Platelet-derived growth factor receptor inhibition and chemotherapy for castration-resistant prostate cancer with bone metastases.

PURPOSE: To further assess preclinical and early clinical evidence that imatinib mesylate, a platelet-derived growth factor receptor (PDGFR) inhibitor, modulates taxane activity in prostate cancer and bone metastases, a randomized study was conducted. EXPERIMENTAL DESIGN: Men with progressive castration-resistant prostate cancer with bone metastases (n = 144) were planned for equal randomization to i.v. 30 mg/m(2) docetaxel on days 1, 8, 15, and 22 every 42 days with 600 mg imatinib daily or placebo, for an improvement in median progression-free survival from 4.5 to 7.5 months (two-sided alpha = 0.05 and beta = 0.20). Secondary end points included differential toxicity and bone turnover markers, tumor phosphorylated PDGFR (p-PDGFR) expression, and modulation of p-PDGFR in peripheral blood leukocytes. RESULTS: Accrual was halted early because of adverse gastrointestinal events. Among 116 evaluable men (57 docetaxel + imatinib; 59 docetaxel + placebo), respective median times to progression were 4.2 months (95% confidence interval, 3.1-7.5) and 4.2 months (95% confidence interval, 3.0-6.8; P = 0.58, log-rank test). Excess grade 3 toxicities (n = 23) in the docetaxel + imatinib group were principally fatigue and gastrointestinal. Tumor p-PDGFR expression was observed in 12 of 14 (86%) evaluable bone specimens. In peripheral blood leukocytes, p-PDGFR reduction was more likely in docetaxel + imatinib-treated patients compared with docetaxel + placebo (P < 0.0001), as were reductions in urine N-telopeptides (P = 0.004) but not serum bone-specific alkaline phosphatase (P = 0.099). CONCLUSIONS: These clinical and translational results question the value of PDGFR inhibition with taxane chemotherapy in prostate cancer bone metastases and are at variance with the preclinical studies. This discordance requires explanation.

Targeting of insulin-like growth factor-I receptor with a monoclonal antibody inhibits growth of hepatic metastases from human colon carcinoma in mice.

BACKGROUND: Colorectal carcinomas (CRC) express high levels of insulin-like growth factor-I/II (IGF-I/II) and the receptor (IGF-IR). We hypothesized that selective inhibition of IGF-IR would inhibit hepatic growth of human CRC in mice. METHODS: Human CRC cells were treated in vitro with anti-IGF-IR monoclonal antibody (MoAB) with and without oxaliplatin to assess cytotoxicity. The effect of anti-IGF-IR MoAB on IGF-I-induced vascular endothelial growth factor (VEGF) production in human CRC cells was assessed by Northern blot and ELISA. We injected human CRC cells intrahepatically in nude mice, and then administered anti-IGF-IR MoAB with and without oxaliplatin. We delayed treatment in one group until large hepatic tumors were present. We assessed tumors for apoptosis, proliferation, and angiogenesis. RESULTS: Anti-IGF-IR MoAB and oxaliplatin inhibited CRC cell growth in vitro and combination treatment was even more effective. IGF-I stimulation of CRC cells resulted in significant upregulation of VEGF and this was completely inhibited by pretreatment with anti-IGF-IR MoAB. Anti-IGF-IR MoAB significantly inhibited hepatic growth of tumors in mice. Anti-IGF-IR MoAB plus oxaliplatin led to a significantly greater inhibition of tumor growth. Anti-IGF-IR MoAB plus oxaliplatin was just as effective at inhibiting growth of larger, more advanced liver tumors. Anti-IGF-IR MoAB, alone and in combination with oxaliplatin, led to a significant increase in tumor cell apoptosis, and a significant inhibition of tumor cell proliferation and angiogenesis. CONCLUSIONS: These findings suggest that IGF-IR is a potential target for therapy in patients with advanced CRC.

Endostatin improves radioresponse and blocks tumor revascularization after radiation therapy for A431 xenografts in mice.

