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Abstract: Translational research is essential to find new therapeutic approaches to improve cancer patient survival. Despite extensive efforts in preclinical studies, many novel therapies fail to turn out to be translational from bench to beside. Therefore, new models better reflecting the conditions in vivo are needed to generate results, which transfer reliably into the clinic. The use of three-dimensional (3D) cell culture models has provided new emerging insights into the understanding of cellular behavior upon cancer therapies. Interestingly, cells cultured in a 3D extracellular matrix are more radio- and chemoresistant than cells grown under conventional 2D conditions. In this review, we summarize and discuss underlying mechanisms of this phenomenon including integrin-mediated cell–matrix interactions, cell shape, nuclear organization and chromatin structure. Identifying the molecular differences between 2D and 3D cultured cells will offer the opportunity to improve our research and widen our therapeutic possibilities against cancer. [Copyright &y& Elsevier]

Abstract: Purpose: Epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) show frequent overexpression and hyperactivity in various human malignancies including head and neck squamous cell carcinomas (HNSCC). To examine effects of dual EGFR/FAK inhibition on cellular radiosensitivity of HNSCC cells in a more physiological environment, we employed a previously established laminin-rich extracellular matrix (lrECM) based three-dimensional (3D) cell culture model. Materials and methods: UTSCC15 and SAS HNSCC cell lines stably transfected with EGFR-CFP or CFP were used. Single or combined EGFR (Cetuximab, siRNA) and FAK (TAE226, siRNA) inhibition were accomplished prior to measuring clonogenic survival and protein expression and phosphorylation. Immunofluorescence enabled visualization of EGFR-CFP and FAK. Results: Cetuximab resulted in higher radiosensitization in EGFR-CFP overexpressing cell lines than CFP controls. Single EGFR or FAK inhibition mediated radiosensitization, while dual EGFR/FAK targeting further augmented this effect. Despite signaling alterations upon Cetuximab and siRNA knockdown, analysis of protein expression and phosphorylation indicates EGFR and FAK signaling coexistence without obvious overlap. Conclusions: Combined EGFR/FAK targeting yielded stronger radiosensitization than either approach alone, which might be based on non-overlapping downstream signaling. Whether dual targeting of EGFR and FAK can reasonably be combined with radiotherapy and chemotherapy needs clarification. [Copyright &y& Elsevier]

Abstract: Interactions of cells with their microenvironment modify the cellular sensitivity of normal and tumor cells for radiation- and drug-induced genotoxic injury. The preexistent or acquired cellular resistance against such agents aggravates anticancer therapies and, therefore, complicates the recovery of patients. Recently, integrin-mediated adhesion was shown to improve cell survival of both normal and cancer cells following DNA damage. Here, I will discuss the role of integrins and integrin-mediated signaling cascades in the survival or death response upon genotoxic stress. Detailed knowledge of the responsible molecular processes might provide implications for putative therapies targeting integrins or integrin-associated molecules to achieve an optimization of anticancer treatments. [Copyright &y& Elsevier]

