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Cilengitide inhibits progression of experimental breast cancer bone metastases as imaged noninvasively using VCT, MRI and DCE-MRI in a longitudinal in vivo study
Abstract
The aim of this study was to investigate the effect of inhibiting αvβ(3)/α(v) β(5) integrins by cilengitide in experimentally induced breast cancer bone metastases using noninvasive imaging techniques. For this purpose, nude rats bearing established breast cancer bone metastases were treated with cilengitide, a small molecule inhibitor of αvβ(3) and αvβ(5) integrins (75 mg/kg, five days per week; n = 12 rats) and compared to vehicle-treated control rats (n = 12). In a longitudinal study, conventional magnetic resonance imaging (MRI) and flat panel volumetric computed tomography were used to assess the volume of the soft tissue tumor and osteolysis, respectively, and dynamic contrast-enhanced (DCE-) MRI was performed to determine functional parameters of the tumor vasculature reflecting blood volume and blood vessel permeability. In rats treated with cilengitide, VCT and MRI showed that osteolytic lesions and the respective bone metastatic soft tissue tumors progressed more slowly than in vehicle-treated controls. DCE-MRI indicated a decrease in blood volume and an increase in vessel permeability and immunohistology revealed increased numbers of immature vessels in cilengitide-treated rats compared to vehicle controls. In conclusion, treatment of experimental breast cancer bone metastases with cilengitide resulted in pronounced antiresorptive and antitumor effects, suggesting that αvβ(3)/αvβ(5) inhibition may be a promising therapeutic approach for bone metastases.
... Several registered, U.S. National Institutes of Health clinical trials have used cilengitide as a single agent or in combination with other agents for treatment of metastatic or unresectable cancers including breast, lung, and prostate [22]. In vivo preliminary results have shown cilengitide to reduce breast cancer bone metastasis [23]. However, the mechanistic basis for this result is unresolved. ...
... High expression of the α V β 3 integrin has been demonstrated in tumors and angiogenic cells [11]. Overexpression of the α V β 3 integrin has been reported in various types of cancers including glioblastoma, melanoma, ovarian, pancreatic, prostate, and breast cancer [10,11,23,[25][26][27]. With regard to ITGAV, only limited reports have evaluated expression levels and the functional role of ITGAV in cancer progression. ...
... Subsequent preclinical studies of various types of cancer have investigated cilengitide as a therapeutic cancer treatment modality. Cilengitide has been shown to inhibit metastasis of breast cancer [23,25,26] and osteosarcoma [39]. Consistent with these previous findings, our results demonstrate cilengitide to inhibit breast cancer proliferation in a dose-dependent manner, inhibit lung cancer metastasis, and reduce Ki67 expression. ...
During tumorigenesis and metastasis, integrins regulate localization and activity of proteolytic enzymes that remodel the extracellular matrix. Previous studies have demonstrated blocking of αVβ3 to effectively inhibit proliferation, angiogenesis, and the survival of various cancer cell types. However, little is known about the functional role of the integrin subunit alpha-V gene (ITGAV) in metastatic breast cancer. In this study, ITGAV knockdown was used to identify the molecular mechanism by which ITGAV promotes tumorigenesis, metastasis, proliferation, invasion, and cellular self-renewal. The effectiveness of an ITGAV antagonist, cilengitide, for breast cancer treatment was investigated in vivo. Analysis of publicly available data demonstrated that overexpression of ITGAV was associated with poor relapse free survival of breast cancer patients. Silencing of ITGAV inhibited cell proliferation, invasion, and self-renewal of breast cancer cell lines by altering expression of BCL2 and PXN. The use of cilengitide significantly reduced lung metastasis in a metastatic breast cancer animal model. In conclusion, overexpression of ITGAV contributes to breast cancer metastasis through upregulation of PXN. Targeting ITGAV is a potential treatment for metastatic breast cancer as well as primary breast tumors with high ITGAV expression. ITGAV expression levels may be useful predictors of patient treatment and outcome responses.
... There are now several ongoing clinical trials and in-vivo studies evaluating the anticancer effect of integrin and SRC antagonists in metastatic cancers [3,94,95]. There are several αvβ3 integrin-targeting drugs, including peptides (S247, ATN-161, cilengitide, IMGN388) and nonpeptidic small molecules (PSK1404, GLPG0187, L-000845704) [3,94,96]. In-vivo studies have shown that these antagonists resulted in the blockade of osteolysis and tumor cell growth in animal models of BM [94,96]. L-000845704, ATN-161, and cilengitide bind to αvβ3 and αvβ5 integrins and block tumor growth and BM in animal models [94,96,97]. ...
... There are several αvβ3 integrin-targeting drugs, including peptides (S247, ATN-161, cilengitide, IMGN388) and nonpeptidic small molecules (PSK1404, GLPG0187, L-000845704) [3,94,96]. In-vivo studies have shown that these antagonists resulted in the blockade of osteolysis and tumor cell growth in animal models of BM [94,96]. L-000845704, ATN-161, and cilengitide bind to αvβ3 and αvβ5 integrins and block tumor growth and BM in animal models [94,96,97]. ...
... In-vivo studies have shown that these antagonists resulted in the blockade of osteolysis and tumor cell growth in animal models of BM [94,96]. L-000845704, ATN-161, and cilengitide bind to αvβ3 and αvβ5 integrins and block tumor growth and BM in animal models [94,96,97]. IMGN388, a human immunoglobulin G 1 antibody conjugated to the maytansinoid DM4, also binds to αv integrin and inhibits its functions [98,99]. ...
Distant metastasis during the advanced stage of malignant tumor progression can cause considerable morbidity in cancer patients. Bone is known to be one of the most common sites of distant metastasis in patients with breast cancer (BC). BC metastases in bone are associated with excessive skeletal complications. These complications can be fatal and reduce quality of life of patients. It is important to understand the metastatic process of BC to bone to improve quality of life and design new therapeutic methods. At present, the molecular mechanisms leading to the BC metastasis to bone are not fully understood. Studying the molecular basis of BC metastasis to bone might improve our insight into this complex process. In addition, it can provide novel approaches for designing advanced and effective targeted therapies. The present article aimed to review the published papers on the molecular basis of the metastatic process of BC to bone, focusing on involved genes and signaling networks. Furthermore, we propose potential therapeutic targets that may be more effective for the inhibition and treatment of BC metastasis to bone. Copyright
... Expression of osteogenic and angiogenic factors, RANKL, VEGF and PTHrP [281][282][283][284][285] Osteoclast maturation and function [286] Cilengitide [281][282][283][284][285][286][287][288][289] Integrin α5β1 antibody antagonists [290] Abituzumab [291] TBK1 ...
... Its efficacy in inhibition of angiogenesis and tumor growth in preclinical in vivo models has been demonstrated [281][282][283][284], and cilengitide has also been shown to inhibit osteoclast maturation and function [286]. Importantly, cilengitide demonstrated efficacy in the inhibition of breast tumor bone metastasis growth and osteolysis in preclinical models [288,289] and demonstrated reduction in levels of angiogenic factors such as VEGF, PTHrP and RANKL [285]. Despite showing good tolerability, clinical testing of cilengitide did not result in efficacy in numerous reported studies [316] including non-metastatic prostate cancer patients [285]. ...
Metastasis to the bone is a common feature of many cancers including those of the breast, prostate, lung, thyroid and kidney. Once tumors metastasize to the bone, they are essentially incurable. Bone metastasis is a complex process involving not only intravasation of tumor cells from the primary tumor into circulation, but extravasation from circulation into the bone where they meet an environment that is generally suppressive of their growth. The bone microenvironment can inhibit the growth of disseminated tumor cells (DTC) by inducing dormancy of the DTC directly and later on following formation of a micrometastatic tumour mass by inhibiting metastatic processes including angiogenesis, bone remodeling and immunosuppressive cell functions. In this review we will highlight some of the mechanisms mediating DTC dormancy and the complex relationships which occur between tumor cells and bone resident cells in the bone metastatic microenvironment. These inter-cellular interactions may be important targets to consider for development of novel effective therapies for the prevention or treatment of bone metastases.
... β1 and β3 integrins are expressed on malignant breast cancer cells and are involved in their adhesion (Das et al. 2018). It is known that integrin αvβ3 has low or undetectable levels of expression in most adult epithelia and resting endothelial cells in non-diseased tissues, whereas in breast and prostate cancers, and in activated tumor endothelial cells it is highly expressed (Desgrosellier and Cheresh 2010; Bauerle et al. 2011). Integrin αvβ3 interacts with the integrin-binding tripeptide (RGD) of secreted proteins from the SIBLING family, as osteopontin (OPN) and bone sialoprotein (BSP) (Weilbaecher et al. 2011). ...
... The cyclic pentapeptide Cilengitide having the sequence c(Arg-Gly-Asp-D-Phe-N-Me-Val) exerts high affinity for αvβ3 and αvβ5 and inhibits their cellular processes. This peptide partially suppressed bone resorption and soft tissue tumor growth in osseous lesions in experimental breast cancer bone metastases (Bauerle et al. 2011). In the current approach, we established a model for better understanding of the role of ITGB3 in breast cancer skeletal metastasis, avoiding the disadvantages of permanent knockout (long-term adaptation to the protein deficiency, which could be basis for potential survival strategies of the tumor cells) and that of a transient knockdown (only short-term protein inhibition). ...
Integrin β3 (ITGB3) is probably related to skeletal metastasis, which is the most frequent complication in breast cancer progression. We aimed to define its role and suitability as target for anti-metastatic therapy. We generated two MDA-MB-231 cell clones with conditional miRNA-mediated ITGB3 knockdown for analyzing the resulting effects in vitro regarding mRNA expression, proliferation and migration, as well the impact on skeletal metastasis in a nude rat model. Furthermore, ITGB3 levels were analyzed in exosomes from plasma of rats with skeletal metastases, and from MDA-MB-231 cells incubated with these vesicles, as well as from exosomes secreted by cells with conditional ITGB3 knockdown. This inhibition of ITGB3 expression decreased cellular proliferation and more distinctly inhibited cellular migration. Reduction and even complete remissions of respective soft tissue and osteolytic lesions were detected after ITGB3 knockdown in vivo. Furthermore, ITGB3 levels were increased in exosomes isolated from plasma of rats harboring MDA-MB-231 lesions as well as in respective cells incubated with these vesicles in vitro. ITGB3 was distinctly decreased in exosomes from cells with ITGB3 knockdown. The observed in vitro and in vivo anti-ITGB3 effects can be explained by downregulation of specific genes, which have roles in angiogenesis (NPTN, RRM2), tumor growth (NPTN), energy metabolism (ISCA1), cytokinesis (SEPT11), migration (RRM2, STX6), cell proliferation, invasiveness, senescence, tumorigenesis (RRM2) and vesicle trafficking (SEPT11, STX6). ITGB3 has a role in breast cancer skeletal metastasis via gene expression modulation, as mirrored for ITGB3 in exosomes, thus it could serve as target for anti-metastatic therapy.
... h us, αvβ3, c-Src and Syk represent an essential signaling complex in the boneresorbing osteoclast, and each member of this complex is a candidate therapeutic target. h ere is preclinical evidence that αvβ3 integrin-targeting drugs, including peptides (S247, ATN-161, cilengitide) and nonpeptidic small molecules (PSK1404), success fully block osteolysis and tumor growth in animal models of bone metastasis [14,15]. Not only are these αvβ3 antagonists inhibitory for osteoclast-mediated bone resorption, but we have shown that PSK1404 prevents bone colonization by αvβ3-expressing human breast cancer cells when using a dosing regimen that did not inhibit bone resorption [14]. ...
... Similarly, IMGN388 (developed by ImmunoGen, Inc.) is a human IgG1 antiαv integrin antibody conjugated to the maytansinoid DM4 that is being tested in patients with advanced solid tumors, including metastatic breast carcinoma (Table 1). Cilengitide (developed by Merck), a RGD-mimetic cyclic peptide inhibitor of both αvβ3 and αvβ5 integrins, inhibits bone metastasis in animals [15], and is currently being tested in patients with glioblastoma and advanced solid tumors, including breast cancer (Table 1) [16]. Finally, L-000845704 (developed by Merck), an αvβ3 integrin non peptide antagonist, inhibits bone resorption in women with postmenopausal osteoporosis [17]. ...
Breast cancer is prone to metastasize to bone. Once metastatic cells are in the bone marrow, they do not, on their own, destroy bone. Instead, they alter the functions of bone-resorbing (osteoclasts) and bone-forming cells (osteoblasts), resulting in skeletal complications that cause pathological fractures and pain. In this review, we describe promising molecular bone-targeted therapies that have arisen from recent advances in our understanding of the pathogenesis of breast cancer bone metastases. These therapies target osteoclasts (receptor activator of nuclear factor kB ligand, integrin αvβ3, c-Src, cathepsin K), osteoblasts (dickkopf-1, activin A, endothelin A) and the bone marrow microenvironment (transforming growth factor β, bone morphogenetic proteins, chemokine CXCL-12 and its receptor CXCR4). The clinical exploitation of these bone-targeted agents will provide oncologists with novel therapeutic strategies for the treatment of skeletal lesions in breast cancer.
