A human IgG-like bispecific antibody co-targeting epidermal growth factor receptor and the vascular endothelial growth factor receptor 2 for enhanced antitumor activity

Both Epidermal Growth Factor Receptor (EGFR) and the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) play critical roles in tumorigenesis. We hypothesized co-targeting EGFR and VEGFR2 using a bispecific antibody might have significant therapeutic potential. Here,we designed and produced a human IgG-like bispecific antibody (Bi-Ab) based on the variable regions of cetuximab (an anti-EGFR antibody) and mAb-04 (an anti-VEGFR2 antibody developed in our lab) . The Bi-Ab was found to inhibit the proliferation, survival and invasion of cancer cells via ablating phosphorylation of receptor and downstream signaling. In vivo efficacy was demonstrated against established HT-29 and SKOV-3 xenografts grown in nude mice. Studies revealed our Bi-Ab was able to restrain xenografted tumor growth and prolong survival of mice through inhibiting cell proliferation,promoting apoptosis and anti-angiogenesis. In contrast to cetuximab or mAb-04 alone, our Bi-Ab exhibits enhanced antitumor activity and has equal or slightly superior activity to their combination (Combi). It shows promise as a therapeutic agent, especially for use against tumors EGFR and/or VEGFR2 over-expressing malignancies.     

Inhibition of renal cell carcinoma angiogenesis and growth by antisense oligonucleotides targeting vascular endothelial growth factor

Angiogenesis is critical for growth and metastatic spread of solid tumours. It is tightly controlled by specific regulatory factors. Vascular endothelial growth factor has been implicated as the key factor in tumour angiogenesis. In the present studies we evaluated the effects of blocking vascular endothelial growth factor production by antisense phosphorothioate oligodeoxynucleotides on the growth and angiogenic activity of a pre-clinical model of renal cell carcinoma (Caki-1). In vitro studies showed that treating Caki-1 cells with antisense phosphorothioate oligodeoxynucleotides directed against vascular endothelial growth factor mRNA led to a reduction in expressed vascular endothelial growth factor levels sufficient to impair the proliferation and migration of co-cultured endothelial cells. The observed effects were antisense sequence specific, dose dependent, and could be achieved at a low, non-toxic concentration of phosphorothioate oligodeoxynucleotides. When vascular endothelial growth factor antisense treated Caki-1 cells were injected into nude mice and evaluated for their angiogenic potential, the number of vessels initiated were approximately half that induced by untreated Caki-1 cells. To test the anti-tumour efficacy of vascular endothelial growth factor antisense, phosphorothioate oligodeoxynucleotides were administrated to nude mice bearing macroscopic Caki-1 xenografts. The results showed that the systemic administration of two doses of vascular endothelial growth factor antisense phosphorothioate oligodeoxynucleotides given 1 and 4 days after the tumours reached a size of approximately 200 mm(3) significantly increased the time for tumours to grow to 1000 mm(3).     

Bevacizumab‐enhanced antitumor effect of 5‐fluorouracil via upregulation of thymidine phosphorylase through vascular endothelial growth factor A/vascular endothelial growth factor receptor 2‐specificity protein 1 pathway

Bevacizumab (Bv) can be used synergistically with fluoropyrimidine‐based chemotherapy to treat colorectal cancer. Whether and how it affects the delivery of fluoropyrimidine drugs is unknown. The present study aimed to explore the effect of Bv on the delivery of 5‐fluorouracil (5‐FU) to tumors and the underlying mechanism from metabolic perspective. Bv enhanced the anti‐tumor effects of 5‐FU in LoVo colon cancer xenograft mice and increased the 5‐FU concentration in tumors without affecting hepatic 5‐FU metabolism. Interestingly, Bv remarkably upregulated thymidine phosphorylase (TP) in tumors, which mediated the metabolic activation of 5‐FU. Although TP is reported to promote angiogenesis and resistance, the combination of Bv and 5‐FU resulted in anti‐angiogenesis and vessel normalization in tumors, indicating that the elevated TP mainly contributed to the enhanced response to 5‐FU. Bv also induced TP upregulation in LoVo cancer cells. Treatment with vascular endothelial growth factor receptor 2 (VEGFR2) antagonist apatinib and VEGFR2 silencing further confirmed TP upregulation. Bv and apatinib both enhanced the cytotoxicity of 5‐FU in LoVo cells, but there was no synergism with adriamycin and paclitaxel. We further demonstrated that the effect of Bv was dependent on VEGFR2 blockade and specificity protein 1 activation via MDM2 inhibition. In summary, Bv enhanced the accumulation of 5‐FU in tumors and the cytotoxicity of 5‐FU via TP upregulation. We provide data to better understand how Bv synergizes with 5‐FU from metabolic perspective, and it may give clues to the superiority of Bv in combination with fluoropyrimidine drugs compared to other chemotherapeutic drugs in colon cancer.     