: PurposeCell–matrix interactions might confer cellular radioresistance in vitro. As a function of radiation, the impact of fibronectin (FN) and phosphatidylinositol-3 kinase (PI3K)-related signaling on survival, the cell cycle, and the β1-integrin signaling kinases integrin-linked kinase (ILK), protein kinase Bα/Akt (PKBα/Akt), and glycogen synthase kinase-3β (GSK-3β) was examined in normal lung fibroblasts in vitro.: Methods and materialsNormal human CCD32 lung fibroblasts grown on polystyrene, FN, or poly-l-lysine were irradiated with 0–8 Gy. Colony forming assays, flow cytometric DNA analysis, immunoblotting (Chk1, Chk2, Cdc25C, Cdk1, 14-3-3, p53, p21), and protein kinase assays (ILK, PKBα/Akt, GSK-3β) were performed with or without PI3K inhibition using LY294002 or wortmannin.: ResultsFN significantly elevated clonogenic survival of CCD32 cells after irradiation compared with polystyrene or poly-l-lysine. FN improved accumulation of irradiated cells in G2/M (60%) compared with polystyrene (43%). LY294002 prevented radiation-dependent G2 blockage on polystyrene; on FN, G2 arrest was only slightly reduced. Radiation- and PI3K inhibition-related changes in expression and phosphorylation of the various cell cycle proteins tested correlated with the cell cycle data acquired. The kinase activities of ILK, PKBα/Akt, and GSK-3β were strongly induced by irradiation on polystyrene, but not on FN, which was a result of a FN-mediated increase of basal kinase activities. In contrast to polystyrene, FN enabled radiation-dependent induction of ILK and GSK-3β in a PI3K-independent manner.: ConclusionThe data indicate a tight convergence of cell–matrix and cell-growth factor interactions that seem to optimize the cellular responsiveness to ionizing radiation in terms of survival and G2 arrest. ILK, PKBα/Akt, and GSK-3β involved in integrin signaling were uncovered as new molecular factors within the cellular radiation response. Our findings might also provide insight into normal tissue effects and cellular radioresistance. [Copyright &y& Elsevier]

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a cancer with unmet needs. The role of highly conformal radiation therapy is still under debate for PDAC. Owing to its desmoplastic nature, integrin-mediated interactions between PDAC cells and extracellular matrix (ECM) profoundly contribute to PDAC therapy resistance. In this study, we investigated the radiochemosensitizing potential of β1 integrin targeting in therapy-naive and radioresistant PDAC cell cultures grown in 3-dimensional (3D) ECM.Methods and Materials: In a panel of 3D, ECM-based PDAC cell cultures, β1 integrin was inhibited by antibodies or siRNA-mediated knockdown. Together with x-ray irradiation and specific chemotherapies, we determined 3D colony formation capacity in therapy-naive and radioresistant PDAC cultures. We used kinome profiling, Western blotting, and immunofluorescence stainings to characterize these cell lines. Various siRNA screens were conducted to identify novel therapeutic targets.Results: We found a significant radiosensitizing potential of β1 integrin inhibition both in therapy-naive and radioresistant PDAC cell cultures. Kinome profiling upon β1 integrin targeting identified a generally declined tyrosine and serine/threonine kinase activity, which presented less prominent in radioresistant than in therapy-naive PDAC cells. siRNA screens employing the top 34 deregulated kinases in combination with β1 integrin inhibition revealed less efficacy and less radiosensitization in radioresistant relative to therapy-naive PDAC cell cultures. Triple inhibition of β1 integrin, protein kinase D1, and rearranged during transfection turned out to be most effective in reducing 3D colony formation of radioresistant PDAC cells.Conclusions: Our study clearly shows that β1 integrins are robust targets for overcoming radioresistance in PDAC. This seems to apply equally to therapy-sensitive and radioresistant cells. Concerning tumor heterogeneity, this dual therapy-sensitizing potential might be exploitable for a significant improvement of patient survival. [ABSTRACT FROM AUTHOR]

Summary Glioblastoma (GBM) is highly refractory to therapy and associated with poor clinical outcome. Here, we reveal a critical function of the promitotic and adhesion-mediating discoidin domain receptor 1 (DDR1) in modulating GBM therapy resistance. In GBM cultures and clinical samples, we show a DDR1 and GBM stem cell marker co-expression that correlates with patient outcome. We demonstrate that inhibition of DDR1 in combination with radiochemotherapy with temozolomide in GBM models enhances sensitivity and prolongs survival superior to conventional therapy. We identify a 14-3-3-Beclin-1-Akt1 protein complex assembling with DDR1 to be required for prosurvival Akt and mTOR signaling and regulation of autophagy-associated therapy sensitivity. Our results uncover a mechanism driven by DDR1 that controls GBM therapy resistance and provide a rationale target for the development of therapy-sensitizing agents. Graphical Abstract Highlights • DDR1 and stem cell marker co-expression in GBM correlates with patient outcome • A DDR1-associated 14-3-3-Beclin-1-Akt1 complex induces prosurvival signaling • DDR1 targeting elicits autophagy-associated therapy sensitization Vehlow and Klapproth et al. identify the discoidin domain receptor 1 (DDR1) to assemble with a 14-3-3-Beclin-1-Akt1 protein complex, which mediates prosurvival Akt and mTOR signaling for regulating autophagy-associated glioblastoma cell sensitivity to therapy. [ABSTRACT FROM AUTHOR]

