Neoadjuvant chemoradiation is associated with decreased lymph node ratio in borderline resectable pancreatic cancer: A propensity score matched analysis.

BACKGROUND: Lymph node ratio (LNR) and margin status have prognostic significance in pancreatic cancer. Herein we examined the pathologic and clinical outcomes in patients with borderline resectable pancreatic cancer (BRPC) following neoadjuvant therapy (NAT) and pancreaticoduodenectomy. METHODS: Patients who underwent treatment between January 1, 2012 and June 30, 2017 were included. Sequential patients in the BRPC group were compared to a propensity score matched cohort of patients with radiographically resectable pancreatic cancer who underwent upfront surgical resection. The BRPC group was also compared to sequential patients with radiographically resectable pancreatic cancer who required vein resection (VR) during upfront surgery. RESULTS: There were 50 patients in the BRPC group, 50 patients in the matched control group, and 38 patients in the VR group. Negative margins (R0) were seen in 72%, 64%, and 34% of the BRPC, control, and VR groups, respectively (P = 0.521 for BRPC vs. control; P = 0.002 for BRPC vs. VR), with 24% of the BRPC group requiring a vascular resection. Nodal stage was N0 in 64%, 20%, and 18% of the BRPC, control, and VR groups, respectively (P < 0.001 for BRPC vs. control or VR). When nodal status was stratified into four groups (N0, or LNR ≤ 0.2, 0.2-0.4, ≥ 0.4), the BRPC group had a more favorable distribution (P < 0.001). The median overall survival were 28.8, 38.6, and 19.0 months for the BRPC, control, and VR groups, respectively (log-rank P = 0.096). CONCLUSIONS: NAT in BRPC was associated with more R0 and N0 resections and lower LNR compared to patients undergoing upfront resection for resectable disease.

Pathologic tumor response to neoadjuvant therapy in borderline resectable pancreatic cancer.

BACKGROUND: Previous studies have demonstrated the prognostic significance of pathologic tumor response in pancreatic adenocarcinoma following neoadjuvant therapy (NAT). The aim of this study was to determine the incidence of significant pathologic response to NAT in borderline resectable pancreatic cancer (BRPC), and association of NAT regimen and other clinico-pathologic characteristics with pathologic response. METHODS: Patients with BRPC who underwent NAT and pancreatic resection between January 2012 and June 2017 were included. Pathologic response was assessed on a qualitative scale based on the College of American Pathologists grading system. Demographics and baseline characteristics, oncologic treatment, pathology, and survival outcomes were compared. RESULTS: Seventy-one patients were included for analysis. Four patients had complete pathologic responses (tumor regression score 0), 12 patients had marked responses (score 1), 42 had moderate responses (score 2), and 13 had minimal responses (score 3). Patients with complete or marked responses were more likely to have received neoadjuvant gemcitabine chemoradiation (62.5%, 38.1%, and 23.1% of the complete/marked, moderate, and minimal response groups, respectively; P = 0.04). Of the complete/marked, moderate, and minimal response groups, margins were negative in 75.0%, 78.6%, and 46.2% (P = 0.16); node negative disease was observed in 87.5%, 54.8%, and 15.4% (P < 0.01); and median overall survival was 50.0 months, 31.7 months, and 23.2 months (P = 0.563). Of the four patients with pathologic complete responses, three were disease-free at 66.1, 41.7 and 31.4 months, and one was deceased with metastatic liver disease at 16.9 months. CONCLUSIONS: A more pronounced pathologic tumor response to NAT in BRPC is correlated with node negative disease, but was not associated with a statistically significant survival benefit in this study.

Predicting early mortality in resectable pancreatic adenocarcinoma: A cohort study.