... Preclinical and clinical research on integrins in cancer, particularly on αvβ3 and αvβ5, revealed their substantial potential as therapeutic targets for the treatment of different cancer types [5,6]. Cilengitide, a cyclic RGD-containing pentapeptide, was recognized as a selective inhibitor of αvβ3 and αvβ5 integrin-mediated cell-cell and cell-matrix interaction that exhibited strong anti-angiogenic, anti-tumor, anti-invasive activity and anti-metastatic activity in various animal models of cancer [7][8][9][10][11][12][13]. Furthermore, cilengitide displayed a favorable safety profile and entered clinical trials for treatment of multiple cancer types [14,15]. ...
... Therefore, in addition to the here reported inhibition of adhesion to the extracellular matrix by cilengitide, it is likely that the compound also inhibited the interaction of osteosarcoma cells with the vasculature and thereby further contributed to the in vivo observed suppression of metastasis. Different from findings in breast carcinoma and multiple myeloma [13,33], in our osteosarcoma model cilengitide did not inhibit osteolysis, examined by micro-computed tomography (data not shown), implying that, under our treatment settings, bone resorption by osteoclasts was not suppressed. The modest anti-tumor effect of cilengitide seen in vivo is in line with the in vitro the G1-cell cycle block, recognized for the first time in our study, and induction of anoikis resulting in a limited effect on viability. ...
Osteosarcoma is an aggressive bone cancer that has a high propensity for metastasis to the lungs. Patients with metastatic disease face a very poor prognosis. Therefore, novel therapeutics, efficiently suppressing the metastatic process, are urgently needed. Integrins play a pivotal role in tumor cell adhesion, motility and metastasis. Here, we evaluated αvβ3 and αvβ5 integrin inhibition with cilengitide as a novel metastasis-suppressive therapeutic approach in osteosarcoma. Immunohistochemical analysis of αvβ3 and αvβ5 integrins expression in a tissue microarray of tumor specimens collected from osteosarcoma patients revealed that αvβ5 integrin is mainly found on tumor cells, whereas αvβ3 is predominantly expressed by stromal cells. In vitro functional assays demonstrated that cilengitide dose-dependently inhibited de novo adhesion, provoked detachment and inhibited migration of osteosarcoma cell lines. Cilengitide induced a decline in cell viability, blocked the cell cycle in the G1 phase and caused anoikis by activation of the Hippo pathway. In a xenograft orthotopic mouse model cilengitide minimally affected intratibial primary tumor growth but, importantly, suppressed pulmonary metastasis. The data demonstrate that targeting αvβ3 and αvβ5 integrins in osteosarcoma should be considered as a novel therapeutic option for patients with metastatic disease.
... [198] Cilengitide Cilengitide is classified as an antiangiogenic drug due to its ability to inhibit the cellular adhesion receptors α v β 3 /α v β 5 integrins. [107,164,199,200] Cilengitide has shown effectiveness in preclinical studies through its induction of cell detachment and reduced proliferation in a panel of four breast cancer cell lines, as well as enhanced treatment effectiveness and cell death and inhibited bone metastasis that was furthered when combined with radiotherapy when utilized in breast cancer xenografts. [200][201][202] Cilengitide has been investigated in glioblastoma, nonsmall cell lung cancer, and prostate cancer with noted tumor reduction and negligible toxicity; however, overall survival was not significantly increased compared to currently-available treatments. ...
... [107,164,199,200] Cilengitide has shown effectiveness in preclinical studies through its induction of cell detachment and reduced proliferation in a panel of four breast cancer cell lines, as well as enhanced treatment effectiveness and cell death and inhibited bone metastasis that was furthered when combined with radiotherapy when utilized in breast cancer xenografts. [200][201][202] Cilengitide has been investigated in glioblastoma, nonsmall cell lung cancer, and prostate cancer with noted tumor reduction and negligible toxicity; however, overall survival was not significantly increased compared to currently-available treatments. [203][204][205][206] Currently, a phase I trial is underway to evaluate cilengitide as a combination therapy with paclitaxel in patients with advanced solid tumors, particularly those diagnosed with high-grade, aggressive breast cancers. ...
Breast cancer is known to metastasize in its latter stages of existence. The different angiogenic mechanisms and factors that allow for its progression are reviewed in this article. Understanding these mechanisms and factors will allow researchers to design drugs to inhibit angiogenic behaviors and control the rate of tumor growth.
... . Weiler et al. confirmed that overexpression of IGTAV is associated with poor clinical outcomes in patients with HCC36 . Cilengitide is an ITGAV antagonist, and Mas-Moruno et al. identified that cilengitide reduces angiogenesis and inhibits breast cancer bone metastasis in vivo preliminary results37,38 . Annexin A5 (ANXA5) was reported to participate in HCC pathogenesis via integrin and MEK-ERK pathways39 . ...
Considering the high fatality of hepatocellular carcinoma (HCC), current prognostic systems are insufficient to accurately forecast HCC patients' outcomes. In our study, nine anoikis‑related genes (PTRH2, ITGAV, ANXA5, BIRC5, BDNF, BSG, DAP3, SKP2, and EGF) were determined to establish a risk scoring model using LASSO regression, which could be validated in ICGC dataset. Kaplan–Meier curves and time-dependent receiver operating characteristic (ROC) curve analysis confirmed the risk score possessed an accurate predictive value for the prognosis of HCC patients. The high-risk group showed a higher infiltration of aDCs, macrophages, T-follicular helper cells, and Th2 cells. Besides, PD-L1 was significantly higher in the high-risk group compared to the low-risk group. Several anoikis‑related genes, such as ANX5, ITGAV, BDNF and SKP2, were associated with drug sensitivity in HCC. Finally, we identified BIRC5 and SKP2 as hub genes among the nine model genes using WGCNA analysis. BIRC5 and SKP2 were over-expressed in HCC tissues, and their over-expression was associated with poor prognosis, no matter in our cohort by immunohistochemical staining or in the TCGA cohort by mRNA-Seq. In our cohort, BIRC5 expression was highly associated with the T stage, pathologic stage, histologic grade and AFP of HCC patients. In general, our anoikis-related risk model can enhance the ability to predict the survival outcomes of HCC patients and provide a feasible therapeutic strategy for immunotherapy and drug resistance in HCC. BIRC5 and SKP2 are hub genes of anoikis‑related genes in HCC.
... Previous studies have demonstrated that cilengitide inhibited angiogenesis and induced endothelial cell apoptosis via the inhibition of integrins and the extracellular matrix [17,19]. Moreover, cilengitide has also been demonstrated to have antitumor activity by blocking integrin function [20,21]. However, the efficacy of cilengitide as an inhibitor of osteoclast adhesion remains to be elucidated. ...
The remodeling of actin cytoskeleton of osteoclasts on the bone matrix is essential for osteoclastic resorption activity. A specific regulator of the osteoclast cytoskeleton, integrin αvβ3, is known to provide a key role in the degradation of mineralized bone matrixes. Cilengitide is a potent inhibitor of integrins and is capable of affecting αvβ3 receptors, and has anti-tumor and anti-angiogenic and apoptosis-inducing effects. However, its function on osteoclasts is not fully understood. Here, the cilengitide role on nuclear factor κB ligand-receptor activator (RANKL)-induced osteoclasts was explored. Cells were cultured with varying concentrations of cilengitide (0,0.002,0.2 and 20 μM) for 7 days, followed by detected via Cell Counting Kit-8, staining for tartrate resistant acid phosphatase (TRAP), F-actin ring formation, bone resorption assays, adhesion assays, immunoblotting assays, and real-time fluorescent quantitative PCR. Results demonstrated that cilengitide effectively restrained the functionality and formation of osteoclasts in a concentration-dependent manner, without causing any cytotoxic effects. Mechanistically, cilengitide inhibited osteoclast-relevant genes expression; meanwhile, cilengitide downregulated the expression of key signaling molecules associated with the osteoclast cytoskeleton, including focal adhesion kinase (FAK), integrin αvβ3 and c-Src. Therefore, this results have confirmed that cilengitide regulates osteoclast activity by blocking the integrin αvβ3 signal pathway resulting in diminished adhesion and bone resorption of osteoclasts.
... In particular, DCE-MRI was capable of diagnosing a decrease in angiogenic activity in experimental bone metastases by semiquantitative measures or pharmacokinetic modeling [28]. Due to inhibition of crucial angiogenic factors VEGF, VEGFR, and integrins alpha v beta 3/5 by tyrosine kinase inhibitors (sunitinib, sorafenib), neutralizing antibodies (avastin), or small molecule inhibitors (cilengitide), significant alterations in DCE-MRI parameters could be quantitatively determined in bone metastases [29][30][31][32]. As angiogenesis was also found to be upregulated in metabolically active adipose tissue in bone [33,34], DCE-MRI parameters present another promising biomarker to assess the progression of bone metastases in adipocyte-rich bone marrow. ...
The growth of primary tumors and metastases is associated with excess body fat. In bone metastasis formation, the bone marrow microenvironment, and particularly adipocytes, play a pivotal role as growth mediators of disseminated tumor cells in the bone marrow. The aim of the present study is to non-invasively characterize the pathophysiologic processes in experimental bone metastasis resulting from accelerated tumor progression within adipocyte-rich bone marrow using multimodal imaging from magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT). To achieve this, we have employed small animal models after the administration of MDA-MB 231 breast cancer and B16F10 melanoma cells into the bone of nude rats or C57BL/6 mice, respectively. After tumor cell inoculation, ultra-high field MRI and µPET/CT were used to assess functional and metabolic parameters in the bone marrow of control animals (normal diet, ND), following a high-fat diet (HFD), and/or treated with the peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist bisphenol-A-diglycidylether (BADGE), respectively. In the bone marrow of nude rats, dynamic contrast-enhanced MRI (DCE-MRI) and diffusion-weighted imaging (DWI), as well as [18F]fluorodeoxyglucose-PET/CT([18F]FDG-PET/CT), was performed 10, 20, and 30 days after tumor cell inoculation, followed by immunohistochemistry. DCE-MRI parameters associated with blood volume, such as area under the curve (AUC), were significantly increased in bone metastases in the HFD group 30 days after tumor cell inoculation as compared to controls (p < 0.05), while the DWI parameter apparent diffusion coefficient (ADC) was not significantly different between the groups. [18F]FDG-PET/CT showed an enhanced glucose metabolism due to increased standardized uptake value (SUV) at day 30 after tumor cell inoculation in animals that received HFD (p < 0.05). BADGE treatment resulted in the inversion of quantitative DCE-MRI and [18F]FDG-PET/CT data, namely a significant decrease in AUC and SUV in HFD-fed animals as compared to ND-fed controls (p < 0.05). Finally, immunohistochemistry and qPCR confirmed the HFD-induced stimulation in vascularization and glucose activity in murine bone metastases. In conclusion, multimodal and multiparametric MRI and [18F]FDG-PET/CT were able to derive quantitative parameters in bone metastases, revealing an increase in vascularization and glucose metabolism following HFD. Thus, non-invasive imaging may serve as a biomarker for assessing the pathophysiology of bone metastasis in obesity, opening novel options for therapy and treatment monitoring by MRI and [18F]FDG-PET/CT.
... In amelanotic hamster melanoma A-Mel-3, cilengitide significantly reduced functional vessel density and decreased tumour growth and metastasis in vivo (Buerkle et al., 2002). In experimental breast cancer bone metastases, cilengitide caused a pronounced antiresorptive and antitumor effects (Bäuerle et al., 2011). Under hypoxic conditions, cilengitide significantly and dose dependently decreased the intracellular level of the hypoxia-inducible factor 1 α (HIF-1 α) in U87MG and SF763 glioblastoma cells (Skuli et al., 2009). ...
Glioblastoma are the most aggressive adult brain tumours for which new therapeutic strategies are highly needed. They almost always recur due to resistance to radio-and chemotherapies. We are interested in integrins, in particular α5β1 integrin, as therapeutic targets in glioblastoma. Previous works showed that it participates to the temozolomide (TMZ) chemotherapy resistance by partly inhibiting the tumour suppressor p53 pathway. Here we investigated a new therapeutic option based on the combination of p53 activators and α5β1 integrin inhibitors in naïve U87MG glioma cells and their TMZ-resistant counterparts. We showed that resistance to TMZ is accompanied by a huge rearrangement of integrin expressions and that α5β1 integrin remains a pertinent target for TMZ-resistant glioblastoma. In addition, TMZ-resistant cells proved sensitive to different blockers of p53/mdm2 complexes able to reactivate the p53 signalling pathways. Our data support the hypothesis that adding p53 activators together with integrin antagonists may represent a pertinent therapeutic strategy for recurrent glioma tumours.
... Cilengitide (50 mg/kg) or PBS was administered intraperitoneally daily. The treatment was determined according to the guide for dose conversion between animals and humans, as well as animal experimental methods from previously published studies [20,26]. Anti-PD1 monoclonal antibody (10 mg/kg) or isotype control (10 mg/kg) was administered intraperitoneally every three days. ...