Numerous natural substances have anti-cancer properties. Especially indigenous people use aqueous plant extracts for tea or ointments including Dioscorea sansibarensis Pax to treat various diseases. The aim of this study was to evaluate the cytotoxic and radiosensitizing potential of aqueous extracts from Dioscorea sansibarensis Pax collected from Kenya in a panel of HPV-negative and -positive head and neck squamous cell carcinoma (HNSCC) cells grown in three-dimensional laminin-rich extracellular matrix (3D lrECM). The results show cytotoxicity, radiosensitization and increased levels of residual double strand breaks (DBS) by Dioscorea sansibarensis Pax extracts in HPV-negative and -positive HNSCC models in a concentration- and cell model-dependent manner. Application of ROS scavengers indicated an association between ROS-induced DSB and radiosensitization through Dioscorea sansibarensis Pax pretreatment. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based characterization of Dioscorea sansibarensis Pax identified the main components of the extract including camptothecin. Overall, Dioscorea sansibarensis Pax aqueous extracts alone and in combination with X-ray irradiation showed effective anticancer properties, which are worthy of further mechanistic investigation. [Display omitted] • D. sansibarensis Pax extracts have cytotoxic and radiosensitising potential in HNSCC cell models. • D. sansibarensis -mediated radiosensitization associates with increased ROS and DSB levels. • HPLC-QTOF MS/MS reveals 108 compounds including camptothecin. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Integrin-mediated adhesion to extracellular matrix (ECM) contributes to the regulation of the cellular radiation response in various tumor entities. To evaluate whether targeting of β1 integrin enhances the radiosensitivity of head and neck SCC cell lines (HNSCC) was assessed using either inhibitory anti-β1 integrin antibodies or specific β1 integrin small interfering RNA (siRNA). Materials and methods: The HNSCC cell lines FaDu, UTSCC15 and UTSCC14 were used. Upon β1 integrin inhibition, colony formation, proliferation, DNA double strand breaks, adhesion, and migration as well as protein expression and phosphorylation of integrin downstream targets like Focal Adhesion Kinase and AKT were determined. Results: We found that siRNA- and antibody-mediated targeting of β1 integrin result in a dose- and cell line-dependent radiosensitization that was accompanied by a decreased cell proliferation and an increased number of radiogenic DNA double strand breaks. Analysis of signal transduction events revealed a dephosphorylation of focal adhesion proteins, prevention of radiation-induced phosphorylation of pro-survival protein kinases and impaired cell adhesion and migration upon blocking of β1 integrins. Conclusions: Our data suggest that β1 integrin critically contributes to the cellular radioresistance of HNSCC. Further studies are warranted to evaluate whether targeting β1 integrin emerges as novel approach to improve radiotherapy patients’ outcome. [Copyright &y& Elsevier]

Abstract: Introduction: Oral mucositis is a severe side effect of radio(chemo)therapy for head and neck tumors, for which β1 integrins have been proposed as potential therapeutic targets. The present study was initiated to determine the effect of selective inhibition of β1 integrin on the oral epithelial radiation response. Materials and methods: Daily fractionated irradiation was given with 5×3Gy/week over 1 or 2weeks with/without the β1 integrin-inhibiting monoclonal antibody AIIB2 or an IgG control. Each protocol was terminated by graded test doses to generate full dose–effect curves for mucosal ulceration. The same technique was used for single dose irradiation. Results: Combined single dose irradiation plus AIIB2 resulted in a significant decrease of the ED50 compared to irradiation alone or control IgG. No effect of AIIB2 was found with fractionated irradiation over 1week. With 2weeks of fractionation, AIIB2 induced a significant increase in the ED50 for the terminating test irradiation when administered in week 2. The time course of the response was largely unaffected by β1 integrin inhibition. Conclusions: A reduction of mucosal reactions by β1 integrin inhibition later in a course of fractionation was observed, i.e. when epithelial repopulation processes were active. Further mechanistic studies are required. [Copyright &y& Elsevier]