BACKGROUND: Survival after surgical resection for pancreatic cancer remains poor. A subgroup of patients die early (<6 months), and understanding factors associated with early mortality may help to identify high-risk patients. The Khorana score has been shown to be associated with early mortality for patients with solid tumors. In the current study, the authors evaluated the role of this score and other prognostic variables in this setting. METHODS: The current study was a cohort study of patients who underwent surgical resection for pancreatic cancer from January 2006 through June 2013. Baseline (diagnosis ±30 days) parameters were used to define patients as high risk (Khorana score ≥3). Statistically significant univariable associations and a priori prognostic variables were tested in multivariable models; adjusted hazard ratios (HR) were calculated. RESULTS: The study population comprised 334 patients. The median age was 67 years, 50% of the study population was female, and 86% of the patients were white. The pancreatic head was the primary tumor site for 73% of patients; 67% of tumors were T3 and 63% were N1. The median Khorana score was 2; 152 patients (47%) were determined to be high risk. Adjunctive treatment included chemotherapy (70%) and radiotherapy (40%). The postoperative (30-day) mortality rate was 0.9%. The 6-month mortality rate for the entire cohort was 9.4%, with significantly higher rates observed for high-risk patients (13.4% vs 5.6%; P = .02). On multivariable analyses (examining a total of 326 patients), the Khorana score (HR for high risk, 2.31; P = .039) and elevated blood urea nitrogen (HR, 4.34; P<.001) were associated with early mortality. CONCLUSIONS: Patients at high risk of early mortality after surgical resection of pancreatic adenocarcinoma can be identified using simple baseline clinical and laboratory parameters. Future studies should address preoperative interventions in these patients at high risk of early mortality.

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.

Short- and long-term costs of laparoscopic colectomy are significantly less than open colectomy.

BACKGROUND: The financial impact of laparoscopic colectomy remains poorly defined. We report the short-term costs of laparoscopic colectomy (LC) as compared with open colectomy (OC) in a high-volume tertiary care hospital, and are the first to incorporate the costs of late, colectomy-related complications in an analysis of long-term costs. METHODS: A retrospective analysis of patients undergoing elective laparoscopic (n = 76) or open (n = 162) colon resection between January 2004 and December 2006 was performed. Primary endpoints were total hospital cost of the index admission and total hospital cost for any subsequent admission for treatment of a colectomy-related complication. RESULTS: Two-hundred thirty-eight patients met inclusion criteria. Mean total hospital cost was significantly greater for patients undergoing OC (US $17,686 per patient versus US $14,518, P = 0.0003). Mean total operative costs were equivalent (US $7,451 OC versus US $7,794 LC, P = 0.274). Average length of stay was shorter for LC (5.2 versus 6.9 days, P < 0.0001). Late complication rates were 5.6% (OC) and 2.6% (LC). Integrating the cost of late complications further increased the disparity between the total cost of OC (US $18,296 per patient, 3.4% increase) as compared with LC (US $14,789, 1.9% increase). CONCLUSION: We demonstrate both short- and long-term financial benefits of LC in a high-volume tertiary care hospital.

Utilization of Quality Improvement Methodology to Standardize Communication of Outside Hospital Transfers in a General Surgery Program.

OBJECTIVE: There is no standardized method of communication regarding the arrival of outside hospital (OSH) transfers at our institution. We utilized quality improvement methodologies to enhance sign-out, benefiting both resident workflow and patient care. DESIGN: A dynamic census log of pending OSH transfers was created. Total number of OSH transfers (with or without prior notification), time to admission orders, and resident self-reported preparedness in receiving/triaging OSH admissions were measured before and after implementation of the census log tool. SETTING: Quaternary referral hospital in Cleveland, Ohio. PARTICIPANTS: The census log was made available to General Surgery residents on receiving surgical teams. After the data collection period, it was made available to all residents in the program. RESULTS: A total of 93 patients were transferred to receiving surgical teams during our 13-week study period. Resident notification of the OSH transfer prior to patient's arrival increased from 44.7% pre- to 70.3% postimplementation of the tool (p = 0.03). When residents received prior notification of pending transfers, time to place admission orders decreased from 81.2 ± 79.9 minutes to 40.4 ± 36.8 minutes (p = 0.0029). Junior residents' self-reported preparedness in admitting an OSH transfer did not significantly differ when they received prior notification versus when they did not. In contrast, senior residents' self-reported higher levels of preparedness in the instances where they received prior notification of a pending transfer. CONCLUSIONS: In light of the recent Clinical Learning Environment Review program set forth by the Accreditation Council for Graduate Medical Education, trainees are expected to engage in improvement processes as it relates to patient safety and transitions of care. The development and implementation of our tool demonstrate that quality improvement methodologies can be effectively applied to resident workflow challenges, improving both trainee education and patient care.

