Cancer Therapy Vol 2, 271-278, 2004

Bundeswehr Institute of Radiobiology, Neuherbergstrasse 11, 80937 Munich, Germany

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*Correspondence: Dr. Nils Cordes, M.D., Bundeswehr Institute of Radiobiology, Neuherbergstrasse 11, 80937 Munich, Germany; Phone: ++89 — 3168 — 3634; Fax: ++89 — 3168 — 2635; E-mail: cordes@radiation-biology.de

Key words: Integrins, radiosensitivity, ECM, tumor cells

Abbreviations: cell adhesion-mediated radioresistance, (CAM-RR); cell adhesion-mediated drug resistance, (CAM-DR); extracellular matrix, (ECM); focal adhesion kinase, (FAK); integrin-linked kinase, (ILK); ionizing radiation, (IR); mitogen-activated protein kinase, (MAPK); phosphatidylinositol-3- kinase, (PI3K); protein kinase B/AKT, (AKT)

Cell-matrix binding is primarily mediated by integrins, a family of 18 a and 8 ß transmembrane glycoproteins (Hynes, 2002). To date, 24 different a ß-heterodimers were identified. Most integrins recognize several ECM proteins and individual matrix proteins bind to several integrins. Activation of integrin-mediated intracellular pathways regulate signaling elements in proliferation, survival, migration, tissue organization and many more (Assoian, 1997; Giancotti and Ruoslahti, 1999; Schwartz, 2001; Watt, 2002; Zahir and Weaver, 2004; Brakebusch and Fässler, 2003). Integrin signaling and assembly of the cytoskeleton are intimately linked. As integrins bind to ECM, they associate with cytoskeletal and signaling complexes to form focal adhesions (Burridge, 1988). In this manner integrins serve as structural integrators between the ECM and the cytoskeleton, the property for which integrins are named. Via diverse cytoplasmic signaling molecules, including focal adhesion kinase (FAK), Src-family kinases and integrin-linked kinase (ILK), the stimuli generated through ligation of integrin receptors are transduced for regulating cellular events (Hannigan et al, 1996; Schaller, 2001).

Integrins were first studied in platelets and thrombocytes (Davignon et al, 1981; Hansen and Clemmensen, 1982). Nowadays e.g. endothelial cells are intensively under investigation to identify potential therapeutic targets for inhibition of angiogenesis in tumors (Wary, 2004). In fact, both ß3 and ß1 integrin subunits are expressed by endothelial cells and regulate critical adhesive interactions with a variety of ECM proteins including fibronectin, vitronectin, laminin, collagens and fibrinogen (Ingber, 2002). Cytokine- and growth factor-stimulated

Figure 1. Schematic diagram of converging and mutually modifying integrin-RTK signaling in focal adhesions. The ligand binding, e.g. extracellular matrix proteins or growth factors, to their receptors activates downstream protein kinases. Stimulation and inhibition of the different signaling pathways as well as the crosstalk between integrin and growth factor receptor pathways optimize the regulation of cell survival, cell cycle progression, apoptosis, DNA repair, adhesion, migration, protein synthesis, and gene transcription. Further structural and signaling molecules of these membranous multiprotein signaling complexes (actin, gelsolin, talin, a -actinin) are not displayed. However, these components are substantial for the regulation of diverse cellular functions. Additionally, most of the delineated signaling events are not located at the cell membrane where they usually occur to avoid overcrowding. RTK, receptor-tyrosine-kinase; GF, growth factor.

