VEGF-mediated Tumor Angiogenesis Research Articles

Combined treatment with Alhagi maurorum and docetaxel inhibits breast cancer progression via targeting HIF-1α/VEGF mediated tumor angiogenesis in vivo

Breast cancer is a challenging disease and leading cause of cancer death in women. There is no effective agent for metastatic breast cancer after surgery and chemotherapy. Alhagi maurorum (A.m) has been reported to exhibit an anticancer effect on various types of cancer cells in vitro. This study aimed to examine the suppressive effect of A.m alone and combined with docetaxel (DTX) on the breast cancer growth in mice models and the possible underlying mechanisms. In the present study, the mice were inoculated subcutaneously with the injections of 4T1 cells. Then, A.m, DTX, and their combination were administered intraperitoneally. The expressions of β-catenin (β-cat), FZD7, MMP2, HIF1-α, and VEGF A (vascular endothelial growth factor A) were investigated using RT-PCR method. Also, plasma alkaline phosphatase (ALP), alanine aminotransferase (GPT or ALT), aspartate transaminase (GOT or AST), serum creatinine, and urea were examined, and histological analyses of the tissues were conducted. The results demonstrated that A.m (500 mg/kg) combined with DTX significantly decreased the expression of β-cat, MMP2, and FZD7 as compared with the negative control group and monotherapies. Also, the mRNA levels of HIF1-α and VEGF A were suppressed significantly by DTX + A.m (500 mg/kg). Tumor weights and sizes were significantly lower and tumor inhibition rate was significantly higher in the DTX + A.m group. The A.m 500 mg/kg + DTX also suppressed the serum GPT level in tumor-bearing mice and decreased the serum urea level. Taken together, our findings suggest that DTX combined with A.m at an optimal dose of 500 mg/kg as the optimal dose can inhibit β-cat, FZD7, MMP2, and breast cancer growth via interrupting HIF-1α/VEGF signaling and might be used as a promising antiangiogenic agent for breast cancer treatment.

Abstract 1415: Osteopontin and matrix metalloproteinase 2 plasma levels are elevated in colorectal cancer (CRC) vs benign disease; The 2 protein combination improves specificity as regards CRC diagnosis

Abstract Introduction: Expression of Osteopontin (OPN) and Matrix Metalloproteinase 2 (MMP 2) are elevated in CRC. OPN is an integrin binding phosphorylated glycoprotein implicated in cell-mediated immunity, inflammation, tumor progression, and cell viability. OPN is secreted by macrophages and leukocytes. OPN activity is associated with the PI3K/AKT and ERK pathways and VEGF mediated tumor angiogenesis. MMP-2 degrades type IV collagen and facilitates tumor cell invasion and metastasis. MMP-2 also promotes tumor angiogenesis. This study’s first purpose was to compare preoperative (PreOp) plasma OPN and MMP2 levels in CRC and benign pathology (BP) patients (pts); the 2nd purpose was to assess the combination of these proteins for diagnosis. Method: PreOp plasma samples obtained from consenting CRC and BP pts undergoing surgery were used for this study. Prospectively gathered demographic, clinical and pathologic data were analyzed. Plasma levels of OPN and MMP2 (ng/ml) were measured via ELISA and reported as median + 95% CI. The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to evaluate the diagnostic value of each protein alone and in combination. Expression levels of OPN and MMP2 were determined in tumor and normal tissue for a subset of pts by QRT-PCR. The Mann-Whitney test was used for analysis. Results: Plasma from 156 CRC (73% colon, 27% rectal) and 102 BP (polyp 32%, diverticulitis 56%, other 12%) pts was studied. CRC stage breakdown was: 1, 26%; 2, 33%, 3, 29%, 4, 12%. CRC median preop plasma levels were significantly higher vs. the BP group for both proteins [OPN; 82.4,CI:75.4, 89.5 vs 59.2, CI:53.8,63.3;MMP2; 203.6,CI:195.0,214.5 vs 160.2,CI:151.9,172.2; P<0.0001). Higher OPN levels were noted in stg 3(p=0.04) and Stg 4(p=0.01) vs stg 1 pts. The AUC value for the ROC curve for OPN and MMP2 were 0.73 and 0.73 respectively with 61% and 70% specificity. The AUC for the 2 protein combination was 0.80 with 65% sensitivity and 80% specificity. OPN and MMP2 expression was elevated in 75% and 50% of CRC tumors tested, respectively (n=12). Conclusion: Median CRC OPN and MMP2 levels were significantly higher (39% and 27%) than BP levels. OPN and MMP2 expression was confirmed in a subset of pts. This 2 protein combination improved the AUC and specificity for CRC diagnosis. Further study with more protein combinations is warranted. Citation Format: Chandana S. k. Herath Mudiyanselage, Hiromichi Miyagaki, Neil Mitra, Yanni Hedjar, Xiaohong Yan, Vesna Cekic, Joseph Martz, Jennifer Agnew, Richard L. Whelan. Osteopontin and matrix metalloproteinase 2 plasma levels are elevated in colorectal cancer (CRC) vs benign disease; The 2 protein combination improves specificity as regards CRC diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1415.