PURPOSE: Clinical trials of antiangiogenic agents used alone for advanced malignancy have been disappointing but preclinical studies suggest that the addition of radiation therapy could improve antitumor efficacy. To test the hypothesis that antiangiogenic therapy combined with radiation therapy can overcome the limitations of antiangiogenic monotherapy, we studied the effects of endostatin combined with radiation on the growth and vascularization of A431 human epidermoid carcinomas growing intramuscularly in the legs of mice. METHODS AND MATERIALS: Mice with established A431 human epidermoid leg tumors were treated with radiation, endostatin, both radiation and endostatin, or vehicle control. The experiment was repeated and mice from each group were killed at 2, 7, and 10 days after irradiation so that tumor tissue could be obtained to further analyze the kinetics of the antitumor, antivascular, and antiangiogenic response to therapy. RESULTS: Endostatin enhanced the antitumor effects of radiation, and prolonged disease-free survival was observed in the combined treatment group. Endothelial cell proliferation was increased in tumors after irradiation but was blocked by the concurrent administration of endostatin, and the combination of endostatin with radiation enhanced endothelial cell apoptosis within 48 h after irradiation. Expression of vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-2 were increased in tumors after irradiation, and this increase was blocked by concurrent administration of endostatin. CONCLUSION: These data indicate that endostatin can block tumor revascularization after radiation therapy and thereby augment radioresponse.

Pathogenesis and vascular integrity of breast cancer brain metastasis.

Dogma dictates that brain metastasis originate from the proliferation of extravasated tumor cells and that the blood-brain barrier (BBB) prevents the delivery of chemotherapeutic drugs to the tumors. The purpose of this study was to clarify the relationship between tumor localization and progression and the involvement of BBB function in a murine model of breast cancer brain metastasis. Green fluorescent protein expressing MDA-MB435 breast cancer cells were injected into the left ventricle of nude mice. At various time points, the entire vasculature was labeled with rhodamine-conjugated albumin. The tumors and vasculature were then imaged by laser-scanning confocal and stereo fluorescence microscopy. About 75% of the cells that reached the brain extravasated and grew perivascularly. Twenty five percent of the cells, however, proliferated within the vasculature and ultimately led to thrombosis-like infarction of the brain parenchyma. The tumorigenic "embolus" served as a sustained release source of tumor cells to downstream sites. Continuing intravascular tumor expansion led to disruption of the BBB and to overflow of cells that progressed along the vessels perivascularly to distant sites that regained protection of the BBB. Breast cancer brain metastases involve both extravascular and intravascular growth of tumor cells. These distinct pathways contribute to different pathological phenotypes that generate a heterogeneous BBB that facilitates or inhibits the delivery of chemotherapeutic drugs to the tumor.

Targeting the expression of platelet-derived growth factor receptor by reactive stroma inhibits growth and metastasis of human colon carcinoma.

The stromal cells within colon carcinoma express high levels of the platelet-derived growth factor receptor (PDGF-R), whereas colon cancer cells do not. Here, we examined whether blocking PDGF-R could inhibit colon cancer growth in vivo. KM12SM human colon cancer cells were injected subcutaneously (ectopic implantation) into the cecal wall (orthotopic implantation) or into the spleen (experimental liver metastasis) of nude mice. In the colon and liver, the tumors induced active stromal reaction, whereas in the subcutis, the stromal reaction was minimal. Groups of mice (n=10) received saline (control), the tyrosine kinase inhibitor imatinib, irinotecan, or a combination of imatinib and irinotecan. Four weeks of treatment with imatinib and irinotecan significantly inhibited tumor growth (relative to control or single-agent therapy) in the cecum and liver but not in the subcutis. The combination therapy completely inhibited lymph node metastasis. Imatinib alone or in combination with irinotecan inhibited phosphorylation of PDGF-Rbeta of tumor-associated stromal cells and pericytes. Combination therapy also significantly decreased stromal reaction, tumor cell proliferation, and pericyte coverage of tumor microvessels and increased apoptosis of tumor cells and tumor-associated stromal cells. These data demonstrate that blockade of PDGF-R signaling pathways in tumor-associated stromal cells and pericytes inhibits the progressive growth and metastasis of colon cancer cells.

