Blocking platelet-derived growth factor-D/platelet-derived growth factor receptor beta signaling inhibits human renal cell carcinoma progression in an orthotopic mouse model

Renal cell carcinoma is a highly malignant and often fatal disease of the kidney. It is difficult to treat, often because metastases are common at the time of presentation. Platelet-derived growth factor-D (PDGF-D) is a newly discovered member of the PDGF family; its function in tumor progression is largely unknown. Here, we examined the expression level of PDGF-D in human renal cell carcinoma by immunohistochemical staining using tissue arrays. We showed that human renal cell carcinoma expresses high levels of PDGF-D protein. The human renal cell carcinoma cell line SN12-C was stably transfected with pdgf-d cDNA. Overexpression of PDGF-D in SN12-C cells promoted tumor growth, angiogenesis, and metastasis of human renal cell carcinoma in an orthotopic severe combined immunodeficient (SCID) mouse model. PDGF-D overproduction in SN12-C cells increased the proliferation and migration of mural cells in vitro and improved perivascular cell coverage in vivo. Overexpression of PDGF-D led to increased expression of angiopoietin-1 and matrix metalloproteinase-9 in tumor tissues. ShRNAi and Gleevec were used to block PDGF-D expression and PDGF receptor beta (PDGFRbeta) signaling. Inhibition of PDGF-D expression by short hairpin RNA interference (shRNAi) and blockage of PDGFRbeta signaling by Gleevec inhibited the growth and lung metastasis of SN12-C cells grown orthotopically in SCID mice. Thus, PDGF-D is a potential candidate for controlling the progression of metastatic renal cell carcinoma. This opens up an avenue of investigation into novel therapeutic strategies for the treatment of renal cell carcinoma, including the use of recently developed tyrosine kinase inhibitors, such as Gleevec, which inhibit PDGF activity through inhibition of its receptor tyrosine kinase.     

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.     

Dual inhibition of epidermal growth factor receptor and vascular endothelial growth factor receptor phosphorylation by AEE788 reduces growth and metastasis of human colon carcinoma in an orthotopic nude mouse model

We studied growth factors and their receptors in tumor cells and tumor-associated endothelial cells as the therapeutic targets in colon cancer. Immunohistochemical analysis of 13 surgical specimens of human colon adenocarcinoma revealed that both tumor cells and tumor-associated endothelial cells in 11 of the 13 specimens expressed the epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), EGF receptor (EGFR), phosphorylated EGFR (pEGFR), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and phosphorylated VEGFR (pVEGFR). HT29 human colon cancer cells growing orthotopically in the cecum of nude mice expressed a high level of EGF, EGFR, pEGFR, VEGF, VEGFR, and pVEGFR. Double-immunofluorescence staining found that tumor-associated mouse endothelial cells also expressed pEGFR and pVEGFR. Tumors in mice treated for 5 weeks with oral AEE788 (an inhibitor of EGFR and VEGFR tyrosine kinase) as a single agent or with CPT-11 alone were smaller (>50%) than those in control mice. Mice treated with the combination of AEE788 and CPT-11 had significantly smaller tumors (P < 0.01) and complete inhibition of lymph node metastasis. AEE788 alone or in combination with CPT-11 inhibited pEGFR, pVEGFR, and phosphorylated Akt expression on tumor-associated endothelial cells as well as on tumor cells. The combination therapy also significantly decreased microvessel density and tumor cell proliferation and increased the level of apoptosis in both tumor cells and tumor-associated endothelial cells. Collectively, these data suggest that the dual inhibition of EGFR and VEGFR signaling pathways in tumor cells and tumor-associated endothelial cells in combination with chemotherapy can provide a new approach to the treatment of colon cancer.     