RNA sequencing profiles and diagnostic signatures linked with response to ramucirumab in gastric cancer.

Gastric cancer (GC) is the fifth-ranked cancer type by associated mortality. The proportion of early diagnosis is low, and most patients are diagnosed at the advanced stages. First-line therapy standardly includes fluoropyrimidines and platinum compounds with trastuzumab for HER2-positive cases. For recurrent disease, there are several alternative options including ramucirumab, a monoclonal therapeutic antibody that inhibits VEGF-mediated tumor angiogenesis by binding with VEGFR2, alone or in combination with other cancer drugs. However, overall response rate following ramucirumab or its combinations is 30%–80% of the patients, suggesting that personalization of drug prescription is needed to increase efficacy of treatment. We report here original tumor RNA sequencing profiles for 15 advanced GC patients linked with data on clinical response to ramucirumab or its combinations. Three genes showed differential expression in the tumors for responders versus nonresponders: CHRM3, LRFN1, and TEX15. Of them, CHRM3 was up-regulated in the responders. Using the bioinformatic platform Oncobox we simulated ramucirumab efficiency and compared output model results with actual tumor response data. An agreement was observed between predicted and real clinical outcomes (AUC ≥ 0.7). These results suggest that RNA sequencing may be used to personalize the prescription of ramucirumab for GC and indicate potential molecular mechanisms underlying ramucirumab resistance. The RNA sequencing profiles obtained here are fully compatible with the previously published Oncobox Atlas of Normal Tissue Expression (ANTE) data.

Identification of signature genes associated with therapeutic resistance to anti-VEGF therapy.

VEGF-mediated tumor angiogenesis is a validated clinical target in many cancers, but modest efficacy and rapid development of resistance are major challenges of VEGF-targeted therapies. To establish a molecular signature of this resistance in ovarian cancer, we developed preclinical tumor models of adaptive resistance to chronic anti-VEGF treatment. We performed RNA-seq analysis and reverse-phase protein array to compare changes in gene and protein expressions in stroma and cancer cells from resistant and responsive tumors. We identified a unique set of stromal-specific genes that were strongly correlated with resistance phenotypes against two different anti-VEGF treatments, and selected the apelin/APJ signaling pathway for further in vitro validation. Using various functional assays, we showed that activation of apelin/APJ signaling reduces the efficacy of a VEGF inhibitor in endothelial cells. In patients with ovarian cancer treated with bevacizumab, increased expression of apelin was associated with significantly decreased disease-free survival. These findings link signature gene expressions with anti-VEGF response, and may thus provide novel targetable mechanisms of clinical resistance to anti-VEGF therapies.

Salinomycin inhibits breast cancer progression via targeting HIF-1α/VEGF mediated tumor angiogenesis in vitro and in vivo

Cancer is a complex disease wherein cells begin to divideabnormally and spread into surrounding tissues. Angiogenesis plays a crucial role in tumor progression as it is required for sustained growth and metastasis, therefore targeting angiogenesis is a promising therapeutic approach for breast cancer management. Salinomycin (SAL) has been reported to exhibit anticancer response on various types of cancer. In the present study, we explored the antiangiogenic and anticancer efficacy of the polyether ionophore SAL in the breast cancer model. It effectively inhibited cell proliferation, invasion, and migration. It also inhibited the expression of pro-angiogenic cell surface marker CD31 in HUVEC, thereby interrupting the endothelial tubulogenesis. It decreased the HIF-1α transcription factor DNA binding activity to HRE sequence in HUVEC and human breast cancer cells. Further, corresponding to our in vitro findings, SAL suppressed neovascularization in the chick chorioallantoic membrane and the Matrigel plug implanted mice model. Bioluminescence and immunofluorescence imaging revealed that SAL treatment in mice inhibits breast cancer growth and tumor angiogenesis. SAL also suppressed the serum VEGFA level in tumor-bearing mice and induced caspase-dependent apoptosis in breast cancer cells. Taken together our findings suggested that SAL inhibits VEGF induced angiogenesis and breast cancer growth via interrupting HIF-1α/VEGF signalling and could be used as a promising antiangiogenic agent for breast cancer treatment.

