The Intermediate Filament Synemin Regulates Non-Homologous End Joining in an ATM-Dependent Manner.

The treatment resistance of cancer cells is a multifaceted process in which DNA repair emerged as a potential therapeutic target. DNA repair is predominantly conducted by nuclear events; yet, how extra-nuclear cues impact the DNA damage response is largely unknown. Here, using a high-throughput RNAi-based screen in three-dimensionally-grown cell cultures of head and neck squamous cell carcinoma (HNSCC), we identified novel focal adhesion proteins controlling DNA repair, including the intermediate filament protein, synemin. We demonstrate that synemin critically regulates the DNA damage response by non-homologous end joining repair. Mechanistically, synemin forms a protein complex with DNA-PKcs through its C-terminal tail domain for determining DNA repair processes upstream of this enzyme in an ATM-dependent manner. Our study discovers a critical function of the intermediate filament protein, synemin in the DNA damage response, fundamentally supporting the concept of cytoarchitectural elements as co-regulators of nuclear events.

Whole exome sequencing identifies mTOR and KEAP1 as potential targets for radiosensitization of HNSCC cells refractory to EGFR and ß1 integrin inhibition.

Intrinsic and acquired resistances are major obstacles in cancer therapy. Genetic characterization is commonly used to identify predictive or prognostic biomarker signatures and potential cancer targets in samples from therapy-naïve patients. By far less common are such investigations to identify specific, predictive and/or prognostic gene signatures in patients or cancer cells refractory to a specific molecular-targeted intervention. This, however, might have a great value to foster the development of tailored, personalized cancer therapy. Based on our identification of a differential radiosensitization by single and combined ß1 integrin (AIIB2) and EGFR (Cetuximab) targeting in more physiological, three-dimensional head and neck squamous cell carcinoma (HNSCC) cell cultures, we performed comparative whole exome sequencing, phosphoproteome analyses and RNAi knockdown screens in responder and non-responder cell lines. We found a higher rate of gene mutations with putative protein-changing characteristics in non-responders and different mutational profiles of responders and non-responders. These profiles allow stratification of HNSCC patients and identification of potential targets to address treatment resistance. Consecutively, pharmacological inhibition of mTOR and KEAP1 effectively diminished non-responder insusceptibility to ß1 integrin and EGFR targeting for radiosensitization. Our data pinpoint the added value of genetic biomarker identification after selection for cancer subgroup responsiveness to targeted therapies.

Head and neck cancer cell radiosensitization upon dual targeting of c-Abl and beta1-integrin.

Integrin-mediated cell adhesion to extracellular matrix (ECM) critically contributes to cancer cell therapy resistance and DNA double strand break (DSB) repair. c-Abl tyrosine kinase has been linked to both of these processes. Based on our previous findings indicating c-Abl hyperphosphorylation on tyrosine (Y) 412 and threonine (T) 735 upon beta1-integrin inhibition, we hypothesized c-Abl tyrosine kinase as an important mediator of beta1-integrin signaling for radioresistance. In a panel of 8 cell lines from different solid cancer types grown in 3D laminin-rich ECM cultures, we targeted beta1 integrin with AIIB2 (mAb) and c-Abl with Imatinib with and without X-ray irradiation and subsequently examined clonogenic survival, residual DSBs, protein expression and phosphorylation. Single or combined treatment with AIIB2 and Imatinib resulted in cell line-dependent cytotoxicity. Intriguingly, we identified a subgroup of this cell line panel that responded with a higher degree of radiosensitization to AIIB2/Imatinib relative to both single treatments. In this subgroup, we observed a non-statistically significant trend between the radioresponse and phospho-c-Abl Y412. Mechanistically, impairment of DNA repair seems to be associated with radiosensitization upon AIIB2/Imatinib and AIIB2/Imatinib-related radiosensitization could be reduced by exogenous overexpression of either wildtype or constitutively active c-Abl forms relative to controls. Our data generated in more physiological 3D cancer cell culture models suggest c-Abl as further determinant of radioresistance and DNA repair downstream of beta1-integrin. For solid cancers, c-Abl phosphorylation status might be an indicator for reasonable Imatinib application as adjuvant for conventional radio(chemo)therapy.

Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by ß1 integrin/JNK co-targeting.

Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor ß1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with ß1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by ß1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a ß1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.

The cancer cell adhesion resistome: mechanisms, targeting and translational approaches.

Cell adhesion-mediated resistance limits the success of cancer therapies and is a great obstacle to overcome in the clinic. Since the 1990s, where it became clear that adhesion of tumor cells to the extracellular matrix is an important mediator of therapy resistance, a lot of work has been conducted to understand the fundamental underlying mechanisms and two paradigms were deduced: cell adhesion-mediated radioresistance (CAM-RR) and cell adhesion-mediated drug resistance (CAM-DR). Preclinical work has evidently demonstrated that targeting of integrins, adapter proteins and associated kinases comprising the cell adhesion resistome is a promising strategy to sensitize cancer cells to both radiotherapy and chemotherapy. Moreover, the cell adhesion resistome fundamentally contributes to adaptation mechanisms induced by radiochemotherapy as well as molecular drugs to secure a balanced homeostasis of cancer cells for survival and growth. Intriguingly, this phenomenon provides a basis for synthetic lethal targeted therapies simultaneously administered to standard radiochemotherapy. In this review, we summarize current knowledge about the cell adhesion resistome and highlight targeting strategies to override CAM-RR and CAM-DR.

BEMER Electromagnetic Field Therapy Reduces Cancer Cell Radioresistance by Enhanced ROS Formation and Induced DNA Damage.

Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of non-invasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF) to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radiosensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease.

Valproic acid modulates radiation-enhanced matrix metalloproteinase activity and invasion of breast cancer cells.

PURPOSE: To evaluate matrix metalloproteinase (MMP) activity and invasion after ionizing radiation (IR) exposure and to determine whether MMP could be epigenetically modulated by histone deacetylase (HDAC) inhibition. MATERIAL AND METHODS: Two human breast cancer cell lines (MDA-MB-231 and MCF-7) were cultured in monolayer (2D) and in laminin-rich extracellular matrix (3D). Invasion capability, collagenolytic and gelatinolytic activity, MMP and TIMP protein and mRNA expression and clonogenic survival were analyzed after IR exposure, with and without a HDAC inhibition treatment [1.5 mM valproic acid (VA) or 1 µM trichostatin-A (TSA)]. RESULTS: IR exposure resulted in cell line-dependent stimulation of invasion capacity. In contrast to MCF-7 cells, irradiated MDA-MB-231 showed significantly enhanced mRNA expression of mmp-1, mmp-3 and mmp-13 and of their regulators timp-1 and timp-2 relative to unirradiated controls. This translated into increased collagenolytic and gelatinolytic activity and could be reduced after valproic acid (VA) treatment. Additionally, VA also mitigated IR-enhanced mmp and timp mRNA expression as well as IR-increased invasion capability. Finally, our data confirm the radiosensitizing effect of VA. CONCLUSION: These results suggest that IR cell line-dependently induces upregulation of MMP mRNA expression, which appears to be mechanistically linked to a higher invasion capability that is modifiable by HDAC inhibition.

EGFR and ß1-integrin targeting differentially affect colorectal carcinoma cell radiosensitivity and invasion.

BACKGROUND AND PURPOSE: Simultaneous targeting of ß1 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 ß1-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. ß1 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 2Gy to 6Gy and 5-fluorouracil (5-FU) concentration was 10µM. RESULTS: Neither ß1-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 ß1 integrin targeting for reducing metastatic CRC cell spread.

Targeting of the EGFR/ß1 integrin connecting proteins PINCH1 and Nck2 radiosensitizes three-dimensional SCC cell cultures.