Anti-stromal therapy with imatinib inhibits growth and metastasis of gastric carcinoma in an orthotopic nude mouse model

Recent studies have revealed that platelet-derived growth factor (PDGF) plays a role in promoting progressive tumor growth in several organs; however, whether PDGF plays such a role in gastric carcinoma is undetermined. We examined whether inhibition of PDGF receptor (PDGF-R) tyrosine kinase signaling by imatinib affects tumor growth and metastasis in an orthotopic nude mouse model of human gastric carcinoma. TMK-1 human gastric carcinoma cells were injected into the gastric wall of nude mice. Groups of mice (n = 10 each) received sterile water (control), low-dose imatinib (50 mg/kg/day), high-dose imatinib (200 mg/kg/day), cancer chemotherapeutic agent irinotecan (5 mg/kg/week), or imatinib (50 mg/kg/day or 200 mg/kg/day) and irinotecan (5 mg/kg/week) in combination for 28 days. Tumor growth and metastasis were assessed. Resected tumors were analyzed immunohistochemically. Carcinoma-associated fibroblasts, pericytes and lymphatic endothelial cells in stroma expressed high levels of PDGF-R; carcinoma cells did not. Treatment with imatinib alone did not inhibit tumor growth and metastasis; however, treatment with irinotecan alone or combined with imatinib significantly inhibited tumor growth. Only treatment with high-dose imatinib and irinotecan in combination inhibited lymph node and peritoneal metastases. Immunohistochemically, only imatinib alone or in combination with irinotecan was shown to significantly decrease the stromal reaction, microvessel area and pericyte coverage of tumor microvessels. These effects were marked with high-dose imatinib. In conclusion, administration of PDGF-R tyrosine kinase inhibitor in combination with irinotecan appears to impair the progressive growth of gastric carcinoma by blockade of PDGF-R signaling pathways in stromal cells.     

Vandetanib inhibits growth of adenoid cystic carcinoma in an orthotopic nude mouse model

PURPOSE: Adenoid cystic carcinoma (ACC) can often be controlled with surgery and postoperative adjuvant radiotherapy but is also characterized by late local recurrence and distant metastasis. No effective systemic therapeutic agents have been found to alter the natural history of ACC. Therefore, new therapeutic approaches are needed. In this study, we evaluated whether vandetanib (Zactima), a potent inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) tyrosine kinases, had antitumor efficacy in vitro and in an orthotopic nude mouse model of human ACC. EXPERIMENTAL DESIGN: The in vitro effects of vandetanib were assessed in three ACC cell lines on cell growth, apoptosis, and VEGFR-2 and EGFR phosphorylation levels. The in vivo antitumor activity of vandetanib was examined in nude mice bearing parotid gland ACC tumors. The mice were treated for 4 weeks with vandetanib (50 mg/kg/d) or placebo (control). Tumors were resected at necropsy, and immunohistochemical and immunofluorescence staining were done. RESULTS: In vitro, vandetanib caused dose-dependent inhibition of VEGFR-2 and EGFR phosphorylation in ACC cells. Vandetanib also inhibited the cell proliferation and induced their dose-dependent apoptosis. In vivo, mice in the vandetanib group had tumor volumes significantly lower than those in the control group (P < 0.01). In addition, immunohistochemical staining showed a decrease in microvessel density and an increase in apoptosis of both tumor cells and endothelial cells within the tumor xenografts. CONCLUSION: These results suggest that vandetanib inhibits the growth of ACC in vitro and in vivo, making it a promising novel agent for the treatment of ACC.     

Zyflamend suppresses growth and sensitizes human pancreatic tumors to gemcitabine in an orthotopic mouse model through modulation of multiple targets

