Nikolaos A. Dallas

Chemoresistant Colorectal Cancer Cells, the Cancer Stem Cell Phenotype, and Increased Sensitivity to Insulin-like Growth Factor-I Receptor Inhibition

5-Fluorouracil (5FU) and oxaliplatin are standard therapy for metastatic colorectal cancer (CRC), but the development of chemoresistance is inevitable. Because cancer stem cells (CSC) are hypothesized to be chemoresistant, we investigated CSC properties in newly developed chemoresistant CRC cell lines and sought to identify targets for therapy. The human CRC cell line HT29 was exposed to increasing doses of 5FU (HT29/5FU-R) or oxaliplatin (HT29/OxR) to achieve resistance at clinically relevant doses. Western blotting and flow cytometry were done to determine molecular alterations. The insulin-like growth factor-I receptor (IGF-IR) monoclonal antibody (mAb) AVE-1642 was used to inhibit signaling in vitro and in vivo using murine xenograft models. HT29/5FU-R and HT29/OxR showed 16- to 30-fold enrichment of CD133(+) cells and 2-fold enrichment of CD44(+) cells (putative CRC CSC markers). Resistant cells were enriched 5- to 22-fold for double-positive (CD133(+)/CD44(+)) cells. Consistent with the CSC phenotype, resistant cells exhibited a decrease in cellular proliferation in vitro (47-59%; P < 0.05). Phosphorylated and total IGF-IR levels were increased in resistant cell lines. HT29/5FU-R and HT29/OxR cells were approximately 5-fold more responsive to IGF-IR inhibition relative to parental cells (P < 0.01) in vitro. Tumors derived from HT29/OxR cells showed significantly greater growth inhibition in response to an IGF-IR mAb than did parental cells (P < 0.05). Chemoresistant CRC cells are enriched for CSC markers and the CSC phenotype. Chemotherapy-induced IGF-IR activation provided for enhanced sensitivity to IGF-IR-targeted therapy. Identification of CSC targets presents a novel therapeutic approach in this disease.

Endoglin (CD105): A Marker of Tumor Vasculature and Potential Target for Therapy

Endoglin (CD105) is an accessory protein of the transforming growth factor-β receptor system expressed on vascular endothelial cells. Mutation of the endoglin gene is associated with hereditary hemorrhagic telangiectasias, or Osler-Weber-Rendu syndrome, and has been studied extensively in the context of this disease. The expression of endoglin is elevated on the endothelial cells of healing wounds, developing embryos, inflammatory tissues, and solid tumors. Endoglin is a marker of activated endothelium, and its vascular expression is limited to proliferating cells. Recent studies identified endoglin expression in several solid tumor types, with the level of expression correlating with various clinicopathologic factors including decreased survival and presence of metastases. Attempts to target endoglin and the cells that express this protein in tumor-bearing mice have yielded promising results.

Therapeutic Targeting of Neuropilin-2 on Colorectal Carcinoma Cells Implanted in the Murine Liver