Novel hKDR mouse model depicts the antiangiogenesis and apoptosis-promoting effects of neutralizing antibodies targeting vascular endothelial growth factor receptor 2

Vascular endothelial growth factor receptor 2 (VEGFR2)/KDR plays a critical role in tumor growth, diffusion, and invasion. The amino acid sequence homology of KDR between mouse and human in the VEGF ligand-binding domain was low, thus the WT mice could not be used to evaluate Abs against human KDR, and the lack of a suitable mouse model hindered both basic research and drug developments. Using the CRISPR/Cas9 technique, we successfully inserted different fragments of the human KDR coding sequence into the chromosomal mouse Kdr exon 4 locus to obtain an hKDR humanized mouse that can be used to evaluate the marketed Ab ramucirumab. In addition, the humanized mAb VEGFR-HK19 was developed, and a series of comparative assays with ramucirumab as the benchmark revealed that VEGFR-HK19 has higher affinity and superior antiproliferation activity. Moreover, VEGFR-HK19 selectively inhibited tumor growth in the hKDR mouse model but not in WT mice. The most important binding epitopes of VEGFR2-HK19 are D257, L313, and T315, located in the VEGF binding region. Therefore, the VEGFR2-HK19 Ab inhibits tumor growth by blocking VEGF-induced angiogenesis, inflammation, and promoting apoptosis. To our best knowledge, this novel humanized KDR mouse fills the gaps both in an animal model and the suitable in vivo evaluation method for developing antiangiogenesis therapies in the future, and the newly established humanized Ab is expected to be a drug candidate possibly benefitting tumor patients.     

Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors.

Chemically stabilized hammerhead ribozymes are nuclease-resistant, RNA-based oligonucleotides that selectively bind and cleave specific target RNAs. Due to their potential for specifically inhibiting gene expression, ribozymes are being investigated for therapeutic applications as well as for the elucidation of gene function. In particular, we have investigated ribozymes that target the mRNA of the vascular endothelial growth factor (VEGF) receptors because VEGF signaling is an important mediator of tumor angiogenesis and metastasis. Here we report pharmacodynamic studies testing anti-Flt-1 (VEGFR-1) and anti-KDR (VEGFR-2) ribozymes in animal models of solid tumor growth and metastasis. Ribozymes targeting either Flt-1 or KDR significantly inhibited primary tumor growth in a highly metastatic variant of Lewis lung carcinoma. However, only treatment with the anti-Flt-1 ribozyme resulted in a statistically significant and dose-dependent inhibition of lung metastasis in this model. The anti-Flt-1 ribozyme was then tested in a xenograft model of human metastatic colorectal cancer in which significant inhibition of liver metastasis was observed. Taken together, these data represent the first demonstration that synthetic ribozymes targeting VEGF receptor mRNA reduced the growth and metastasis of solid tumors in vivo.     