Integrins play an important role in multiple stages of tumor progression and metastasis. Previous studies have shown synergistic effects of combined αvβ6-integrin and αvβ8-integrin inhibitors with immunotherapy. However, the role of αvβ3-integrin inhibitor in tumor immunity is still unclear. In this study, we aimed to elucidate the impact of the αvβ3-integrin inhibitor on PD-L1 expression and sensitivity to immune checkpoint blockade in melanoma. We investigated the effects of cilengitide, an αvβ3-integrin inhibitor, on cell viability and apoptosis of melanoma cell lines. And we explored how cilengitide regulated the expression of PD-L1 in melanoma cells in vitro and in vivo, using immunofluorescence, flow cytometry, Western blotting, and immunohistochemistry. A subcutaneous B16 murine melanoma model was utilized to determine whether combining cilengitide with anti-PD1 therapy inhibited tumor growth and positively regulated tumor microenvironment (TME). Our results showed that cilengitide inhibited cell viability and induced apoptosis in B16 and A375 cell lines. Furthermore, cilengitide decreased PD-L1 expression by reducing STAT3 phosphorylation in two melanoma cell lines. Cilengitide also reduced subcutaneous tumor PD-L1 expression in the B16 murine melanoma model. Accordingly, cilengitide positively regulated antitumor immune responses and provided durable therapy when combined with anti-PD1 monoclonal antibody in the murine melanoma model. This combination therapy reduced tumor growth and extended survival. Our study highlights that cilengitide enhances the efficacy of anti-PD1 therapy and produces a stronger antitumor immune response. This combination therefore represents a novel therapeutic regimen that may improve immunotherapy treratment.
... While the impact of the physical microenvironment on tumor progression is now well established, targeting these phenomena in the clinic remains challenging. For example, cilengitide, a peptide inhibitor of integrins αvβ3 and αvβ5, initially demonstrated anti-invasive effects in preclinical tumor models and phase I/II clinical trials (208,209) but failed to improve patient survival in a phase III clinical trial (210). Perhaps the most successful example of targeting physical tissue properties thus far is the use of anti-angiogenic agents, which were shown to normalize tumor vasculature, reduce IFP, and improve drug penetration in preclinical studies (23,211). ...
The success of anticancer therapies is often limited by heterogeneity within and between tumors. While much attention has been devoted to understanding the intrinsic molecular diversity of tumor cells, the surrounding tissue microenvironment is also highly complex and coevolves with tumor cells to drive clinical outcomes. Here, we propose that diverse types of solid tumors share common physical motifs that change in time and space, serving as universal regulators of malignancy. We use breast cancer and glioblastoma as instructive examples and highlight how invasion in both diseases is driven by the appropriation of structural guidance cues, contact-dependent heterotypic interactions with stromal cells, and elevated interstitial fluid pressure and flow. We discuss how engineering strategies show increasing value for measuring and modeling these physical properties for mechanistic studies. Moreover, engineered systems offer great promise for developing and testing novel therapies that improve patient prognosis by normalizing the physical tumor microenvironment.
Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 24 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Anti-α V β 3 inhibitory antibodies, based on a humanized form of the mouse monoclonal LM609 (etaracizumab; abegrin; vitaxin), were tested and well tolerated [70,71], as were two pan-α V integrin antibodies, abituzumab [72] and intetumumab [73]. In addition, the cyclic peptide cilengitide, which specifically inhibits α V β 3 and α V β 5 integrins, performed well in preclinical studies [74,75] and progressed to Phase 3 trials for cancer indications, including glioblastoma [76]. However, despite compelling preclinical results demonstrating the therapeutic potential of integrin inhibition, clinical trials repeatedly failed to demonstrate therapeutic benefits in cancer patients [73,[77][78][79][80]. Furthermore, low concentrations of cilengitide were found to induce, rather than inhibit, α V β 3 signaling, resulting in stimulated angiogenesis [81]. ...
To improve tumor selectivity of cytotoxic agents, we designed VIP236, a small molecule–drug conjugate consisting of an αVβ3 integrin binder linked to a modified camptothecin payload (VIP126), which is released by the enzyme neutrophil elastase (NE) in the tumor microenvironment (TME). The tumor targeting and pharmacokinetics of VIP236 were studied in tumor-bearing mice by in vivo near-infrared imaging and by analyzing tumor and plasma samples. The efficacy of VIP236 was investigated in a panel of cancer cell lines in vitro, and in MX-1, NCI-H69, and SW480 murine xenograft models. Imaging studies with the αVβ3 binder demonstrated efficient tumor targeting. Administration of VIP126 via VIP236 resulted in a 10-fold improvement in the tumor/plasma ratio of VIP126 compared with VIP126 administered alone. Unlike SN38, VIP126 is not a substrate of P-gp and BCRP drug transporters. VIP236 presented strong cytotoxic activity in the presence of NE. VIP236 treatment resulted in tumor regressions and very good tolerability in all in vivo models tested. VIP236 represents a novel approach for delivering a potent cytotoxic agent by utilizing αVβ3 as a targeting moiety and NE in the TME to release the VIP126 payload—designed for high permeability and low efflux—directly into the tumor stroma.
... Tumor cells can also express these integrins (Desgrosellier and Cheresh, 2010) and some inhibitors have been suggested, including cilengitide (CLG or EMD121974), with promising results (Harjunpää et al., 2019). CLG is a small pentapeptide that inhibits both integrins and has been used in preclinical studies in breast cancer, supporting its efficacy against bone metastasis (Bäuerle et al., 2011), and in clinical trials for other malignancies (Gilbert et al., 2012;Haddad et al., 2017;Vansteenkiste et al., 2015). However, CLG is rapidly cleared from the bloodstream and from tumor sites which has impeached this drug to reach the market so far (Stupp et al., 2014b). ...
Treatment in children with high-risk neuroblastoma remains largely unsuccessful due to the development of metastases and drug resistance. The biological complexity of these tumors and their microenvironment represent one the many challenges to face. Matrix glycoproteins such as vitronectin act as bridge elements between extracellular matrix and tumor cells and can promote tumor cell spreading. In this study, we established through a clinical cohort and preclinical models that the interaction of vitronectin and its ligands, such as αv integrins, are related to the stiffness of the extracellular matrix in high-risk neuroblastoma. These marked alterations found in the matrix led us to specifically target tumor cells within these altered matrices by employing nanomedicine and combination therapy. Loading the conventional cytotoxic drug etoposide into nanoparticles significantly increased its efficacy in neuroblastoma cells. We noted high synergy between etoposide and cilengitide, a high-affinity cyclic pentapeptide αv integrin antagonist. The results of this study highlight the need to characterize cell-extracellular matrix interactions, to improve patient care in high-risk neuroblastoma.
... Cilengitide administration was observed to inhibit partial regrowth of bone and bone resorption. Therefore, cilengitide is a feasible choice for breast cancer patient's discomforts with bone metastasis [147]. ...
Breast cancer is a multifactorial, heterogeneous disease and the second most frequent cancer amongst women worldwide. Metastasis is one of the most leading causes of death in these patients. Early-stage or locally advanced breast cancer is limited to the breast or nearby lymph nodes. When breast cancer spreads to farther tissues/organs from its original site, it is referred to as metastatic or stage IV breast cancer. Normal breast development is regulated by specific genes and signalling pathways controlling cell proliferation, cell death, cell differentiation and cell motility. Dysregulation of genes involved in various signalling pathways not only leads to the formation of primary tumour but also to the metastasis as well. The metastatic cascade is represented by a multi-step process including invasion of the local tumour cell followed by its entry into the vasculature, exit of malignant cells from the circulation and ultimately their colonization at the distant sites. These stages are referred to as formation of primary tumour, angiogenesis, invasion, intravasation and extravasation, respectively. The major sites of metastasis of breast cancer are the lymph nodes, bone, brain and lung. Only about 28% five-year survival rate has been reported for stage IV breast cancer. Metastasis is a serious concern for breast cancer and therefore, various therapeutic strategies such as tyrosine kinase inhibitors have been developed to target specific dysregulated genes and various signalling pathways involved in different steps of metastasis. In addition, other therapies like hyperbaric oxygen therapy, RNA interference and CRISPR/Cas9 are also being explored as novel strategies to cure the stage IV/metastatic breast cancer. Therefore, the current review has been compiled with an aim to evaluate the genetic basis of stage IV breast cancer with a focus on the molecular mechanisms. In addition, the therapeutic strategies targeting these dysregulated genes involved in various signalling pathways have also been discussed. Genome editing technologies that can target specific genes in the affected areas by making knock-in and knock-out alternations and thereby bring significant treatment outcomes in breast cancer have also been summarized.
... Appropriate algorithms for the management of BD mainly derive from extensive prospective trials with patients harboring tumors that frequently metastasize to the skeleton, such as breast, prostate, lung, and kidney cancers (21). Otherwise, a definite evidencebased strategy for the treatment of BMs from other malignancies endowed with lower osteotropic propensity does not exist, while apparently innovative drugs such as cilengitide failed to provide satisfactory results to be applied for routine clinical practice (22,23). Particularly, the impact of BD in melanoma has been poorly investigated, as well as the potential therapeutic strategies such as either RT or BTA use. ...
Background
We performed a multicenter retrospective observational study to investigate the impact of clinical–pathological features and therapeutic strategies on both the complications and survival of patients with bone metastases (BMs) from malignant melanoma.Patients and MethodsA total of 305 patients with melanoma and radiological evidence of BMs were retrospectively enrolled from 19 Italian centers. All patients received conventional treatments in accordance with each own treating physician’s practice. Both univariate and multivariate models were used to explore the impact of melanoma features, including skeletal-related events (SREs), and different treatments on both overall survival (OS) and time-to-SREs. The chi-squared test evaluated the suitability of several parameters to predict the occurrence of SREs.ResultsEighty-three percent of patients had metachronous BMs. The prevalent (90%) bone metastatic site was the spine, while 45% had involvement of the appendicular skeleton. Forty-seven percent experienced at least one SRE, including palliative radiotherapy (RT) in 37% of cases. No melanoma-associated factor was predictive of the development of SREs, although patients receiving early treatment with bone-targeted agents showed 62% lower risk and delayed time of SRE occurrence. Median OS from the diagnosis of bone metastasis was 10.7 months. The multivariate analysis revealed as independent prognostic factors the number of BMs, number of metastatic organs, baseline lactate dehydrogenase levels, and treatment with targeted therapy or immunotherapy. Subgroup analyses showed the best OS (median = 16.5 months) in the subset of patients receiving both immunotherapy and palliative RT.Conclusion
Based on our results, patients undergoing immunotherapy and palliative RT showed an OS benefit suggestive of a possible additive effect. The apparent protective role of bone targeting agent use on SREs observed in our analysis should deserve prospective evaluation.
... Similarly, drugs targeting integrins inhibit the adhesions of cancer cells to fibronectin and collagen in laboratory experiments [61]. Cilengitide, a small molecule that inhibits avb3 and avb5 integrins, has shown significant antiresorptive and antitumor effects in breast cancer metastases to bones in a phase II clinical trial [62]. Furthermore, the combination of this molecule with radiation in the treatment of breast tumors in vivo induced cell detachment and reduced proliferation via cilengitide-induced apoptosis and enhanced radiation response [63]. ...
Purpose of the Review
Breast cancer research has long focused on breast epithelium in cancer development. This traditional view is being challenged by more recent work which suggests that stromal components such as fibroblasts, immune cells, endothelial cells, and others may play a central role in cancer development. The purpose of this review is to investigate the elements of the breast stroma and their interaction with the epithelium in creating the necessary pro-tumorigenic environment.
Recent Findings
Several studies have revealed that breast cancer stroma differs significantly from normal breast stroma, with altered cellular and molecular factors that may serve as potential therapeutic targets in breast cancer treatment.
Summary
Studies of the complex interactions within the cancer ecosystem have revealed tumor-associated stromal changes that promote cell growth and invasiveness. They also reveal specific targets for novel strategies to inhibit inflammation, immunosuppression, angiogenesis, and distant invasion.
... This agrees with our suggestion that targeting survival signaling from cell-ECM adhesion could be a valuable treatment option. There is promising preclinical evidence of efficacy of cilengitide as an anti-metastatic drug [72][73][74], and co-targeting dormant cells with cilengitide and chemotherapy may prevent metastatic outgrowth by eliminating dormant cells at the minimal residual disease stage. ...
Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, non-proliferative state before reactivation and outgrowth. For a patient, these post-remission tumors are often drug resistant and highly aggressive, resulting in poor prognosis. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system that combines carefully controlled ECM substrates with nutrient deprivation to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle arrested, and actively proliferating cells. Cell populations that endured extended periods of serum-deprivation-induced dormancy formed an organized, fibrillar fibronectin matrix via αvβ3 and α5β1 integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation. We surmised that the fibronectin matrix was primarily a mediator of cell survival, not proliferation, during the serum-deprivation stress, bacause cancer cell outgrowth after dormancy required MMP-2-mediated fibronectin degradation. Given the difficulty of animal models in observing entrance and exit from dormancy in real-time, we propose this approach as a new, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation.
... This agrees with our suggestion that targeting survival signaling from cell-ECM adhesion could be a valuable treatment option. There is promising preclinical evidence of efficacy of cilengitide as an anti-metastatic drug [72][73][74], and co-targeting dormant cells with cilengitide and chemotherapy may prevent metastatic outgrowth by eliminating dormant cells at the minimal residual disease stage. ...
Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, non-proliferative state before reactivation and outgrowth. For a patient, these post-remission tumors are often drug resistant and highly aggressive, resulting in poor prognosis. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system that combines carefully controlled ECM substrates with nutrient deprivation to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle arrested, and actively proliferating cells. Cell populations that endured extended periods of serum-deprivation-induced dormancy formed an organized, fibrillar fibronectin matrix via avb3 and a5b1 integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation. We surmised that the fibronectin matrix was primarily a mediator of cell survival, not proliferation, during the serum-deprivation stress, bacause cancer cell outgrowth after dormancy required MMP-2-mediated fibronectin degradation. Given the difficulty of animal models in observing entrance and exit from dormancy in real-time, we propose this approach as a new, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation.
... S247, cilengitide, ATN-161) and non-peptidic (e.g. PSK1404) compounds that target ανβ3 could inhibit osteolysis and tumor growth in bone metastasis animal models [95,96]. These ανβ3 inhibitors, not only antagonized the osteoclast-mediated bone resorption but PSK1404 also prevented bone colonization by cancer cells expressing ανβ3 integrins at the dosage regimen that does not block bone resorption [95]. ...
Background:
Breast cancer is the most frequently diagnosed malignancy in women worldwide. Breast cancer tends to metastasize to bone. Around 70% of the breast cancer patients eventually develop bone metastasis. After the bone invasion, metastatic cells disrupt the balance between osteoblastic and osteoclastic activities, leading to skeletal complications, characterized by pain and pathological fractures and hence worsening the patient's quality of life. Once tumor invades the bone, it is hard to treat it with, the so-far available treatments options (e.g. bisphosphonates and denosumab). Bone metastasis should be essentially controlled, in cancer treatment and there is a strong need to explore new, more efficient therapeutic targets. This review discusses the bone physiological processes and the recent advances in exploring different pathways involved in bone metastasis. Furthermore, some novel treatment options, which are under preclinical and clinical investigations, are highlighted.
Conclusion:
A deeper understanding of these metastatic pathways can provide oncology researchers with novel avenues for treating bone metastasis, one of the main challenges to cure breast cancer. The restoration of healthy bone environment will not only improve the patient's quality of life but also reduces the tumor burden.
... It is the first small molecule anti-angiogenic drug ( Figure 5) and targets the integrins αvβ3, αvβ5, and α5β1. It works as an anti-proliferation entity and is useful in cell necrosis and in stopping the bone metastasis [146][147][148][149]. The drug is helpful in tumor-size reduction with negligible toxicity. ...
ANTI-ANGIOGENIC MECHANISM, BIOCHEMICAL FACTORS’ ROLES, THERAPEUTIC AGENTS, AND UNDER CLINICAL TRIAL DRUGS FOR BREAST AND PROSTATE CANCERS
Varsha M. Singh1, Shilpa J. Newati1, Mohammad Yusuf2, Alexandre Bridoux3,4, Salman A. A. Mohammed5, Naseem Akhtar6, and Riaz A. Khan1,5*
1 Manav Rachna International Institute of Research & Study (MRIIRS)-Manav Rachna International University (MRIU), Faridabad, HR 121 001, India; 2University of Taif, Taif Al-Haweiah, KSA; 3Integral University, Lucknow 226 026, India; 4Laboratoire Centre Atlantique, Zone Industrielle Chef de Baie, 1 rue Samuel de Champlain, Cs 41074, 17000 la Rochelle, France; 5College of Pharmacy, Qassim University; 6Boraida Colleges, Qassim, KSA.
ABSTRACT
The genesis of new blood vessels which is the culmination of angiogenic activity is responsible for the spread of the tumor, and other cancerous masses wherein blood supply provides nourishment to these tissues and helps in their maintenance, growth and further proliferation. Biological factors that favor the angiogenesis processes include vascular endothelial growth factors (VEGFs), tumor necrosis factors (TNFs), and fibroblast growth factors (FGFs), significantly contributing towards angiogenesis. The disruption and inhibition of angiogenic growth factors and their biochemical pathways during cancer build-up are among the obvious choices to control the growth and proliferation of cancers. The current work dwells in details about the growth factors, their roles, contextual biomechanics, and approaches to control angiogenesis through different inhibitory acts on biochemical pathways, growth factors, and other structural motifs involved in the angiogenesis process. The approaches to find novel molecular templates, new chemical entity, bio-macromolecular substrates, and probable drug leads for anti-angiogenic pharmacology are discussed. The chapter enlists various anti-angiogenesis based drugs, under clinical trial candidate drugs, new chemical entities, and other biochemical and recombinant therapeutic agents, either as monotherapy or as combination drugs, used for the treatment of various cancers, especially the breast and prostate cancers.
KEYWORDS
Angiogenesis; TNFα; VEGF; VEGF-A; VEGF-R; FGF; Ephrin; Angiogenin; Integrin; Endostatin, Interferon; Decorin; Thrombospondin; Interleukin; Matrix Metallo-Proteinases (MMP); Angiostatin; Antiangiogensis; CAM Model; Bevacizumab; Cilengitide; Vitaxin
... This reaction could be initiated by binding of BSP to integrin α V β 3 , which is amply present on MDA-MB-231 cells ( Figure 3B). Based on this, integrin α V β 3 has been successfully targeted by its inhibitor cilengitide and this treatment correlated with significant growth inhibition of MDA-MB-231 skeletal metastases [29]. of a T-cell deficient model for screening immunotherapeutic agents has restrictions as these models are not applicable for studying checkpoint blockers [32]. However, the use of syngeneic mouse models was excluded due to the lack of IDK1 cross reactivity with mouse BSP (unpublished own data) and, likewise, the use of humanised models was precluded by their availability. ...
Changes in glycosylation are salient features of cancer cells. Here we report on the diagnostic and therapeutic properties of IDK1, an antibody against tumour associated, hypoglycosylated bone sialoprotein (hypo-BSP). The affinity of the rat monoclonal antibody IDK1 for hypo-BSP,as determined by microscale thermophoresis, was three orders of magnitude higher than for mature BSP, whereasthe mouse monoclonal antibody used had similar affinity for both BSP forms. IDK1 showed no activity against the proliferation or migration of normal or cancer cells growing in vitro. In vivo, however, IDK1 caused dose-dependent regression of soft tissue and skeletal lesions in nude ratsharbouring human MDA-MB-231 cells. At optimal dose, 80% of the treated rats showed complete remission of all tumour lesions. Analysis of BSP expression in vitro by FACS and immunocytochemistry showed basal levels of this protein,which were visible only in a fraction of these cells. Cells of the metastatic cell lines MDA-MB-231 and PC-3 were more often positive for hypo-BSP. In addition, there was co-expression of both forms in some cells, but almost no co-localization; rather, hypo-BSP was present in the nucleus and mature BSP was detected extracellularly. Normal osteoblasts and osteoclasts were negative for hypo-BSP. Breast cancer tissue, however, showed strong expression of mature BSP, which was present intracellularly as well as in vesicles outside cells. Hypo-BSP was present mainly in lesions from skeletal sites, thus explaining the antineoplastic activity of IDK1, which was high in lesions growing in the vicinity of the skeleton but low in lesions growing subcutaneously. Finally, hypo-BSP was detected in specimens from breast cancer patients, with a significantly greater intensity in skeletal metastases as compared to the respective primary cancers. In conclusion, IDK-1 is an antibody with diagnostic and therapeutic applicationsin skeletal metastases of breast cancer. This article is protected by copyright. All rights reserved.
... Preclinical studies supported its efficacy as an anti-angiogenic and anti-tumor agent-and it was active in αvβ3 and αvβ5-driven cellular assays at~1 µM [108,131]. It suppressed breast metastasis to bone [132,133], and enhanced radiotherapy in orthotopic brain tumor models [134]. After unsuccessful early trials in pancreatic carcinoma and melanoma [135,136], a dose escalation PH1/2 trial in late-stage glioblastoma (GBM) showed some unexpected long-lasting responses as monotherapy [137] as well as an activity signal in combination with temozolomide in newly diagnosed GBM patients. ...
Integrins are transmembrane receptors that are central to the biology of many human pathologies. Classically mediating cell-extracellular matrix and cell-cell interaction, and with an emerging role as local activators of TGFβ, they influence cancer, fibrosis, thrombosis and inflammation. Their ligand binding and some regulatory sites are extracellular and sensitive to pharmacological intervention, as proven by the clinical success of seven drugs targeting them. The six drugs on the market in 2016 generated revenues of some US$3.5 billion, mainly from inhibitors of α4-series integrins. In this review we examine the current developments in integrin therapeutics, especially in cancer, and comment on the health economic implications of these developments.
... For the first time, Hood et al. (2002) reported to specifically remit a mutant RAF1 gene by targeting integrin αvβ3 to the tumor vasculature, ensuing in apoptosis of endothelial cells and tumor regression (Bauerle et al., 2011). Besides the integrin targeted therapeutic approaches, a range of other treatment approaches are made currently that are developed to relief cancer pain. ...
Breast cancer is found to be the most prevalent neoplasm in women worldwide. Despite the function of physically tethering cells to the matrix, transmembrane protein integrins are crucially involved in diverse cellular functions such as cell differentiation, proliferation, invasion, migration and metastasis. Dysregulation of integrins and their interactions with the cells and their microenvironment can trigger several signalling cues that determine the cell fate decision. In this review we spotlight all pre-existing integrin molecules, their structure, molecular interactions motifs, and function through several cross talks with kinase receptors. We also discuss the role of these integrins as potential prognostic and therapeutic targets and also in the regulation of breast cancer cells differentiation. Understanding of integrin structure and their motifs for ligand interactions in this context will enable the development of new therapeutic approaches to sensitize the tumours and their microenvironment to conventional therapy and overall suppress their metastatic phenotype. This article is protected by copyright. All rights reserved.
... The most prominent compound targeting integrins might be cilengitide, a cyclic RGD peptide that targets integrins αvβ3 and αvβ5 [214]. We and other groups could show in vitro and in vivo that this peptide inhibits attachment and invasion of different tumor cells [215][216][217][218][219][220][221]. However, cilengitide failed to provide benefit in a clinical trial phase III (CENTRIC study) [222]. ...
Integrins are heterodimeric cell surface receptors that bind to different extracellular ligands depending on their composition and regulate all processes which enable multicellular life. In cancer, integrins trigger and play key roles in all the features that were once described as the Hallmarks of Cancer. In this review, we will discuss the contribution of integrins to these hallmarks, including uncontrolled and limitless proliferation, invasion of tumor cells, promotion of tumor angiogenesis and evasion of apoptosis and resistance to growth suppressors, by highlighting the latest findings. Further on, given the paramount role of integrins in cancer, we will present novel strategies for integrin inhibition that are starting to emerge, promising a hopeful future regarding cancer treatment.
... Odanacatib is a promising compound under phase II clinical investigation in women with BCa and bone metastases [27]. Cilengitide is an RGD-mimetic cyclic pentapeptide inhibitor of both avb3 and avb5 integrins, inhibits bone metastasis in animals and tested in patients [28]. Saracatinib, a dual inhibitor of Src/Abl, has been shown to decrease levels of bone resorption markers in a phase I study in patients with solid tumors [29]. ...
Breast cancer is the most prominent cause of cancer death in women worldwide. The highlights of this review are to provide an overview of the targeted therapeutic agents, challenges with metastatic breast cancer (MBCa), mechanisms of action through Hedgehog/Gli 1 signaling pathway and future prospective. Over a decade of success, several drugs have been approved and are in the advanced stages of clinical trials that target the receptors such as estrogen receptor, growth factor receptor, receptor activator of nuclear factor kappa-B, etc. Currently, several monoclonal antibodies are also used for the treatment of breast cancer. Advances in understanding tumor biology, particularly signaling pathways such as Notch signaling pathway, Hedgehog/Gli 1 signaling pathway and inhibitors are considered to be important for bone metastasis. These studies may provide vital information for the design and development of new strategies with respect to efficacy, reduction of the side effects and treatment strategies.
... MRI-based approaches have been successfully exploited to monitor neovascularity or antivascular treatments. 33,34 In our study, we used Dynamic Contrast Enhanced (DCE)-MRI technique which reports about ...
Tumour progression depends on several sequential events that include the microenvironment remodelling processes and the switch to the angiogenic phenotype, leading to new blood vessels recruitment. Non-invasive imaging techniques allow the monitoring of functional alterations in tumour vascularity and cellularity. The aim of this work was to detect functional changes in vascularisation and cellularity through Dynamic Contrast Enhanced (DCE) and Diffusion Weighted (DW) Magnetic Resonance Imaging (MRI) modalities during breast cancer initiation and progression of a transgenic mouse model (BALB-neuT). Histological examination showed that BALB-neuT mammary glands undergo a slow neoplastic progression from simple hyperplasia to invasive carcinoma, still preserving normal parts of mammary glands. DCE-MRI results highlighted marked functional changes in terms of vessel permeability (K(trans) , volume transfer constant) and vascularisation (vp , vascular volume fraction) in BALB-neuT hyperplastic mammary glands if compared to BALB/c ones. When breast tissue progressed from simple to atypical hyperplasia, a strong increase in DCE-MRI biomarkers was observed in BALB-neuT in comparison to BALB/c mice (K(trans) = 5.3±0.7E-4 and 3.1±0.5E-4; vp = 7.4±0.8E-2 and 4.7±0.6E-2 for BALB-neuT and BALB/c, respectively) that remained constant during the successive steps of the neoplastic transformation. Consistent with DCE-MRI observations, microvessel counting revealed a significant increase in tumour vessels. Our study showed that DCE-MRI estimates can accurately detect the angiogenic switch at early step of breast cancer carcinogenesis. These results support the view that this imaging approach is an excellent tool to characterize microvasculature changes, despite only small portions of the mammary glands developed neoplastic lesions in a transgenic mouse model. This article is protected by copyright. All rights reserved.