Agents that can potentiate the efficacy of standard chemotherapy against pancreatic cancer are of great interest. Because of their low cost and safety, patients commonly use a variety of dietary supplements, although evidence of their efficacy is often lacking. One such commonly used food supplement is Zyflamend, a polyherbal preparation with potent anti-inflammatory activities and preclinical efficacy against prostate and oral cancer. Whether Zyflamend has any efficacy against human pancreatic cancer alone or in combination with gemcitibine, a commonly used agent, was examined in cell cultures and in an orthotopic mouse model. In vitro, Zyflamend inhibited the proliferation of pancreatic cancer cell lines regardless of p53 status and also enhanced gemcitabine-induced apoptosis. This finding correlated with inhibition of NF-κB activation by Zyflamend and suppression of cyclin D1, c-myc, COX-2, Bcl-2, IAP, survivin, VEGF, ICAM-1 and CXCR4. In nude mice, oral administration of Zyflamend alone significantly inhibited the growth of orthotopically transplanted human pancreatic tumors, and when combined with gemcitabine, further enhanced the antitumor effects. Immunohistochemical and Western blot analyses of tumor tissue showed that the suppression of pancreatic cancer growth correlated with inhibition of proliferation index marker (Ki-67), COX-2, MMP-9, NF-κB and VEGF. Overall, these results suggest that the concentrated multiherb product Zyflamend alone can inhibit the growth of human pancreatic tumors and, in addition, can sensitize pancreatic cancers to gemcitabine through the suppression of multiple targets linked to tumorigenesis.     

Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator in tumor-stromal interactions through enhancing tumor invasion and metastasis. We found recently that NK4, a four-kringle fragment of HGF, functions as both an HGF-antagonist and an angiogenesis inhibitor. We have now determined whether blockade of the HGF-c-Met/HGF receptor pathway and tumor angiogenesis by administration of recombinant NK4 would inhibit growth, invasion, and metastasis of human pancreatic carcinoma implanted into the pancreas of nude mice. When treatment with NK4 or anti-HGF neutralizing antibody was initiated from the third day after orthotopic injection of SUIT-2 human pancreatic cancer cells, both NK4 and anti-HGF antibody suppressed the conversion of orthotopic pancreatic tumors from carcinoma in situ to aberrantly invading cancers during days 3-14. On the other hand, when the treatment was begun on day 10, a time when cancer cells were already invading surrounding tissues, NK4 but not anti-HGF antibody inhibited tumor growth, peritoneal dissemination, and ascites accumulation at 4 weeks after the inoculation. Antitumor effects of NK4 correlated with decreased microvessel density in pancreatic tumors thereby indicating that the antiangiogenic activity of NK4 may have mainly contributed to its antitumor effects. Moreover, although NK4-treatment was initiated from the end stage (day 24 after tumor inoculation), NK4 prolonged survival time of mice, and the suppression of peritoneal dissemination, ascites accumulation, and invasion of metastasized cancer cells into the peritoneal wall were remarkable. We propose that simultaneous targeting of both tumor angiogenesis and the HGF-mediated invasion-metastasis may prove to be a new approach to treating patients with pancreatic cancer.     

Therapeutic synergism of gemcitabine and CpG-oligodeoxynucleotides in an orthotopic human pancreatic carcinoma xenograft

CpG-oligodeoxynucleotides (CpG-ODN) exhibit potent immunostimulatory activity by binding with Toll-like receptor 9 (TLR9). Based on the finding that TLR9 is highly expressed and functional in pancreatic tissue, we evaluated the antitumor effects of chemotherapy combined with CpG-ODNs in the orthotopic mouse model of a human pancreatic tumor xenograft. Chemotherapy consisted of the maximum tolerated dose of gemcitabine (i.v., 100 mg/kg, q3dx4). CpG-ODNs were delivered (i.p., 20 microg/mouse), weekly, after the end of chemotherapy. CpG-ODNs alone had little effect on tumor growth, whereas gemcitabine alone significantly delayed the median time of disease onset (palpable i.p. tumor) and of bulky disease development (extensive peritoneal tumor burden), but did not enhance survival time. When the gemcitabine regimen was followed by administration of the immunostimulator, development of bulky disease was delayed, survival time was significantly improved (median survival time, 106 days; P < 0.02 versus gemcitabine-treated mice). Autoptic examination showed that tumor spread in the peritoneal cavity was reduced to a greater extent than with gemcitabine alone. All treatment regimens were well-tolerated. The use of nude mice excluded a T cell-mediated immune response, whereas the high pancreatic expression of TLR9 might have contributed to the tumor response. The clear improvement of survival observed in an orthotopic murine model of human pancreatic cancer by the combined use of CpG-ODNs with chemotherapy suggests the promise of this therapeutic regimen in the clinical setting.     