Novel strategy of liver cancer treatment with modified antisense oligonucleotides targeting human vasohibin-2

Vasohihibin-2 (VASH2) is a homolog of vasohibin-1 (VASH1) and is overexpressed in various cancers. Vasohihibin-2 acts on both cancer cells and cancer microenvironmental cells. Previous analyses have shown that VASH2 promotes cancer progression and abrogation of VASH2 results in significant anticancer effects. We therefore propose VASH2 to be a practical molecular target for cancer treatment. Modifications of antisense oligonucleotide (ASO) such as bridged nucleic acids (BNA)-based modification increases the specificity and stability of ASO, and are now applied to the development of a number of oligonucleotide-based drugs. Here we designed human VASH2-ASOs, selected an optimal one, and developed 2′,4′-BNA-based VASH2-ASO. When systemically administered, naked 2′,4′-BNA-based VASH2-ASO accumulated in the liver and showed its gene-silencing activity. We then examined the effect of 2′,4′-BNA-based VASH2-ASO in liver cancers. Intraperitoneal injection of naked 2′,4′-BNA-based VASH2-ASO exerted a potent antitumor effect on orthotopically inoculated human hepatocellular carcinoma cells. The same manipulation also showed potent antitumor activity on the splenic inoculation of human colon cancer cells for liver metastasis. These results provide a novel strategy for the treatment of primary as well as metastatic liver cancers by using modified ASOs targeting VASH2.     

Novel human Ab against vascular endothelial growth factor receptor 2 shows therapeutic potential for leukemia and prostate cancer

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor‐associated endothelial cells, where it modulates tumor‐promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti‐VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti‐VEGFR2‐AF, which shows excellent VEGFR2 binding activity. Anti‐VEGFR2‐AF bound Ig‐like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF‐A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti‐VEGFR2‐AF inhibited capillary structure formation and exerted Ab‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC‐3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC‐3 xenograft mouse model, treatment with anti‐VEGFR2‐AF repressed tumor growth and angiogenesis as effectively and safely as US FDA‐approved anti‐VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti‐VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL‐60 human leukemia‐xenografted mice, anti‐VEGFR2‐AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti‐VEGFR2‐AF has strong potential as a cancer therapy that could directly target VEGFR2‐expressing tumor cells in addition to its anti‐angiogenic action.     

Angiogenesis and radiation response modulation after vascular endothelial growth factor receptor-2 (VEGFR2) blockade

The formation of new blood vessels (angiogenesis) represents a critical factor in the malignant growth of solid tumors and metastases. Vascular endothelial cell growth factor (VEGF) and its receptor VEGFR2 represent central molecular targets for antiangiogenic intervention, because of their integral involvement in endothelial cell proliferation and migration. In the current study, we investigated in vitro and in vivo effects of receptor blockade on various aspects of the angiogenic process using monoclonal antibodies against VEGFR2 (cp1C11, which is human specific, and DC101, which is mouse specific). Molecular blockade of VEGFR2 inhibited several critical steps involved in angiogenesis. VEGFR2 blockade in endothelial cells attenuated cellular proliferation, reduced cellular migration, and disrupted cellular differentiation and resultant formation of capillary-like networks. Further, VEGFR2 blockade significantly reduced the growth response of human squamous cell carcinoma xenografts in athymic mice. The growth-inhibitory effect of VEGFR2 blockade in tumor xenografts seems to reflect antiangiogenic influence as demonstrated by vascular growth inhibition in an in vivo angiogenesis assay incorporating tumor-bearing Matrigel plugs. Further, administration of VEGFR2-blocking antibodies in endothelial cell cultures, and in mouse xenograft models, increased their response to ionizing radiation, indicating an interactive cytotoxic effect of VEGFR2 blockade with radiation. These data suggest that molecular inhibition of VEGFR2 alone, and in combination with radiation, can enhance tumor response through molecular targeting of tumor vasculature.     

可溶性VEGF受体基因sflt-1与反义VEGF核苷酸对新生血管形成的影响

背景与目的:肿瘤组织中新生血管提供的大量营养物质和生长因子是肿瘤快速生长的关键,因此如何抑制肿瘤组织中新生血管的形成、促使肿瘤组织坏死也是肿瘤治疗中的一条值得探讨的途径。本文拟研究和观察可溶性血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)受体基因sflt-1与反义VEGF对新生血管形成的影响。方法:用Ad-反义VEGF感染肝癌细胞株MM45T.Li后,观察反义VEGF对MM45T.Li分泌VEGF的影响;将人工重组的3'ΔFlt-1蛋白(C末端缺失的Flt-1蛋白)加入条件培养液中,观察3'ΔFlt-1对VEGF刺激的脐静脉血管内皮细胞(humanumbilicalvascularendotheliumcell,HUVEC)增殖的影响;并将Ad-反义VEGF、Ad-sflt-1注射于鸡胚尿囊绒毛膜中,观察sflt-1、反义VEGF对鸡胚新生血管形成的影响。结果:反义VEGF重组腺病毒感染MM45T.Li细胞后,细胞培养上清中VEGF浓度仅为对照组中VEGF浓度的15%(P<0.01);在含有3'ΔFlt-1蛋白的调价培养液中,HUVEC的增殖明显减慢,在一定的范围内与剂量呈负相关;两者都能有效地抑制鸡胚新生血管的形成。结论:sflt-1与反义VEGF均能有效抑制新生血管的形成,两者联合能增强抑制效果,但其作用机制不同。     