BACKGROUND Neuropilin-2 (NRP2) is a high-affinity kinase-deficient receptor for vascular endothelial growth factor (VEGF) and semaphorin 3F. We investigated its function in human colorectal cancers. METHODS Immunohistochemistry and immunoblotting were used to assess NRP2 expression levels in colorectal tumors and colorectal cancer cell lines, respectively. HCT-116 colorectal cancer cells stably transfected with short hairpin RNA (shRNAs) against NRP2 or control shRNAs were assayed for proliferation by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and for activation of the VEGFR1 pathway by immunoblotting. Soft agar assays, Annexin V staining, and Boyden chamber assays were used to examine anchorage-independent growth, apoptosis in response to hypoxia, and cell migration/invasion, respectively, in HCT-116 transfectants. Tumor growth and metastasis were analyzed in mice (groups of 10) injected with shRNA-expressing HCT-116 cells. The effect of in vivo targeting of NRP2 by small interfering RNA (siRNA) on the growth of hepatic colorectal tumors derived from luciferase-expressing HCT-116 cells was assessed by measuring changes in bioluminescence and final tumor volumes. All statistical tests were two-sided. RESULTS NRP2 expression was substantially higher in tumors than in adjacent mucosa. HCT-116 transfectants with reduced NRP2 levels had reduced VEGFR1 signaling, but proliferation was unchanged. Anchorage-independent growth, survival under hypoxic conditions, and motility/invasiveness were also reduced. In vivo, HCT-116 transfectants with reduced NRP2 demonstrated decreased tumor growth, fewer metastases, and increased apoptosis compared with control cells. Hepatic colorectal tumors in mice treated with NRP2 siRNAs were statistically significantly smaller than those in mice treated with control siRNAs (at 28 days after implantation, mean control siRNAs = 420 mm3, mean NRP2 siRNAs = 36 mm3, NRP2 vs control: difference = 385 mm3, 95% confidence interval = 174 mm3 to 595 mm3, P = .005). CONCLUSION NRP2 on colorectal carcinoma cells is important for tumor growth and is a potential therapeutic target in human cancers where it is expressed.

Role of class 3 semaphorins and their receptors in tumor growth and angiogenesis.

Class 3 semaphorins (SEMA3) were first identified as glycoproteins that negatively mediate neuronal guidance by binding to neuropilin and repelling neurons away from the source of SEMA3. However, studies have shown that SEMA3s are also secreted by other cell types, including tumor cells, where they play an inhibitory role in tumor growth and angiogenesis (specifically SEMA3B and SEMA3F). SEMA3s primarily inhibit the cell motility and migration of tumor and endothelial cells by inducing collapse of the actin cytoskeleton via neuropilins and plexins. Besides binding to SEMA3s, neuropilin also binds the protumorigenic and proangiogenic ligand vascular endothelial growth factor (VEGF). Although some studies attribute the antitumorigenic and antiangiogenic properties of SEMA3s to competition between SEMA3s and VEGF for binding to neuropilin receptors, several others have shown that SEMA3s display growth-inhibitory activity independent of competition with VEGF. A better understanding of these molecular interactions and the role and signaling of SEMA3s in tumor biology will help determine whether SEMA3s represent potential therapeutic agents. Herein, we briefly review (a) the role of SEMA3s in mediating tumor growth, (b) the SEMA3 receptors neuropilins and plexins, and (c) the potential competition between SEMA3s and VEGF family members for neuropilin binding. (Clin Cancer Res 2009;15(22):676370)

Effect of Molecular Therapeutics on Liver Regeneration in a Murine Model

PURPOSE Unresectable metastatic colorectal cancer (CRC) can be rendered resectable with systemic chemotherapy in approximately 20% of cases. Most patients with metastatic CRC receive chemotherapy with the addition of targeted therapy with anti-vascular endothelial growth factor (VEGF) or anti-epidermal growth factor receptor (EGFR) antibodies. We sought to determine whether anti-VEGF receptor (VEGFR) or anti-EGFR therapy would impair liver regeneration after partial hepatectomy (PH) in mice. MATERIALS AND METHODS Mice underwent either 66% PH or sham laparotomy. In the first experiment, mice in the PH group were randomly assigned to receive daily intraperitoneal injections of monoclonal antibodies (MoABs) to murine VEGFR-2 or nonspecific MoABs (control). In the second experiment, mice in the PH group were randomly assigned to receive intraperitoneal injections of antimurine EGFR or nonspecific (control) MoABs. In both experiments, therapy was initiated the day before surgery and continued until the mice were killed on day 5. Livers were collected and processed. RESULTS Anti-VEGFR-2 therapy slightly impaired liver regeneration and hepatic cell proliferation compared with control. Hematoxylin and eosin staining showed no differences in liver morphology. CD105 staining showed decreased levels of activated endothelium in livers in the VEGFR-2 MoAB group. VEGFR-2 MoAB therapy decreased the levels of the cell cycle regulators cyclin D1 and cyclin D3 and the regenerative cytokine interleukin-6. Anti-EGFR therapy had no effect on liver regeneration or cellular proliferation. CONCLUSION Anti-VEGFR-2 therapy slightly impaired liver regeneration in this murine model, whereas anti-EGFR therapy had no effect on liver regeneration.

