George Van Buren

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.

Chronic Oxaliplatin Resistance Induces Epithelial-to-Mesenchymal Transition in Colorectal Cancer Cell Lines

Purpose: Epithelial-to-mesenchymal transition (EMT) is a process whereby cells acquire molecular alterations that facilitate cell motility and invasion. In preliminary studies, we observed that oxaliplatin-resistant (OxR) colorectal cancer (CRC) cells underwent morphologic changes suggestive of a migratory phenotype, leading us to hypothesize that OxR CRC cells undergo EMT. Experimental Design: The human CRC cell lines KM12L4 and HT29 were exposed to increasing doses of oxaliplatin to establish stable cell lines resistant to oxaliplatin. Migration and invasion were assessed by modified Boyden chamber assays. Morphologic and molecular changes characteristic of EMT were determined by immunofluorescence staining and Western blot analyses. Results: The OxR cells showed phenotypic changes consistent with EMT: spindle-cell shape, loss of polarity, intercellular separation, and pseudopodia formation. KM12L4 and HT29 OxR cells exhibited an ∼8- to 15-fold increase in migrating and invading cells, respectively (P < 0.005 for both). Immunofluorescence staining of OxR cells revealed translocation of E-cadherin and β-catenin from their usual membrane-bound complex to the cytoplasm and nucleus, respectively. The OxR cells also had decreased expression of the epithelial adhesion molecules E-cadherin and plakoglobin and an increase in the mesenchymal marker vimentin. The KM12L4 OxR cells exhibited increased nuclear expression of Snail, an EMT-regulatory transcription factor, whereas the HT29 OxR cells exhibited an increase in nuclear expression of the EMT-associated transcription factor nuclear factor κB. Conclusion: We hypothesize that induction of EMT may contribute to the decreased efficacy of therapy in chemoresistant CRC, as the tumor cells switch from a proliferative to invasive phenotype. Further understanding of the mechanisms of chemoresistance in CRC will enable improvements in chemotherapy for metastatic disease.

A Randomized Prospective Multicenter Trial of Pancreaticoduodenectomy With and Without Routine Intraperitoneal Drainage

Objective:To test by randomized prospective multicenter trial the hypothesis that pancreaticoduodenectomy (PD) without the use of intraperitoneal drainage does not increase the frequency or severity of complications. Background:Some surgeons have abandoned the use of drains placed during pancreas resection. Methods:We randomized 137 patients to PD with (n = 68, drain group) and without (n = 69, no-drain group) the use of intraperitoneal drainage and compared the safety of this approach and spectrum of complications between the 2 groups. Results:There were no differences between drain and no-drain cohorts in demographics, comorbidities, pathology, pancreatic duct size, pancreas texture, baseline quality of life, or operative technique. PD without intraperitoneal drainage was associated with an increase in the number of complications per patient [1 (0-2) vs 2 (1-4), P = 0.029]; an increase in the number of patients who had at least 1 ≥grade 2 complication [35 (52%) vs 47 (68%), P = 0.047]; and a higher average complication severity [2 (0-2) vs 2 (1-3), P = 0.027]. PD without intraperitoneal drainage was associated with a higher incidence of gastroparesis, intra-abdominal fluid collection, intra-abdominal abscess (10% vs 25%, P = 0.027), severe (≥grade 2) diarrhea, need for a postoperative percutaneous drain, and a prolonged length of stay. The Data Safety Monitoring Board stopped the study early because of an increase in mortality from 3% to 12% in the patients undergoing PD without intraperitoneal drainage. Conclusions:This study provides level 1 data, suggesting that elimination of intraperitoneal drainage in all cases of PD increases the frequency and severity of complications.

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.

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.

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.

The Development and Characterization of a Human Midgut Carcinoid Cell Line

Purpose: Gastrointestinal neuroendocrine tumors (NET) are rare heterogeneous tumors that hypersecrete neuropeptides. The scarcity of good gastrointestinal NET models has limited the ability to study potential therapeutic agents. We describe and characterize the establishment of a human midgut carcinoid tumor cell line carcinoid tumor 2 (CNDT2). Experimental Design: Tumor cells (CNDT2) were isolated from a liver metastasis from a patient with a primary ileal carcinoid. After 9 weeks in culture, the cells were plated in soft agar, and cells from a single colony were put back in culture (CNDT2.1). Those CNDT2.1 cells were injected s.c. into nude mice. Cells were isolated from a single resultant tumor (CNDT2.5), cultured, and characterized by electron microscopy, reverse transcription-PCR, serotonin enzyme immunoassay, Western blotting, and immunohistochemical analysis for NET markers and potential therapeutic targets. Results: CNDT2 cells grew in monolayers in vitro, formed colonies in soft agar, and formed tumors in mice. Electron microscopy revealed round, pleomorphic, electron-dense neurosecretory granules characteristic of NETs. Tumor xenografts exhibited the appearance of NETs with small “salt-and-pepper” nuclei on H&E staining and chromogranin A, synaptophysin, and CD56 on immunohistochemical staining. CNDT2.5 cells produced serotonin and expressed insulin-like growth factor receptor-I, platelet-derived growth factor receptor-β, vascular endothelial growth factor receptor-1, cMET, epidermal growth factor receptor, neuropilin-1, and somatostatin receptors 1 to 5. Cytogenetic analysis revealed the presence of deletions at 2p and 6q and numerous translocations. Conclusion: The establishment of this human midgut carcinoid tumor cell line may serve as a useful model system for studying cell biology and novel targeted agents in preclinical models.

Pancreatic cancer: Advances in treatment

Pancreatic cancer is a leading cause of cancer mortality and the incidence of this disease is expected to continue increasing. While patients with pancreatic cancer have traditionally faced a dismal prognosis, over the past several years various advances in diagnosis and treatment have begun to positively impact this disease. Identification of effective combinations of existing chemotherapeutic agents, such as the FOLFIRINOX and the gemcitabine + nab-paclitaxel regimen, has improved survival for selected patients although concerns regarding their toxicity profiles remain. A better understanding of pancreatic carcinogenesis has identified several pre-malignant precursor lesions, such as pancreatic intraepithelial neoplasias, intraductal papillary mucinous neoplasms, and cystic neoplasms. Imaging technology has also evolved dramatically so as to allow early detection of these lesions and thereby facilitate earlier management. Surgery remains a cornerstone of treatment for patients with resectable pancreatic tumors, and advances in surgical technique have allowed patients to undergo resection with decreasing perioperative morbidity and mortality. Surgery has also become feasible in selected patients with borderline resectable tumors as a result of neoadjuvant therapy. Furthermore, pancreatectomy involving vascular reconstruction and pancreatectomy with minimally invasive techniques have demonstrated safety without significantly compromising oncologic outcomes. Lastly, a deeper understanding of molecular aberrations contributing to the development of pancreatic cancer shows promise for future development of more targeted and safe therapeutic agents.

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]