-negative prostate cancer (PC)3 cell line to gefitinib resulted in a sustained growth inhibition of 50% for about 2 months, but afterwards the surviving cells resumed their usual proliferation rate. During chronic treatment, gefitinib-treated cells developed drug resistance undergoing a G0/G1 cell cycle arrest, with a corresponding reduction in the G2/M cells without evident cell apoptosis, and thus a tyrosine kinase inhibitor-resistant (TKI-R) PC3 cell subline was isolated. TKI-R cells show i) an increment in basal ERK activation, ii) an epidermal growth factor-mediated and gefitinib insensitive ERK phosporylation, iii) increased levels of Her2/Neu, iv) a significant decrement in epidermal growth factor receptor (EGFR) expression, v) a very low sensitivity against EGFR TKIs and blocking antibodies, vi) a moderate increase in the sensitivity to growth inhibition by the Her2 inhibitor, AG825 or by 2C4, the humanized monoclonal antibody which blocks Her2 heterodymerization, vii) an increased expression of the neutrophine receptors, TrkA and TrkB, and viii) a significantly increased sensitivity to growth inhibition by the TrkA inhibitor, CEP701. Treatment with a mitogen-activated-protein kinase inhibitor abolished gefitinib resistance completely. Therefore, the ability of tumor cells to maintain high ERK activity under EGFR inhibition could represent a potential mechanism of the resistance to gefitinib.     

Inhibition of the T790M gatekeeper mutant of the epidermal growth factor receptor by EXEL-7647.

PURPOSE: Agents inhibiting the epidermal growth factor receptor (EGFR) have shown clinical benefit in a subset of non-small cell lung cancer patients expressing amplified or mutationally activated EGFR. However, responsive patients can relapse as a result of selection for EGFR gene mutations that confer resistance to ATP competitive EGFR inhibitors, such as erlotinib and gefitinib. We describe here the activity of EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, against both wild-type (WT) and mutant EGFR in vitro and in vivo. EXPERIMENTAL DESIGN: The activity of EGFR inhibitors against WT and mutant EGFRs and their effect on downstream signal transduction was examined in cellular assays and in vivo using A431 and MDA-MB-231 (WT EGFR) and H1975 (L858R and T790M mutant EGFR) xenograft tumors. RESULTS: EXEL-7647 shows potent and long-lived inhibition of the WT EGFR in vivo. In addition, EXEL-7647 inhibits cellular proliferation and EGFR pathway activation in the erlotinib-resistant H1975 cell line that harbors a double mutation (L858R and T790M) in the EGFR gene. In vivo efficacy studies show that EXEL-7647 substantially inhibited the growth of H1975 xenograft tumors and reduced both tumor EGFR signaling and tumor vessel density. Additionally, EXEL-7647, in contrast to erlotinib, substantially inhibited the growth and vascularization of MDA-MB-231 xenografts, a model which is more reliant on signaling through vascular endothelial growth factor receptors. CONCLUSIONS: These studies provide a preclinical basis for clinical trials of XL647 in solid tumors and in patients bearing tumors that are resistant to existing EGFR-targeted therapies.     

PTEN/Akt signaling through epidermal growth factor receptor is prerequisite for angiogenesis by hepatocellular carcinoma cells that is susceptible to inhibition by gefitinib

Hepatocellular carcinoma (HCC) is one of the most common tumor-related causes of death worldwide for which there is still no satisfactory treatment. We previously reported the antiangiogenic effect of gefitinib, a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has been used successfully to treat lung cancer. In this study, we investigated the effects of gefitinib on tumor-induced angiogenesis by using HCC cell lines (HCC3, CBO12C3, and AD3) in vitro as well as in vivo. Oral administration of gefitinib inhibited angiogenesis induced by HCC3 and CBO12C3, but not by AD3 in the mouse dorsal air sac model. Production of both vascular endothelial growth factor (VEGF) and chemokine C-X-C motif ligand 1 (CXCL1) by EGF-stimulated HCC was more markedly inhibited by gefitinib in HCC3 and CBO12C3 cells than in AD3 cells. EGF stimulated the phosphorylation of EGFR, Akt, and extracellular signal-regulated kinase 1/2 (ERK1/2) in HCC3 and CBO12C3 cells, whereas EGF stimulated phosphorylation of EGFR and ERK1/2, but not Akt in AD3 cells. In fact, Akt was constitutively activated in the absence of EGF in AD3 cells. Gefitinib inhibited Akt phosphorylation in all three cell lines, but it was about five times less effective in AD3 cells. The concentration of PTEN in AD3 cells was about a half that in HCC3 and CBO12C3 cells. Transfection of HCC3 cells with PTEN small interfering RNA reduced their sensitivity to gefitinib in terms of its inhibitory effect on both Akt phosphorylation and the production of VEGF and CXCL1. In conclusion, effect of gefitinib on HCC-induced angiogenesis depends on its inhibition of the production of angiogenic factors, probably involving a PTEN/Akt signaling pathway.