E. Ramsay Camp

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.

Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells

Our laboratory has shown that vascular endothelial growth factor receptor-1 (VEGFR-1) expression on human pancreatic cancer cell lines mediates cell migration and invasion. Because epithelial to mesenchymal transition (EMT) also plays a role in cell motility by altering the cell phenotype and morphology, we hypothesized that VEGFR-1 activation induces molecular alterations that mediate EMT. Our treatment of the human pancreatic cancer cell line L3.6pl with the VEGFR-1 ligands VEGF-A and VEGF-B led to morphologic changes characteristic of EMT, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Immunofluorescent staining with antibodies to E-cadherin and beta-catenin showed that VEGFR-1 activation led to translocation of E-cadherin and beta-catenin from their usual cell membrane-bound location to the cytoplasm and nucleus, respectively. Western blotting showed that VEGFR-1 activation led to decreased expression of the epithelial markers E-cadherin and plakoglobin, increased expression of the mesenchymal markers vimentin and N-cadherin, and increased nuclear expression of beta-catenin. Pretreatment of tumor cells with a VEGFR-1 blocking antibody inhibited the VEGFR-1-induced immunohistochemical and molecular changes in E-cadherin. VEGFR-1 activation led to an increase in expression of the EMT-associated transcription factors Snail, Twist, and Slug. The changes mediated by VEGFR-1 in this pancreatic carcinoma cell line are highly consistent with the changes characteristic of EMT. Given our previous finding of VEGFR-1-mediated tumor cell invasion and migration in pancreatic carcinoma cells, we hypothesize that VEGFR-1 plays a role in tumor progression in pancreatic cancer through the induction of EMT.

RON, a tyrosine kinase receptor involved in tumor progression and metastasis

Tyrosine kinase receptors mediate many critical cellular functions that contribute to tumor progression and metastasis and thus are potential targets for molecular-based cancer therapy. As has been found for many receptor tyrosine kinases, RON (recepteur d’origine nantais) and its ligand, macrophage-stimulating protein, have recently been implicated in the progression and metastasis of tumors. In in vitro experiments using colon and breast cancer cell lines, overexpression of RON led to increased invasion and migration of cancer cells and prevented apoptosis and anoikis. In addition, transgenic mice engineered to overexpress RON in the lung epithelium developed multiple pulmonary tumors, suggesting a role for RON in tumorigenesis. In human cancer specimens, increased RON expression has been demonstrated in colon, breast, ovarian, and lung tumors. Therefore, therapies designed to inhibit RON activation may hinder critical tumor survival mechanisms and play a role in the treatment of advanced disease.

Tyrosine kinase receptor RON in human pancreatic cancer: expression, function, and validation as a target.

Specific tyrosine kinase receptors such as c‐MET mediate epithelial‐mesenchymal (EMT) transition, leading to phenotypic alterations associated with increased cell motility. It was hypothesized that RON, a tyrosine kinase receptor related to c‐MET, would be expressed in human pancreatic cancer cells, induce EMT, and would thus serve as a target for therapy in a preclinical model.

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.

Improving delivery of antineoplastic agents with anti-vascular endothelial growth factor therapy

It is believed that impairments in delivery of antineoplastic agents to solid tumors result from abnormalities of the tumor microenvironment. Vascular endothelial growth factor (VEGF), the prototypical angiogenic molecule, is one of the main factors responsible for the development and maintenance of the aberrant tumor vascular network, which is characterized by chaotic, leaky blood vessels with high interstitial fluid pressure and inefficient blood flow. The authors proposed that anti‐VEGF therapy would reduce the elevated interstitial fluid pressure in tumors, thereby improving blood flow and potentially improving delivery of cytotoxic agents to tumor cells. For the current report, the authors reviewed characteristics of the abnormal tumor vasculature created under the influence of VEGF, the resulting tumor microenvironment, how the tumor microenvironment may impede delivery of antineoplastic agents, and how the combination of anti‐VEGF and cytotoxic therapy may maximize the efficacy of antineoplastic treatment regimens. Cancer 2005.

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]

Roles of Nitric Oxide Synthase Inhibition and Vascular Endothelial Growth Factor Receptor-2 Inhibition on Vascular Morphology and Function in an In vivo Model of Pancreatic Cancer

Purpose: Both nitric oxide (NO) and vascular endothelial growth factor (VEGF) mediate tumor vascular function. Because these molecules regulate one anothers expression, we hypothesized that NO synthase (NOS) inhibition produces effects comparable to those of anti-VEGF therapy on human pancreatic cancer xenografts. Experimental Design: L3.6pl human pancreatic cancer cells were s.c. implanted in nude mice. On day 6, mice were randomized to receive (a) PBS (control), (b) DC101 [VEGF receptor 2 (VEGFR-2) antibody] by i.p. injection, (c) N-nitro-l-arginine (NNLA; NOS inhibitor) in the drinking water, or (d) both DC101 and NNLA. Mice were killed on day 20. Results: DC101 and NNLA as single agents inhibited tumor growth by ∼50% to 60% (P < 0.008 for both). Furthermore, combined therapy inhibited mean tumor growth by 89% (P < 0.008). Combined inhibition of VEGFR-2 and NOS also decreased mean vessel counts by 65% (P < 0.03) and vessel area by 80% versus controls (P < 0.001). In contrast to DC101 where vessel diameter was similar to control, NNLA decreased mean vessel diameter by 42% (P < 0.001). NNLA also led to a 54% (P < 0.03) decrease in tumor uptake of the perfusion marker Hoechst 33342 versus controls whereas DC101 decreased Hoechst 33342 staining by 43% (P < 0.03). The combination of inhibitors decreased perfusion by 73% (P < 0.03). Conclusions: Although VEGFR-2 can mediate NOS activity, the combination of VEGFR-2 and NOS inhibition significantly increased the antivascular effect over single agent therapy. The addition of NOS inhibition led to an even further alteration of tumor vessel morphology and vascular perfusion compared with VEGFR-2 blockade, suggesting that NO and VEGFR-2 have distinct but complementary effects on the tumor vasculature.

The role of nitric oxide in mediating tumour blood flow

Nitric oxide (NO) is a ubiquitous molecule with a myriad of physiological and pathophysiological roles. It has numerous direct and indirect effects on tumour vasculature as both a regulatory and effector molecule. NO affects tumour blood flow through its effects on tumour angiogenesis, vascular tone and vascular permeability, partly via its interaction with vascular endothelial growth factor. In this review, the authors examine the basic tenants of NO biology, the association of NO with tumour progression, and the role NO plays in mediating alterations in vascular functions in tumours.