Magnetic resonance imaging in an orthotopic rat model: blockade of epidermal growth factor receptor with EMD72000 inhibits human pancreatic carcinoma growth

The purpose of our research was to investigate the antiangiogenic effect of the epidermal growth factor receptor monoclonal antibody (anti-EGF-R MAB) EMD72000, in an orthotopic human pancreatic carcinoma model in rats, assessed by magnetic resonance (MR) imaging using angiogenic surrogate markers in comparison with histopathologic findings. Human pancreatic adenocarcinoma cells L3.6pl were injected orthotopically in the pancreas of 12 athymic nude rats. Through a 21-day course, groups of 6 rats were treated intraperitoneally with either EMD72000 or with saline solution for control animals. Dynamic contrast-enhanced MR imaging was performed before and after the treatment to assess microvascular permeability, estimated by the endothelial transfer coefficient (KPS) and fractional plasma volumes (fPV) of the pancreatic tumors. EMD72000-treated animals showed significantly less tumor volume progression (1,080 mm3 +/- 1,244; p = 0.012) and significantly lower values for microvascular permeability (KPS = 4.2 ml min(-1) 100 ml(-1) of tissue +/- 2.8; p = 0.015), fractional plasma volume (fPV = 0.018 ml ml(-1) of tissue +/- .015; p = 0.003) and microvessel density (MVD = 13 +/- 4 (0.159 mm2); p = 0.001) than saline-treated animals (6,544 mm3 +/- 5,202; 9.5 ml min(-1) 100 ml(-1) of tissue +/- 4.3, 0.056 ml ml(-1) of tissue +/- 0.019 and 25 +/- 5 (0.159 mm2), respectively). KPS and fPV values showed moderate positive correlation with MVD (r = 0.5, p = 0.103; r = 0.6, p = 0.065, respectively). Intraperitoneal injection of EMD72000 inhibits orthotopic human pancreatic carcinoma growth in rats. Antiangiogenic effects of anti-EGF-R MAB EMD72000 can be quantified and monitored noninvasively by dynamic MR imaging.     

The guanine nucleotide exchange factor Tiam1 increases colon carcinoma growth at metastatic sites in an orthotopic nude mouse model

Alterations in migration and adhesion are critical to invasion and metastasis. To examine signaling pathways important for colon tumor metastasis, cells of increased migratory potential from the low migratory SW480 human colorectal carcinoma parental cell line were biologically selected by serial migration through modified Boyden chambers. Several sublines were obtained with statistically significantly increased migration relative to the parental cell line. One highly migratory population was single-cell cloned and characterized. The migratory clones exhibit a four- to five-fold increase in protein and mRNA expression of T-lymphoma invasion and metastasis gene 1 (Tiam1), a guanine nucleotide exchange factor. To determine directly the role of Tiam1 in the migration of these migratory sublines, the parental SW480 cell line was transfected with a plasmid encoding the Tiam1 protein, and single cell clones were established. Ectopic expression of Tiam1 in these clones led to morphologic changes identical to biologically selected clones and increased migration. Finally, the implantation of clones that overexpress Tiam1 into the cecum of athymic mice resulted in tumor growth in the spleen, liver, and lung, whereas parental cells do not form tumors by this route of injection. These results demonstrate that overexpression of Tiam1 contributes to the metastatic phenotype of colon cancer cells.     

Targeting the platelet-derived growth factor receptor in antivascular therapy for human ovarian carcinoma.