Phosphoinositide 3-kinase (PI3K)-α represents a key intracellular signal transducer involved in the regulation of key cell functions such as cell survival and proliferation. Excessive activation of PI3Kα is considered one of the major determinants of cancer therapy resistance. Despite preclinical and clinical evaluation of PI3Kα inhibitors in various tumor entities, including head and neck squamous cell carcinoma (HNSCC), it remains elusive how conventional radiochemotherapy can be enhanced by concurrent PI3K inhibitors and how PI3K deactivation mechanistically exerts its effects. Here, we investigated the radiochemosensitizing potential and adaptation mechanisms of four PI3K inhibitors, Alpelisib, Copanlisib, AZD8186, and Idelalisib in eight HNSCC models grown under physiological, three-dimensional matrix conditions. We demonstrate that Alpelisib, Copanlisib and AZD8186 but not Idelalisib enhance radio- and radiochemosensitivity in the majority of HNSCC cell models (= responders) in a manner independent of PIK3CA mutation status. However, Alpelisib promotes MAPK signaling in non-responders compared to responders without profound impact on Akt, NFκB, TGFβ, JAK/STAT signaling and DNA repair. Bioinformatic analyses identified unique gene mutations associated with extracellular matrix to be more frequent in non-responder cell models than in responders. Finally, we demonstrate that targeting of the cell adhesion molecule β1 integrin on top of Alpelisib sensitizes non-responders to radiochemotherapy. Taken together, our study demonstrates the sensitizing potential of Alpelisib and other PI3K inhibitors in HNSCC models and uncovers a novel β1 integrin-dependent mechanism that may prove useful in overcoming resistance to PI3K inhibitors. • PI3Kα inhibition by Alpelisib sensitizes HNSCC cells to radio(chemo)therapy. • Alpelisib regulates MAPK differentially in responder and non-responder HNSCC cells. • ECM-associated gene mutations are more frequent in HNSCC non-responder cells. • β1 integrin blockade helps to overcome resistance to PI3K inhibitor in HNSCC cells. [ABSTRACT FROM AUTHOR]

Background and purpose Simultaneous targeting of β1 integrin receptor and epidermal growth factor receptor (EGFR) showed higher level of radiosensitization in head and neck cancers than monotherapies. As EGFR inhibition is similarly performed in colorectal cancer (CRC), we investigated the radiosensitizing and anti-invasive potential of β1-integrin/EGFR inhibition in CRC cell lines grown in more physiological three-dimensional (3D) matrix-based cell cultures. Materials and methods DLD-1 and HT-29 cells were used for 3D-colony formation, invasion and proliferation assays and Western blotting. β1 integrin, focal adhesion kinase and EGFR were inhibited by AIIB2, TAE226 and Cetuximab, respectively. KRAS and BRAF knockdown were accomplished using small-interfering RNA technology. Single doses of X-rays ranged from 2 Gy to 6 Gy and 5-fluorouracil (5-FU) concentration was 10 μM. Results Neither β1-integrin/EGFR inhibition nor KRAS or BRAF depletion nor 5-FU significantly modified CRC cell radiosensitivity. Cetuximab, AIIB2 and Cetuximab/AIIB2 differentially modulated MAPK, JNK and AKT phosphorylation. AIIB2 and TAE226 significantly decreased cell invasion. Conclusions Our data show inefficiency of Cetuximab and AIIB2 on top of radiochemotherapy. The functions of KRAS and BRAF in therapy resistance remain unanswered and warrant further preclinical molecular-driven investigations. One promising approach might be β1 integrin targeting for reducing metastatic CRC cell spread. [ABSTRACT FROM AUTHOR]