Expression of activated platelet-derived growth factor receptor in stromal cells of human colon carcinomas is associated with metastatic potential.

Platelet-derived growth factor receptor (PDGF-R) expression has been reported in a variety of cancers, including colorectal, breast, lung, ovarian and pancreatic cancers, but the role of PDGF-R expression in the development and progression of colon carcinoma has not yet been elucidated. The purpose of this study was to examine the expression of PDGF and PDGF-R in human colon carcinomas. The expression of PDGF, PDGF-R and phosphorylated PDGF-R (p-PDGF-R) was examined by immunofluorescence in 12 surgical specimens of colon carcinoma and in human colon carcinoma cells growing in the subcutis (ectopic site) and the cecal wall (orthotopic site) of nude mice. In most surgical specimens, tumor cells expressed PDGF-A and -B subunits, without corresponding levels of PDGF-Ralpha and PDGF-Rbeta. PDGF-Rbeta was predominantly expressed by tumor-associated stromal cells and pericytes of tumor vasculature. The expression of PDGF-Rbeta in the stroma was associated with advanced stage disease. Under culture conditions, human colon carcinoma cell lines expressed PDGF-A and -B, but not PDGF-R. In orthotopic tumors, the KM12 cells (Duke's stage B) expressed PDGF-A and -B, but PDGF-Rbeta was expressed only by stromal cells and pericytes in the tumor vasculature. This expression of PDGF-Rbeta by stromal cells and pericytes was higher in tumors growing at the orthotopic site than in those at the ectopic site. The expression of PDGF-Rbeta in the stroma was higher in highly metastatic KM12SM tumors than in low metastatic KM12C tumors. In conclusion, the expression of PDGF-Rbeta in stromal cells is influenced by the organ-specific microenvironment and is associated with metastatic potential.

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 approximately 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.

Simultaneous inhibition of EGFR, VEGFR, and platelet-derived growth factor receptor signaling combined with gemcitabine produces therapy of human pancreatic carcinoma and prolongs survival in an orthotopic nude mouse model.

Although gemcitabine has been approved as the first-line chemotherapeutic reagent for pancreatic cancer, its response rate is low and average survival duration is still only marginal. Because epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) modulate tumor progression, we hypothesized that inhibition of phosphorylation of all three on tumor cells, tumor-associated endothelial cells, and stroma cells would improve the treatment efficacy of gemcitabine in an orthotopic pancreatic tumor model in nude mice and prolong survival. We implanted L3.6pl, a human pancreatic cancer cell, in the pancreas of nude mice. We found that tumor-associated endothelial cells in this model highly expressed phosphorylated EGFR, VEGFR, and PDGFR. Oral administration of AEE788, a dual tyrosine kinase inhibitor against EGFR and VEGFR, decreased phosphorylation of EGFR and VEGFR. PDGFR phosphorylation was inhibited by STI571. Although i.p. injection of gemcitabine did not inhibit tumor growth, its combination with AEE788 and STI571 produced >80% inhibition of tumor growth and prolonged survival in parallel with increases in number of tumor cells and tumor-associated endothelial cell apoptosis, decreased microvascular density, decreased proliferation rate, and prolonged survival. STI571 treatment also decreased pericyte coverage on tumor-associated endothelial cells. Thus, inhibiting phosphorylation of EGFR, VEGFR, and PDGFR in combination with gemcitabine enhanced the efficacy of gemcitabine, resulting in inhibition of experimental human pancreatic cancer growth and significant prolongation of survival.

Targeting of urokinase plasminogen activator receptor in human pancreatic carcinoma cells inhibits c-Met- and insulin-like growth factor-I receptor-mediated migration and invasion and orthotopic tumor growth in mice.