PURPOSE: We sought to determine whether blockade of platelet-derived growth factor receptor (PDGF-R) activation by oral administration of a PDGF-R tyrosine kinase inhibitor (STI571) alone or in combination with i.p. paclitaxel can inhibit the progression of tumors caused by human ovarian carcinoma cells growing in the peritoneal cavity of female nude mice. EXPERIMENTAL DESIGN: In several different experiments, paclitaxel-sensitive and paclitaxel-resistant metastatic human ovarian carcinoma cells were injected into the peritoneal cavity of nude mice. Seven days later, groups (n = 10) of mice began receiving a control treatment, STI571 alone, paclitaxel alone, or a combination of STI571 and paclitaxel. The mice were necropsied after 45 days of treatment. RESULTS: Treatment with combination therapy significantly reduced tumor weight (relative to control or single-agent therapy) in all three human ovarian cancer cell lines. Immunohistochemical analyses revealed that PDGF-R activation was blocked by STI571 administered alone or in combination with paclitaxel. Tumor-associated endothelial cells expressed both PDGF-R and phosphorylated PDGF-R. In mice receiving combination therapy, tumor-associated endothelial cells underwent apoptosis, leading to decreases in microvessel density and tumor cell proliferation relative to control and single-agent therapy. CONCLUSIONS: These results show that administration of a PDGF-R tyrosine kinase inhibitor in combination with paclitaxel impairs the progression of ovarian cancer in the peritoneal cavity of nude mice, in part, by blockade of PDGF, an endothelial cell survival factor, which results in the increased apoptosis of tumor-associated endothelial cells.     

Optimization for the blockade of epidermal growth factor receptor signaling for therapy of human pancreatic carcinoma.

We determined the optimal administration schedule of a novel epidermal growth factor receptor (EGFR) protein tyrosine kinase inhibitor (PKI), PKI 166 (4-(R)-phenethylamino-6-(hydroxyl)phenyl-7H-pyrrolo[2.3-d]-pyrimidine), alone or in combination with gemcitabine (administered i.p.) for therapy of L3.6pl human pancreatic carcinoma growing in the pancreas of nude mice. Seven days after orthotopic implantation of L3.6pl cells, the mice received daily oral doses of PKI 166. PKI 166 therapy significantly inhibited phosphorylation of the EGFR without affecting EGFR expression. EGFR phosphorylation was restored 72 h after cessation of therapy. Seven days after orthotopic injection of L3.6pl cells, groups of mice received daily or thrice weekly oral doses of PKI 166 alone or in combination with gemcitabine. Treatment with PKI 166 (daily), PKI 166 (3 times/week), or gemcitabine alone produced a 72%, 69%, or 70% reduction in the volume of pancreatic tumors in mice, respectively. Daily oral PKI 166 or thrice weekly oral PKI 166 in combination with injected gemcitabine produced 97% and 95% decreases in volume of pancreatic cancers and significant inhibition of lymph node and liver metastasis. Daily oral PKI 166 produced a 20% decrease in body weight, whereas treatment 3 times/week did not. Decreased microvessel density, decreased proliferating cell nuclear antigen staining, and increased tumor cell and endothelial cell apoptosis correlated with therapeutic success. Collectively, our results demonstrate that three weekly oral administrations of an EGFR tyrosine kinase inhibitor in combination with gemcitabine are sufficient to significantly inhibit primary and metastatic human pancreatic carcinoma.     

Peritumoral injection of adenovirus vector expressing NK4 combined with gemcitabine treatment suppresses growth and metastasis of human pancreatic cancer cells implanted orthotopically in nude mice and prolongs survival