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.

Diagnostic Laparoscopy Prior to Neoadjuvant Therapy in Pancreatic Cancer Is High Yield: an Analysis of Outcomes and Costs.

BACKGROUND: There is currently no standardized regimen for management of borderline resectable pancreatic cancer (BRPC), and treatment includes varying sequences of surgery, chemotherapy, and/or radiation. This study examines the diagnostic yield and cost of performing staging diagnostic laparoscopy (SDL) prior to neoadjuvant therapy (NAT) in BRPC. METHODS: Sequential patients treated for BRPC between January 2010 and October 2013 were included. SDL was adopted in a staged fashion due to surgeon preference, and included biopsy of visible lesions and washings for cytology. Cost ratios (CRs) were calculated to compare the direct cost of the SDL versus no-SDL groups and to compare patients with positive versus negative SDL. RESULTS: Of 116 patients evaluated for BRPC, 75 patients underwent SDL and 19 (25%) revealed occult metastatic disease. Sixteen patients had a positive biopsy and three had positive cytology alone. There was no difference in overall treatment cost (CR 0.95, 95% CI 0.62-1.37), oncologic treatment (CR 0.66, 95% CI 0.32-1.23), or remaining surgical treatment (CR 1.14, 95% CI 0.77-1.71) for patients who underwent SDL compared to those who did not. Patients with a positive SDL incurred lower overall cost compared to those with a negative SDL (CR 0.23, 95% CI 0.16-0.32) due to lack of further surgery or radiation, and less intensive chemotherapy regimens. CONCLUSIONS: SDL prior to NAT is a useful adjunct to CT to diagnose occult metastatic disease in BRPC.

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-kappaB 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.

Angiopoietin-1 inhibits vascular permeability, angiogenesis, and growth of hepatic colon cancer tumors.

Angiopoietin (Ang)-1 and -2 are critical regulators of embryonic and postnatal neovascularization. Ang-1 activates the endothelial cell-specific tyrosine kinase receptor Tie-2, which in turn leads to enhanced endothelial cell survival and stabilization. The effects of Ang-1 on tumor angiogenesis remain controversial; although we have previously demonstrated that Ang-1 overexpression in colon cancer cells leads to a decrease in s.c. tumor growth, others have shown that Ang-1 may be proangiogenic. Few studies have addressed the role of the Angs in tumors growing in the organ of metastatic growth. We hypothesized that overexpression of Ang-1 may inhibit the growth of colon cancers growing in the liver by inhibition of angiogenesis. We also wanted to investigate the mechanisms by which Ang-1 affects angiogenesis in vivo. Human colon cancer cells (HT29) were stably transfected with an Ang-1 construct or an empty vector (pcDNA) and injected directly into the livers of nude mice. After 37 days, livers were harvested and weighed, and tumor sizes were measured. In an additional experiment, to validate the paracrine effect of Ang-1, various mixtures of control cells and Ang-1-transfected cells were injected into livers, and tumor growth was assessed. Direct effects of recombinant Ang-1 on angiogenesis were studied with an in vivo Gelfoam angiogenesis assay. The impact of Ang-1 on vascular permeability was investigated using an intradermal Miles assay with conditioned media from transfected cells. Liver weights (P < 0.05), tumor volumes (P < 0.05), vessel counts (P < 0.01), and tumor cell proliferation (P < 0.01) in the Ang-1 group were significantly lower than those in the control (pcDNA) group. Tumor vessels in the Ang-1 group developed a significantly higher degree of pericyte coverage (P < 0.02) than vessels in pcDNA tumors. In the cell mixture experiment, even as few as a 1:10 mixture of Ang-1-transfected cells/control cells resulted in a significant reduction of hepatic tumor volumes (P < 0.04). In the angiogenesis assay, vessel counts in Gelfoam implants were significantly decreased by the addition of Ang-1 (P < 0.01). Finally, conditioned medium from Ang-1-transfected cells decreased vascular permeability more than that from control cells (P < 0.05). Our results suggest that Ang-1 is an important regulator of angiogenesis and vascular permeability and that this effect may be secondary to increasing periendothelial support and vessel stabilization. Thus, Ang-1 could potentially serve as an antineoplastic or anti-permeability agent for patients with metastatic colorectal cancer.