Pancreatic carcinomas express high levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), both of which mediate cell migration and invasion. We investigated the hypotheses that (a) insulin-like growth factor-I (IGF-I)- and hepatocyte growth factor (HGF)-mediated migration and invasion of human pancreatic carcinoma cells require uPA and uPAR function and (b) inhibition of uPAR inhibits tumor growth, retroperitoneal invasion, and hepatic metastasis of human pancreatic carcinomas in mice. Using transwell assays, we investigated the effect of IGF-I and HGF on L3.6pl migration and invasion. We measured the induction of uPA and uPAR following treatment of cells with IGF-I and HGF using immunoprecipitation and Western blot analysis. The importance of uPA and uPAR on L3.6pl cell migration and invasion was studied by inhibiting their activities with amiloride and antibodies before cytokine treatment. In an orthotopic mouse model of human pancreatic carcinoma, we evaluated the effect of anti-uPAR monoclonal antibodies with and without gemcitabine on primary tumor growth, retroperitoneal invasion, and hepatic metastasis. IGF-I and HGF mediated cell migration and invasion in L3.6pl cells. In addition, IGF-I and HGF induced uPA and uPAR expression in L3.6pl cells. In vitro, blockade of uPA and uPAR activity inhibited IGF-I- and HGF-mediated cell migration and invasion. Treatment of mice with anti-uPAR monoclonal antibody significantly decreased pancreatic tumor growth and hepatic metastasis and completely inhibited retroperitoneal invasion. Our study shows the importance of the uPA/uPAR system in pancreatic carcinoma cell migration and invasion. These findings suggest that uPAR is a potential target for therapy in patients with pancreatic cancer.

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.

AEE788, a dual tyrosine kinase receptor inhibitor, induces endothelial cell apoptosis in human cutaneous squamous cell carcinoma xenografts in nude mice.

PURPOSE: We investigated whether concomitant blockade of the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways by AEE788, a dual inhibitor of EGFR and VEGFR tyrosine kinases, would inhibit the growth of cutaneous squamous cell carcinoma (SCC) cells and human cutaneous cancer xenografts in nude mice. EXPERIMENTAL DESIGN: We examined the effects of AEE788 on the phosphorylation of EGFR and VEGFR-2 in cutaneous SCC cells expressing EGFR and VEGFR-2 and cutaneous SCC cell growth and apoptosis. We assessed the in vivo antitumor effects of AEE788 in a xenograft model in nude mice. AEE788 (50 mg/kg) was given orally thrice weekly to mice that had been s.c. injected with Colo16 tumor cells. Mechanisms of in vivo AEE788 activity were determined by immunohistochemical analysis. RESULTS: Treatment of cutaneous SCC cells with AEE788 led to dose-dependent inhibition of EGFR and VEGFR-2 phosphorylation, growth inhibition, and induction of apoptosis. In mice treated with AEE788, tumor growth was inhibited by 54% at 21 days after the start of treatment compared with control mice (P < 0.01). Immunohistochemical analysis revealed that AEE788 inhibited phosphorylation of EGFR and VEGFR and induced apoptosis of tumor cells and tumor-associated endothelial cells. CONCLUSIONS: In addition to inhibiting cutaneous cancer cell growth by blocking EGFR and VEGFR signaling pathways in vitro, AEE788 inhibited in vivo tumor growth by inducing tumor and endothelial cell apoptosis.

Expression of interleukin-8 gene in radical prostatectomy specimens is associated with advanced pathologic stage.