NK4 or adenovirus vector expressing NK4 (Ad-NK4) can act bifunctionally as a hepatocyte growth factor antagonist and angiogenesis inhibitor and has potential value in cancer therapy. The aim of this study was to evaluate the therapeutic efficacy of Ad-NK4 in combination with gemcitabine (GEM) against pancreatic cancer. In vitro study showed a strong antiproliferative effect of GEM and a potent anti-invasive effect of Ad-NK4 against pancreatic cancer cells. In in vivo experiments, SUIT-2 human pancreatic cancer cells were implanted into the pancreas of nude mice. Mice were treated with Ad-NK4 by injection into the peritumoral region of the pancreas on day 5 after implantation followed by weekly i.p. injections of GEM. On day 28 after implantation, pancreatic tumor volume was significantly smaller than that in mice treated with Ad-LacZ, Ad-NK4 alone, or GEM alone. Furthermore, combination therapy completely suppressed peritoneal dissemination and liver metastases, leading to significantly increased survival. Histologic and immunohistochemical assays of primary tumors indicated that combination therapy prohibited both cell proliferation and angiogenesis, resulting in high levels of apoptosis. These results suggest that peritumoral injection of Ad-NK4 plus GEM is a potent combination therapy for pancreatic cancer.     

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.     

Blockade of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling for therapy of metastatic human pancreatic cancer

We determined whether concurrent blockage of vascular endothelial growth factor (VEGF) receptor and epidermal growth factor (EGF) receptor signaling by two novel tyrosine kinase inhibitors, PTK 787 and PKI 166, respectively, can inhibit angiogenesis and, hence, the growth and metastasis of human pancreatic carcinoma in nude mice. Highly metastatic human pancreatic carcinoma L3.6pl cells were injected into the pancreas of nude mice. Seven days later, groups of mice began receiving oral doses of PTK 787 and PKI 166 three times weekly. Some groups of mice also received i.p. injections of gemcitabine twice a week. The mice were necropsied when the control mice became moribund. Treatment with PTK 787 and PKI 166, with gemcitabine alone, or with the combination of PTK 787, PKI 166, and gemcitabine produced 69, 50, and 97% reduction in the volume of pancreatic tumors, respectively. Administration of protein tyrosine kinase inhibitors and gemcitabine also significantly decreased the incidence of lymph node and liver metastasis. The therapeutic efficacy directly correlated with a decrease in circulating proangiogenic molecules (VEGF, interleukin-8), a decrease in microvessel density, a decrease in proliferating cell nuclear antigen staining, and an increase in apoptosis of tumor cells and endothelial cells. Therapies produced by combining gemcitabine with either PKI 166 or PTK 787 were similar to those produced by combining gemcitabine with both PKI 166 and PTK 787. These results suggest that blockade of either epidermal growth factor receptor or VEGF receptor signaling combined with chemotherapy provides an effective approach to the therapy of pancreatic cancer.     

Inhibition of platelet-derived growth factor receptor signaling restricts the growth of human breast cancer in the bone of nude mice.

PURPOSE: Bone is a common site for breast cancer metastasis. Platelet-derived growth factor (PDGF) and PDGF receptors (PDGFR) are involved in the regulation of bone resorption. This study examined the effects of STI571 (imatinib mesylate), which inhibits PDGFR tyrosine kinase signaling, on the growth of human breast cancer cells in the bone of nude mice with consequent osteolysis. EXPERIMENTAL DESIGN: Human breast cancer MDA-MB-435 cells were injected into the tibia of female nude mice. Two weeks later the mice were treated with p.o. and injected water (control), daily p.o. STI571, weekly injection of paclitaxel, or daily STI571, plus weekly paclitaxel, for up to 8 weeks. Growth of tumors in bones and osteolysis were monitored by digital radiography and tumors were collected for histochemical analysis. RESULTS: Mice treated with STI571 or STI571 plus paclitaxel had smaller bone tumors with less lytic bone destruction than did mice treated with water or paclitaxel alone. The results of treatment with paclitaxel plus STI571 did not differ from those with STI571 alone. Immunohistochemistry showed that PDGF-A, PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in the bone tumors. STI571 treatment inhibited PDGFR phosphorylation in tumor cells and tumor-associated endothelial cells, coincident with increased apoptosis, reduced proliferation, and lower microvessel density in the tumors. CONCLUSIONS: Activated PDGFRs are expressed by endothelial and tumor cells in breast cancer tumors growing in the bone of nude mice. Interfering with PDGFR signaling may be an approach to control the progressive growth of breast cancer cells and thus reduce bone lysis.     