Numerous factors determine the current poor prognosis of pancreatic ductal adenocarcinoma (PDAC). One of the greatest challenges to overcome is treatment resistance. Among a large repertoire of intrinsic resistance mechanisms, integrin-mediated cell adhesion to extracellular matrix (ECM) has been identified to be fundamental. Coalesced in focal adhesion complexes, integrins, receptor tyrosine kinases, protein kinases and adapter proteins mediate prosurvival signaling. Four and a half LIM domains protein 2 (FHL2) is one of these adapter proteins, which operates through protein–protein interactions and shows tumor-specific expression. Based on this, we investigated FHL2 expression in PDAC specimens and three-dimensionally grown cell lines and how FHL2 mechanistically contributes to cell survival, cell cycling and radiation resistance. PDAC exhibited a significantly increased and heterogeneous FHL2 expression. Upon FHL2 depletion, pancreatic cancer cell lines showed significantly decreased cell survival, proliferation and radioresistance as well as enhanced apoptosis and MEK/ERK signaling and cyclin D1, E, A and B1 expression were strongly induced. Targeting of FHL2 and MEK1 was similarly effective than FHL2 depletion alone, suggesting MEK1 as a downstream signaling mediator of FHL2. Taken together, our results provide evidence for the importance of the focal adhesion protein FHL2 in pancreatic cancer cell survival, proliferation and radiosensitivity. [ABSTRACT FROM AUTHOR]

Integrin cell adhesion molecules play a crucial role in tumor cell resistance to radio- and chemotherapy and are therefore considered attractive targets for cancer therapy. Here, we assessed the role of β1 integrin-interacting α integrin subunits in more physiological three-dimensional extracellular matrix grown head and neck squamous cell carcinoma (HNSCC) cell cultures for evaluating cytotoxic and radiosensitizing potential. α2, α3, α5 and α6 integrins, which are overexpressed in HNSCC according to Oncomine database analysis, were coprecipitated with β1 integrin. More potently than α2, α5 or α6 integrin inhibition, siRNA-based α3 integrin targeting resulted in reduced clonogenic cell survival, induced apoptosis and enhanced radiosensitivity. These events were associated with diminished phosphorylation of Akt, Cortactin and Paxillin. Cell line-dependently, simultaneous α3 and β1 integrin inhibition led to higher cytotoxicity and radiosensitization than α3 integrin blocking alone. Stable overexpression of wild-type and constitutively active forms of the integrin signaling mediator focal adhesion kinase (FAK) revealed FAK as a key determinant of α3 integrin depletion-mediated radiosensitization. Our findings show that α3 integrin is essentially involved in HNSCC cell radioresistance and critical for a modified cellular radiosensitivity along with β1 integrins. [ABSTRACT FROM AUTHOR]

Abstract: Background and purpose: Inhibition of histone deacetylases (HDACs) has preclinically and clinically shown promise to overcome radio- and chemoresistance of tumor cells. NDACI054 is a novel HDAC inhibitor, which has been evaluated here for its effects on cell survival and radiosensitization of human tumor cell lines from different origins cultured under more physiological three-dimensional (3D), extracellular matrix (ECM)-based conditions. Material and methods: A549 lung, DLD-1 colorectal, MiaPaCa2 pancreatic and UT-SCC15 head and neck squamous cell carcinoma cells were treated with increasing NDACI054 concentrations (0–50nM, 24h) either alone or in combination with X-rays (single dose, 0–6Gy). Subsequently, 3D clonogenic cell survival, HDAC activity, histone H3 acetylation, apoptosis, residual DNA damage (γH2AX/p53BP1 foci assay 24h post irradiation) and phosphorylation kinetics of Ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), Caspase-3 and Poly(ADP-ribose)-Polymerase 1 (PARP 1) cleavage were analyzed. Results: NDACI054 potently decreased HDAC activity with concomitant increase in acetyl-histone H3 levels, mediated significant cytotoxicity and radiosensitization. These effects were accompanied by a significant increase of residual γH2AX/p53BP1-positive foci, slightly elevated levels of Caspase-3 and PARP 1 cleavage but no induction of apoptosis. Conclusions: Our data show potent antisurvival and radiosensitizing effects of the novel HDAC inhibitor NDACI054 encouraging further preclinical examinations on this compound for future clinical use. [Copyright &y& Elsevier]

