Marya F. McCarty

Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma

Interleukin-8 (IL-8) has recently been shown to contribute to human melanoma progression by functioning as a mitogenic and angiogenic factor. In the present study, we investigated whether targeting IL-8 by a fully human anti-IL-8 antibody (ABX-IL8) could be a potential therapeutic strategy to control angiogenesis, growth, and metastasis of melanoma. The human melanoma cells A375SM (high IL-8 producer) and TXM-13 (intermediate IL-8 producer) were injected subcutaneously into nude mice, which were then treated with ABX-IL8 (1 mg/3 times weekly, i.p., for 3 weeks). Tumor growth of both melanomas in ABX-IL8-treated mice was significantly inhibited when compared with control IgG-treated animals. ABX-IL8 treatment also suppressed experimental metastasis when the melanoma cells were injected intravenously. IL-8 blockade by ABX-IL8 significantly inhibited the promoter activity and the collagenase activity of matrix metalloproteinase-2 in human melanoma cells, resulting in decreased invasion through reconstituted basement membrane in vitro. In vivo, ABX-IL8 treatment resulted in decreased expression of matrix metalloproteinase-2, and decreased vascularization (angiogenesis) of tumors concomitant with increased apoptosis of tumor cells. Moreover, in an in vitro vessel formation assay, ABX-IL8 directly interfered with the tubule formation by human umbilical vein endothelial cells. Taken together, these results point to the potential utility of ABX-IL8 as a modality to treat melanoma and other solid tumors either alone or in combination with conventional chemotherapy or other anti-tumor agents.

Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells.

Vascular endothelial growth factor (VEGF) is associated with tumor angiogenesis and poor prognosis in human colorectal cancer (CRC). VEGF receptor-1 (VEGFR-1 or Flt-1) is a high-affinity receptor for VEGF and is typically considered specific to endothelial cells. Here we report the expression and function of VEGFR-1 in CRC cell lines. VEGFR-1 was expressed in all CRC cell lines studied as determined by RT–PCR, Western blot analysis, FACS, and ELISA. Treatment of the human CRC cell lines HT-29 and SW480 with VEGF-A (a ligand for both VEGFR-1 and -2) or VEGF-B (a ligand specific for VEGFR-1) led to activation of Erk-1/2, SAPK/JNK, and translocation of the p65 subunit of nuclear factor-κB into the nucleus. Both VEGF-A and -B led to significant induction of cell motility and invasiveness of CRC cells. Stimulation of cells with VEGF-A or -B also led to larger and more numerous colonies in soft agar. However, activation of VEGFR-1 did not increase CRC cell proliferation. In contrast to the previous paradigm that VEGFRs are not present on tumor cells of epithelial origin, we found that VEGFR-1 is present and functional on CRC cells, and activation by VEGF family ligands can activate processes involved in tumor progression and metastasis.

Mekk3 is essential for early embryonic cardiovascular development.

The early development of blood vessels consists of two phases, vasculogenesis and angiogenesis, which involve distinct and also overlapping molecular regulators, but the intracellular signal transduction pathways involved in these processes have not been well defined. We disrupted Map3k3 −/− (also known as Mekk3), which encodes Mekk3, a member of the Mekk/Ste11 family, in mice. Map3k3 −/− embryos died at approximately embryonic day (E) 11, displaying disruption of blood vessel development and the structural integrity of the yolk sac. Angiogenesis was blocked at approximately E9.5 in mutant embryos. Map3k3 disruption did not alter the expression of the genes encoding Vegf-1, angiopoietin or their receptors. The development of embryonic, but not maternal, blood vessels in the placentas of Map3k3 −/− embryos was impaired, revealing an intrinsic defect in Map3k3−/− endothelial cells. Moreover, Mekk3 activated myocyte-specific enhancer factor 2C (Mef2c), a transcription factor crucial for early embryonic cardiovascular development through the p38 mitogen-activated protein kinase (Mapk) cascade. We conclude that Mekk3 is necessary for blood vessel development and may be a possible target for drugs that control angiogenesis.

Inhibition of integrin α5β1 function with a small peptide (ATN‐161) plus continuous 5‐FU infusion reduces colorectal liver metastases and improves survival in mice

Integrin α5β1 is expressed on activated endothelial cells and plays a critical role in tumor angiogenesis. We hypothesized that a novel integrin α5β1 antagonist, ATN‐161, would inhibit angiogenesis and growth of liver metastases in a murine model. We further hypothesized that combining ATN‐161 with 5‐fluorouracil (5‐FU) chemotherapy would enhance the antineoplastic effect. Murine colon cancer cells (CT26) were injected into spleens of BALB/c mice to produce liver metastases. Four days thereafter, mice were given either ATN‐161 (100 mg/kg, every 3rd day) or saline by intraperitoneal injection, with or without combination of continuous‐infusion 5‐FU (100 mg/kg/2 weeks), which was started on day 7. On day 20 after tumor cell inoculation, mice were killed and liver weights and number of liver metastases were determined. A follow‐up study on survival was also conducted in which mice were randomized to receive ATN‐161, 5‐FU or ATN‐161+5‐FU. Combination therapy with ATN‐161+5‐FU significantly reduced tumor burden (liver weight) and number of liver metastases (p<0.02). Liver tumors in the ATN‐161 and ATN‐161+5‐FU groups had significantly fewer microvessels (p<0.05) than tumors in the control or 5‐FU‐treated groups. Unlike treatment with either agent alone, ATN‐161+5‐FU significantly increased tumor cell apoptosis and decreased tumor cell proliferation (p<0.03) and improved overall survival (p<0.03, log‐rank test). Targeting integrin α5β1 in combination with 5‐FU infusion reduced liver metastases formation and improved survival in this colon cancer model. The enhancement of antineoplastic activity from the combination of anti‐angiogenic therapy and chemotherapy may be a promising approach for treating metastatic colorectal cancer.