Proteasome inhibition activates epidermal growth factor receptor (EGFR) and EGFR-independent mitogenic kinase signaling pathways in pancreatic cancer cells.

PURPOSE: In the current study, we investigate the activation of antiapoptotic signaling pathways in response to proteasome inhibitor treatment in pancreatic cancer and evaluate the use of concomitant inhibition of these pathways to augment proteasome inhibitor treatment responses. EXPERIMENTAL DESIGN: Pancreatic cancer cell lines and mouse flank xenografts were treated with proteasome inhibitor alone or in combination with chemotherapeutic compounds (gemcitabine, erlotinib, and bevacizumab), induction of apoptosis and effects on tumor growth were assessed. The effect of bortezomib (a first-generation proteasome inhibitor) and NPI-0052 (a second-generation proteasome inhibitor) treatment on key pancreatic mitogenic and antiapoptotic pathways [epidermal growth factor receptor, extracellular signal-regulated kinase, and phosphoinositide-3-kinase (PI3K)/AKT] was determined and the ability of inhibitors of these pathways to enhance the effects of proteasome inhibition was assessed in vitro and in vivo. RESULTS: Our data showed that proteasome inhibitor treatment activates antiapoptotic and mitogenic signaling pathways (epidermal growth factor receptor, extracellular signal-regulated kinase, c-Jun-NH2-kinase, and PI3K/AKT) in pancreatic cancer. Additionally, we found that activation of these pathways impairs tumor response to proteasome inhibitor treatment and inhibition of the c-Jun-NH2-kinase and PI3K/AKT pathways increases the antitumor effects of proteasome inhibitor treatment. CONCLUSION: These preclinical studies suggest that targeting proteasome inhibitor-induced antiapoptotic signaling pathways in combination with proteasome inhibition may augment treatment response in highly resistant solid organ malignancies. Further evaluation of these novel treatment combinations in clinical trials is warranted.     

Blockade of epidermal growth factor receptor signaling leads to inhibition of renal cell carcinoma growth in the bone of nude mice

Renal cell carcinoma (RCC) frequently produces metastases to the musculoskeletal system that are a major source of morbidity in the form of pain, immobilization, fractures, neurological compromise, and a decreased ability to perform activities of daily living. Patients with metastatic RCC therefore have a dismal prognosis because there is no effective adjuvant treatment for this disease. Because the epidermal growth factor receptor (EGF-R) signaling cascade is important in the growth and metastasis of RCC, its blockade has been hypothesized to inhibit tumor growth and hence prevent resultant bone destruction. We determined whether blockade of EGF-R by the tyrosine kinase inhibitor PKI 166 inhibited the growth of RCC in bone. We use a novel cell line, RBM1-IT4, established from a human RCC bone metastasis. Protein and mRNA expression of the ligands and receptors was assessed by Western and Northern blots. The stimulation of RBM1-IT4 cells with epidermal growth factor or transforming growth factor alpha resulted in increased cellular proliferation and tyrosine kinase autophosphorylation. PKI 166 prevented these effects. First, RBM1-IT4 cells were implanted into the tibia of nude mice, where they established lytic, progressively growing lesions, after which the mice were treated with PKI 166 alone or in combination with paclitaxel (Taxol). Immunohistochemical analysis revealed that tumor cells and tumor-associated endothelial cells in control mice expressed activated EGF-R. Treatment of mice with PKI 166 alone or in combination with Taxol produced a significant decrease in the incidence and size of bone lesions as compared with the results in control or Taxol-treated mice (P < 0.001). Treatment with PKI 166 also decreased the expression of phosphorylated EGF-R by tumor cells and tumor-associated endothelial cells, and this was even more pronounced with PKI 166 plus Taxol treatment. The PKI 166 plus Taxol combination produced apoptosis of tumor cells and tumor-associated endothelial cells. Tumor cell proliferation, shown by proliferating cell nuclear antigen positivity, was decreased in all treatment groups. In addition, the integrity of the bone was maintained in mice treated with PKI 166 or PKI 166 plus Taxol, whereas massive bone destruction was seen in control and Taxol-treated mice. These results suggest that blockade of EGF-R signaling inhibits growth of RCC in the bone by its effect on tumor cells and tumor-associated endothelial cells.     