Chronic exposure of colorectal cancer cells to bevacizumab promotes compensatory pathways that mediate tumour cell migration.

Background:Bevacizumab (Bev), a monoclonal antibody to vascular endothelial growth factor (VEGF), is used in combination with chemotherapy for the treatment of metastatic colorectal cancer (CRC). The effects of Bev on angiogenesis have been well described, but the direct effect of Bev on tumour cells is unknown. This study was carried out to determine the molecular and phenotypic changes in CRC cells after chronic Bev exposure in vitro.Methods:Human CRC cell lines were chronically exposed (3 months) to Bev in vitro to develop Bev-adapted (Bev-A) cell lines. Vascular endothelial growth factor family members were determined by reverse transcription–polymerase chain reaction and western blotting. Migration and invasion was determined using standard in vitro assays. Intravenous injection of tumour cells was carried out to evaluate metastatic potential in mice.Results:Bevacizumab-adapted cells were found to be more migratory and invasive than control cells (P<0.001). Bevacizumab-adapted cells showed higher levels of VEGF-A, -B, -C, placental growth factor (PlGF), VEGF receptor-1 (VEGFR-1) and phosphorylation of VEGFR-1. Furthermore, treatment with SU5416, a VEGFR protein tyrosine kinase inhibitor, led to significantly decreased cell migration in vitro (P<0.001). Bevacizumab-adapted cells were more metastatic in vivo (P<0.05).Conclusion:Chronic exposure of CRC cells to Bev (1) increased expression of VEGF-A, -B, -C, PlGF, VEGFR-1 and VEGFR-1 phosphorylation, (2) increased tumour cell migration and invasion, and (3) metastatic potential in vivo. Our study shows the functional significance of autocrine VEGF signalling in CRC cells.

Neuropilin-2-mediated tumor growth and angiogenesis in pancreatic adenocarcinoma.

Purpose. Neuropilin-2 (NRP-2) is a coreceptor for vascular endothelial growth factor (VEGF) on endothelial cells. NRP-2 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) cells relative to nonmalignant ductal epithelium. This study determined the role of NRP-2 in PDAC cells. Experimental Design. NRP-2 expression was reduced in PDAC cells with stable short-hairpin RNA (shRNA) transfection. Western blotting was done to evaluate signaling intermediates. Migration and invasion studies were carried out in Boyden chambers. Anchorage-independent growth was assessed by soft-agar colony formation. In vivo growth was evaluated using murine subcutaneous and orthotopic xenograft models. Immunohistochemical analysis evaluated in vivo proliferation and angiogenesis. Results. shRNA-NRP-2 decreased NRP-2 levels without affecting neuropilin-1 levels. Akt activation was decreased in clones with reduced NRP-2 (shRNA-NRP-2). shRNA-NRP-2 cells showed decreased migration, invasion, and anchorage-independent growth compared with control cells. In vitro proliferation rates were similar in control- and shRNA-transfected cells. Subcutaneous and orthotopic xenografts from shRNA-transfected cells were significantly smaller than those resulting from control-transfected cells (P < 0.05). Furthermore, shRNA-NRP-2 tumors exhibited less cellular proliferation and decreased microvascular area relative to control tumors (P < 0.05). Constitutive expression of the angiogenic mediator Jagged-1 was reduced in shRNA-NRP-2 cells, whereas vascular endothelial growth factor levels were unchanged. Conclusion. Reduction of NRP-2 expression in PDAC cells decreased survival signaling, migration, invasion, and ability to grow under anchorage-independent conditions. In vivo, reduction of NRP-2 led to decreased growth of xenograft tumors and decreased vascular area, which was associated with decreased Jagged-1 levels. NRP-2 is a potential therapeutic target on PDAC cells.