... Inhibition of ECM receptors could moreover provide important opportunities in treating cancer. In mouse models for breast cancer metastasis to bone, the inhibition of aVb3 and aVb5 integrins represses bone resorption and metastasis growth in the bone [103]. Finally, the matrix composition at invasive fronts of tumors can be used to deliver drugs or radiation specifically to active sites of cancer. ...
... Intetumumab, a monoclonal antibody blocking integrin αvβ3, decreased metastasis and increased survival in a rat model for breast cancer metastasis to brain [212]. Similarly in rats, a reduction in bone metastases was observed upon administration of cilengitide, an integrin αvβ3/αvβ5 inhibitor [213]. ...
The extracellular matrix (ECM) is increasingly recognized as an important regulator in breast cancer. ECM in breast cancer development features numerous changes in composition and organization when compared to the mammary gland under homeostasis. Matrix proteins that are induced in breast cancer include fibrillar collagens, fibronectin, specific laminins and proteoglycans as well as matricellular proteins. Growing evidence suggests that many of these induced ECM proteins play a major functional role in breast cancer progression and metastasis. A number of the induced ECM proteins have moreover been shown to be essential components of metastatic niches, promoting stem/progenitor signaling pathways and metastatic outgrowth. ECM remodeling enzymes are also greatly increased, leading to major changes in the matrix structure and biomechanical properties. Importantly, several ECM components and ECM remodeling enzymes are specifically induced in breast cancer or during tissue regeneration while healthy tissues under homeostasis express exceedingly low levels. This may indicate that ECM and ECM-associated functions may represent promising drug targets against breast cancer, providing important specificity that could be utilized when developing therapies.
... 49 Recent studies showed that cilengitide, an inhibitor of a v b 3 and a v b 5 integrin, decreased vessel density and changed the vessel phenotype in breast tumor bone metastasis already at an early stage whereas it changed tumor cell metabolism, as well as decreased bone resorption in a later stage. [50][51][52] Thus, targeting signaling pathways that are not only important for angiogenesis but also for osteoclast activity as well as tumor cell behavior may provide an interesting approach. ...
Emerging evidence indicates that the interactions between tumor cells and the bone microenvironment have a crucial role in the pathogenesis of bone metastasis and that they can influence tumor cell dissemination, quiescence and tumor growth in the bone. The vasculature is known to be critical for primary tumor growth, and anti-angiogenesis drugs are approved for the treatment of certain tumor types. The role of the vasculature in bone metastasis is less well known, but recent evidence shows that blood vessels in the bone are a key component of the local microenvironment for the tumor cells and contribute to the different consecutive phases of bone metastasis. A better insight in the importance of the vasculature for bone metastasis may help develop novel treatment modalities that either slow down tumor growth or, preferably, prevent or cure bone metastasis.
... In addition, the recent finding that integrin signaling in breast TICs drives resistance to EGFR inhibitors suggests potential novel strategies for sensitizing breast tumors to receptor tyrosine kinase inhibition (Seguin et al. 2014). Cilengitide, an integrin antagonist currently being tested in clinical trials (Goodman & Picard 2012), has been shown to reduce bone metastasis in a mouse mammary tumor model (Bauerle et al. 2011). A more recent study also reported that the increased stiffness of the extracellular matrix in tumors can activate integrin signaling to drive tumor progression, which suggests another new intervention method (Mouw et al. 2014). ...
Breast cancer is the most common cancer in women (exclusive of skin cancer), and is the second leading cause of cancer-related deaths. Although conventional and targeted therapies have improved survival rates, there are still considerable challenges in treating breast cancer, including treatment resistance, disease recurrence, and metastasis. Treatment resistance can be either de novo - due to traits that tumor cells possess prior to treatment, or acquired, - due to traits that tumor cells gain in response to treatment. A recently proposed mechanism of de novo resistance invokes existence of a specialized subset of cancer cells defined as tumor-initiating cells (TICs), or cancer stem cells (CSC). TICs have the capacity to self-renew and regenerate new tumors that consist of all clonally-derived cell types present in the parental tumor. There are data to suggest that TICs are resistant to many conventional cancer therapies, and survive treatment in spite of dramatic shrinkage of the tumor. Residual TICs can then eventually regrow resulting in disease relapse. It is also hypothesized that TIC may be responsible for metastatic disease. If these hypotheses are correct, targeting TICs, particularly in metastases, may be imperative to achieve cure. Herein, we discuss evidence for breast TICs and their apparent resistance to conventional chemotherapy and radiotherapy, as well as to various targeted therapies. We also address the potential impact of breast TIC plasticity and metastatic potential on therapeutic strategies. Finally, we describe several genes and signaling pathways that appear important for TIC function that may represent promising therapeutic targets.
... There is preclinical evidence that ␣V3 integrin inhibition is able to prevent bone colonization by ␣V3 expressing human breast cancer cells [148]. Several ongoing clinical trials are evaluating the anticancer effect of integrin antagonists in advanced refractory and metastatic cancers, but only one phase I clinical trial is evaluating integrin antagonists (GLPG0187) in cancer patients with bone metastasis (NCT01313598) [149,150]. ...
Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. While advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarizes recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. This review lists the disruption of E-cadherin and tight junctions, key signaling pathways, including urokinase type plasminogen activator (uPA), phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT), focal adhesion kinase (FAK), β-catenin/zinc finger E-box binding homeobox 1 (ZEB-1) and transforming growth factor beta (TGF-β), together with inactivation of activator protein-1 (AP-1) and suppression of matrix metalloproteinase-9 (MMP-9) activity as key targets and the use of phytochemicals, or natural products, such as those from Agaricus blazei, Albatrellus confluens, Cordyceps militaris, Ganoderma lucidum, Poria cocos and Silybum marianum, together with diet derived fatty acids gamma linolenic acid (GLA) and eicosapentanoic acid (EPA) and inhibitory compounds as useful approaches to target tissue invasion and metastasis as well as other hallmark areas of cancer. Together, these strategies could represent new, inexpensive, low toxicity strategies to aid in the management of cancer metastasis as well as having holistic effects against other cancer hallmarks.
Copyright © 2015. Published by Elsevier Ltd.
... There is preclinical evidence that ␣V3 integrin inhibition is able to prevent bone colonization by ␣V3 expressing human breast cancer cells [148]. Several ongoing clinical trials are evaluating the anticancer effect of integrin antagonists in advanced refractory and metastatic cancers, but only one phase I clinical trial is evaluating integrin antagonists (GLPG0187) in cancer patients with bone metastasis (NCT01313598) [149,150]. ...
... There is preclinical evidence that ␣V3 integrin inhibition is able to prevent bone colonization by ␣V3 expressing human breast cancer cells [148]. Several ongoing clinical trials are evaluating the anticancer effect of integrin antagonists in advanced refractory and metastatic cancers, but only one phase I clinical trial is evaluating integrin antagonists (GLPG0187) in cancer patients with bone metastasis (NCT01313598) [149,150]. ...
Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. Whilst advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarises recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. Together with the Capstone paper of this issue, this review aims to provide useful references for this poorly understood area, highlighting the need and potential for new therapeutics.
Breast cancer is one of the most common cancers in women, which can metastasize to other organs and has a high mortality rate. Previous studies have shown that angiogenic factors can contribute to tumor growth, development, and metastasis by altering the tumor microenvironment (TME). These angiogenic factors include a wide range of molecules, and in contrast, anti-angiogenic factors also inhibit angiogenesis and inhibit tumor growth. Evidence suggests that an imbalance between angiogenic and anti-angiogenic factors leads to angiogenesis, facilitating the migration of tumor cells from the source tissue in the breast to other organs such as the lung, liver, bone, and brain. By supplying blood through these neomicrovascular vessels, the nutrients and oxygen needed to grow tumor cells are provided. Due to the significant anti-tumor role of anti-angiogenesis factors, cancer researchers have always considered these molecules, and it is believed that anti-angiogenesis factors can be employed in cancer treatment approaches. This review discusses the role of anti-angiogenesis agents in breast cancer pathogenesis and reviews therapeutic approaches based on anti-angiogenesis factors.
Tumor metastasis is the major cause of mortality from cancer. Metabolic rewiring and the metastatic cascade are highly intertwined, co-operating to promote multiple steps of cancer metastasis. Metabolites generated by cancer cells influence the metastatic cascade, encompassing epithelial-mesenchymal transition (EMT), survival of cancer cells in circulation, and metastatic colonization at distant sites. A variety of molecular mechanisms underlie the prometastatic effect of tumor-derived metabolites, such as epigenetic deregulation, induction of matrix metalloproteinases (MMPs), promotion of cancer stemness, and alleviation of oxidative stress. Conversely, metastatic signaling regulates expression and activity of rate-limiting metabolic enzymes to generate prometastatic metabolites thereby reinforcing the metastasis cascade. Understanding the complex interplay between metabolism and metastasis could unravel novel molecular targets, whose intervention could lead to improvements in the treatment of cancer. In this review, we summarized the recent discoveries involving metabolism and tumor metastasis, and emphasized the promising molecular targets, with an update on the development of small molecule or biologic inhibitors against these aberrant situations in cancer.
Accumulating evidence has disclosed effective anti-angiogenic strategies should simultaneously inhibit endothelium-dependent vessels (EDV) and tumor cell-mediated vasculogenic mimicry (VM). The αvβ3 integrin-targeting peptide cRGD has the ability to inhibit EDV and we have found cRGD can also suppress the formation of VM in ovarian cancer cells. Herein, a cRGD-based combination strategy was developed to suppress the proliferation of tumor cells by anti-EDV and anti-VM. We firstly engineered two cRGD functionalized nanoparticles (cRGD-NPs1 and cRGD-NPs2) by self-assembly using heparin conjugated with cRGD and folate. In vitro experiments demonstrated cRGD-NPs2 exhibited more significant cytotoxicity and higher intracellular uptake ability than cRGD-NPs1. Also, cRGD-NPs2 could efficiently discourage EDV, VM and proliferation in HUVECs and SKOV3 (VM+) cells. In vivo studies showed cRGD-NPs2 could specifically accumulate in ovarian cancer tissues and exerted a superior anti-tumor effect in SKOV3 xenografts. The mechanisms responsible for creating anti-EDV and anti-VM action of cRGD-NPs2 related to combined effects of cRGD, heparin and folate. The results demonstrated cRGD-NPs2 represented a versatile anti-angiogenic medicine via their combined inhibitory effect.
Statement of Significance
Accumulating documents indicate tumor cell-mediated vasculogenic mimicry (VM) is positively correlated with poor prognosis, occurrence of distant metastasis and low survival rate in cancer patients, suggesting VM is a potential therapeutic target for cancer treatment. Thus, effective anti-angiogenic strategies should simultaneously inhibit VM as well as endothelium-dependent vessels (EDV). Integrin αvβ3 is a crucial inducer involved in the formation of both EDV and VM. In this study, we engineered αvβ3 integrin-targeting peptide cRGD functionalized nanoparticles (cRGD-NPs) by self-assembly using heparin conjugated with cRGD and folate. The prepared cRGD-NPs represent a promising anti-angiogenic medicine in that they are able to inhibit endothelial sprouting angiogenesis and tumor cell-mediated VM. This work may provide useful information with which to construct effective anti-angiogenic nanomedicines.
Exosomes are important contributors to cell-cell communication and their role as diagnostic markers for cancer and the pathogenesis for cancer is under intensive investigation. Here, we focus on their role in metastasis-related processes. We discuss their impact regarding promotion of invasion and migration of tumor cells, conditioning of lymph nodes, generation of premetastatic niches and organotropism of metastasis. Furthermore, we highlight interactions of exosomes with bone marrow and stromal components such as fibroblasts, endothelial cells, myeloid- and other immune-related cells in the context of metastases. For all processes as described above, we outline molecular and cellular components for therapeutic intervention with metastatic processes.
Noninvasive multimodal imaging of tumor blood vessels allows the qualitative and quantitative assessment of morphological, functional, and molecular features of tumor angiogenesis longitudinally in a living organism. In this chapter we focus on the application of magnetic resonance imaging (MRI), computed tomography (CT), ultrasound (US), and positron emission tomography (PET) in tumor blood vessel visualization on the example of breast cancer bone metastasis in a nude rat model. Thereby, materials and methods are described that are needed to obtain complementary data on tumor vascularization from these imaging techniques.