前列腺癌细胞株VEGF及其受体KDR,bFGF及其受体FGFR2的表达及意义

目的:探讨人前列腺癌细胞株血管内皮细胞生长因子(VEGF)及其受体(KDR),碱性成纤维细胞生长因子(bFGF)及其受体(FGFR2)的表达,进一步阐明前列腺癌细胞中VEGF和bFGF的自分泌机制。方法:以小鼠成纤维细胞系L929作为对照,选取三种前列腺癌细胞株(PC3、LNcap和DU145),采用免疫组化染色、RT-PCR及Westernblot,检测VEGF及其受体KDR,bFGF及其受体FGFR2的表达。结果:三种前列腺癌细胞株(PC3、LNcap和DU145)中均有VEGF、KDR及bFGF、FGFR2的表达,但表达水平略有差别。结论:在前列腺癌的血管形成中可能存在VEGF和bFGF的自分泌机制。     

Treatment of the T98G glioblastoma cell line with antisense oligonucleotides directed toward mRNA encoding transforming growth factor-α and the epidermal growth factor receptor

Antisense oligonucleotides (oligos) complementary to mRNA encoding transforming growth factor-α (TGF-α) and its target, the epidermal growth factor receptor (EGFR), are efficacious against human prostate and breast cancers carried in athymic nude mice. Glioblastomas, also regulated by EGFR expression, would appear to be similarly susceptible, and we now employ them against the T98G tumor model. T98G cells were distributed into wells and allowed to adhere prior to addition of oligos (12.5 μM) directed against TGF-α and/or EGFR for 6 d of treatment before thymidine radiolabeling. Supplemental media and oligos (25 μM final concentration) were added after d 3. Statistically significant inhibition by oligos directed against TGF-α, EGFR, and their combination was 13.8%, 26.3%, and 18.1%, respectively. In a subsequent experiment cells were incubated with increasing amounts of each oligo and their combination for 3 d prior to radiolabeling. Statistically significant inhibition of growth for either oligo at every concentration was found. Cells incubated with 6.25, 12.5, 25, and 50 μM antisense directed against TGF-α had a mean inhibition of 29.3%, 33.3%, 21.7%, and 46.6%, respectively. Cells similarly treated with oligos against EGFR had a mean inhibition of 77.9%, 80.3%, 82.0%, and 83.7%, respectively, and cells incubated with 6.25, 12.5, 25 and 50 μM of each oligo had a mean inhibition of 74.7%, 70.6%, 70.8%, and 76.3%, respectively. Lastly, in a paired experiment, cells treated with 0, 0.39, 0.78, 1.56, 3.125, and 6.25 μM of oligos, either specifically directed against EGFR or a random control, for 3 d were evaluated for both thymidine incorporation and EGFR expression. Statistically significant inhibition of ³H-thymidine incorporation was seen in cells with the oligo specifically directed against EGFR at 3.125 μM and 6.25 μM when compared to non-oligo containing controls. This was accompanied by a comparable significantly decreased expression of a low-MW reactive derivative of EGFR at 3.125 μM and 6.25 μM in Western blots, and of a high-MW reactive EGFR at 6.25 μM. The significant effect against high-MW EGFR was observed vs both the non-oligo containing control and the random sequence. Oligo concentrations between 0.78 and 1.5 μM also resulted in decreased expression of the low-MW form, but not significant differences in thymidine radiolabeling. In recovery experiments, cells treated initially with greater oligo concentrations required significantly increased time to recover, particularly in cells treated with EGFR directed oligos. Intracellular uptake and nuclear localization was demonstrated with FITC tagged oligos. In summary, even at relatively low oligo concentrations and short exposure, oligos against TGF-α, and particularly EGFR, significantly inhibit in vitro growth of the T98G glioblastoma, possibly mediated by decreased EGFR expression.     

