Pathways for aberrant angiogenesis in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Although the specific mechanisms that dictate its biological aggressiveness are not clearly established, it is characterized by a variety of molecular alterations as well as by the overexpression of mitogenic and angiogenic growth factors and their receptors. PDACs also express high levels of vascular endothelial growth factor (VEGF). Recent studies indicate that suppression of VEGF expression attenuates pancreatic cancer cell tumorigenicity in a nude mouse model, and that VEGF can exert direct mitogenic effects on some pancreatic cancer cells. These findings suggest that cancer cell derived VEGF promotes pancreatic cancer growth in vivo via a paracrine angiogenic pathway and an autocrine mitogenic pathway, and provide novel opportunities for therapeutic intervention in this deadly disease.     

Pancreatic cancer cell-derived vascular endothelial growth factor is biologically active in vitro and enhances tumorigenicity in vivo

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator that acts by binding to high-affinity transmembrane receptors. Although both VEGF and its receptors are overexpressed in human pancreatic ductal adenocarcinoma (PDAC), this malignancy is not generally considered to be highly vascular. It is not known, therefore, whether the abundance of VEGF in PDAC is biologically relevant. To address this issue, we measured the angiogenic effects of pancreatic cancer cell-derived VEGF in an in vitro endothelial cell proliferation assay and characterized the consequences of suppressing VEGF expression on pancreatic tumor growth in an athymic nude mouse model. We found that human pancreatic cancer cell lines secrete large quantities of biologically active VEGF into conditioned medium (CM). Stable transfection of an anti-sense VEGF(189) (AS-VEGF(189)) expression construct into PANC-1 pancreatic cancer cells resulted in decreased VEGF expression and secretion, a decreased capacity of the resultant CM to enhance endothelial cell proliferation and a significant attenuation of tumor cell proliferation in vitro. Furthermore, when injected into athymic nude mice, AS-VEGF(189)-expressing cells exhibited an 80% decrease in tumor growth compared with control cells. These results support the hypothesis that VEGF promotes pancreatic cancer growth in vivo and suggest that anti-VEGF therapy may be useful in the treatment of this disease.     

Safety profile and tolerability of antiangiogenic agents in non-small-cell lung cancer

Recent advances in understanding the importance of angiogenesis to tumor growth and distant metastasis has driven the development of antiangiogenic therapies for the treatment of non-small-cell lung cancer (NSCLC). The anti-vascular endothelial growth factor (VEGF) monoclonal antibody, bevacizumab, is the only US Food and Drug Administration-approved antiangiogenic agent for advanced NSCLC. Accumulated safety data with bevacizumab in NSCLC shows that patients are at risk for hemorrhage, venous thromboembolism, hypertension, and proteinuria. Investigational agents that target VEGF via a different mechanism (such as aflibercept [VEGF Trap]) or simultaneously inhibit multiple molecular pathways involved in angiogenesis (ie, multitargeted tyrosine kinase inhibitors [TKIs]) and vascular disrupting agents (VDAs) that target existing tumor vasculature are in various stages of clinical development for NSCLC, and safety profiles are emerging for these classes of agents. This review describes the molecular rationale for targeting angiogenic pathways in anticancer therapy and summarizes safety and tolerability data from clinical trials of bevacizumab or aflibercept in combination with chemotherapy and the investigational TKIs and VDAs in patients who have advanced NSCLC.     

Apoptotic effect of tolfenamic acid in androgen receptor-independent prostate cancer cell and xenograft tumor through specificity protein 1

Tolfenamic acid (Tol) is a non-steroidal anti-inflammatory drug that was reported to exhibit anticancer activity in pancreatic and colorectal cancer models. This study examined the role of Tol in the death regulation of PC-3 and DU145 human androgen-independent prostate cancer cells. The results showed that Tol inhibited cell growth and induced apoptosis, as evidenced by nuclear fragmentation and cleaved caspase 3 and poly(ADP-ribose) polymerase. Tol suppressed the specificity protein 1 (Sp1) protein in both PC-3 and DU145 cells. Tol also attenuated Sp1 mRNA and its promoter activity in DU145 cells, but did not alter them in PC-3 cells, indicating that Tol degrades Sp1 protein in these cells. Tol also downregulated protein levels, mRNA levels and promoter activities of survivin and myeloid cell leukemia-1, which are downstream targets of Sp1. The expressions of survivin and Mcl-1 and cancer cell growth were lower in the PC-3 cells treated with Sp1 interfering RNA and mithramycin A. Moreover, an oral injection of Tol decreased tumor growth and downregulated the Sp1 protein in athymic nude mice bearing DU145 cell xenografts without hepatotoxicity. Overall, Tol downregulates the Sp1 protein to inhibit growth and induce apoptosis in androgen-refractory prostate cancers, both in vitro and in vivo, that show resistance against many chemotherapeutic agents.     