Cancer metastasis to the bone develops commonly in patients with various malignancies, and is a major cause of morbidity and diminished quality of life in many affected patients. Emerging treatments for metastatic bone disease have arisen from advances in our understanding of the unique cellular and molecular mechanisms that contribute to the bone metastases.
In this chapter, we discussed the relevant preclinical and clinical studies of targeted therapies in bone metastatic prostate cancer.
We also focused on studies optimizing bisphosphonate use and recent clinical data on denosumab in the treatment of bone disease.
In addition, we described new drugs that target both osteoblast and osteoclast deregulated pathways exerting, concomitantly, bone anabolic and anticatabolic therapeutic effects (c-Src inhibitors, activin A, transforming growth factor β inhibitors, integrins inhibitors, cathepsin K inhibitors, endothelin 1 inhibitors, and WNT signaling pathway modulators).
Finally, we reported the main preclinical and clinical studies of targeted therapies in myeloma bone disease that represents a typical osteolytic disease with concurrent reduction of osteoblast function.
The clinical exploitation of these bone-targeted agents will provide oncologists with novel therapeutic strategies for the treatment of bone disease.
Bone metastasis is frequently observed in the most relevant types of solid tumors representing an important imaging target for detection and follow-up. For this purpose, morphologic aspects of skeletal lesions are assessed by conventional X-rays, CT, and MRI, whereas bone scintigraphy and SPECT reveal changes of bone remodeling. However, more recently developed approaches reflecting functional and metabolic characteristics in bone metastasis can be used for early detection and treatment response assessment of these lesions that include dynamic contrast-enhanced, vessel size, and diffusion-weighted imaging as well as MR spectroscopy, different PET tracers, and hybrid techniques. The focus of this chapter is to review functional and metabolic techniques and the underlying pathogenic processes resulting in the respective imaging findings.
Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.
Bone metastasis is a common burden in many types of cancer and has a severe impact on the quality of life in patients. Hence, specific therapeutic strategies inhibiting tumor induced osteolysis are urgently needed. In this study, we aimed to interfere with integrin adhesion receptors, which are central players of the bone resorption process. For this purpose, we used cilengitide, a cyclic RGD peptide, which blocks integrin αVβ3 and αVβ5-ligand binding. Our results revealed that cilengitide blocked osteoclast maturation in a dose-dependent manner. In detail, pre-osteoclasts treated with cilengitide exhibited reduced cell spreading, cell migration and cell adhesion on RGD-containing matrix proteins, which are ligands of integrin αVβ3. The activation of the most upstream signal transduction molecules of the integrin receptor-initiated pathway, FAK and c-Src, were consistently blocked by cilengitide. First evidence suggests that cilengitide might interfere with metastatic bone disease in vivo and this study describes a potential underlying mechanism of the inhibitory effect of cilengitide on αV-integrin expressing pre-osteoclasts by blocking integrin ligand binding and interfering with osteoclast maturation and cell behavior. In conclusion, our findings suggest that cilengitide, which interferes with αV-integrins on osteoclasts, may represent a novel therapeutic strategy in the treatment of malignant bone disease.
Copyright © 2015. Published by Elsevier Inc.
Angiogenesis is a requirement for tumor growth and metastasis. The angiogenic process depends on vascular endothelial cell migration and invasion, and is regulated by various cell adhesion receptors. Integrins are such a family of receptors that facilitate the cellular adhesion to and migration on extracellular matrix proteins in the intercellular spaces and basement membranes. Among 24 members of the integrin family, αvβ3 is studied most extensively for its role in tumor angiogenesis and metastasis. The v3 is expressed at relatively low levels on epithelial cells and mature endothelial cells, but it is highly expressed on the activated endothelial cells of tumor neovasculature and some tumor cells. This restricted expression makes v3 an excellent target to develop antiangiogenic drugs and diagnostic molecular imaging probes. Since v3 is a receptor for extracellular matrix proteins with one or more RGD tripeptide sequence, many radiolabeled cyclic RGD peptides have been evaluated as "v3-targeted" radiotracers for tumor imaging over the last decade. This article will use the dimeric and tetrameric cyclic RGD peptides developed in our laboratories as examples to illustrate basic principles for development of v3-targeted radiotracers. It will focus on different approaches to maximize the radiotracer tumor uptake and tumor/background ratios. This article will also discuss some important assays for pre-clinical evaluations of integrin-targeted radiotracers. In general, multimerization of cyclic RGD peptides increases their integrin binding affinity and the tumor uptake and retention times of their radiotracers. Regardless of their multiplicity, the capability of cyclic RGD peptides to bind other integrins (namely αvβ5, α5β1, α6β4, α4β1 and αvβ6) is expected to enhance the radiotracer tumor uptake due to the increased integrin population. The result from preclinical and clinical studies clearly show that radiolabeled cyclic RGD peptides (such as 99mTc-3P-RGD2, 18F-Alfatide-I and 18F-Alfatide-II) are useful as the molecular imaging probes for early cancer detection and noninvasive monitoring of the tumor response to antiangiogenic therapy.
Purpose:
To validate imaging parameters from diffusion-weighted imaging and dynamic contrast-enhanced MRI with immunohistology and to non-invasively assess microstructure of experimental breast cancer bone metastases.
Materials and methods:
Animals bearing breast cancer bone metastases were imaged in a clinical 1.5 T MRI scanner. HASTE sequences were performed to calculate apparent diffusion coefficients. Saturation recovery turbo FLASH sequences were conducted while infusing 0.1 mmol/l Gd-DTPA for dynamic contrast-enhanced MRI to quantify parameters amplitude A and exchange rate constant kep. After imaging, bone metastases were analyzed immunohistologically.
Results:
We found correlations of the apparent diffusion coefficients from diffusion-weighted imaging with tumor cellularity as assessed with cell nuclei staining. Histological vessel maturity was correlated negatively with parameters A and kep from dynamic contrast-enhanced MRI. Tumor size correlated inversely with cell density and vessel permeability as well as positively with mean vessel calibers. Parameters from the rim of bone metastases differed significantly from values of the center.
Conclusion:
In vivo diffusion-weighted imaging and dynamic contrast-enhanced MRI in experimental bone metastases provide information about tumor cellularity and vascularity and correlate well with immunohistology.
The development of drugs to treat breast and other cancers proceeds through phase I dose finding, phase II efficacy, and phase III comparative studies in the metastatic setting, only then asking if metastasis can be prevented in adjuvant trials. Compounds without overt cytotoxic activity, such as those developed to inhibit metastatic colonization, will likely fail to shrink established lesions in the metastatic setting and never be tested in a metastasis prevention scenario where they were preclinically validated. We and others have proposed phase II primary and secondary metastasis prevention studies to address this need. Herein, we have asked whether preclinical metastasis prevention data agrees with the positive adjuvant setting trials. The data are limited but complimentary. We also review fundamental pathways involved in metastasis, including Src, integrins, focal adhesion kinase (FAK), and fibrosis, for their clinical progress to date and potential for metastasis prevention. Issues of inadequate preclinical validation and clinical toxicity profiles are discussed.
Early osteoclast precursors, in the form of murine bone marrow macrophages (BMMs), while expressing no detectable avb3 integrin, contain abundant avb5 and attach to matrix in an av integrin-depen- dent manner. Furthermore, avb5 expression by osteoclast precursors progressively falls as they assume the resorptive phenotype. We find the osteoclastogenic agent, tumor necrosis factor-a, (TNF) down-reg- ulates avb5 expression by BMMS via attenuation of b5 messenger RNA (mRNA) t1/2. Using BMMs from TNF receptor knockout mice we establish the p55 receptor transmits the b5 suppressive effect. The functional implications of TNF-mediated avb5 down-regulation are underscored by the capacity of an av inhibitory peptide mimetic to prevent spreading by BMMs expressing abundant avb5 while failing to impact those in which the integrin has been diminished by TNF. Finally, b5 mRNA in BMMs of wild-type mice administered lipopoly- saccharide (LPS) progressively falls with time of in vivo treatment. Alternatively, b5 mRNA does not decline in BMMs of LPS-treated mice lacking both TNF receptors, documenting down-regulation of the b5 integrin subunit, in vivo, is mediated by TNF. Thus, matrix at- tachment of osteoclast precursors and mature osteoclasts are gov- erned by distinct av integrins which are differentially regulated by specific cytokines. (Endocrinology 141: 284 -290, 2000)
Background: Bone sialoprotein (BSP) interacts separately with both matrix metalloproteinase 2 (MMP-2) and integrin αvβ3 and is overexpressed in many metastatic tumors. Its role in tumor biology, however, remains unclear. We investigated whether
BSP enhances cancer cell invasiveness by forming a trimolecular complex with MMP-2 and cell-surface integrin αvβ3. Methods: Invasiveness of breast, prostate, lung, and thyroid tumor cell lines was measured with a modified Boyden chamber assay. Binding
and co-localization of BSP, MMP-2, and integrin αvβ3 were investigated with immunoprecipitation and in situ hybridization. All statistical tests were two-sided. Results: Treatment with BSP increased invasiveness of many breast, prostate, lung, and thyroid cancer cells through Matrigel in a
dose-dependent manner. BSP at 50 nM increased the invasiveness of SW-579 thyroid cancer cells (95.2 units, 95% confidence interval [CI] = 90.4 to 100 units)
by approximately 10-fold compared with that of untreated control SW-579 cells (9.1 units, 95% CI = 5.7 to 12.5 units) (P<.001). Addition of an inactive mutated BSP, in which BSP's integrin-binding RGD tripeptide was altered, or addition of integrin
αvβ3-blocking antibodies resulted in invasiveness equivalent to that of untreated cells. Inhibiting cellular MMP-2 activity with
chemical inhibitors or a specific antibody also blocked BSP-enhanced invasiveness. Osteopontin and dentin matrix protein 1,
proteins related to BSP that also bind integrin αvβ3 and form complexes with other MMPs (but not MMP-2), did not enhance invasiveness. Immunoprecipitation showed that a complex
containing BSP, integrin αvβ3, and MMP-2 formed in vitro. Addition of BSP increased the amount of MMP-2 bound by cells in an integrin-dependent fashion. Co-expression of BSP, integrin
αvβ3, and MMP-2 in papillary thyroid carcinoma cells was shown by in situ hybridization. Conclusion: Cancer cells appear to become more invasive when BSP forms a cell-surface trimolecular complex by linking MMP-2 to integrin
αvβ3.
Unlabelled:
The vitronectin receptor integrin alphavbeta3 promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSCs) in vitro. Expecting antifibrotic synergism, we studied the effect of alphavbeta3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by way of bile duct ligation (BDL) for 6 weeks or thioacetamide (TAA) injections for 12 weeks. A specific alphavbeta3 (alphavbeta5) inhibitor (Cilengitide) was given intraperitoneally twice daily at 15 mg/kg during BDL or after TAA administration. Liver collagen was determined as hydroxyproline, and gene expression was quantified by way of quantitative polymerase chain reaction. Liver angiogenesis, macrophage infiltration, and hypoxia were assessed by way of CD31, CD68 and hypoxia-inducible factor-1alpha immunostaining. Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and up-regulation of profibrogenic genes and matrix metalloproteinase-13. Treatment increased gamma glutamyl transpeptidase in both models, while other serum markers remained unchanged. alphavbeta3 inhibition resulted in mild liver hypoxia, as evidenced by up-regulation of hypoxia-inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in tumor necrosis factor alpha, interleukin-18, and cyclooxygenase-2 messenger RNA indicated modest macrophage activation.
Conclusion:
Specific inhibition of integrin alphavbeta3 (alphavbeta5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSCs in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis.
Molecular imaging of angiogenesis is urgently needed for diagnostic purposes such as early detection, monitoring of (angiostatic)
therapy and individualized therapy. Multimodality molecular imaging is a promising and refined technique to study tumor angiogenesis,
which has so far been largely unexplored due to the lack of suitable multimodal contrast agents. Here, we report on the application
of a novel αvβ3-specific quantum dot-based nanoparticle, which has been optimized for both optical and magnetic resonance
detection of tumor angiogenesis. Upon intravenous injection of RGD-pQDs in tumor-bearing mice, intravital microscopy allowed
the detection of angiogenically activated endothelium at cellular resolution with a small scanning window and limited penetration
depth, while magnetic resonance imaging was used to visualize angiogenesis at anatomical resolution throughout the entire
tumor. Fluorescence imaging allowed whole-body investigation of angiogenic activity. Using these quantum dots and the aforementioned
imaging modalities, the angiogenic tumor vasculature was readily detected with the highest angiogenic activity occurring in
the periphery of the tumor. This nanoparticle may be employed for multimodality imaging of a variety of diseases that are
accompanied by activation of endothelial cells. Furthermore, the current technology might be developed for molecular imaging
of other pathophysiological processes.