Absence of a Periampullary Mass on Cross-sectional Imaging Delays Diagnosis and Time to Pancreatoduodenectomy But Does Not Impair Outcome.

BACKGROUND: The aim of this study was to assess whether the lack of a radiological mass in patients with periampullary malignancies led to protracted diagnosis, delayed resection, and an inferior outcome. METHODS: The departmental database was interrogated to identify all patients undergoing pancreatoduodenectomy during the period 2000-2014. The absence of a mass on cross-sectional and endoscopic ultrasound was noted. The interval between imaging and surgery was evaluated and related to the absence of a mass. The relationship between mass/no mass and the pathological profile was also assessed. RESULTS: Among 490 patients who underwent pancreatoduodenectomy for periampullary malignancies, masses were detected in 299 patients. Patients with undetected mass on either endoscopic ultrasonography (EUS) or computed tomography (CT)/magnetic resonance imaging (MRI) had a longer median interval from initial imaging to resection than detected mass with no difference in survival (66 vs. 41 days, p = 0.001). The absence of a mass was more common in cholangiocarcinomas (p < 0.001). The absence of a mass on imaging was associated with smaller size on final histopathology (2.4 vs. 2.8 cm; p < 0.001). CONCLUSIONS: The absence of a mass with all modalities in patients with a periampullary malignancy leads to a delayed diagnosis without a significant effect on survival.

Frailty predicts risk of life-threatening complications and mortality after pancreatic resections.

BACKGROUND: To assess the effect of frailty on morbidity and mortality after partial pancreatectomy. METHODS: A retrospective analysis of National Surgical Quality Improvement Project from 2005-2010 was conducted. A modified frailty index was created based on previously validated methodology. Patients were classified as nonfrail, low frailty, intermediate frailty, and frail. Outcomes of pancreatoduodenectomy and distal pancreatectomy were examined. RESULTS: In the study, 13,020 patients were analyzed (8,729 pancreatoduodenectomy and 4,291 distal pancreatectomy). Among the pancreatoduodenectomy and distal pancreatectomy patients, frail patients regardless of the degree of frailty were older, more likely male, had a greater body mass index, lower serum albumin, and greater weight loss compared with the nonfrail patients (all P ≤ .05). Postoperatively, a stepwise increased risk of grade 4 complications (Clavien/Dindo) and mortality was noted from nonfrail to frail patients. Every 1-point increase in modified frailty index was associated with a significantly increased risk of grade 4 complications (∼2-6 times) and mortality (∼2-10 times) from low-frail to frail (adjusted for age, sex, body mass index, albumin, weight loss, and type of pancreatectomy). An abbreviated frailty index incorporating 8 variables was as predictive as the modified frailty index (P = .68). CONCLUSION: An 11-point frailty index as measured in National Surgical Quality Improvement Project predicts serious complications and death after pancreatectomy. A modification of this index with 8 factors continues to have similar predictive ability. Consideration of frailty may be beneficial prior to the pancreatic surgeon and particularly in discussion of operative risk and selection of patients who might receive benefit from pre-operative optimization.

ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor with additional activity against epidermal growth factor receptor tyrosine kinase, inhibits orthotopic growth and angiogenesis of gastric cancer.

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) have been strongly implicated in the growth and metastasis of gastric cancer. The purpose of this study was to examine the effects of ZD6474, an inhibitor of inhibitor of VEGF receptor (VEGFR) tyrosine kinase with additional activity against EGF receptor (EGFR), on tumor growth and angiogenesis in an orthotopic model of gastric cancer. In vitro, ZD6474 inhibited human umbilical vascular endothelial cell and TMK-1 human gastric tumor cell proliferation in a dose-dependent fashion. EGF-mediated activation of EGFR and Erk-1/2 was decreased in tumor cells after ZD6474 treatment. In addition, VEGF-mediated activation of VEGFR2 and Erk-1/2 was decreased in human umbilical vascular endothelial cells. TMK-1 human gastric adenocarcinoma cells were injected into the gastric wall of nude mice. ZD6474 therapy was initiated on day 10. Mice (n = 14 per group) were treated p.o. with (a) 1% Tween 80 (control), (b) 50 mg/kg/d ZD6474, or (c) 100 mg/kg/d ZD6474. Mice were sacrificed on day 33. Tumors from each group were stained for markers of blood vessels, pericytes, proliferation, and apoptosis. ZD6474 at both 50 and 100 mg/kg/d led to marked inhibition of tumor growth (P < 0.05). ZD6474 reduced tumor cell proliferation by 48% in the 50 mg/kg/d group and 65% in the 100 mg/kg/d group (P < 0.03) and increased tumor cell apoptosis (P < 0.001) in vivo. ZD6474 led to a 69% decrease in microvessel density in the 50 mg/kg/d group (P < 0.001) and a 62% decrease in the 100 mg/kg/d group (P < 0.001). Although microvessel density was decreased by ZD6474, the remaining vessels showed a relatively higher percentage of pericyte coverage (3-fold increase; P < 0.001), perhaps reflecting selective loss of uncovered vessels in the ZD6474 group. In conclusion, therapies such as ZD6474 that target two distinct aspects of tumor growth, angiogenesis and tumor cell proliferation, warrant further investigation.

Vascular endothelial growth factor receptors: expression and function in solid tumors.

Vascular endothelial growth factor (VEGF) and its family members are important mediators of tumor angiogenesis. The multiple functions of the VEGF are mediated through complex and selective interactions between the ligands, their high-affinity tyrosine kinase receptors, and co-receptors (neuropilins). While these receptors are historically described as being exclusively expressed on endothelial cells, emerging evidence has documented expression of these receptors on a number of nonendothelial cells, including tumor cells. The VEGF receptors (VEGFR) have also been shown to be functional in a number of nonendothelial systems, where they may be targets for anti-VEGF therapy. This article will review the basic effects and interactions of the VEGFRs on endothelial cells and the evidence for their expression and function on tumor cells. The novel expression of VEGFRs on tumor cells contributes to the understanding of the complex roles of VEGF within the tumor microenvironment, potentially affecting both endothelial cells and tumor cells expressing the VEGFRs. This elucidation of VEGF activity may further refine antineoplastic regimens for solid malignancies.

Upregulation of neuropilin-1 by basic fibroblast growth factor enhances vascular smooth muscle cell migration in response to VEGF.