Abstract: Background and purpose: In the present study, we aimed to investigate the effect of single and double knockdown of the inhibitor of apoptosis proteins (IAP) Survivin and X-linked IAP (XIAP) on three-dimensional (3D) clonogenic survival, migration capacity and underlying signaling pathways. Materials and methods: Colorectal cancer cell lines (HCT-15, SW48, SW480, SW620) were subjected to siRNA-mediated single or Survivin/XIAP double knockdown followed by 3D colony forming assays, cell cycle analysis, Caspase activity assays, migration assays, matrigel transmigration assays and Western blotting (Survivin, XIAP, Focal adhesion kinase (FAK), p-FAK Y397, Akt1, p-Akt1 S473, Extracellular signal-regulated kinase (ERK1/2), p-ERK1/2 T202/Y204, Glycogen synthase kinase (GSK)3β, p-GSK3β S9, nuclear factor (NF)-κB p65). Results: While basal cell survival was altered cell line-dependently, Survivin or XIAP single and Survivin/XIAP double knockdown enhanced cellular radiosensitivity of all tested cancer cell lines grown in 3D. Particularly double knockdown conditions revealed accumulation of cells in G2/M, increased subG1 fraction, elevated Caspase 3/7 activity, and reduced migration. Intracellular signaling showed dephosphorylation of FAK and Akt1 upon Survivin and/or Survivin/XIAP silencing. Conclusions: Our results strengthen the notion of Survivin and XIAP to act as radiation resistance factors and further indicate that these apoptosis-regulating proteins are also functioning in cell cycling and cell migration. [Copyright &y& Elsevier]

Purpose: Focal adhesion kinase (FAK), a main regulator of integrin signaling and cell migration, is frequently overexpressed and hyperphosphorylated in human head-and-neck squamous cell carcinoma (HNSCC). We have previously shown that pharmacologic FAK inhibition leads to radiosensitization of 3-dimensionally grown HNSCC cell lines. To further evaluate the role of FAK in radioresistance and as a potential cancer target, we examined FAK and FAK downstream signaling in HNSCC cell lines grown in more physiologic extracellular matrix-based 3-dimensional cell cultures. Methods and Materials: Seven HNSCC cell lines were grown in 3-dimensional extracellular matrix and the clonogenic radiation survival, expression, and phosphorylation of FAK, paxillin, Akt1, extracellular signal-regulated kinase (ERK)1/2, and MEK1/2 were analyzed after siRNA-mediated knockdown of FAK, Akt1, MEK1, FAK+Akt1, or FAK+MEK1 compared with controls or stable overexpression of FAK. The role of MEK1/2 for clonogenic survival and signaling was investigated using the MEK inhibitor U0126 with or without irradiation. Results: FAK knockdown moderately or significantly enhanced the cellular radiosensitivity of 3-dimensionally grown HNSCC cells. The FAK downstream targets paxillin, Akt1, and ERK1/2 were substantially dephosphorylated under FAK depletion. FAK overexpression, in contrast, increased radiation survival and paxillin, Akt1, and ERK1/2 phosphorylation. The degree of radiosensitization upon Akt1, ERK1/2, or MEK1 depletion or U0126 was superimposable to FAK knockdown. Combination knockdown conditions (ie, Akt1/FAK, MEK1/FAK, or U0126/FAK) failed to provide additional radiosensitization. Conclusions: Our data provide further evidence for FAK as important determinant of radiation survival, which acts in the same signaling axis as Akt1 and ERK1/2. These data strongly support our hypothesis that FAK is a relevant molecular target for HNSCC radiotherapy. [ABSTRACT FROM AUTHOR]