Early osteoclast precursors, in the form of murine bone marrow macrophages (BMMs), while expressing no detectable alpha(v)beta3 integrin, contain abundant alpha(v)beta5 and attach to matrix in an alpha(v) integrin-dependent manner. Furthermore, alpha(v)beta5 expression by osteoclast precursors progressively falls as they assume the resorptive phenotype. We find the osteoclastogenic agent, tumor necrosis factor-alpha, (TNF) down-regulates alpha(v)beta5 expression by BMMS via attenuation of beta5 messenger RNA (mRNA) t1/2. Using BMMs from TNF receptor knockout mice we establish the p55 receptor transmits the beta5 suppressive effect. The functional implications of TNF-mediated alpha(v)beta5 down-regulation are underscored by the capacity of an alpha(v) inhibitory peptide mimetic to prevent spreading by BMMs expressing abundant alpha(v)beta5 while failing to impact those in which the integrin has been diminished by TNF. Finally, beta5 mRNA in BMMs of wild-type mice administered lipopolysaccharide (LPS) progressively falls with time of in vivo treatment. Alternatively, beta5 mRNA does not decline in BMMs of LPS-treated mice lacking both TNF receptors, documenting down-regulation of the beta5 integrin subunit, in vivo, is mediated by TNF. Thus, matrix attachment of osteoclast precursors and mature osteoclasts are governed by distinct alpha(v) integrins which are differentially regulated by specific cytokines.
Inhibitors of alpha(v)beta(3) and alpha(v)beta(5) integrin have entered clinical trials as antiangiogenic agents for cancer treatment but generally have been unsuccessful. Here we present in vivo evidence that low (nanomolar) concentrations of RGD-mimetic alpha(v)beta(3) and alpha(v)beta(5) inhibitors can paradoxically stimulate tumor growth and tumor angiogenesis. We show that low concentrations of these inhibitors promote VEGF-mediated angiogenesis by altering alpha(v)beta(3) integrin and vascular endothelial growth factor receptor-2 trafficking, thereby promoting endothelial cell migration to VEGF. The proangiogenic effects of low concentrations of RGD-mimetic integrin inhibitors could compromise their efficacy as anticancer agents and have major implications for the use of RGD-mimetic compounds in humans.
Cilengitide is a high-affinity cyclic pentapeptdic alphaV integrin antagonist previously reported to suppress angiogenesis by inducing anoikis of endothelial cells adhering through alphaVbeta3/alphaVbeta5 integrins. Angiogenic endothelial cells express multiple integrins, in particular those of the beta1 family, and little is known on the effect of cilengitide on endothelial cells expressing alphaVbeta3 but adhering through beta1 integrins. Through morphological, biochemical, pharmacological and functional approaches we investigated the effect of cilengitide on alphaVbeta3-expressing human umbilical vein endothelial cells (HUVEC) cultured on the beta1 ligands fibronectin and collagen I. We show that cilengitide activated cell surface alphaVbeta3, stimulated phosphorylation of FAK (Y(397) and Y(576/577)), Src (S(418)) and VE-cadherin (Y(658) and Y(731)), redistributed alphaVbeta3 at the cell periphery, caused disappearance of VE-cadherin from cellular junctions, increased the permeability of HUVEC monolayers and detached HUVEC adhering on low-density beta1 integrin ligands. Pharmacological inhibition of Src kinase activity fully prevented cilengitide-induced phosphorylation of Src, FAK and VE-cadherin, and redistribution of alphaVbeta3 and VE-cadherin and partially prevented increased permeability, but did not prevent HUVEC detachment from low-density matrices. Taken together, these observations reveal a previously unreported effect of cilengitide on endothelial cells namely its ability to elicit signaling events disrupting VE-cadherin localization at cellular contacts and to increase endothelial monolayer permeability. These effects are potentially relevant to the clinical use of cilengitide as anticancer agent.
Cilengitide, an inhibitor of alphavbeta3 and alphavbeta5 integrin receptors, demonstrated minimal toxicity and durable activity across a wide range of doses administered to adults with recurrent glioblastoma multiforme (GBM) in a prior phase I study. The current multicenter phase II study was conducted to evaluate the activity and safety of cilengitide in GBM patients at first recurrence.
Eligible patients were randomly assigned to receive either 500 or 2,000 mg of cilengitide twice weekly on a continuous basis. Patients were assessed every 4 weeks. The primary end point was 6-month progression-free survival (PFS) rate. Secondary end points included PFS, overall survival (OS), and radiographic response, as well as quality-of-life and pharmacokinetic assessments.
Eighty-one patients were enrolled, including 41 on the 500-mg arm and 40 on the 2,000-mg arm. The safety profile of cilengitide was excellent, with no significant reproducible toxicities observed on either arm. Antitumor activity was observed in both treatment cohorts but trended more favorably among patients treated with 2,000 mg, including a 6-month PFS of 15% and a median OS of 9.9 months.
Cilengitide monotherapy is well tolerated and exhibits modest antitumor activity among recurrent GBM patients. Additional studies integrating cilengitide into combinatorial regimens for GBM are warranted.
M21 human melanoma cells express an Arg-Gly-Asp-directed adhesion receptor composed of noncovalently associated alpha and beta chains. To establish the structural and functional properties of this receptor on M21 human melanoma cells, stable variant cell lines were selected that express altered alpha chain levels. One of these variants, M21-L, fails to synthesize alpha chain protein or its mRNA, yet does produce normal levels of the beta chain. In these cells the beta chain does not reach the cell surface but rather accumulates within the cell. M21-L cells lacking the alpha chain are incapable of attaching to vitronectin, von Willebrand factor, fibrinogen, or an Arg-Gly-Asp-containing heptapeptide yet attach normally to fibronectin, whereas the unselected M21 cells attach to all of these adhesive proteins. In addition, a monoclonal antibody, LM609 generated to a functional site on the intact receptor, is capable of preventing M21 cell attachment to vitronectin, von Willebrand factor, fibrinogen, and the Arg-Gly-Asp peptide but not to fibronectin. Following a 2-min biosynthetic pulse-label, the newly synthesized alpha chain remains in free form for 5 min and then associates with previously synthesized beta chain present in an intracellular pool. Once oligomerization takes place, the receptor gains the capacity to recognize Arg-Gly-Asp, and at this time the epitope recognized by monoclonal antibody LM609 is formed.
A series of murine monoclonal antibodies were raised against purified human αvβ3 integrin and against M21 human melanoma cells. Five notable hybridomas were identified by ELISA on purified integrins, and the isolated antibodies bound the αv-chain. These antibodies, 17E6, 20A9, 23G5, 14D9.F8 and 10G2, recognised the extracellular domains of the integrin, and were shown to be reactive in FACS, immunoprecipitation, ELISA, and ELISA on fixed cells with M21, M21-L4, and UCLA-P3, but not with the αv-deficient M21-L or M21-L-IIb (M21-L transfected with GpIIb integrin).
One antibody, 17E6, strongly perturbed cell attachment mediated by αv integrins, reacting at least with αvβ3, αvβ5, and αvβ1, and strongly inhibiting cell attachment to αv-ligands vitronectin and fibronectin with an IC50 of ∼0.1 µg ml-1. Furthermore, 17E6 at this concentration could induce cell retraction from the substrate, while LM609 (anti-αvβ3) and control antibody 14E2 (anti-200 kDa melanoma surface protein) at 1,000-fold higher concentrations had minimal effects on cell morphology. The action of 17E6 was reversible and was not due to toxic effects: in vitro 17E6 at 0.1 mg ml-1 did not affect either cell proliferation or DNA synthesis.
In two nude-mouse tumour models, subcutaneous tumour development and a lung colonisation (‘experimental metastasis’) assay, injection of 17E6 strongly inhibited tumour development, while isotype-matched controls had no effect. There was no obvious mechanism of cell or of complement-mediated tumour cytotoxicity; the antibody did not mediate ADCC or AECDC, or complement fixation. The data strongly support previous studies which have indicated the importance of αv-integrins, and especially αvβ3, in the tumour progression of human melanoma.
The integrin alpha v beta 6 has been shown to be a fibronectin-binding protein. To determine whether the cytoplasmic and transmembrane domains of alpha v beta 6 are necessary for binding to fibronectin, a truncated, secreted form of the integrin lacking these domains was engineered and expressed in Chinese hamster ovary cells. Fibronectin affinity chromatography demonstrated that the secreted integrin, like its full-length counterpart, was capable of binding fibronectin. Monoclonal antibodies were made to secreted alpha v beta 6 and to beta 6-transfected NIH 3T3 cells. In experiments designed to determine whether alpha v beta 6 can mediate cell attachment to fibronectin, full-length human beta 6 was expressed in Chinese hamster ovary cells and in the human colon carcinoma cell line SW480. beta 6-expressing cells were identified by alpha v beta 6-specific antibodies, and the beta 6-transfectants were used in cell-adhesion assays. In Chinese hamster ovary cells, human beta 6 associated with hamster alpha v but was incapable of mediating cell attachment to fibronectin. However, expression of beta 6 in these cells had the dominant negative effect of decreasing alpha v beta 5-dependent adhesion to vitronectin. In SW480 cells, beta 6 expression conferred the ability to bind to fibronectin even in the presence of inhibitory antibodies against beta 1 integrins. In such cells, fibronectin binding ability could be blocked by an antibody to alpha v integrins. These results constitute the first direct evidence that alpha v beta 6 mediates cell attachment to fibronectin.
Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.
Anti-angiogenetic cancer therapy is a potential new form for treatment of solid tumours. The αv-integrins (αvβ3, αvβ5) mediate the contact of activated endothelial cells to proteins of the extracellular matrix during tumour angiogenesis as a prerequisite for survival of endothelial cells. The aim of this study was to investigate the effects of application of a methylated cyclic RGD-peptide as an αv-integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. Experiments were performed in the dorsal skinfold preparation of Syrian Golden hamsters bearing the amelanotic hamster melanoma A-Mel-3. Animals were injected intraperitoneally with a methylated cyclic RGD-peptide every 12 h, the control group received an inactive peptide. Microcirculatory parameters of tumour angiogenesis including functional vessel density, red blood cell velocity, vessel diameter and leucocyte–endothelium interaction were analysed using intravital microscopy. In an additional study the effects on growth and metastasis of subcutaneous A-Mel-3 were quantified. Functional vessel density was markedly reduced on day 3 in treated animals compared to controls (37.2±12.1 vs 105.2±11.2 cm⁻¹; mean±s.e.m.; P<0.05) and increased subsequently in both groups. Red blood cell velocity at day 3 was below values of controls (0.026±0.01 vs 0.12±0.03 mm s⁻¹; P<0.05). No differences were observed in vessel diameters and leucocyte–endothelium interaction was almost absent in both groups. Furthermore, growth and metastasis of subcutaneous tumours after administration of the cyclic RGD-peptide was significantly delayed in comparison to controls (P<0.05). Inhibition of αv-integrins by a cyclic RGD-peptide resulted in significant reduction of functional vessel density, retardation of tumour growth and metastasis in vivo. Taken together, these results implicate RGD-peptides as agents which have anti-tumour and anti-metastatic activity in vivo.
British Journal of Cancer (2002) 86, 788–795. DOI: 10.1038/sj/bjc/6600141 www.bjcancer.com
© 2002 Cancer Research UK
The structural basis for the divalent cation–dependent binding of heterodimeric αβ integrins to their ligands, which contain
the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements
in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin
αVβ3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between
the αV and β3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence
of ligand. The tertiary rearrangements take place in βA, the ligand-binding domain of β3; in the complex, βA acquires two
cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding
induces small changes in the orientation of αV relative to β3.
Vascular endothelial growth factor (VEGF) promotes vascular permeability (VP) and neovascularization, and is required for development. We find that VEGF-stimulated Src activity in chick embryo blood vessels induces the coupling of focal adhesion kinase (FAK) to integrin alpha(v)beta5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with beta1 and beta3 integrins in the presence or absence of growth factors. In cultured endothelial cells, VEGF, but not basic fibroblast growth factor, promotes the Src-mediated phosphorylation of FAK on tyrosine 861, which contributes to the formation of a FAK/alpha(v)beta5 signaling complex. Moreover, formation of this FAK/alpha(v)beta5 complex is significantly reduced in pp60c-src-deficient mice. Supporting these results, mice deficient in either pp60c-src or integrin beta5, but not integrin beta3, have a reduced VP response to VEGF. This FAK/alpha(v)beta5 complex was also detected in epidermal growth factor-stimulated epithelial cells, suggesting a function for this complex outside the endothelium. Our findings indicate that Src can coordinate specific growth factor and extracellular matrix inputs by recruiting integrin alpha(v)beta5 into a FAK-containing signaling complex during growth factor-mediated biological responses.
Expression of αvβ3- or αvβ5-integrins and selectins is widespread on blood cells and endothelial cells. Here we report that human tumor cells injected s.c. into mice lacking β3- or β3/β5-integrins or various selectins show enhanced tumor growth compared with growth in control mice. There was increased angiogenesis in mice lacking β3-integrins, but no difference in structure of the vessels was observed by histology or by staining for NG2 and smooth muscle actin in pericytes. Bone marrow transplants suggest that the absence of β3-integrins on bone marrow-derived host cells contributes to the enhanced tumor growth in β3-null mice, although few, if any, bone marrow-derived endothelial cells were found in the tumor vasculature. Tumor growth also was affected by bone marrow-derived cells in mice lacking any one or all three selectins, implicating both leukocyte and endothelial selectins in tumor suppression. Reduced infiltration of macrophages was observed in tumors grown in mice lacking either β3-integrins or selectins. These results implicate cells of the innate immune system, macrophages or perhaps natural killer cells, in each case dependent on integrins and selectins, in tumor suppression.