BACKGROUND: We previously reported that relative expression of E-cadherin, matrix metalloproteinases (MMPs)-2 and -9, and vascular endothelial growth factor (VEGF)/vascular permeability factor in radical prostatectomy specimens (RP) can distinguish organ-confined cancers from advanced prostate cancers. Here, we evaluate the expression of interleukin-8 (IL-8) and basic fibroblast growth factor (bFGF), two other genes involved in angiogenesis and metastasis, in RP specimens. METHODS: The expression level of IL-8 and bFGF mRNA in the invasive edge of 41 prostate cancers of different stages was determined using a rapid colorimetric in situ hybridization (ISH) technique. Gene expression levels of IL-8 and bFGF were correlated with the Gleason score and pathologic stage to ascertain their relationship to prostate cancer progression. RESULTS: The expression of IL-8 and bFGF genes was detected by ISH in histologically normal prostate gland epithelium as well as in glands with foci of cancer. Increased mRNA expression of IL-8 was associated with both the Gleason score and pathologic stage of tumors and distinguished organ-confined from non-confined tumors (P = 0.002). In contrast, the expression of bFGF mRNA did not correlate with the Gleason score or pathologic stage. CONCLUSIONS: Overexpression of Il-8 mRNA, but not bFGF mRNA, in RP specimens is directly associated with progression of prostate cancer.

Tumor cell and endothelial cell therapy of oral cancer by dual tyrosine kinase receptor blockade.

Expression of the epidermal growth factor (EGF) and activation of its receptor (EGFR), a tyrosine kinase, are associated with progressive growth of head and neck cancer. Expression of the vascular endothelial growth factor (VEGF) is associated with angiogenesis and progressive growth of tumor. The tyrosine kinase inhibitor NVP-AEE788 (AEE788) blocks the EGF and VEGF signaling pathways. We examined the effects of AEE788 administered alone, or with paclitaxel (Taxol), on the progression of human head and neck cancer implanted orthotopically into nude mice. Cells of two different human oral cancer lines, JMAR and MDA1986, were injected into the tongues of nude mice. Mice with established tumors were randomized to receive three times per week oral AEE788, once weekly injected paclitaxel, AEE788 plus paclitaxel, or placebo. Oral tumors were resected at necropsy. Kinase activity, cell proliferation, apoptosis, and mean vessel density were determined by immunohistochemical immunofluorescent staining. AEE788 inhibited cell growth, induced apoptosis, and reduced the phosphorylation of EGFR, VEGFR-2, AKT, and mitogen-activated protein kinase in both cell lines. Mice treated with AEE788 and AEE788 plus paclitaxel had decreased microvessel density, decreased proliferative index, and increased apoptosis. Hence, AEE788 inhibited tumor vascularization and growth and prolonged survival. Inhibition of EGFR and VEGFR phosphorylation by AEE788 effectively inhibits cellular proliferation of squamous cell carcinoma of the head and neck, induces apoptosis of tumor endothelial cells and tumor cells, and is well tolerated in mice. These data recommend the consideration of patients with head and neck cancer for inclusion in clinical trials of AEE788.

Role of hypoxia-inducible factor 1alpha in gastric cancer cell growth, angiogenesis, and vessel maturation.