Suppression of angiogenesis and tumor growth by recombinant T4 phages displaying extracellular domain of vascular endothelial growth factor receptor 2.

Tumor growth, invasion and metastasis are dependent on angiogenesis. The Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway plays a pivotal role in tumor angiogenesis and therefore represents a reasonable target for anti-angiogenesis/anti-tumor therapy. In the present study, we generated T4 recombinant phages expressing the extracellular domain of VEGFR2 (T4-VEGFR2) and investigated their anti-angiogenic activity. The T4-VEGFR2 phages were able to bind to VEGF specifically and inhibit VEGF-mediated phosphorylation of VEGFR2 and its downstream kinases such as extracellular signal-regulated kinase (ERK) and p38 mitogen activated protein kinase (MAPK). The in vitro experiments showed that the T4-VEGFR2 phages could inhibit VEGF-stimulated cell proliferation and migration of endothelial cells. Finally, administration of T4-VEGFR2 phages was able to suppress tumor growth and decrease microvascular density in murine models of Lewis lung carcinoma and colon carcinoma, and prolong the survival of tumor bearing mice. In conclusion, this study reveals that the recombinant T4-VEGFR2 phages generated using T4-based phage display system can inhibit VEGF-mediated tumor angiogenesis and the T4 phage display technology can therefore be used for the development of novel anti-cancer strategies.

PF-2341066 combined with celecoxib promotes apoptosis and inhibits proliferation in human cholangiocarcinoma QBC939 cells.

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with high incidence and an average age of onset of 50-70 years old. However, at present there is no effective treatment for this disease. The aim of the present study was to investigate the effects of a c-Met inhibitor, PF-2341066 and a cyclooxygenase-2 (COX-2) inhibitor, celecoxib, on c-Met and COX-2 expression, proliferation and apoptosis. The results demonstrated that c-Met and COX-2 are highly expressed in hepatobiliary calculus with cholangiocarcinoma. PF-2341066 was able to downregulate the expression of c-Met and COX-2 in a dose-dependent manner at the mRNA and protein levels in human cholangiocarcinoma QBC939 cells. Furthermore, combined treatment with PF-2341066 with celecoxib downregulated the mRNA expression of both genes, inhibited cell proliferation and promoted cell apoptosis. It was also demonstrated that PF-2341066 and celecoxib treatment was able to restrict the expression of vascular endothelial growth factor (VEGF). The results of the present study suggest that PF-2341066 and celecoxib may inhibit the development of cholangiocarcinoma by downregulating the expression of c-Met and COX-2 to inhibit cell proliferation, promote apoptosis and prevent VEGF-mediated tumor angiogenesis. Co-treatment with PF-2341066 and celecoxib may be a potential therapeutic strategy for hepatobiliary calculus with cholangiocarcinoma.

Potent and long‐term antiangiogenic efficacy mediated by FP3‐expressing oncolytic adenovirus

Various ways to inhibit vascular endothelial growth factor (VEGF), a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic adenovirus (Ad) expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases.

328. FP3, a Novel VEGF Blocker, Expressing Oncolytic Adenovirus Induces Potent Antiangiogenic and Antitumor Efficacy

Various ways to inhibit VEGF, a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic Ad expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density, and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases.

The synergistic regulation of VEGF-mediated angiogenesis through miR-190 and target genes.

VEGF is a major contributor to angiogenesis, a vital process in normal growth and development and tumor transition. However, the current clinical efficacy of VEGF inhibitors is limited, and the molecular mechanism underlying VEGF regulation remains to be elucidated. Here we show that miR-190 directly targets a group of angiogenic effectors besides VEGF per se. Noted, these effectors can transcriptionally regulate VEGF expression in an intracellular or intercellular manner, thus demonstrating that miR-190 modulates the VEGF-mediated tumor angiogenesis at three levels. The synergistic effect of miR-190 and its target genes demonstrates a complex but apparently more stable system, allowing for the tight control of the level of VEGF. Finally, we showed that miR-190 significantly suppresses tumor metastasis, especially angiogenesis. Together, these results indicate that miR-190 is a promising antitumor target in clinical applications.