Neuropilin-1 (NRP-1) is a co-receptor for vascular endothelial growth factor (VEGF). During neovascularization, vascular smooth muscle cells (VSMCs) and pericytes modulate the function of endothelial cells. Factors that mediate NRP-1 in human VSMCs (hVSMCs) remain to be elucidated. We studied various angiogenic cytokines to identify factors that increase NRP-1 expression in hVSMCs. Treatment of hVSMCs with basic fibroblast growth factor (b-FGF) induced expressions of NRP-1 mRNA and protein whereas epidermal growth factor, insulin-like growth factor-1, and interleukin-1beta did not. b-FGF induced phosphorylation of Erk-1/2 and JNK. MEK1/2 and nuclear factor kappa B (NF-kappaB) inhibitors (U0126 and TLCK, respectively) blocked the ability of b-FGF to induce NRP-1 mRNA expression, but inhibition of JNK (SP600125) or PI3-kinase activity (wortmannin) did not. Further, the increase in NRP-1 expression by b-FGF enhanced hVSMCs migration in response to VEGF(165). This effect was dependent on the binding of VEGF(165) to VEGFR-2, as blocking antibodies to VEGFR-2, but not VEGFR-1, inhibited VEGF(165)-induced migration. In conclusion, b-FGF increased NRP-1 expression in hVSMCs that in turn enhance the effect of VEGF(165) on cell migration. The enhanced migration of hVSMCs was mediated through binding of VEGF(165) to both NRP-1 and VEGFR-2, as inhibition of VEGFR-2 on these cells blocked the effect of VEGF-mediated cell migration.

Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines.

BACKGROUND: Vascular endothelial growth factor receptor-1 (VEGFR-1) is one of three receptor tyrosine kinases for VEGF, a key regulator of angiogenesis in cancer. Although VEGFRs initially were believed to be expressed exclusively on endothelial cells (ECs), recent studies have demonstrated the presence of VEGFR-1 on non-EC types. The authors hypothesized that VEGFR-1 is present and functional in pancreatic carcinoma cells, contributing to the malignant phenotype. METHODS: The authors assessed the expression of VEGFR-1 and its ligands in 11 pancreatic carcinoma cell lines by reverse-transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and/or Western blot analysis. The function of VEGFR-1 was evaluated by treating two representative cell lines with VEGF-B, a selective ligand for VEGFR-1, and/or a specific anti-VEGFR-1 antibody and assessing the effects on signaling, migration, invasion, and proliferation. RESULTS: All 11 pancreatic carcinoma cell lines expressed VEGFR-1 mRNA and protein, as well as the VEGFR-1 ligands VEGF-A and VEGF-B. Two representative cell lines (L3.6 and Panc-1) exhibited VEGF-B-induced mitogen-activated protein kinase signaling. A VEGFR-1 neutralizing antibody abrogated signaling, confirming that the ligand effect was mediated through VEGFR-1. VEGFR-1 stimulation by VEGF-A or VEGF-B was found to promote migration in both cell lines. Panc-1 cells also demonstrated enhanced Matrigel invasion after VEGFR-1 stimulation. VEGFR-1-dependent migration and invasion were blocked by the VEGFR-1 neutralizing antibody. VEGFR-1 activation did not appear to enhance cell proliferation. CONCLUSIONS: VEGFR-1 appears to be expressed ubiquitously in pancreatic carcinoma cell lines, in which it induces signaling and promotes migration and invasion. Overexpression of VEGF in tumors may activate tumor cells bearing VEGFR-1 via an autocrine pathway. Agents targeting VEGF or its receptors may have a dual inhibitory effect on tumor growth by suppressing both angiogenesis and tumor cell function.

The role of endogenous heme synthesis and degradation domain cysteines in cellular iron-dependent degradation of IRP2.

