Jane S. Wey

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.

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.

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.

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.

HIF-lα als Regulator von Tumorangiogenese, Gefäßmorphologie und Wachstum des Adenokarzinoms des Magens

The hypoxia inducible factor-1α (HIF-lα) is an important transcription factor for mediating vascular endothelial growth factor (VEGF) expression in solid tumors. We hypothesized that inhibition of HIF-1α in human gastric cancers cells would significantly impair angiogenesis and tumor growth in vivo. Gastric cancer cells (TMK-1) were stably transfected with a dominant-negative HIF-lα construct (HIF-lαDN) or vector alone (pCEP4) as control. Transfected cells were screened for nuclear HIF-lα expression by Western blot. Differences in VEGF secretion were detected by ELISA. Growth rates of HIF-lαDN and pCEP4 cells were first investigated in a subcutaneous (SQ) xenograft model, and subsequently in an orthotopic model of gastric cancer in mice. Vessel area (measure of functional vascular volume), proliferating tumor cells, and vessel pericyte coverage were evaluated by immunohistochemistry. HIF-1α inhibition led to a 60 - 70% reduction of constitutive VEGF secretion (P < 0.05). VEGF secretion was also blunted in DN cells in response to hypoxia (P< 0.05). In the SQ model, HIF-lαDN cells exhibited significantly reduced tumor growth, vessel area, and tumor cell proliferation (P < 0.05 for all). Similar findings were observed in the orthotopic model, where tumor growth, vessel area and vessel morphology (pericyte coverage) were significantly impaired in the HIF-lαDN groups (P < 0.05 for both). In conclusion, HIF-lα may be a viable molecular target for inhibiting angiogenesis and growth of gastric cancer.

Therapeutic targeting of the insulin-like growth factor-I receptor (IGF-IR) plus oxaliplatin decreases hepatic growth of human colon cancer

S: PLENARY and PARALLEL SESSIONS 5O Systemic administration of an anti-VEGF monoclonal antibody inhibits growth of human pancreatic adenocarcinoma in an orthotopic mouse model S.E. Holloway,* A. Beck, M. Davis, T. Hart, R.A. Brekken, J.B. Fleming. University of Texas Southwestern Medical