Effects of MDM2 inhibitors on vascular endothelial growth factor-mediated tumor angiogenesis in human breast cancer

Mouse double minute 2 (MDM2) is overexpressed in many malignant tumors, and MDM2 levels are associated with poor prognosis of several human cancers, including breast cancer. In the present study, we investigated the function of MDM2 in vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis of breast cancer and the potential value of MDM2 as an anti-angiogenic therapy target for cancer therapy by inhibiting MDM2 with antisense oligonucleotides (ASO) or other antagonist nutlin-3. Anti-MDM2 ASO and nutlin-3 were evaluated for their in vitro and in vivo anti-angiogenesis activities in different human breast cancer models with a different p53 status: MCF-7 cell line containing wild-type p53 and MDA-MB-468 cell line containing mutant p53. MCF-7 and MDA-MB-468 cells were incubated with different concentrations of ASO or nutlin-3 for various periods of time. VEGF gene and protein expression in tumor cells was measured by qPCR and Western blot. The level of VEGF protein secreted in the culture supernatant of treated cells was quantified by enzyme-linked immunosorbent assay (ELISA). Nude mouse xenograft models were further established to determine their effects on tumor growth and angiogenesis. Serum levels of VEGF were measured by ELISA. VEGF expression and microvessel density in tumor tissues were studied by immunohistochemistry. Both angiogenesis and tumor growth were digitally quantified. In both MCF-7 and MDA-MB-468 cells, VEGF expression and secretion were reduced, resulting from specific inhibition of MDM2 expression by ASO. In vivo assay, after administration of ASO, VEGF production reduced and anti-angiogenesis activity occurred in nude mice bearing MCF-7 or MDA-MB-468 xenograft. However, in both models treated with nutlin-3, VEGF production was not changed and anti-angiogenesis activity was not observed. In summary, the ASO construct targeting MDM2 specifically suppresses VEGF expression in vitro and VEGF-mediated tumor angiogenesis in vivo in breast cancer. Furthermore, the suppression of VEGF expression subsequent to inhibition of MDM2 in p53 mutant cells suggests that MDM2 has a regulatory role on VEGF expression through a p53-independent mechanism.

Overexpression of human papillomavirus (HPV) type 16 oncoproteins promotes angiogenesis via enhancing HIF-1α and VEGF expression in non-small cell lung cancer cells

HPV-16 infection may play an important role in the development of non-small cell lung cancer (NSCLC) among never-smokers. Due to the critical role of angiogenesis in NSCLC development, we describe here the effect of HPV-16 oncoproteins on angiogenesis in NSCLC and the underlying mechanisms. We found that overexpression of HPV-16 E6 and E7 oncoproteins in NSCLC cells significantly promoted angiogenesis both in vitro and in vivo, and correspondingly, an enhanced expression of HIF-1α and VEGF, important pro-angiogenic factors in tumor angiogenesis. Meanwhile, overexpression of HPV-16 oncoproteins also led to HIF-1α-dependent increases in the secretion of several other pro-angiogenic factors, including IL-8. Our findings suggest that HPV-16 oncoproteins contribute to the development of NSCLC possibly by promoting HIF-1α/VEGF-mediated tumor angiogenesis.

Selective aldosterone blocker, eplerenone, attenuates hepatocellular carcinoma growth and angiogenesis in mice

The renin-angiotensin-aldosterone system (RAAS) has become known as a prerequisite for tumor angiogenesis, including hepatocellular carcinoma (HCC). Although angiotensin II is known to play an important role in tumor growth and angiogenesis, the role of aldosterone (Ald) is still obscure. The aim of our current study was to elucidate the effect of eplerenone, a clinically used selective Ald blocker (SAB), on murine HCC development especially in conjunction with angiogenesis. To create an allograft model, we injected 1 x 10(6) of BNL-HCC cells into the flanks of BALB/c mice. After the tumor was established, SAB was administrated at dose of 100 mg/kg per day. Administration of SAB significantly suppressed HCC development along with inhibition of angiogenesis and expression of the vascular endothelial growth factor (VEGF), a potent angiogenic factor. SAB treatment resulted in a marked increase of apoptosis in the tumor, whereas tumor cell proliferation was not altered. Our in vitro study showed that SAB significantly suppressed the Ald-induced endothelial proliferation and tubular formation through inhibition of phosphorylation of the extracellular signal-regulated kinase 1/2. On the contrary, neither Ald nor SAB affected the proliferation of HCC cells in vitro. Ald plays a pivotal role in HCC development through VEGF-mediated tumor angiogenesis, and SAB may be a potential new strategy in HCC therapy in the future.