Abstract: Background and purpose: Focal adhesion kinase (FAK), a main player in integrin signaling and survival, is frequently overexpressed in human cancers and therefore postulated as potential target in cancer therapy. The aim of this study was to evaluate the radiosensitizing potential of the FAK inhibitor TAE226 in three-dimensional (3D) tumor cell cultures. Materials and methods: Head and neck squamous cell carcinoma (HNSCC) cells (FaDu, UT-SCC15, UT-SCC45), lung cancer cells (A549), colorectal carcinoma cells (DLD-1, HCT-116) and pancreatic tumor cells (MiaPaCa2, Panc1) were treated with different concentrations of TAE226 (0–1μm; 1 or 24h) without or in combination with irradiation (0–6Gy, X-ray, single dose). Subsequently, 3D clonogenic survival assays (laminin-rich extracellular matrix) and Western blotting (expression/phosphorylation, e.g. FAK, Akt, ERK1/2) were performed. Results: All investigated 3D cell cultures showed a dose-dependent reduction in clonogenic survival by TAE226. Intriguingly, TAE226 only significantly radiosensitized 3D HNSCC cell cultures accompanied by a pronounced dephosphorylation of FAK, Akt and ERK1/2. Conclusions: Our data demonstrate TAE226 as potent FAK inhibitor that enhances the cellular radiosensitivity particularly of HNSCC cells grown in a 3D cell culture model. Future in vitro and in vivo investigations will clarify, to which extent this approach might be clinically relevant for radiotherapy of HNSCC. [Copyright &y& Elsevier]

Abstract: Background and purpose: Resistance of pancreatic ductal adenocarcinoma (PDAC) to chemo- and radiotherapy is a major obstacle. The integral membrane protein Caveolin-1 (Cav-1) has been suggested as a potent target in human pancreatic carcinoma cells. Materials and methods: Human pancreatic tumor cells were examined in a three-dimensional (3D) cell culture model with regard to clonogenic survival, apoptosis, radiogenic DNA-double strand breaks and protein expression and phosphorylation under siRNA-mediated knockdown of Cav-1 without and in combination with irradiation (X-rays, 0–6Gy). Immunohistochemistry was used to assess Cav-1 expression in biopsies from patients with PDAC. Results: Tumor cells in PDAC showed significantly higher Cav-1 expression relative to tumor stroma. Cav-1 knockdown significantly reduced β1 integrin expression and Akt phosphorylation, induced Caspase 3- and Caspase 8-dependent apoptosis and enhanced the radiosensitivity of 3D cell cultures. While cell cycling and Cav-1 promoter activity remained stable, Cav-1 knockdown-induced radiosensitization correlated with elevated numbers of residual DNA-double strand breaks. Conclusions: Our data strongly support the concept of Cav-1 as a potent target in pancreatic carcinoma cells due to radiosensitization and Cav-1 overexpression in tumor cells of PDAC. 3D cell cultures are powerful and useful tools for the testing of novel targeting strategies to optimize conventional radio- and chemotherapy regimes for PDAC. [Copyright &y& Elsevier]

Abstract: Purpose: Cancer treatment by conventional radiotherapy is limited by normal tissue side-effects. Fibroblasts as “non-target” stromal cell type are considered as strong promoter of tumor growth and for developing a therapy resistant phenotype. Regarding application of novel molecular therapeutics combined with radiotherapy, evaluation of a specific targeted molecule in both tumor and normal cells is mandatory for efficacy and tolerability assessment. Previous work showed integrin-linked kinase (ILK), a mediator of β-integrin signals and putative phosphorylator of AKT, as potent anti-survival regulator in human cancer cell lines. Materials and methods: To evaluate the role of ILK in normal fibroblast survival, ILK-wild-type (ILKfl/fl), ILK−/− and ILKN-terminal and ILKC-terminal domain expressing fibroblasts were irradiated with X-rays on different substrata or in three-dimensional laminin-rich extracellular matrix (lrECM). Results: On control substrata, ILK-deficient and ILK-mutant fibroblasts showed significant increase in radiation survival relative to ILK-wild-type cells. This effect was compensated by growth on ECM proteins and in 3D lrECM. ILK regulated AKT activity in a phosphatidylinositol-3 kinase (PI3K)-dependent manner. Upon PI3K inhibition, only ILK-wild-type fibroblasts showed significant radiosensitization. Conclusions: These findings obtained in 3D cell cultures suggest ILK to be dispensable for the radiation survival response of normal fibroblasts. However, targeting the PI3K/AKT signaling axis pharmacologically might be critical for survival of normal fibroblasts exposed to ionizing radiation. [Copyright &y& Elsevier]