BACKGROUND: Hypoxia-inducible factor 1 (HIF-1), a heterodimer comprising the oxygen-regulated subunit, HIF-1alpha, and HIF-1beta, mediates transcription of the gene for vascular endothelial growth factor (VEGF). Overexpression of HIF-1alpha is associated with tumor angiogenesis and tumor cell proliferation and invasion. We examined the effects of inhibiting HIF-1alpha activity on angiogenesis and human gastric cancer growth in vivo. METHODS: Human gastric cancer TMK-1 cells were stably transfected with pHIF-1alphaDN, an expression plasmid encoding a dominant-negative form of HIF-1alpha that dimerizes with endogenous HIF-1beta to produce HIF-1 complexes that cannot activate transcription, or with the empty expression vector (pCEP4). Two clones of pHIF-1alphaDN-transfected cells, DN2 and DN3, were tested in all experiments. We used an enzyme-linked immunosorbent assay to measure VEGF secretion by transfected cells cultured in hypoxic (1% O2) or nonhypoxic (20% O2) conditions. We used subcutaneous and orthotopic mouse tumor models to examine the growth of tumors derived from injected pHIF-1alphaDN-or pCEP4-transfected cells. Tumor cell proliferation, vessel area (a measure of functional vascular volume), and tumor endothelial cell association with pericyte-like cells (a measure of vessel maturation) were analyzed by immunohistochemical or immunofluorescent staining. All statistical tests were two-sided. RESULTS: DN2 cells and DN3 cells secreted less VEGF than pCEP4-transfected TMK-1 cells when cultured in nonhypoxic or hypoxic conditions (e.g., DN2 versus pCEP4 in nonhypoxic conditions: 645 pg of VEGF/10(6) cells versus 1591 pg of VEGF/10(6) cells, difference = 946 pg of VEGF/10(6) cells [95% confidence interval [CI] = 640 to 1251 pg of VEGF/10(6) cells; P =.006]; DN2 versus pCEP4 in hypoxic conditions: 785 pg of VEGF/10(6) cells versus 2807 pg of VEGF/10(6) cells, difference = 2022 pg of VEGF/10(6) cells [95% CI = 1871 to 2152 pg of VEGF/10(6) cells; P<.001]). In the subcutaneous tumor model, tumors derived from DN2 or DN3 cells had lower final volumes, weights, and vessel areas, less tumor endothelial cell association with desmin-positive cells, and fewer proliferating tumor cells than tumors derived from pCEP4-transfected cells. In the orthotopic tumor model, tumors derived from DN2 cells had smaller volumes and less vessel area and maturation than tumors derived from pCEP4-transfected cells. CONCLUSIONS: Inhibition of HIF-1alpha activity impairs gastric tumor growth, angiogenesis, and vessel maturation.

Neuropilin-1 in human colon cancer: expression, regulation, and role in induction of angiogenesis.

Neuropilin-1 (NRP-1), a recently identified co-receptor for vascular endothelial growth factor, is expressed by several nongastrointestinal tumor types and enhances prostate cancer angiogenesis and growth in preclinical models. We investigated the expression and regulation of NRP-1 and the effect of NRP-1 overexpression on angiogenesis and growth of human colon adenocarcinoma by immunohistochemistry and in situ hybridization. NRP-1 was expressed in 20 of 20 human colon adenocarcinoma specimens but not in the adjacent nonmalignant colonic mucosa. By reverse transcriptase-polymerase chain reaction analysis, NRP-1 mRNA was expressed in seven of seven colon adenocarcinoma cell lines. Subcutaneous xenografts of stably transfected KM12SM/LM2 human colon cancer cells overexpressing NRP-1 led to increased tumor growth and angiogenesis in nude mice. In in vitro assays, conditioned medium from NRP-1-transfected cell lines led to an increase in endothelial cell migration, but did not affect endothelial cell growth. Epidermal growth factor (EGF) led to induction of NRP-1 in human colon adenocarcinoma cells and selective blockade of the epidermal growth factor receptor (EGFR) decreased constitutive and EGF-induced NRP-1 expression. Blockade of the Erk 1/2 and P38 mitogen-activated protein kinase signaling pathways also led to a decrease in constitutive and EGF-induced NRP-1 expression. These findings demonstrate the ubiquitous expression of NRP-1 in human colon cancer and suggest that NRP-1 may contribute to colon cancer angiogenesis and growth. This study also suggests that EGF and mitogen-activated protein kinase signaling pathways play an important role in NRP-1 regulation in colon cancer cells.

Expression of platelet-derived growth factor and activated receptor in clinical specimens of epithelial ovarian cancer and ovarian carcinoma cell lines.