Antiangiogenesis Therapy in Breast Cancer

Increased expression of the vascular endothelial growth factor (VEGF) is a poor prognostic factor in breast cancer, indicating that antiangiogenic therapies may improve outcomes. Novel antiangiogenic agents targeting the proangiogenic VEGF ligand and receptor tyrosine kinase inhibitors have been developed. Of these, bevacizumab, a humanized monoclonal antibody directed against VEGF, is very promising in breast cancer. A large phase 3 clinical trial demonstrated a statistically significant improvement in progression-free survival with the addition of bevacizumab to paclitaxel as first-line treatment of advanced breast cancer, establishing the benefit of antiangiogenic therapy in breast cancer. Additional studies of bevacizumab in the metastatic, adjuvant, and neoadjuvant settings are underway. Ongoing trials are also evaluating the efficacy of multitargeted tyrosine kinase inhibitors in advanced breast cancer. This article reviews the results of the key trials evaluating antiangiogenic agents in breast cancer with particular emphasis on bevacizumab and future directions of antiangiogenic therapy.     

Neoadjuvant treatment of colorectal cancer with bevacizumab: the perioperative angiogenic balance is sensitive to systemic thrombospondin-1 levels.

PURPOSE: Colorectal cancer patients receiving neoadjuvant treatment with bevacizumab, a monoclonal antibody neutralizing vascular endothelial growth factor (VEGF), may suffer from wound healing complications after surgery as the antibody persists in patient blood. We characterized the systemic angiogenic balance in the perioperative period to evaluate its effect on physiologic angiogenesis. EXPERIMENTAL DESIGN: Nineteen patients receiving combination chemotherapy and bevacizumab for six neoadjuvant cycles were compared with 14 patients receiving chemotherapy without bevacizumab. Plasma from perioperative days -1, +1, +7, and +21 was analyzed for VEGF, thrombospondin-1 (TSP-1), and PD-ECGF concentrations. The angiogenic capacity was further tested in an in vitro assay of endothelial cell proliferation and migration. RESULTS: On day +1, the onset of wound healing was reflected in a change of balance, i.e., an increase of proangiogenic factors VEGF and platelet-derived endothelial cell growth factor compared with low TSP-1 inhibitor levels in both treatment groups. Patients with bevacizumab therapy showed significantly higher blood levels of total VEGF throughout the evaluation period. However, most VEGF molecules were inactive, i.e., complexed with antibody. Nevertheless, the capacity to stimulate endothelial growth was higher for these plasma samples and was reflected in low TSP-1 levels and an altered TSP-1 sensitivity. When purified TSP-1 protein was added, plasma samples of the bevacizumab but not the chemotherapy group showed reduced endothelial growth. CONCLUSIONS: Feedback mechanisms of bevacizumab therapy are not restricted to VEGF expression but seem to involve additional factors, such as TSP-1, which influences the systemic angiogenic balance and permits endothelial growth.     

靶向VEGF治疗恶性肿瘤的研究进展

血管生成是肿瘤生长、侵袭、转移的基础。目前,研究表明VEGF及其家族成员在肿瘤的血管生成中发挥着重要作用。抑制VEGF的表达、活性和其下游信号传递可取得明显的抗肿瘤效应,表明VEGF是一个有效的肿瘤治疗靶点。     

Role of Sp proteins in regulation of vascular endothelial growth factor expression and proliferation of pancreatic cancer cells

Sp proteins play an important role in angiogenesis and growth of cancer cells, and specificity protein 1 (Sp1) has been linked to vascular endothelial growth factor (VEGF) expression in pancreatic cancer cells. RNA interference was used to investigate the role of Sp family proteins on regulation of VEGF expression and proliferation of Panc-1 pancreatic cancer cells. Using a series of constructs containing VEGF promoter inserts, it was initially shown that Sp1 and Sp3 were required for transactivation, and this was primarily dependent on proximal GC-rich motifs. We also showed that Sp4 was expressed in Panc-1 cells, and RNA interference assays suggested that Sp4 cooperatively interacted with Sp1 and Sp3 to activate VEGF promoter constructs in these cells. However, the relative contributions of Sp proteins to VEGF expression were variable among different pancreatic cancer cell lines. Small inhibitory RNAs for Sp3, but not Sp1 or Sp4, inhibited phosphorylation of retinoblastoma protein, blocked G0/G1-->S-phase progression, and up-regulated p27 protein/promoter activity of Panc-1 cells; similar results were observed in other pancreatic cancer cells, suggesting that Sp3-dependent growth of pancreatic cancer cells is caused by inhibition of p27 expression.     