Blockade of Epidermal Growth Factor Receptor Signaling on Tumor Cells and Tumor-Associated Endothelial Cells for Therapy of Human Carcinomas

The purpose of this study was to determine whether the expression of epidermal growth factor receptor (EGF-R) and activated EGF-R by tumor-associated endothelial cells is influenced by interaction with specific growth factors in the microenvironment. Different human carcinoma cell lines expressing EGF-R with low or high levels of EGF/transforming growth factor (TGF)-alpha were implanted into orthotopic organs of nude mice. In the EGF/TGF-alpha-positive bladder cancer (253J-BV), pancreatic cancer (L3.6pl), and renal cancer (RBM1-IT) but not in the EGF/TGF-alpha-negative renal cancer SN12-PM6, tumor-associated endothelial cells expressed EGF-R and activated EGF-R. Mice were implanted with human 253J-BV bladder tumors (EGF+) or human SN12-PM6 renal tumors (EGF-). Treatment with oral PKI 166 (a specific inhibitor of EGF-R phosphorylation) alone, intraperitoneal paclitaxel alone (253J-BV), gemcitabine alone (SN12-PM6), or combination of PKI 166 and chemotherapy produced a 60%, 32%, or 81% reduction in the volume of 253J-BV bladder tumors, respectively, and 26%, 23%, or 51% reduction in the volume of SN12-PM6 kidney tumors, respectively. Immunohistochemical analyses demonstrated down-regulation of activated EGF-R in EGF/TGF-alpha-positive and EGF/TGF-alpha-negative lesions from mice treated with PKI 166, although apoptosis of tumor-associated endothelial cells was found only in EGF/TGF-alpha-positive tumors. Collectively, these data suggest that expression of activated EGF-R by tumor-associated endothelial cells provides an important target for therapy.

Overexpression of PDGF-BB decreases colorectal and pancreatic cancer growth by increasing tumor pericyte content

We hypothesized that overexpression of PDGF-BB in colorectal cancer (CRC) and pancreatic cancer cells would result in increased pericyte coverage of ECs in vivo, rendering the tumor vasculature more resistant to antiangiogenic therapy. We stably transfected the cDNA for the PDGF-B into HT-29 human CRC and FG human pancreatic cancer cells. Surprisingly, when HT-29 or FG parental and transfected cells were injected into mice (subcutaneously and orthotopically), we observed marked inhibition of tumor growth in the PDGF-BB-overexpressing clones. In the PDGF-BB-overexpressing tumors, we observed an increase in pericyte coverage of ECs. Treatment of PDGF-BB-overexpressing tumors with imatinib mesylate (PDGFR inhibitor) resulted in increased growth and decreased total pericyte content compared with those in untreated PDGF-BB-overexpressing tumors. In vitro studies demonstrated the ability of VSMCs to inhibit EC proliferation by approximately 50%. These data show that increasing the pericyte content of the tumor microenvironment inhibits the growth of angiogenesis-dependent tumors. Single-agent therapy targeting PDGF receptor must be used with caution in tumors when PDGFR is not the target on the tumor cell itself.

Induction of neuropilin-1 and vascular endothelial growth factor by epidermal growth factor in human gastric cancer cells

The epidermal growth factor receptor (EGF-R) pathway plays a pivotal role in the progression of human gastric cancer. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be induced by EGF in various cancer cell lines. Neuropilin-1 (NRP-1) acts as a coreceptor for VEGF-165 and increases its affinity for VEGF receptor 2 (VEGFR-2) in endothelial cells. Furthermore, NRP-1 has been found to be expressed by tumour cells and has been shown to enhance tumour angiogenesis and growth in preclinical models. We examined the expression of NRP-1 mRNA and EGF-R protein in seven human gastric cancer cell lines. NRP-1 expression was expressed in five of seven cell lines, and EGF-R expression closely mirrored NRP-1 expression. Moreover, in EGF-R-positive NCI-N87 and ST-2 cells, EGF induced both NRP-1 and VEGF mRNA expression. C225, a monoclonal antibody to EGF-R, blocked EGF-induced NRP-1 and VEGF expression in NCI-N87 cells in a dose-dependent manner. The treatment of NCI-N87 cells with EGF resulted in increases in phosphorylation of Erk1/2, Akt, and P38. Blockade of the Erk, phosphatidylinositol-3 kinase/Akt, or P38 pathways in this cell line prevented EGF induction of NRP-1 and VEGF. These results suggest that regulation of NRP-1 expression in human gastric cancer is intimately associated with the EGF/EGF-R system. Activation of EGF-R might contribute to gastric cancer angiogenesis by a mechanism that involves upregulation of VEGF and NRP-1 expression via multiple signalling pathways.

Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells

Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). Neuropilin-1 is expressed in pancreatic cancer, but not in nonmalignant pancreatic tissue. We hypothesised that NRP-1 expression by pancreatic cancer cells contributes to the malignant phenotype. To determine the role of NRP-1 in pancreatic cancer, NRP-1 was stably transfected into the human pancreatic cancer cell line FG. Signal transduction was assessed by Western blot analysis. Susceptibility to anoikis (detachment induced apoptosis) was evaluated by colony formation after growth in suspension. Chemosensitivity to gemcitabine or 5-fluorouracil (5-FU) was assessed by MTT assay in pancreatic cancer cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. Differential expression of apoptosis-related genes was determined by gene array and further evaluated by Western blot analysis. Neuropilin-1 overexpression increased constitutive mitogen activated protein kinase (MAPK) signalling, possibly via an autocrine loop. Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by >30% after exposure to clinically relevant levels of gemcitabine and 5-FU. In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing >50% more cell death at clinically relevant concentrations of gemcitabine. Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1. Neuropilin-1 overexpression in pancreatic cancer cell lines is associated with (a) increased constitutive MAPK signalling, (b) inhibition of anoikis, and (c) chemoresistance. Targeting NRP-1 in pancreatic cancer cells may downregulate survival signalling pathways and increase sensitivity to chemotherapy.

Up-regulation of Flotillin-2 Is Associated with Melanoma Progression and Modulates Expression of the Thrombin Receptor Protease Activated Receptor 1

Flotillin 2 (flot-2) is a highly conserved protein isolated from caveolae/lipid raft domains that tether growth factor receptors linked to signal transduction pathways. Flot-2 protein and mRNA were increased in tumorigenic and metastatic melanoma cell lines in vitro, and the immunostaining intensity increased substantially across a tissue array of melanocytic lesions. Flot-2 transfection transformed SB2 melanoma cells from nontumorigenic, nonmetastatic to highly tumorigenic and metastatic in a nude mouse xenograft model. SB2 cells stably transfected with the flot-2 cDNA (SB2-flot)–2 cells proliferated faster in the absence of serum, and their migration through Matrigel was additionally enhanced by thrombin. When SB2-flot–2 cells were compared with SB2-vector–control cells on a cancer gene pathway array, SB2-flot–2 cells had increased expression of protease activated receptor 1 (PAR-1) mRNA, a transmembrane, G-protein–coupled receptor involved in melanoma progression. PAR-1 and flot-2 were coimmunoprecipitated from SB2-flot–2 cells. Up-regulation of PAR-1 was additionally confirmed in SB2-flot–2 cells and melanoma cell lines. SB2-flot–2 cells transfected with flot-2–specific small-interfering RNAs made substantially less flot-2 and PAR-1 mRNA. In conclusion, flot-2 overexpression is associated with melanoma progression, with increased PAR-1 expression, and with transformation of SB2 melanoma cells to a highly metastatic line. Flot-2 binds to PAR-1, a known upstream mediator of major signal transduction pathways implicated in cell growth and metastasis, and may thereby influence tumor progression.

Neuropilin-1 Suppresses Tumorigenic Properties in a Human Pancreatic Adenocarcinoma Cell Line Lacking Neuropilin-1 Coreceptors

Neuropilin-1 (NRP-1) was first described as a coreceptor implicated in neuronal guidance that bound members of the semaphorin/collapsin family. NRP-1 is also expressed in endothelial cells and is believed to promote angiogenesis by acting as a coreceptor with vascular endothelial growth factor (VEGF) receptor 2. Recent studies suggest that NRP-1 can function through both a VEGF-dependent and VEGF-independent fashion. Expression of NRP-1 has been shown in many human tumors, including pancreatic adenocarcinomas. The exact role of NRP-1 in tumor cells is unknown, particularly in cells that lack the NRP-1 coreceptors VEGF receptor 2 and Plexin-A1. To discern the regulatory role(s) of NRP-1 in pancreatic adenocarcinoma that lack these coreceptors, we overexpressed both full-length NRP-1 and a deletion form of NRP-1 that does not interact with semaphorin or VEGF. Overexpression of either isoform reduced several key tumorigenic properties, including anchorage-independent cell growth and migration in vitro, and resulted in reduced tumor incidence and tumor volume in vivo. Conversely, reduction of NRP-1 expression by small interfering RNA targeting led to enhanced tumor growth. Thus, NRP-1 may play distinct growth regulatory roles in different tumor types, and altering NRP-1 expression or function may be a means of influencing the growth of pancreatic cancers.