OBJECTIVES: We determined the expression of platelet-derived growth factor (PDGF), PDGF-receptor (PDGF-R), and phosphorylated PDGF-R (p-PDGF-R) on tumor cells and tumor-associated endothelial cells in clinical specimens of human ovarian carcinoma and human ovarian cancer cells growing in culture and in the peritoneal cavity of nude mice. METHODS: Ten specimens of high-grade serous ovarian carcinoma were analyzed using immunohistochemistry (IHC). IHC was used to detect ligand and receptor expression in the human ovarian cancer cells from Hey A8 and SKOV3ip1 growing in culture. Cells from these lines were also implanted orthotopically into the peritoneal cavity of nude mice. IHC was used to determine ligand and receptor expression in tumors that formed in the peritoneal cavity. RESULTS: All 10 evaluable samples expressed both PDGF AA and BB on tumor cells. Tumor cells were positive for PDGF-Ralpha in 10/10 samples, PDGF-Rbeta in 8/10 samples, p-PDGF-Ralpha in 6/10 samples, and p-PDGF-Rbeta in 4/10 samples. p-PDGF-Ralpha was positive in 4/10 tumor-associated endothelial cell samples and p-PDGF-Rbeta was positive in 3/10 samples. Human ovarian cancer cells expressed PDGF, PDGF-R, and p-PDGF-R when growing in culture or in the peritoneal cavity of nude mice. PDGF-R and p-PDGF-R were also present on tumor-associated endothelial cells as demonstrated by simultaneous staining with CD31 antibody. CONCLUSIONS: PDGF and the corresponding receptors were expressed in autochthonous human ovarian cancer lesions on both tumor cells and tumor-associated endothelial cells. The ligand and receptor were also present on Hey A8 and SKOV3ip1 human ovarian cancer cells growing in vitro and in the peritoneal cavity of nude mice.

Synchronous overexpression of epidermal growth factor receptor and HER2-neu protein is a predictor of poor outcome in patients with stage I non-small cell lung cancer.

PURPOSE: Despite maximal therapy, surgically treated patients with stage I non-small cell lung cancer (NSCLC) are at risk for developing metastatic disease. Histopathologic findings cannot adequately predict disease progression, so there is a need to identify molecular factors that serve this purpose. Because the ErbB receptors play an important role in lung cancer progression, we analyzed the expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR, transforming growth factor alpha (TGFalpha), and HER2-neu as potential prognostic factors in stage I NSCLC. EXPERIMENTAL DESIGN: Using immunohistochemical techniques, we retrospectively analyzed formalin-fixed, paraffin-embedded samples from 111 patients with resected pathological stage I NSCLC. Then we correlated these data with patient clinical outcome. RESULTS: Median follow-up was 69.3 months. EGFR overexpression (defined as >10% membranous staining) was found in 66 tumors (59.5%). It was significantly more common in T(2) tumors than in T(1) tumors (P = 0.001), and in more squamous cell carcinomas than in adenocarcinomas (P = 0.07). HER2-neu overexpression was found in 19 tumors (17.1%) and was significantly more common in adenocarcinomas than in squamous cell carcinomas (P = 0.035). Synchronous overexpression of EGFR and HER2-neu was found in 11 tumors (9.9%). Patients with these tumors had a significantly shorter time to recurrence (P = 0.006) and a trend toward shorter overall survival (P = 0.093). Phosphorylated EGFR and transforming growth factor alpha were detected but were not related to prognosis. CONCLUSIONS: Synchronous overexpression of EGFR and HER2-neu at the protein level predicts increased recurrence risk and may predict decreased survival in patients with stage I NSCLC. This suggests that important interactions take place among the different members of the ErbB family during tumor development and suggests a method for choosing targeted therapy. A prospective study is planned.

Bioassay using epidermal keratinocytes to determine phosphorylation status of the epidermal growth factor receptor in distant neoplasms.

We developed a bioassay to evaluate the phosphorylation status of a fibrosarcoma following systemic administration of the protein tyrosine kinase inhibitor PKI 166. Samples of subcutaneous fibrosarcomas and distant skin were fixed in formalin, sectioned, and stained with several fluorescent antibodies against the epidermal growth factor receptor (EGF-R) and phosphorylated EGF-R. In mice given different doses of PKI 166, the dose-dependent inhibition of phosphorylation of EGF-R in epidermal keratinocytes paralleled that in fibrosarcomas growing subcutaneously, suggesting that skin biopsies can be used as surrogate tissues for distant neoplasms to determine the phosphorylation status of protein tyrosine kinase receptors.