VEGF及其受体Flt和KDR水平变化与胶质瘤关系及临床意义

 目的 探讨胶质瘤患者血清血管内皮生长因子（VEGF）及其受体Flt-1和KDR水平变化，为评价胶质瘤治疗效果，判断预后寻找一种科学的生物学标志物。方法 收集治疗前后胶质瘤患者、脑转移癌患者和健康对照者血清，进行VEGF，Flt-1和KDR水平的检测，SPSS11.5统计软件包对数据进行t检验和相关分析。结果 （1）治疗前脑胶质瘤和脑转移瘤患者血清VEGF水平明显高于健康对照组； 脑胶质瘤患者组和脑转移瘤患者组血清Flt-1水平明显高于健康对照组；脑胶质瘤患者组血清KDR水平明显高于健康对照组； 治疗后缓解的胶质瘤患者组VEGF和Flt-1水平明显低于治疗前。差异均有统计学意义。（2） VEGF与Flt-1和KDR有显著的相关性。结论 胶质瘤患者血清中VEGF及其受体的检测，可作为胶质瘤辅助诊断、治疗效果监测和预后判断的重要生物学指标。     

Preclinical characterization of anlotinib,a highly potent and selective vascular endothelial growth factor receptor‐2 inhibitor

Abrogating tumor angiogenesis by inhibiting vascular endothelial growth factor receptor‐2 (VEGFR2) has been established as a therapeutic strategy for treating cancer. However, because of their low selectivity, most small molecule inhibitors of VEGFR2 tyrosine kinase show unexpected adverse effects and limited anticancer efficacy. In the present study, we detailed the pharmacological properties of anlotinib, a highly potent and selective VEGFR2 inhibitor, in preclinical models. Anlotinib occupied the ATP‐binding pocket of VEGFR2 tyrosine kinase and showed high selectivity and inhibitory potency (IC₅₀ <1 nmol/L) for VEGFR2 relative to other tyrosine kinases. Concordant with this activity, anlotinib inhibited VEGF‐induced signaling and cell proliferation in HUVEC with picomolar IC₅₀ values. However, micromolar concentrations of anlotinib were required to inhibit tumor cell proliferation directly in vitro. Anlotinib significantly inhibited HUVEC migration and tube formation; it also inhibited microvessel growth from explants of rat aorta in vitro and decreased vascular density in tumor tissue in vivo. Compared with the well‐known tyrosine kinase inhibitor sunitinib, once‐daily oral dose of anlotinib showed broader and stronger in vivo antitumor efficacy and, in some models, caused tumor regression in nude mice. Collectively, these results indicate that anlotinib is a well‐tolerated, orally active VEGFR2 inhibitor that targets angiogenesis in tumor growth, and support ongoing clinical evaluation of anlotinib for a variety of malignancies.     

Antiangiogenic and antitumor activity of anti-epidermal growth factor receptor C225 monoclonal antibody in combination with vascular endothelial growth factor antisense oligonucleotide in human GEO colon cancer cells.