Abstract 2468: Selective aldosterone blocker inhibits murine hepatocellular carcinoma development via angiogenesis suppression

Abstract It is now widely recognized that angiogenesis is a crucial step in the development of tumors, including hepatocellular carcinoma (HCC). Therapies targeting the tumor vessels have proven successful for cancer treatment in experimental models and, in the clinical practice, several anti-angiogenic agents have already been employed. The renin-angiotensin-aldosterone system (RAAS) has also become known as a prerequisite for tumor angiogenesis, including HCC. We previously reported that angiotensin-II (AT-II) plays an important role in HCC development, and that suppression of AT-II by angiotensin-converting enzyme (ACE) inhibitor attenuated the tumor growth of HCC via suppression of intra-tumoral angiogenesis. Recent studies have also suggested the possible involvement of aldosterone (Ald) in neovascularization, although the actual role of Ald in tumor angiogenesis is still vague. The aim of our current study was to elucidate the effect of eplerenone (Epl), a clinically used selective Ald blocker, on HCC development, especially in conjunction with angiogenesis, using subcutaneous HCC model. To create an allograft model, we injected 1×106 of BNL-HCC cells into the flanks of BALB/c mice. The administration of Epl by daily gavage at a dose of 100 mg/kg/day was started on day 14, when the mean tumor volume was ∼ 200 mm3. The tumor volume was measured twice a week, and the mice were killed 35 days after tumor cell implantation. Administration of Epl at clinically comparable low dose could suppress HCC development as compared with the non-treated control. Epl treatment resulted in a marked increase of TUNEL-positive apoptosis in the tumor, whereas Ki67-positive tumor cell proliferation was not altered. Both CD31-immunopositive neo-vessels and VEGF expression in the tumor were markedly suppressed by this treatment in a magnitude similar to the inhibitory effect on the tumor growth. Our in-vitro study showed that Epl significantly suppressed the Ald-induced endothelial proliferation and tubular formation via inhibition of phosphorylation of the extracellular signal-regulated kinase 1/2. On the other hand, neither Ald nor Epl affected the proliferation of HCC cells. In conclusion, these results indicated that aldosterone plays a pivotal role in HCC development via VEGF-mediated tumor angiogenesis, and that Epl may be a potential new strategy for HCC therapy in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2468.

Bevacizumab (BEV) and continuous low-dose granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with advanced solid tumors: final results of a prospective clinical trial

e14544 Background: BEV is potent inhibitor of VEGF-mediated tumor angiogenesis. VEGF is also crucial in antineoplastic immunortolerance by blocking the generation of dendritic cells (DC). VEGF inhibition may thus result in an improved anti-cancer immune response which may be further augmented by GM-CSF, a powerful stimulator of DC generation. In a previous study, GM-CSF was able to produce clinical responses in patients (pts) suffering from refractory carcinomas (Kurbacher et al., Oncology 2005). In this trial, we investigated BEV+GM-CSF in pts with advanced solid neoplasms. Methods: 26 pts have been enrolled: epithelial ovarian cancer, n=12; breast cancer, n= 6; primary peritoneal carcinoma, n=3; hormone-refractory prostate cancer, n=2; miscellaneous, n=3. All pts have been considered refractory to or non-treatable by chemotherapy due to their baseline bone marrow and/or organ functions. Fourteen pts had previously received BEV as part of other antineoplastic regimens. BEV was applied at a fixed dose of 6 mg/kg q2w. The starting dose of GM-CSF (sargramostim) was 125 μg/d. In cases not responding and/or not achieving moderate leukocytosis (< 20 G/L), GM-CSF was increased every 4 weeks at 25 μg increments up to a maximum of 250 μg/d. Toxicity was documented according to the NCI-CTC score, responses were recorded according to the RECIST or Rustin criteria. Results: Grade (G) 3–4 hypertension was seen in 3 pts, with 1 pt experiencing cerebral stroke 6 weeks after cessation of BEV+GM-CSF. Other G3–4 toxicities were fatigue in two pts, and fever, pain, myalgia, and central nervous symptoms in either one pt. Other side-effects like injection-site reactions, proteinuria, anemia, thrombocytopenia, and constipation did not exceed G1–2. The objective response rate was 31% (1 CR, 7 PR, 10 SD, and 8 PD); the rate of pts benefiting was 69%. Eight pts failing prior BEV benefited from BEV+GM-CSF. Until today, 13 pts have died. The median time to progression is 92 days and the median overall survival is 302 days. Conclusions: BEV+GM-CSF is an active and generally well tolerated non-cytotoxic regimen in pts with advanced solid tumors. Thus, large-scaled studies of this promising treatment are warranted. No significant financial relationships to disclose.