Abstract: Background and purpose: Integrin-linked kinase (ILK) functions in cooperative integrin-growth factor receptor-mediated signaling to control cell survival and proliferation. The effect of tyrosine kinase (tk) inhibition of the epidermal growth factor receptor (EGFR) on radiation survival and growth was evaluated in human FaDu squamous cell carcinoma cells expressing different forms of ILK. Material and methods: ILK-wild-type (wk) and -hyperactive kinase (hk) transfected cells were grown on fibronectin (Fn) under serum presence or depletion, irradiated (0–6Gy) and/or treated with the EGFR-tk inhibitor BIBX1382BS. Results: ILK-wk and -hk transfectants showed significant radiosensitization compared to vector control cells. Antisurvival and antiproliferative effects of EGFR-tk inhibition plus/minus irradiation were counteracted by adhesion to Fn relative to the control substratum, poly-l-lysine. Similar to vector controls, ILK transfectants exhibited a strong decrease in cell proliferation but no enhanced radiation sensitivity after EGFR-tk inhibition. This decrease was accompanied by changes in cyclin D1 and phosphorylated MAPK persisting to day 10 following transient drug exposure. Conclusions: Our data demonstrate a prosurvival role of adhesion and an antisurvival role of ILK upon irradiation. Inhibition of EGFR-tk using BIBX1382BS does not affect the intrinsic cellular radiosensitivity of cells grown on fibronectin. Thus, simultaneous targeting of adhesion and growth factor receptor-mediated signaling might potently improve anticancer strategies. [Copyright &y& Elsevier]

Abstract: Background and purpose: Cell adhesion-mediated radioresistance is a common phenomenon particularly relevant in tumor cells, which might hamper anticancer therapies. To analyze the role of adhesion-mediating β1-integrins, stably transfected functional β1A-integrin-expressing GD25β1A and GD25β1B cells, which express mutant β1B-integrins, were compared in terms of radiation survival and β1-integrin signaling. Materials and methods: Cells grown on fibronectin, collagen-III, laminin, vitronectin, anti-β1-integrin-IgG (β1-IgG) or poly-l-lysine were irradiated with 0–6Gy in presence or absence of growth factors or inhibitors for phosphatidylinositol-3 kinase (PI3K), i.e. Ly294002 and wortmannin. In addition to colony formation, protein kinase B/Akt (PKB/Akt) kinase activity, focal adhesion kinase (FAK), p130Cas, paxillin and c-Jun N2-terminal kinase (JNK) expression and phosphorylation were analyzed by Western blot technique. Results: Adhesion of GD25β1A cells to extracellular matrix proteins or β1-IgG resulted in growth factor-independent radiation survival. In contrast, serum starved GD25β1B cells showed a significant (P<0.01) reduction in radiation survival on all substrates. PI3K inhibition moderately or strongly radiosensitized GD25β1A or GD25β1B cells, respectively. The pro-survival effects detected in serum starved GD25β1A cells were due to direct, PI3K-mediated stimulation of PKB/Akt activity by β1-integrins and induced p130Cas and paxillin phosphorylation. Phosphorylated p130Cas and paxillin subsequently prevented activation of cell death-regulating JNK. Conclusions: The data show that β1-integrin-mediated signaling through the cytoplasmic integrin domains is critical for efficient pro-survival regulation after irradiation. Profound knowledge of the underlying mechanisms of integrin-mediated cellular radioresistance could foster the design of new molecular-targeted anticancer therapies. [Copyright &y& Elsevier]

Purpose: Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. Methods and Materials: Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks (γH2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg, β1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. Results: Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the β1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. Conclusions: These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy. [ABSTRACT FROM AUTHOR]