The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells

There is evidence that specificity proteins (Sp), such as Sp1, Sp3, and Sp4, are overexpressed in tumors and contribute to the proliferative and angiogenic phenotype associated with cancer cells. Sp1, Sp3, and Sp4 are expressed in a panel of estrogen receptor (ER)-positive and ER-negative breast cancer cell lines, and we hypothesized that regulation of their expression may be due to microRNA-27a (miR-27a), which is also expressed in these cell lines and has been reported to regulate the zinc finger ZBTB10 gene, a putative Sp repressor. Transfection of ER-negative MDA-MB-231 breast cancer cells with antisense miR-27a (as-miR-27a) resulted in increased expression of ZBTB10 mRNA and decreased expression of Sp1, Sp3, and Sp4 at the mRNA and protein levels and also decreased activity in cells transfected with constructs containing Sp1 and Sp3 promoter inserts. In addition, these responses were accompanied by decreased expression of Sp-dependent survival and angiogenic genes, including survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1). Moreover, similar results were observed in MDA-MB-231 cells transfected with ZBTB10 expression plasmid. Both as-miR-27a and ZBTB10 overexpression decreased the percentage of MDA-MB-231 cells in S phase of the cell cycle; however, ZBTB10 increased the percentage of cells in G(0)-G(1), whereas as-miR-27a increased the percentage in G(2)-M. This latter response was associated with induction of Myt-1 (another miR-27a target gene), which inhibits G(2)-M through enhanced phosphorylation and inactivation of cdc2. Thus, the oncogenic activity of miR-27a in MDA-MB-231 cells is due, in part, to suppression of ZBTB10 and Myt-1.     

Antiangiogenic mechanisms of PJ-8, a novel inhibitor of vascular endothelial growth factor receptor signaling

Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.     

The vascular-targeting fusion toxin VEGF121/rGel inhibits the growth of orthotopic human bladder carcinoma tumors

Vascular endothelial growth factor (VEGF) and its receptors (FLT-1 and KDR) are overexpressed by human bladder cancer cells and tumor endothelial cells, respectively. Strategies that target VEGF receptors hold promise as antiangiogenic therapeutic approaches to bladder cancer. A fusion protein of VEGF121 and the plant toxin gelonin (rGel) was constructed, expressed in bacteria, and purified to homogeneity. Cytotoxicity experiments of VEGF121/rGel on the highly metastatic 253J B-V human bladder cancer cell line demonstrated that the VEGF121/rGel does not specifically target these cells, whereas Western blot analysis showed no detectable expression of KDR. Treatment with VEGF121/rGel against orthotopically implanted 253J B-V xenografts in nude mice resulted in a significant suppression of bladder tumor growth (approximately 60% inhibition; P < .05) compared to controls. Immunohistochemistry studies of orthotopic 253J B-V tumors demonstrated that KDR is highly overexpressed in tumor vasculature. Immunofluorescence staining with antibodies to CD-31 (blood vessel endothelium) and rGel demonstrated a dramatic colocalization of the construct on tumor neovasculature. Treated tumors also displayed an increase in terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining compared to controls. Thus, VEGF121/rGel inhibits the growth of human bladder cancer by cytotoxic effects directed against the tumor vascular supply and has significant potential as a novel antiangiogenic therapeutic against human bladder cancer.     

The evolving role of VEGF-targeted therapies in the treatment of metastatic colorectal cancer

Therapies that target angiogenesis and the VEGF pathway are a component of treatment for patients with metastatic colorectal cancer (mCRC). Bevacizumab is a humanized monoclonal antibody that binds to VEGFA. Chemotherapy plus bevacizumab has led to improved outcomes for mCRC patients. Despite these benefits, progressive disease invariably ensues. Multiple members of the VEGF family can potentially contribute to tumor angiogenesis and/or evasion of antiangiogenic therapy if one pathway should be inhibited. Aflibercept, a new biological agent, is a multiple angiogenic factor trap that prevents not only VEGFA, but also VEGFB and PlGF from activating their native receptors. Key clinical data for bevacizumab and aflibercept for treatment of mCRC, clinical evidence for use of these agents beyond progression, and the search for angiogenic biomarkers to better define patients most likely to benefit from these interventions will be reviewed.