Angiogenesis plays a key role in tumor growth and metastasis. The transforming growth factor alpha (TGF-alpha)-epidermal growth factor receptor (EGFR) autocrine pathway controls in part the production of angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in cancer cells. In this study, we have evaluated the antiangiogenic and antitumor activity of monoclonal antibody (MAb) C225, an anti-EGFR chimeric human-mouse MAb, alone and in combination with a human VEGF antisense (AS) 21-mer phosphorothioate oligonucleotide (VEGF-AS) in human GEO colon cancer cells. MAb C225 treatment determined a dose-dependent inhibition of VEGF, bFGF, and TGF-alpha production by GEO cells in vitro. Treatment with VEGF-AS caused a selective inhibition in VEGF expression by GEO cells in vitro. Treatment of immunodeficient mice bearing established, palpable GEO xenografts for 3 weeks with VEGF-AS or with MAb C225 determined a cytostatic reversible inhibition of tumor growth. In contrast, a prolonged inhibition of tumor growth was observed in all mice treated with the two agents, in combination with a significant improvement in mice survival compared with controls (P < .001), to MAb C225 (P < .001), or to VEGF-AS (P < .001) treated mice. All mice died within 4, 6, and 8 weeks after tumor cell injection in the control, VEGF-AS and MAb C225 groups, respectively. In contrast, 50% of mice treated with the combination of VEGF-AS and MAb C225 were alive at 13 weeks. Ten % of mice treated with VEGF-AS plus MAb C225 were alive at 20 weeks and had no histological evidence of GEO tumors. Immunohistochemical analysis of GEO tumor xenografts demonstrated a significant reduction of VEGF expression after treatment with VEGF-AS with a parallel reduction in microvessel count. MAb C225 treatment determined a reduction in the expression of VEGF, bFGF, and TGF-alpha with a reduction in microvessel count. Finally, a significant potentiation in inhibition of VEGF expression and little or no microvessels were observed in GEO tumors after the combined treatment with the two agents.     

AEE788: a dual family epidermal growth factor receptor/ErbB2 and vascular endothelial growth factor receptor tyrosine kinase inhibitor with antitumor and antiangiogenic activity

Aberrant epidermal growth factor receptor (EGFR) and ErbB2 expression are associated with advanced disease and poor patient prognosis in many tumor types (breast, lung, ovarian, prostate, glioma, gastric, and squamous carcinoma of head and neck). In addition, a constitutively active EGFR type III deletion mutant has been identified in non-small cell lung cancer, glioblastomas, and breast tumors. Hence, members of the EGFR family are viewed as promising therapeutic targets in the fight against cancer. In a similar vein, vascular endothelial growth factor (VEGF) receptor kinases are also promising targets in terms of an antiangiogenic treatment strategy. AEE788, obtained by optimization of the 7H-pyrrolo[2,3-d]pyrimidine lead scaffold, is a potent combined inhibitor of both epidermal growth factor (EGF) and VEGF receptor tyrosine kinase family members on the isolated enzyme level and in cellular systems. At the enzyme level, AEE788 inhibited EGFR and VEGF receptor tyrosine kinases in the nm range (IC(50)s: EGFR 2 nm, ErbB2 6 nm, KDR 77 nm, and Flt-1 59 nm). In cells, growth factor-induced EGFR and ErbB2 phosphorylation was also efficiently inhibited (IC(50)s: 11 and 220 nm, respectively). AEE788 demonstrated antiproliferative activity against a range of EGFR and ErbB2-overexpressing cell lines (including EGFRvIII-dependent lines) and inhibited the proliferation of epidermal growth factor- and VEGF-stimulated human umbilical vein endothelial cells. These properties, combined with a favorable pharmacokinetic profile, were associated with a potent antitumor activity in a number of animal models of cancer, including tumors that overexpress EGFR and or ErbB2. Oral administration of AEE788 to tumor-bearing mice resulted in high and persistent compound levels in tumor tissue. Moreover, AEE788 efficiently inhibited growth factor-induced EGFR and ErbB2 phosphorylation in tumors for >72 h, a phenomenon correlating with the antitumor efficacy of intermittent treatment schedules. Strikingly, AEE788 also inhibited VEGF-induced angiogenesis in a murine implant model. Antiangiogenic activity was also apparent by measurement of tumor vascular permeability and interstitial leakage space using dynamic contrast enhanced magnetic resonance imaging methodology. Taken together, these data indicate that AEE788 has potential as an anticancer agent targeting deregulated tumor cell proliferation as well as angiogenic parameters. Consequently, AEE788 is currently in Phase I clinical trials in oncology.     

Colorectal carcinoma cell production of transforming growth factor beta decreases expression of endothelial cell vascular endothelial growth factor receptor 2

BACKGROUND:

Vascular endothelial growth factor (VEGF) signaling is a target for antiangiogenic cancer therapy. The authors have previously observed that up to 40% of vessels in colorectal carcinoma (CRC) tumors are negative for VEGF receptor 2 (VEGFR2) expression. Differential activity of transforming growth factor beta (TGF‐β) is a potential contributor to this receptor heterogeneity because TGF‐β contributes to both angiogenesis and CRC tumor progression.