endothelial cells express a wide spectrum of cell adhesion receptors as compared to quiescent cells (Ingber, 1992). In particular, the function of a vß3 integrins was elucidated in survival and apoptotic processes with regard to the identification of a specific target molecule for innovative anti-tumor therapies (Kumar et al, 2001). This integrin is only minimally expressed on quiescent blood vessels, but highly upregulated on cells undergoing angiogenesis (Ingber, 1992). Recent studies provided promising evidence that a vß3 integrins may also serve as a useful diagnostic indicator. In animal studies, anti-a vß3 antibodies coupled to a gadolinium-containing liposome or iodine radioisotopes specifically targeted to tumor-associated blood vessels and, thus, proved useful in MRI and scintigraphy (Blankenberg et al, 2002). However, blockade of this integrin disturbed angiogenic processes in the chicken chorioallantoic membrane, mouse retina and rabbit cornea (Friedlander et al, 1995; Luna et al, 1996; Kumar et al, 2001). Moreover, a vß3 or ß1 integrin knockout mice developed serious vascular malformations (Hodivala-Dilke et al, 1999). These observations led to the conclusion that a vß3 integrin facilitates the survival of stimulated endothelial cells. In fact, systemic administration of a vß3 antagonists to animals with ongoing angiogenesis showed blood vessels containing high levels of apoptotic endothelial cells. Similar effects were observed in vitro by comparing endothelial cells of different origins. Testing of a first anti-angiogenic compound, which is a specific a vß3 integrin antibody, has recently started in clinical oncological trials (Eskens et al, 2003).

In contrast to normal cells, cancer cells are often able to proliferate and survive in the absence of adhesion to ECM proteins (Schwartz, 1997). These characteristics are important for cancer progression and metastases formation. Intense studies performed in a wide spectrum of cancer cells including melanoma, breast, prostate or colon gave rise to the hypothesis that resistance of cancer cells to medical treatment is partly due to alteration or loss of cell-matrix interactions (Lewis et al, 2002). Because of difficulties in demonstrating a positive or negative correlation between tumor progression and integrin expression, which was due to the heterogeneity of tumors and to the fact that changes in the expression level of a single integrin subunit always had to be judged against the background of the expression levels of all other integrins, the results are still controversial. For example, a 2ß1 integrin expression seems to promote invasion in pancreatic carcinomas but is downregulated in bladder and colon cancer (Orian-Rousseau et al, 1998; Arao et al, 2000). On the other hand, a strong correlation between the enhancement of a vß3 integrin expression and the metastatic potential of melanoma cells was detected, thus rating expression of a vß3 as prognostic factor (Hieken et al, 1996). Contrary, expression of ß8 integrin was only observed in normal airway epithelium but not in lung cancer cells (Fjellbirkeland et al, 2003). Tumor progression and ability to form distant metastases has been correlated with the elevation of integrin-dependent protein kinases activity such as FAK or ILK (Gabarra-Niecko et al, 2003; Persad and Dedhar, 2003). A major observation in these studies was a strong converging communication between oncogenic Ras and Raf with components of integrin signaling cascades (Kinbara et al, 2003). Ras and Raf activate prosurvival pathways and are able to induce a significant degree of anchorage-independent growth.

Considering the role of integrins in malignant disease, this family of cell adhesion molecules presents as attractive drug target but the wide range of functions in different cell types is daunting. Understanding of integrin-mediated signaling, function and cross-talk with growth factor receptor-regulated pathways is crucial for the optimization and development of innovative chemo- and radiotherapeutic approaches. Nevertheless, a handful of therapeutic approaches have been initiated that implicate engineered anti-integrin antibodies or peptidomimetics with clinical success (Mousa, 2002). Most of the compounds tested with high degree of specificity were anti-a vß3 or anti-RGD peptides for blocking the RGD receptor subset of integrins (Tucker, 2002). While a 5ß1 integrins are predominantly involved in survival signalling and anti-RGD peptides possess anti-migratory potential, a vß3-integrins mainly promote angiogenic events (Tucker, 2003). Interestingly, neither systemic increase in bleeding nor impairment of wound healing repair have yet been observed. These findings provide first successful results that can serve as basis for further detailed preclinical and clinical trials.