Functional significance of vascular endothelial growth factor receptors on gastrointestinal cancer cells

Vascular endothelial growth factor (VEGF) has been shown to be the major mediator of physiologic and pathologic angiogenesis. VEGF was initially thought to be an endothelial cell specific ligand, but recently, VEGF has been shown to mediate tumor cell function via activation of receptors on tumor cells themselves. Here, we review the expression patterns and binding profiles of the VEGF receptors and their ligands on gastrointestinal tumor cells. Furthermore, we describe the current knowledge in regards to the function of these receptors on tumor cells. Elucidating the function of VEGF receptors on tumor cells should help us to better understand the potential mechanisms of action of anti-VEGF therapies.

Chemoresistant colorectal cancer cells and cancer stem cells mediate growth and survival of bystander cells

Background:Recent studies suggest that cancer stem cells (CSCs) mediate chemoresistance, but interestingly, only a small percentage of cells in a resistant tumour are CSCs; this suggests that non-CSCs survive by other means. We hypothesised that chemoresistant colorectal cancer (CRC) cells generate soluble factors that enhance survival of chemonaive tumour cells.Methods:Chemoresistant CRC cells were generated by serial passage in oxaliplatin (Ox cells). Conditioned media (CM) was collected from parental and oxaliplatin-resistant (OxR) cells. CRC cells were treated with CM and growth and survival were assessed. Tumour growth rates were determined in nude mice after cells were treated with CM. Mass spectrometry (MS) identified proteins in CM. Reverse phase protein microarray assays determined signalling effects of CM in parental cells.Results:Oxaliplatin-resistant CM increased survival of chemo-naive cells. CSC CM also increased growth of parental cells. Parental and OxR mixed tumours grew larger than tumours composed of parental or OxR cells alone. Mass spectrometry detected unique survival-promoting factors in OxR CM compared with parental CM. Cells treated with OxR CM demonstrated early phosphorylation of EGFR and MEK1, with later upregulation of total Akt .We identified progranulin as a potential mediator of chemoresistance.Conclusion:Chemoresistant tumour cells and CSCs may promote resistance through soluble factors that mediate survival in otherwise chemosensitive tumour cells.

Effect of chemotherapeutic stress on induction of vascular endothelial growth factor family members and receptors in human colorectal cancer cells

Vascular endothelial growth factor (VEGF) is induced by stress. We determined whether chemotherapy (genotoxic stress) could induce expression of VEGF and VEGF receptors (VEGFR) in human colorectal cancer cells. The colorectal cancer cell lines HT29, RKO, and HCT116 were acutely exposed to increasing doses of oxaliplatin or 5-fluorouracil for 2, 6, and 24 h in vitro. Expression of VEGF ligand family members, VEGFRs, and signaling intermediates was determined by reverse transcription-PCR and Northern and Western blotting. The effect of oxaliplatin on VEGF-A transcriptional activity was determined by promoter assays. Acute exposure of human colorectal cancer cells to oxaliplatin led to a marked induction of VEGF-A mRNA and protein, whereas 5-fluorouracil alone or when added to oxaliplatin did not cause a further increase in VEGF levels. VEGF-A promoter activity was induced by oxaliplatin exposure. Expression of VEGF-C, placental growth factor, VEGFR-1, and neuropilin-1 levels were also increased when cells were treated with oxaliplatin. Oxaliplatin led to an increase in Akt and Src activation in HT29 cells. In contrast, Akt activation did not change in RKO cells whereas phospho-Src and phospho-p44/42 mitogen-activated protein kinase was dramatic increased by oxaliplatin. Inhibition of Akt or Src activation with wortmannin or PP2 blocked induction of VEGF-A by oxaliplatin in HT29 or RKO cells, respectively. VEGFRs may reflect the adaptive stress responses by which tumor cells attempt to protect themselves from genotoxic stress. Neutralization of prosurvival responses with anti-VEGF therapy might explain, in part, some of the beneficial effects of anti-VEGF therapy when added to chemotherapy. [Mol Cancer Ther 2008;7(9):3064–70]