Therapy resistance mechanisms in human cancers are determined by intracellular factors and interactions of cells with ECM or neighboring cells as well as the tumor microenvironment. While integrin cell adhesion receptors are overexpressed in the majority of human cancers, how tumor cells are sensitized to radiotherapies or chemotherapies upon integrin inhibition remains elusive. Here, we provide direct evidence in 3D lrECM cell culture models and in tumor xenografts that β1 integrin targeting is a promising approach to sensitize tumor cells to radiotherapy and that the susceptibility of HNSCC cells to anti–β1 integrin treatment depends on the activity of FAK. Further, to our knowledge, this study identified new regulatory motifs of FAK kinase activity and cortactin as a novel binding partner of FAK. Mechanistically, dissociation of cortactin from FAK, resulting in JNK1 deactivation, is a critical event leading to radiosensitization of tumor cells upon β1 integrin inhibition.
Prior studies implicated that targeting integrins may be potentially useful to induce death of tumor cells or endothelial cells of the tumor vasculature and prevent neoangiogenesis (9
). Additionally, tumor cell invasion and metastasis could be efficiently targeted, as these processes, at least partially, depend on integrins (49
). Compounds facilitating effective integrin inhibition include monoclonal antibodies like volociximab, synthetic peptides like cilengitide, and peptidomimetics, which are directed against different αβ integrin heterodimers or specific ECM-binding motifs on integrins (51
). Due to their expression on tumor cells and endothelium, a large body of preclinical and clinical work on αvβ3
integrin inhibition indicates substantial tumor cell death in a variety of tumor entities, such as glioblastoma and lung cancer, in addition to providing imaging possibilities prior and during the course of therapy. Most importantly, all studies accomplished to date come to the same conclusion: monotherapeutic anti-integrin approaches are by far less efficient alone than in combination with chemotherapy and, particularly, radiotherapy, a strategy in line with modern multimodal treatment regimes (11
Despite strong evidence for an enhancement of tumor cell chemosensitivity and radiosensitivity by β1
integrin inhibition (8
), the underlying mechanisms are unclear. To address this issue, we chose the 3D lrECM based cell culture model and tumor xenografts in nude mice. Intriguingly, 8 out of 10 HNSCC cell lines exhibited susceptibility to antibody-mediated β1
integrin inhibition. The same 8 cell lines showed enhanced radiosensitization in response to β1
integrin inhibition, which was confirmed by similar results from siRNA-mediated β1
integrin knockdown. In a proof-of-principle study on tumors in vivo, we successfully demonstrated that a triple i.p. application of the inhibitory monoclonal anti–β1
integrin antibody AIIB2 plus irradiation with 20 Gy significantly delayed the growth of UTSCC15 tumor xenografts. These promising results will now undergo further validation in preclinical translational studies using clinically relevant fractionated radiotherapy and permanent local tumor control as the end point. Interestingly, except for a reduction in the mitotic index, neither changes in BrdU, Ki-67, animal weight, or necrotic/hypoxic tumor areas nor increased metastasis were observable in AIIB2-treated mice.
To evaluate the responsible mechanisms and discriminate between responder and nonresponder HNSCC cell lines, a phosphoproteome array analysis was performed. Aside from specific modifications of cell proliferation-associated proteins, such as GSK3 and ERK2, we found strong downregulation of FAK phosphorylation. Further analysis revealed a deactivation of the FAK/paxillin/p130Cas signaling cascade by β1
integrin inhibition in the responder cell lines UTSCC15 and XF354 and in UTSCC15 xenografts in contrast to the nonresponder cell line SAS. Although reduced phosphorylation of the FAK/paxillin/p130Cas protein complex is in line with results from many other studies (50
), the reduced phosphorylation of JNK1 and JNK2 as well as absent Src modifications appear to be unique for HNSCC and particularly for 3D lrECM and in vivo growth conditions. Thus, signaling alterations induced by β1
integrin inhibition seem to vary between tumor entities. For example, AIIB2 treatment of 3D lrECM–grown breast carcinoma cell lines mainly mediated deactivation of PI3K/Akt signaling (21
), which was also found in lung carcinoma cells (55
) but not in HNSCC as presented. Strong support for our hypothesis that FAK is most upstream to β1
integrin signaling came from in situ PLAs, showing that the interaction between β1
integrins and FAK is immediately released upon β1
integrin blocking. Intriguingly, expression of either wild-type or constitutively active forms of FAK bypassed the effect of AIIB2 treatment and imparted significant radioprotection. Conclusively, these findings demonstrate that FAK is a key determinant of β1
integrin downstream signaling, clonogenic cell survival, and radiosensitization by β1
integrin inhibition of HNSCC cells.