Experimental data demonstrated that cancer cells are often more resistant against cell killing by conventional antimitotic drugs in the presence of ECM proteins compared to standard in vitro culture conditions. Cell adhesion-mediated drug resistance (CAM-DR) is thought to be partially caused by integrin signaling (Damiano et al, 1999; Cordes et al, 2004). Sethi et al, (1999) showed that adhesion of small-cell lung cancer cells to ECM protects from the chemotherapeutic agents doxorubicin, etoposide, cyclophosphamide, and cisplatin. This effect is mediated by ß1 integrins, which activate intracellular protein kinases in response to therapy-induced DNA-damage. PI3K was uncovered to participate in drug resistance of breast cancer cells (Clark et al, 2002) and increased drug resistance could also be correlated with overexpressed a 4ß1 integrin in human myeloma cells (Damiano et al, 1999).

Similar findings were seen in cell adhesion-mediated radioresistance (CAM-RR) studies and the specific impact of integrin-mediated signaling on decreased cellular radiosensitivity will be outlined in detail below.

The cytoprotective effects of physiological substrata were detected both in irradiated normal cells like skin and lung fibroblasts, endothelial cells, and keratinocytes as well as in transformed cells of different origin (lung, pancreas, skin, brain, breast, melanoma, colon) (Figure 2; Rose et al, 1999; Cordes and Meineke, 2003; Cordes and van Beuningen, 2003; Cordes and van Beuningen, 2004). Clonogenic survival experiments provided evidence that the interaction of cells with the physiological integrin ligands fibronectin, laminin, collagen-III and vitronectin before and at the time of irradiation essentially promotes survival compared to cells irradiated on culture plastic. Interestingly, this effect demonstrated only marginal variation between the different matrix proteins, complex matrix compositions like Matrigel (matrix extract from the Englebreth-Holm-Swarm mouse sarkoma) or matrix produced by bovine and human endothelial cells. Except from a variety of human glioblastoma cell lines (Cordes et al, 2003), our studies clearly show that the transformed cell lines used are all susceptible to matrix signals and that these signals strongly impact on the cellular resistance to IR. In agreement with our studies on different ILK overexpressing mutants (Cordes, 2004), Lewis et al, (2002) and Truong et al, (2003) observed adhesion to matrix as one of the major determinants for the ability to execute apoptosis after induction of genotoxic stress.

Cell survival and cell cycling are closely linked cell functions. After irradiation, cells are blocked in the G1- or G2-phase of the cell cycle, which is hypothesized to provide time for DNA-repair (O`Connor, 1997). These cellular phenomena have been intensively investigated in radiobiology and are regulated by cyclins, cyclin-dependent kinases (CDK) and their inhibitors (Bernhard et al, 1999) (Figure 3). Unfortunately, only few studies have considered an impact of cell-matrix interactions on the regulation of cell cycling in irradiated cells (Gadbois et al, 1997; Dimitrijevic-Bussod et al, 1999). Recently, we showed that adhesion of normal lung fibroblasts to fibronectin enabled pronounced radiation-induced accumulation of cells in the G2/M phase of the cell cycle relative to culture plastic (Cordes and van Beuningen, 2004). Whether improved ECM-dependent survival is caused by optimization of DNA repair mechanisms at fibronectin presence is currently not known but studies to clarify this aspect are ongoing in our laboratory. Confirmatory observations were reported by Gadbois et al. (1997) and Dimitrijevic-Bussod et al. (1999) who showed that fibronectin and collagen-IV significantly reduced the portion of cells arrested in G1 and S and that radiation-induced cell cycle arrest of human normal fibroblasts is substratum-dependent.

In vitro experiments using primary and permanent cell cultures of diverse origin demonstrated that IR is able to induce significant up-regulation of cell surface ß1-, ß3-,

Figure 2. Fibronectin-mediated radiation resistance. In contrast to polystyrene (Poly), growth of cells on the matrix protein fibronectin (FN) improves survival of irradiated human normal and transformed cells. Radiation doses ³ 2 Gy for normal skin (HSF1) and lung (CCD32) fibroblasts and for radiation doses ³ 4 Gy for A172 (glioblastoma) and A549 (lung carcinoma) cells grown on FN resulted in a p < 0.05 compared to cells grown on polystyrene. * p < 0.05.