METHODS:

The authors analyzed VEGFR2 expression by Western blotting, and TGF‐β expression in endothelial and CRC cell lines, respectively. In addition, they immunostained endothelial cells in CRC xenografts to find an association between VEGFR2 and TGF‐β levels or activity.

RESULTS:

In bovine aortic endothelial cells (BAECs), TGF‐β1 significantly repressed VEGFR2 protein in a time‐dependent and dose‐dependent fashion (P < .05). Serum‐free conditioned media from various malignant human CRC cell lines (HCT116, 379.2, Dks8, and DLD1) induced down‐regulation of VEGFR2 in BAECs. This effect was proportional to the total levels of TGF‐β1 and TGF‐β2 and was blocked by SB‐431542 and SD‐208, TGF‐β receptor I inhibitors. Immunofluorescence staining of subcutaneous mouse xenografts of HCT116, 379.2, Dks8, and SW480 cells revealed vessels with an inverse relationship between TGF‐β activity and VEGFR2 expression. Oxygen and bone morphogenetic protein 9 levels were shown to modulate TGF‐β–induced VEGFR2 down‐regulation.

CONCLUSIONS:

In combination with other factors, TGF‐β may contribute to the vascular heterogeneity in human colorectal tumors. Cancer 2011;. © 2011 American Cancer Society.     

The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma

Vascular endothelial growth factor receptor 1 (VEGFR-1) is present on endothelial cells and subsets of human tumor cells, raising the hypothesis that angiogenic factors may promote tumor growth both by inducing angiogenesis and directly signaling through activation of VEGFR-1 on tumor cells. Here, we report that VEGFR-1 is expressed on a panel of 16 human breast tumor cell lines, and the vasculature and the tumor cell compartment of a subset of breast carcinoma lesions, and that selective signaling through VEGFR-1 on breast cancer cells supports tumor growth through downstream activation of the p44/42 mitogen-activated protein kinase (MAPK) or Akt pathways. Ligand-stimulated proliferation of breast tumor cells was inhibited by specific blockade with an anti-VEGFR-1 neutralizing monoclonal antibody. Treatment with anti-VEGFR-1 mAb significantly suppressed the growth of DU4475, MCF-7, BT-474 and MDA-MB-231 breast xenografts in athymic mice. Histological examination of anti-VEGFR-1 mAb treated tumor xenografts showed a significant reduction of activation of the p44/42 MAPK or Akt pathways in tumor cells resulting in an increase in tumor cell apoptosis. Importantly, cotreatment with mAbs targeting human VEGFR-1 on tumor cells and murine VEGFR-1 on vasculature led to more potent growth inhibition of breast tumor xenografts. The results suggest that VEGF receptors may not only modulate angiogenesis, but also directly influence the growth of VEGF receptor expressing tumors.     

Pharmacodynamic behavior of liposomal antisense oligonucleotides targeting Her-2/neu and vascular endothelial growth factor in an ascitic MDA435/LCC6 human breast cancer model

The nature of anti-cancer therapeutics is currently undergoing a paradigm change, with biologic agents slowly being introduced into the therapeutic armory, displacing or complimenting the traditionally used cytotoxic agents. These new agents include monoclonal antibodies, recombinant DNA, antisense oligonucleotides (ASO) and others. To assess the new therapeutics, new predictive models are required. Utilizing the MDA435/LCC6 human breast cancer xenograft model, the pharmacokinetic behavior of antisense oligonucleotides targeted against vascular endothelial growth factor and HER-2/neu was assessed. For pharmacodynamic analysis, ASO in buffer or encapsulated in a liposomal formulation were injected intravenously or intraperitoneally into MDA435/LCC6 ascites tumor-bearing mice. Plasma antisense elimination, tissue distribution, total peritoneal antisense and peritoneal cell associated antisense levels were determined. Liposomal encapsulation led to significant decreases in the plasma elimination rate, as evidenced by an approximate 10-fold increase in mean AUC over 24 hours, as well as enhanced peritoneal cell delivery in mice bearing ascites tumors. Tissue distribution studies of both free and liposome encapsulated ASO indicated that ASO distribution was dictated primarily by the liposomal carrier when administered in liposomal form.     