Inhibition of platelet-derived growth factor receptor phosphorylation by STI571 (Gleevec) reduces growth and metastasis of human pancreatic carcinoma in an orthotopic nude mouse model.

PURPOSE: We evaluated the expression of platelet-derived growth factor (PDGF) ligands and receptors in clinical specimens of human pancreatic adenocarcinomas and determined the therapeutic effect of STI571 (Gleevec), a protein tyrosine kinase inhibitor of PDGF receptor (PDGFR), on human pancreatic carcinoma cells growing in the pancreas and liver of nude mice. EXPERIMENTAL DESIGN: Immunohistochemical staining for PDGF-AA and -BB ligands, PDGFR-alpha and -beta, and phosphorylated PDGFR-alpha and -beta was performed on 31 specimens of human pancreatic cancer and L3.6pl human pancreatic adenocarcinoma cell line. To determine the in vivo effects of STI571, nude mice with L3.6pl cells injected into the pancreas were randomized 7 days later to receive one of the following treatments: sterile water p.o. (control), STI571, gemcitabine, or a combination of STI571 and gemcitabine. RESULTS: In 29 of 31 clinical specimens of human pancreatic adenocarcinoma, both tumor cells and tumor-associated endothelial cells expressed phosphorylated PDGFR-alpha and -beta. L3.6pl cells growing in culture expressed moderate amounts of PDGF-AA and little to no PDGFR-alpha or -beta, whereas L3.6pl cells growing in the pancreas of nude mice expressed a high level of PDGF and receptors. Colocalization immunohistochemical analysis demonstrated expression of activated PDGFR-beta by tumor-associated endothelial cells in both the pancreas and in liver metastases. Tumors of mice treated for 4 weeks with STI571 (50 mg/kg or 100 mg/kg p.o. daily) were slightly smaller than controls. Tumors treated with gemcitabine and STI571 (50 mg/kg) were >70% smaller than tumors in control mice and 36% smaller than those in mice treated with gemcitabine only (P < 0.0002 and P < 0.04, respectively). Combination therapy also inhibited spontaneous metastasis to the liver. Tumors from mice treated with both STI571 and gemcitabine had decreased expression of activated (phosphorylated) PDGFR-alpha and -beta, decreased mean vessel density, decreased cell proliferation, and increased apoptosis of tumor cells. CONCLUSIONS: Collectively, these data show that activated PDGFR on tumor cells and tumor-endothelial cells can be a novel target for therapy of pancreatic carcinoma.

Regulation of hypoxia-inducible factor-1alpha, vascular endothelial growth factor, and angiogenesis by an insulin-like growth factor-I receptor autocrine loop in human pancreatic cancer.

Activation of the insulin-like growth factor-I receptor (IGF-IR) was recently shown to modulate angiogenesis by up-regulating the expression of vascular endothelial growth factor (VEGF). We hypothesized that inhibiting IGF-IR function would inhibit angiogenesis and growth of pancreatic cancer in vivo and sought to identify major signaling pathways regulated by IGF-IR in pancreatic cancer cells. Human pancreatic cancer cells (L3.6pl) were stably transfected with a dominant-negative form of IGF-IR (IGF-IR DN) or an empty vector (pcDNA). In vitro, IGF-IR DN cells exhibited a decrease in both constitutive and inducible phosphorylation of IGF-IR and Erk1/2. Constitutive expression of nuclear hypoxia-inducible factor-1alpha and secreted VEGF (P < 0.01) protein levels also were significantly lower in IGF-IR DN cells than in pcDNA cells. In vivo, IGF-IR inhibition led to decreases in pancreatic tumor volume and weight, vessel density, and tumor cell proliferation (P < 0.01 for all) and increases in tumor cell apoptosis (P < 0.02). Our results suggest that autocrine activation of the IGF-IR system significantly affects VEGF expression and angiogenesis in human pancreatic cancer. Thus, IGF-IR may be a valid target in the treatment of pancreatic cancer.