Another obvious discrepancy between responder and nonresponder cells was cell rounding and FA disassembly upon AIIB2-mediated β1 integrin blocking. To address this issue, we sought actin binding proteins in a complex with FAK. Mass spectrometry of FAK immunoprecipitates assisted us in identifying the actin organization regulators cortactin, cofilin, and ezrin/moesin. From this panel, only cortactin, a FAK-interacting protein, was unphosphorylated after AIIB2 treatment; this was associated with its dissociation from FAK, which remained until at least 24 hours. Further proof for FAK-cortactin binding as a key step downstream of β1 integrin came from FAK, Src, JNK1, JNK2, or cortactin knockdown experiments and transfectants expressing the constitutively active kinase form of FAK that perpetuated FAK-cortactin binding upon AIIB2-mediated β1 integrin inhibition.
To provide further evidence for a direct physical interaction between FAK and cortactin, to identify putative cortactin binding sequences on FAK, and to assess whether loss of cortactin binding to FAK impacts on radiosensitivity of 3D HNSCC cells, we mutated 2 PXΨP or 2 PXXPXXP motifs of FAK. These sequences function as SH3 consensus binding motifs for cortactin, as shown previously for cortactin and BPGAP1 (45
). These motifs are located at very different regions of the FAK protein: the FERM and kinase domain linker domain, the kinase domain, the kinase-FAT linker domain, and the FAT domain. According to recent work on FAK activity regulation by cooperative FERM-kinase domain interactions (reviewed in ref. 46
), we hypothesized that inactivating mutations at these motifs modify both FAK-cortactin binding and FAK phosphorylation and thus FAK activity. Indeed, mutations of the 2 PXΨP motifs located at the FERM-kinase domain linker and the FAT domain completely abrogated cortactin binding to FAK similar to the dominant-negative FRNK. The mutated PXXPXXP motif within the kinase domain led to strong reduction of this protein interaction. It can be speculated that both a sound regulatory interaction between the FERM and kinase domain and correct FAK targeting to FAs determine cortactin linkage to FAK. Importantly, these binding motifs represent essential regulatory sites for FAK activity that we believe to be novel. In contrast to the mutated PXXPXXP motif at the kinase domain-FAT linker, the other mutations caused pronounced dephosphorylation of FAK, cortactin, and JNK1. Strikingly, perturbation of FAK kinase activity and loss of FAK/cortactin interaction translated into significant enhancement of radiosensitivity. The lesser degree of radiosensitization as compared with that in controls and FRNK transfectants might be due to spatiotemporal patterns of specific fractions of FAK, cortactin, and the FAK/cortactin protein complex, which differentially govern additional functions of FAK and cortactin.
In summary, this study reveals mechanistic evidence for prosurvival and radioresistance-mediating signals via a β1 integrin/FAK/cortactin/JNK1 pathway in HNSCC cells (Supplemental Figure 25). Moreover, these findings underscore β1 integrin targeting as an attractive approach in combination with radiotherapy and radiochemotherapy for patients suffering from HNSCC. Consisting of a myriad of prosurvival signaling molecules, FA signaling hubs serve as major promoters of tumor growth and progression, whose specific targeting may be used to overcome therapy resistance for increasing cancer patient survival rates.