The antitumor and antiangiogenic activity of vascular endothelial growth factor receptor inhibition is potentiated by ErbB1 blockade.

PURPOSE: Receptor tyrosine kinases of the ErbB family play important roles in the control of tumor growth. Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation, enhances vascular permeability, and plays an important role in tumor vascularization. We evaluated the effects of selective VEGF receptor (VEGFR; PTK787/ZK222584) and ErbB (PKI166 and ZD1839) inhibitors on tumor growth and angiogenesis and asked whether additional therapeutic benefit was conferred by combination treatment. EXPERIMENTAL DESIGN: The antitumor activity of each inhibitor alone or in combination was assessed in human cancer models in immunocompromised mice. ErbB receptor expression and activation of downstream signaling pathway was evaluated in both tumor and endothelial cells. RESULTS: Both ErbB inhibitors significantly enhanced the antitumor activity of PTK787/ZK222584. In vitro, ErbB1 inhibition blocked VEGF release by tumor cells and proliferation of both tumor and endothelial cells. In an in vitro angiogenesis assay, epidermal growth factor (EGF) stimulated the release of VEGF by smooth muscle cells resulting in increased angiogenesis, a response blocked by administration of PTK787/ZK222584. Under basal condition, both ZD1839 and PTK787/ZK222584 blocked sprouting, likely via inhibition of an autocrine ErbB1 loop and VEGFR signaling, respectively, in endothelial cells. In conditions of limiting VEGF, EGF plays an important role in endothelial cell proliferation, survival, and sprouting. CONCLUSION: We have shown that activation of ErbB1 triggers a plethora of effects, including direct effects on tumor and endothelial cells and indirect effects mediated via induction of VEGF release. Simultaneous blockade of ErbB1 and VEGFR pathways results in a cooperative antitumor effect, indicating that this combination may represent a valid therapeutic strategy.     

Relationship between Co-expression of Vascular Endothelial Growth Factor and its Receptor, Kinase Insert Domain Containing Receptor and Tumor Angiogenesis in Invasive Carcinoma of the Cervix

Objective The aim of the present study was to determine the expression of vascular endothelial growth factor (VEGF) and its receptor, kinase insert domain containing receptor (KDR), and their significance in regulating tumor angiogenesis in the early stages of cervical cancer. Methods Using the immunohistochemical SP method, the expression of VEGF and KDR was determined in the cancer cells. In addition, the microvessel density (MVD), labeled by CD34 in the tumor stroma, was examined in 18 cases of cervical intraepithelial neoplasms (CIN), 75 cases of early invasive cervix carcinomas (ICC) and 15 specimens of normal cervical epithelium (NCE). Results In ICC cases, VEGF and KDR were mainly expressed in the cellular membrane and/or cytoplasm of tumor cells, while expression of CD34 was found mainly in the vascular epithelial cells of the tumor stroma. The positive expression rate of VEGF and KDR, and the MVD increased remarkably from NCE through CIN to ICC (P< 0.01). For the ICC group, in the patients with positive expression of VEGF and KDR, the MVD was significantly higher than those with negative expression of VEGF and KDR (P<0.05). Expression of VEGF in ICC was positively related to KDR expression (r=0.56,P<0.01 ). The MVD was also positively related to both the expression of VEGF (r=0.60,P<0.01), and KDR (r=0.33,P<0.01). In the cases with both positive expression of VEGF and KDR, the MVD was significantly higher than those in which there was negative expression of both(P<0.01). Conclusion Expression of VEGF and its receptor KDR plays a key role in up-regulating tumor angiogenesis in cervical carcinoma. Co-overexpression of VEGF and KDR results in rapid tumor vasculogenesis. Detection of co-expression of VEGF and KDR may be of value in further understanding tumor angiogenesis and in searching for new targets for anti-angiogenesis therapy in invasive carcinoma of the cervix. This work was supported by the Department of Education of Fujian Province (No. 01B017).