Figure 3. Schematic diagram delineating the regulation of the G2 checkpoint response after genotoxic stress (e.g. ionizing radiation) in vitro. During G2/M-phase transition, the activity of various cell cycle proteins is tightly regulated by phosphorylation, dephosphorylation and protein interactions, mechanisms that are modulated by cell-matrix interactions. Chk1 and Chk2 phosphorylate Cdc25C, which is proposed to be critical for G2 checkpoint regulation. After binding of 14-3-3, the 14-3-3-Cdc25C complex is degraded in the cytoplasm thus preventing the activation of the Cdc25C substrate and G2-phase driving protein kinase Cdk1 (p34^cdc2) by dephosphorylation. Furthermore, Chk1 and Chk2 are able to phosphorylate p53 on serine-20, a site implicated in p53 stabilization through p53-MDM2 interactions. While subsequent induction of CDK-inhibitor p21 prevents cleavage of the pRb-E2F-complex via inhibition of cyclin D1/CDK4/CDK6 activation responsible for G1-phase arrest, p53 is also required to sustain but not to initiate a G2 arrest.

and a 5-integrin expression within 48 to 96 h (Meineke et al, 2002; Cordes et al, 2002). This effect presented always dose- and frequently matrix-dependent. Interestingly, in most cell lines there was a transient decrease in ß1 or ß3 integrin cell surface presentation detectable during the first 6 h following IR. Evaluation of the adhesive functionality of the newly expressed integrin receptors revealed a strong dose-dependent elevation of cell adhesion to various matrix proteins. To assess the importance of single integrin subunits in this process, blocking experiments were carried out and identified both ß1- and ß3-integrins to play an essential role in adhesion of irradiated cells. Similar observations were reported by other investigators in endothelial cells (Hallahan et al, 2003). One elegant therapeutic approach used enhancement of a vß3 integrin expression on sublethally irradiated endothelial cells undergoing angiogenesis to deliver drugs or immuno-radioisotopes directly into the irradiated endothelium of a malign tumor (Hallahan et al, 2003). This might be a reasonable strategy for tumor-specific integrin trageting in selected tumor entities.

Besides adhesion, integrins serve in invasion and migration events of normal but especially cancer cells. The impact of irradiation on these two cell functions is still controversial. Wild-Bode et al, (2001) showed enhanced migration and invasiveness of glioma cells, which correlated with elevated expression of a vß3 integrins and activity of matrix-metalloproteinases (MMPs). In contrast, we showed differential invasion-impairing potential of irradiation in glioma cells (Cordes et al, 2003). Radiation-induced ß1 and ß3 integrin cell surface presentation in combination with elevated MMP-2 activity suggested that the invasion-inhibiting effect in irradiated cells is in part due to integrin-mediated local anchorage and disproportional MMP-2-mediated degradation of ECM proteins. Further controversy data on this issue indicated that exposure of endothelial cells to 6 Gy of X-rays promotes migration of endothelial cells in vitro (Sonveaux et al, 2003). Studies on the area vasculosa or the chorioallantoic membrane of the fertilized egg clearly showed that angiogenesis is inhibited after low doses of IR such as 2 Gy but is stimulated after higher doses like 10 Gy (Hatjikondi et al, 1996; Plasswilm et al, 1999).

In many cases refractory tumor responses are clinically observable during both chemo- and radiotherapy. The resistance of tumors or tumor cell clones is still a major problem to be solved and integrin-mediated cell-ECM interactions are very likely to play an essential role in this context. It is widely accepted that a combination of molecularly targeted anticancer therapies may be more effective than conventional approaches. The presented data on the role of integrin receptors in CAM-RR could provide a new, challenging, and effective basis for the development of innovative multi-targeted oncological strategies that are able to augment the cytotoxic powers against the primary tumor as well as its metastases. Further investigations on tumor-specific integrin-mediated signaling and cross-talk between integrins and growth factor receptor signaling may contribute to the identification of new molecular targets and the design of interacting substances for adjuvant chemo- and radiotherapy strategies.