Iron regulatory protein 2 (IRP2) is a mammalian cytosolic iron-sensing protein that regulates expression of iron metabolism proteins, including ferritin and transferrin receptor 1. IRP2 is ubiquitinated and degraded by the proteasome in iron-replete cells but is relatively stable in iron-depleted cells. Recent work has shown that IRP2 contains a unique 73-amino-acid domain that binds iron in vitro and undergoes iron-dependent oxidation and cleavage (J. Biol. Chem. 278 (2003), 14857). Several cysteines in the 73-amino-acid domain function as an in vitro iron-binding site. To assess the role of these cysteines in cellular iron- dependent degradation of IRP2, we mutagenized these cysteines in various combinations in the context of full-length protein and generated cell lines in which recombinant IRP2 expression was inducible. Iron-dependent degradation of IRP2 mutagenized at any or all of the cysteines of the putative degradation domain in cells was comparable to wild-type (WT). Both WT and cysteine mutant protein were stabilized in 3% oxygen. Treatment with sodium nitroprusside (SNP), an NO+ donor, caused a decrease in cellular IRP2 concentrations, but the SNP effect was abrogated by simultaneous addition of the iron chelator desferal and was not affected by cysteine mutations. Inhibition of endogenous heme synthesis with succinylacetone significantly inhibited iron- dependent degradation of IRP2. Addition of cobalt chloride inhibited degradation of both WT and mutagenized IRP2. Thus, we could not discern a role for the recently defined in vitro cysteine-dependent iron-binding site of IRP2 in cellular physiology. The early molecular events in iron-dependent degradation of IRP2 remain to be elucidated.

Role of hypoxia-inducible factor 1alpha in gastric cancer cell growth, angiogenesis, and vessel maturation.

BACKGROUND: Hypoxia-inducible factor 1 (HIF-1), a heterodimer comprising the oxygen-regulated subunit, HIF-1alpha, and HIF-1beta, mediates transcription of the gene for vascular endothelial growth factor (VEGF). Overexpression of HIF-1alpha is associated with tumor angiogenesis and tumor cell proliferation and invasion. We examined the effects of inhibiting HIF-1alpha activity on angiogenesis and human gastric cancer growth in vivo. METHODS: Human gastric cancer TMK-1 cells were stably transfected with pHIF-1alphaDN, an expression plasmid encoding a dominant-negative form of HIF-1alpha that dimerizes with endogenous HIF-1beta to produce HIF-1 complexes that cannot activate transcription, or with the empty expression vector (pCEP4). Two clones of pHIF-1alphaDN-transfected cells, DN2 and DN3, were tested in all experiments. We used an enzyme-linked immunosorbent assay to measure VEGF secretion by transfected cells cultured in hypoxic (1% O2) or nonhypoxic (20% O2) conditions. We used subcutaneous and orthotopic mouse tumor models to examine the growth of tumors derived from injected pHIF-1alphaDN-or pCEP4-transfected cells. Tumor cell proliferation, vessel area (a measure of functional vascular volume), and tumor endothelial cell association with pericyte-like cells (a measure of vessel maturation) were analyzed by immunohistochemical or immunofluorescent staining. All statistical tests were two-sided. RESULTS: DN2 cells and DN3 cells secreted less VEGF than pCEP4-transfected TMK-1 cells when cultured in nonhypoxic or hypoxic conditions (e.g., DN2 versus pCEP4 in nonhypoxic conditions: 645 pg of VEGF/10(6) cells versus 1591 pg of VEGF/10(6) cells, difference = 946 pg of VEGF/10(6) cells [95% confidence interval [CI] = 640 to 1251 pg of VEGF/10(6) cells; P =.006]; DN2 versus pCEP4 in hypoxic conditions: 785 pg of VEGF/10(6) cells versus 2807 pg of VEGF/10(6) cells, difference = 2022 pg of VEGF/10(6) cells [95% CI = 1871 to 2152 pg of VEGF/10(6) cells; P<.001]). In the subcutaneous tumor model, tumors derived from DN2 or DN3 cells had lower final volumes, weights, and vessel areas, less tumor endothelial cell association with desmin-positive cells, and fewer proliferating tumor cells than tumors derived from pCEP4-transfected cells. In the orthotopic tumor model, tumors derived from DN2 cells had smaller volumes and less vessel area and maturation than tumors derived from pCEP4-transfected cells. CONCLUSIONS: Inhibition of HIF-1alpha activity impairs gastric tumor growth, angiogenesis, and vessel maturation.