Synergistic antitumor activity of ZD6474, an inhibitor of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling, with gemcitabine and ionizing radiation against pancreatic cancer.

PURPOSE: Standard treatments have modest effect against pancreatic cancer, and current research focuses on agents targeting molecular pathways involved in tumor growth and angiogenesis. This study investigated the interactions between ZD6474, an inhibitor of tyrosine kinase activities of vascular endothelial growth factor receptor-2 and epidermal growth factor receptor (EGFR), gemcitabine, and ionizing radiation in human pancreatic cancer cells and analyzed the molecular mechanisms underlying this combination. EXPERIMENTAL DESIGN: ZD6474, ionizing radiation, and gemcitabine, alone or in combination, were given in vitro to MIA PaCa-2, PANC-1, and Capan-1 cells and in vivo to MIA PaCa-2 tumor xenografts. The effects of treatments were studied by the evaluation of cytotoxicity, apoptosis, cell cycle, EGFR and Akt phosphorylation, modulation of gene expression of enzymes related to gemcitabine activity (deoxycytidine kinase and ribonucleotide reductase), as well as vascular endothelial growth factor immunohistochemistry and microvessel count. RESULTS: In vitro, ZD6474 dose dependently inhibited cell growth, induced apoptosis, and synergistically enhanced the cytotoxic activity of gemcitabine and ionizing radiation. Moreover, ZD6474 inhibited phosphorylation of EGFR and Akt and triggered cell apoptosis. PCR analysis showed that ZD6474 increased the ratio between gene expression of deoxycytidine kinase and ribonucleotide reductase. In vivo, ZD6474 showed significant antitumor activity alone and in combination with radiotherapy and gemcitabine, and the combination of all three modalities enhanced MIA PaCA-2 tumor growth inhibition compared with gemcitabine alone. CONCLUSIONS: ZD6474 decreases EGFR and Akt phosphorylation, enhances apoptosis, favorably modulates gene expression in cancer cells, and acts synergistically with gemcitabine and radiotherapy to inhibit tumor growth. These findings support the investigation of this combination in the clinical setting.     

Induction of apoptosis in tumor-associated endothelial cells and therapy of orthotopic human pancreatic carcinoma in nude mice

Although gemcitabine has been accepted as the first-line chemotherapeutic reagent for advanced pancreatic cancer, improvement of response rate and survival is not sufficient and patients often develop resistance. We hypothesized that the inhibition of phosphorylation of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) on tumor cells and tumor-associated endothelial cells, combined with gemcitabine, would overcome the resistance to gemcitabine in orthotopic pancreatic tumor animal model. L3.6pl, human pancreatic cancer cells growing in the pancreas, and tumor-associated endothelial cells in microorgan environment highly expressed phosphorylated EGFR, VEGFR, and Akt, which regulates antiapoptotic mechanism. Oral administration of AEE788 (dual tyrosine kinase inhibitor against EGFR and VEGFR) inhibited the phosphorylation of EGFR, VEGFR, and Akt on tumor-associated endothelial cells as well as tumor cells. Although intraperitoneal (i.p.) injection of gemcitabine showed limited inhibitory effect on tumor growth, combination with AEE788 and gemcitabine produced nearly 95% inhibition of tumor growth in parallel with a high level of apoptosis on tumor cells and tumor-associated endothelial cells, and decreased microvascular density and proliferation rate. Collectively, these data indicate that dual inhibition of phosphorylation of EGFR and VEGFR, in combination with gemcitabine, produces apoptosis of tumor-associated endothelial cells and significantly suppresses human pancreatic cancer in nude mice.