637. Suppression of VEGF-Mediated Tumor Angiogenesis by Cellular Relocalization of Its Transcriptional Activator HIF-1α

637. Suppression of VEGF-Mediated Tumor Angiogenesis by Cellular Relocalization of Its Transcriptional Activator HIF-1α

CXCR4/CXCL12 axis promotes VEGF-mediated tumor angiogenesis through Akt signaling pathway

CXC chemokine receptor 4 (CXCR4) has been shown to play a critical role in chemotaxis and homing, which are key steps in cancer metastasis. There is also increasing evidence that links this receptor to angiogenesis; however, its molecular basis remains elusive. Vascular endothelial growth factor (VEGF), one of the major angiogenic factors, promotes the formation of leaky tumor vasculatures that are the hallmarks of tumor progression. Here, we investigated whether CXCR4 induces the expression of VEGF through the PI3K/Akt pathway. Our results showed that CXCR4/CXCL12 induced Akt phosphorylation, which resulted in upregulation of VEGF at both the mRNA and protein levels. Conversely, blocking the activation of Akt signaling led to a decrease in VEGF protein levels; blocking CXCR4/CXCL12 interaction with a CXCR4 antagonist suppressed tumor angiogenesis and growth in vivo. Furthermore, VEGF mRNA levels correlated well with CXCR4 mRNA levels in patient tumor samples. In summary, our study demonstrates that the CXCR4/CXCL12 signaling axis can induce angiogenesis and progression of tumors by increasing expression of VEGF through the activation of PI3K/Akt pathway. Our findings suggest that targeting CXCR4 could provide a potential new anti-angiogenic therapy to suppress the formation of both primary and metastatic tumors.

Increased Melanoma Growth and Metastasis Spreading in Mice Overexpressing Placenta Growth Factor

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family, plays an important role in adult pathological angiogenesis. To further investigate PlGF functions in tumor growth and metastasis formation, we used transgenic mice overexpressing PlGF in the skin under the control of the keratin 14 promoter. These animals showed a hypervascularized phenotype of the skin and increased levels of circulating PlGF with respect to their wild-type littermates. Transgenic mice and controls were inoculated intradermally with B16-BL6 melanoma cells. The tumor growth rate was fivefold increased in transgenic animals compared to wild-type mice, in the presence of a similar percentage of tumor necrotic tissue. Tumor vessel area was increased in transgenic mice as compared to controls. Augmented mobilization of endothelial and hematopoietic stem cells from the bone marrow was observed in transgenic animals, possibly contributing to tumor vascularization. The number and size of pulmonary metastases were significantly higher in transgenic mice compared to wild-type littermates. Finally, PlGF promoted tumor cell invasion of the extracellular matrix and increased the activity of selected matrix metalloproteinases. These findings indicate that PlGF, in addition to enhancing tumor angiogenesis and favoring tumor growth, may directly influence melanoma dissemination.

Vascular endothelial growth factor and osteopontin in stage I lung adenocarcinoma.

Tumor growth and metastasis are angiogenesis-dependent processes initiated and regulated by a number of cytokines. Vascular endothelial growth factor (VEGF) is a potent angiogenic protein with a selective mitogenic effect on vascular endothelial cells. Osteopontin (OPN) induces endothelial cell migration and upregulates endothelial cell migration induced by VEGF. To clarify the cooperative role of VEGF and OPN in tumor angiogenesis, we stained VEGF, OPN, and CD34 immunohistochemically in 87 cases of stage I non-small cell lung cancer (adenocarcinoma, 55, and squamous cell carcinoma, 32). Of the 87 patients studied, 27 patients had postoperative relapse and 60 patients did not. VEGF was found in 34 of 55 cases of adenocarcinomas and 14 of 32 squamous cell carcinomas, and OPN was found in 30 of 55 adenocarcinomas and 10 of 32 squamous cell carcinomas. In adenocarcinoma, microvessel counts of VEGF-positive and OPN-positive tumors were significantly higher than VEGF-negative and OPN-negative tumors, respectively, whereas in squamous cell carcinoma they were not. More importantly, patients with VEGF- and OPN-positive stage I lung adenocarcinoma had significantly worse prognosis as compared with other groups. Cooperation of OPN is important in VEGF-mediated tumor angiogenesis in stage I lung adenocarcinoma.