• tumors
• bone marrow transplants
• cell adhesion
• innate immunity
Bone sialoprotein (BSP) interacts separately with both matrix metalloproteinase 2 (MMP-2) and integrin alpha(v)beta3 and is overexpressed in many metastatic tumors. Its role in tumor biology, however, remains unclear. We investigated whether BSP enhances cancer cell invasiveness by forming a trimolecular complex with MMP-2 and cell-surface integrin alpha(v)beta3.
Invasiveness of breast, prostate, lung, and thyroid tumor cell lines was measured with a modified Boyden chamber assay. Binding and co-localization of BSP, MMP-2, and integrin alpha(v)beta3 were investigated with immunoprecipitation and in situ hybridization. All statistical tests were two-sided.
Treatment with BSP increased invasiveness of many breast, prostate, lung, and thyroid cancer cells through Matrigel in a dose-dependent manner. BSP at 50 nM increased the invasiveness of SW-579 thyroid cancer cells (95.2 units, 95% confidence interval [CI] = 90.4 to 100 units) by approximately 10-fold compared with that of untreated control SW-579 cells (9.1 units, 95% CI = 5.7 to 12.5 units) (P<.001). Addition of an inactive mutated BSP, in which BSP's integrin-binding RGD tripeptide was altered, or addition of integrin alpha(v)beta3-blocking antibodies resulted in invasiveness equivalent to that of untreated cells. Inhibiting cellular MMP-2 activity with chemical inhibitors or a specific antibody also blocked BSP-enhanced invasiveness. Osteopontin and dentin matrix protein 1, proteins related to BSP that also bind integrin alpha(v)beta3 and form complexes with other MMPs (but not MMP-2), did not enhance invasiveness. Immunoprecipitation showed that a complex containing BSP, integrin alpha(v)beta3, and MMP-2 formed in vitro. Addition of BSP increased the amount of MMP-2 bound by cells in an integrin-dependent fashion. Co-expression of BSP, integrin alpha(v)beta3, and MMP-2 in papillary thyroid carcinoma cells was shown by in situ hybridization.
Cancer cells appear to become more invasive when BSP forms a cell-surface trimolecular complex by linking MMP-2 to integrin alpha(v)beta3.
The involvement of alpha(v)beta3 and alpha(v)beta5 integrins in angiogenesis and the use of integrin antagonists as effective antiangiogenic agents are documented. Radiotherapy is an important therapy option for cancer. It has been shown that ionizing radiation exerts primarily antiangiogenic effects in tumors but has also proangiogenic effects as the reaction of the tumor to protect its own vasculature from radiation damage. Here, we show that combined treatment with S247, an Arg-Gly-Glu peptidomimetic antagonist of alpha(v)beta3 integrin, and external beam radiotherapy are beneficial in local tumor therapy. We found that radiation up-regulates alpha(v)beta3 expression in endothelial cells and consecutively phosphorylates Akt, which may provide a tumor escape mechanism from radiation injury mediated by integrin survival signaling. In the presence of S247, the radiation-induced Akt phosphorylation is strongly inhibited. Our studies on endothelial cell proliferation, migration, tube formation, apoptosis, and clonogenic survival show that the radiosensitivity of endothelial cells is enhanced by the concurrent administration of the integrin antagonist. The in vitro data are successfully translated into human glioma (U87), epidermoid (A431), and prostate cancer (PC3) xenograft models growing s.c. on BALB/c-nu/nu mice. In vivo, the combination of S247 treatment and fractionated radiotherapy (5 x 2.5 Gy) leads to enhanced antiangiogenic and antitumor effects compared with either monotherapies. These results underline the importance of alpha(v)beta3 integrin when tumors protect their microvasculature from radiation-induced damage. The data also indicate that the combination of integrin antagonists and radiotherapy represents a rational approach in local cancer therapy.
The extracellular bone matrix protein bone sialoprotein (BSP) is considered to play an important role in the pathogenesis of lytic skeletal lesions which are associated with severe morbidity in breast, prostate or lung cancer patients. In addition to in vitro studies, nude rats were implanted with 10(5) MDA-MB-231 cells transfected with GFP into a small branch of the femoral artery. Osteolytic lesions of the respective hind leg were detected by X-ray and CT analysis as well as by immunohistochemistry. Exposure of MDA-MB-231GFP cells in vitro to an antibody against BSP (0-400 microg/ml) decreased proliferation, colony formation and migration of these cells by up to 95, 83 and 89 T/C%, respectively. In nude rats, pre-incubation of MDA-MB-231GFP cells prior to inoculation (25-100 microg/ml) reduced the mean osteolytic lesion size to 22 T/C% after 90 days of observation (p<0.05). Treatment of overt lytic metastasis with the anti-BSP antibody (10 mg/kg) resulted in a significantly smaller mean lesion size of 57 T/C% at the end of the observation period (p<0.05) as well as in new bone formation. Immunohistochemical analysis revealed the presence of BSP in MDA-MB-231GFP cells and in vessel endothelium cells during processes such as migration and invasion. In conclusion, an anti-BSP antibody decreased proliferation, colony formation and migration of MDA-MB-231GFP cells in vitro and reduced osteolysis besides inducing bone formation in a nude rat model.
Studies in xenograft models and experimental models of metastasis have implicated several beta3 integrin-expressing cell populations, including endothelium, platelets and osteoclasts, in breast tumor progression. Since orthotopic human xenograft models of breast cancer are poorly metastatic to bone and experimental models bypass the formation of a primary tumor, however, the precise contribution of tumor-specific alphavbeta3 to the spontaneous metastasis of breast tumors from the mammary gland to bone remains unclear.
We used a syngeneic orthotopic model of spontaneous breast cancer metastasis to test whether exogenous expression of alphavbeta3 in a mammary carcinoma line (66cl4) that metastasizes to the lung, but not to bone, was sufficient to promote its spontaneous metastasis to bone from the mammary gland. The tumor burden in the spine and the lung following inoculation of alphavbeta3-expressing 66cl4 (66cl4beta3) tumor cells or control 66cl4pBabe into the mammary gland was analyzed by real-time quantitative PCR. The ability of these cells to grow and form osteolytic lesions in bone was determined by histology and tartrate-resistant acid phosphatase staining of bone sections following intratibial injection of tumor cells. The adhesive, migratory and invasive properties of 66cl4pBabe and 66cl4beta3 cells were evaluated in standard in vitro assays.
The 66cl4beta3 tumors showed a 20-fold increase in metastatic burden in the spine compared with 66cl4pBabe. A similar trend in lung metastasis was observed. alphavbeta3 did not increase the proliferation of 66cl4 cells in vitro or in the mammary gland in vivo. Similarly, alphavbeta3 is not required for the proliferation of 66cl4 cells in bone as both 66cl4pBabe and 66cl4beta3 proliferated to the same extent when injected directly into the tibia. 66cl4beta3 tumor growth in the tibia, however, increased osteoclast recruitment and bone resorption compared with 66cl4 tumors. Moreover, alphavbeta3 increased 66cl4 tumor cell adhesion and alphavbeta3-dependent haptotactic migration towards bone matrix proteins, as well as their chemotactic response to bone-derived soluble factors in vitro.
These results demonstrate for the first time that tumor-specific alphavbeta3 contributes to spontaneous metastasis of breast tumors to bone and suggest a critical role for this receptor in mediating chemotactic and haptotactic migration towards bone factors.
Integrin αvβ3 (vitronectin receptor) has been implicated in human malignant melanoma progression and angiogenesis as a receptor that provides survival signals. However, little is known about the therapeutic potential of antagonists of αvβ3. In this report, we characterize the activities of 2 antagonists of αvβ3 integrins: a human specific monoclonal antibody (MAb), 17E6, and a cyclic RGD peptide that blocked cell adhesion and induced detachment of previously substrate-attached cells in vitro. In vivo, αvβ3 antagonists behaved as anti-tumor drugs in a dose- and time-dependent manner. Moreover, different therapeutic treatments proved to be effective even in the therapy of established macroscopic tumor masses, thus supporting the use of these antagonists in clinical therapy. Using a panel of 6 human melanomas and 5 carcinomas, MAb 17E6 efficiently blocked the in vivo tumor growth of melanomas expressing αvβ3 as xenografts but did not affect the αvβ3-negative (although αv integrin-positive) tumors. This demonstrated that αvβ3 is a pivotal integrin for the growth of human melanomas. Furthermore, since MAb 17E6 does not recognize murine αvβ3, the effect is due only to the direct anti-tumor activity and not to the well-known anti-angiogenic activity of αv-integrin antagonists. Taken together, our results confirm the essential role of αvβ3 integrin in the growth of human malignant melanoma in vivo and provide strong evidence of the therapeutic potential of αv-integrin antagonists for the treatment of such tumors. Int. J. Cancer 87:716–723, 2000. © 2000 Wiley-Liss, Inc.
Expression of the αvβ3 integrin is upregulated on sprouting endothelia. Systemic application of antibody or peptidic inhibitors of αvβ3 function disrupts tumor angiogenesis and reduces growth and invasiveness of human tumors in animal models. We systematically investigated αvβ3 expression on tumor-associated vessels of 4 different human epithelial tumors and the corresponding normal tissues by means of immunohistochemistry on frozen sections using the αvβ3-complex specific monoclonal antibody LM609. Variable levels of LM609 staining were found in all carcinoma lesions. A considerable number of tumor tissues (35/50) expressed αvβ3 on more than 50% of their vessels. Inflammatory infiltrates and the possibly hypoxic conditions near necrotic areas of tumors were accompanied by an increased αvβ3 expression. Remarkably, the vasculature in apparently normal tissue also stained for αvβ3. However, the percentages of stained vessels and their staining intensity were lower than in neoplastic tissues. Besides the vascular αvβ3 expression, several extravascular cell types stained positive, in both normal and tumor specimens. Taken together, our findings show a considerable number of colon, pancreas, lung and breast carcinoma lesions with many αvβ3-expressing vessels that could be targets for anti-αvβ3-therapy. Int. J. Cancer 71:320-324, 1997. © 1997 Wiley-Liss Inc.
Introduction: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three
out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The αvβ3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption.
Therefore, we hypothesized that the αvβ3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. Materials and methods: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle)
injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with
vehicle (saline) or S247 (1, 10, or 100mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor
cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases
were recorded. Results and conclusions: IC50 for S247 adhesion to αvβ3 or αIIBβ3a substrates was 0.2nM vs. 244nM, respectively. Likewise, S247 was not toxic at doses up to 1000μM. However, osteoclast
cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247
was administered prior to tumor cells, there was a significant, dose-dependent reduction (25–50% of vehicle-only-treated mice;
P=0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but
the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the αvβ3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data
suggest that αvβ3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.
In the adult, angiogenesis, the formation of new blood vessels from pre-existing vasculature contributes to the pathogenesis of many disorders including cancer. The role of adhesion molecules, especially integrins, in pathological angiogenesis has long been the subject of investigation, mostly because of their potential as anti-angiogenic targets. Recent studies have highlighted the complexities connected with understanding the roles of one particular integrin, alphavbeta3, in neovascularization. This integrin is notoriously promiscuous and its precise functions in angiogenesis are unclear. Here, I have firstly summarized some of the salient features of the roles played by alphavbeta3 during angiogenesis; secondly attempted to address the apparently conflicting issues surrounding this topic; and finally raised some questions that appear to be unanswered.
Molecular imaging of angiogenesis is urgently needed for diagnostic purposes such as early detection, monitoring of (angiostatic) therapy and individualized therapy. Multimodality molecular imaging is a promising and refined technique to study tumor angiogenesis, which has so far been largely unexplored due to the lack of suitable multimodal contrast agents. Here, we report on the application of a novel alpha v beta 3-specific quantum dot-based nanoparticle, which has been optimized for both optical and magnetic resonance detection of tumor angiogenesis. Upon intravenous injection of RGD-pQDs in tumor-bearing mice, intravital microscopy allowed the detection of angiogenically activated endothelium at cellular resolution with a small scanning window and limited penetration depth, while magnetic resonance imaging was used to visualize angiogenesis at anatomical resolution throughout the entire tumor. Fluorescence imaging allowed whole-body investigation of angiogenic activity. Using these quantum dots and the aforementioned imaging modalities, the angiogenic tumor vasculature was readily detected with the highest angiogenic activity occurring in the periphery of the tumor. This nanoparticle may be employed for multimodality imaging of a variety of diseases that are accompanied by activation of endothelial cells. Furthermore, the current technology might be developed for molecular imaging of other pathophysiological processes.
Bone metastases are the most common skeletal complication of malignancy. Tumor cells disrupt normal bone remodeling to promote bone destruction and its associated morbidity. In the August 15, 2009, issue of Genes & Development, Lu and colleagues (pp. 1882-1894) propose a novel molecular mechanism by which tumor-produced metalloproteinases release epidermal growth factor (EGF) ligands to activate the central osteoclastogenic pathway receptor activator of NF-kappaB ligand (RANKL) to promote breast cancer osteolysis. This work has important therapeutic applications that may quickly translate to more effective treatment for bone metastases.