Epidermal growth factor receptor (EGFR) signaling plays an important role in tumor cell resistance to therapy. In addition to ligand binding, mutual and cooperative interactions of EGFR with integrin cell adhesion receptors critically influence proper downstream signaling through a number of bridging adapter proteins. In the present study, we analyzed the role of two of these adapter proteins, called PINCH1 and Nck2, for cellular radioresistance in combination with EGFR-targeting using the monoclonal antibody cetuximab. siRNA-mediated knockdown of PINCH1 or Nck2 resulted in enhanced radiosensitivity of 3D grown human squamous cell carcinoma cell lines FaDu (head and neck) and A431 (epidermis) comparable with effects seen after cetuximab treatment. Combination of knockdown and cetuximab did not result in additive nor synergistic effects regarding clonogenic radiation survival. Modifications in MAPK, Akt and FAK phosphorylation occurred upon cetuximab treatment as well as PINCH1 or Nck2 depletion. We further found this tumor cell radiosensitization to be due to attenuated repair of DNA double strand breaks and altered Rad50 and Nbs1 expression but without changes in other DNA repair proteins such as ATM, DNA-PK and Mre11. Our data suggest that the adaptor proteins PINCH1 and Nck2 critically contribute to cellular radioresistance and proper EGFR signaling in 3D lrECM grown human squamous cell carcinoma cells. Further investigations are warranted to identify the intracellular signaling network controlled by EGFR, PINCH1 and Nck2.

Simultaneous ß1 integrin-EGFR targeting and radiosensitization of human head and neck cancer.

BACKGROUND: Signaling from integrins and receptor tyrosine kinases (RTKs) contributes substantially to therapy resistance of malignant tumors. We investigated simultaneous ß1 integrin-epidermal growth factor receptor (EGFR) targeting plus radiotherapy in human head and neck squamous cell carcinomas (HNSCCs). METHODS: Ten HNSCC cell lines were grown in three-dimensional laminin-rich extracellular matrix cell cultures and two of them as tumor xenografts in nude mice (n = 12-16 per group). Targeting of ß1 integrin and EGFR with monoclonal inhibitory antibodies (AIIB2 and cetuximab, respectively) was combined with x-ray irradiation. Clonogenic survival, tumor growth, and tumor control (evaluated by Kaplan-Meier analysis), apoptosis, phosphoproteome (interactome, network betweeness centrality analysis), receptor expression (immunohistochemistry), and downstream signaling (western blotting) were assessed. Various mutants of the integrin signaling mediator focal adhesion kinase (FAK) were employed for mechanistic studies. All statistical tests were two-sided. RESULTS: Compared with ß1 integrin or EGFR single inhibition, combined ß1 integrin-EGFR targeting resulted in enhanced cytotoxicity and radiosensitization in eight out of 10 tested HNSCC cell lines, which responded with an FAK dephosphorylation after ß1 integrin inhibition. In vivo, simultaneous anti-ß1 integrin/anti-EGFR treatment and radiotherapy of UTSCC15 responder xenografts enabled better tumor control compared with anti-EGFR monotherapy and irradiation (hazard ratio [HR] = 6.9, 95% confidence interval [CI] = 1.6 to 30.9, P = .01), in contrast to the SAS nonresponder tumor model (HR = 0.9, 95% CI = 0.4 to 2.3, P = .83). Mechanistically, a protein complex consisting of FAK- and Erk1-mediated prosurvival signals for radiation resistance, which was effectively compromised by ß1 integrin and EGFR blocking. CONCLUSIONS: Concomitant targeting of ß1 integrin and EGFR seems a powerful and promising approach to overcome radioresistance of HNSCCs.

Enhanced radiosensitivity of head and neck squamous cell carcinoma cells by ß1 integrin inhibition.

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 ß1 integrin enhances the radiosensitivity of head and neck SCC cell lines (HNSCC) was assessed using either inhibitory anti-ß1 integrin antibodies or specific ß1 integrin small interfering RNA (siRNA). MATERIALS AND METHODS: The HNSCC cell lines FaDu, UTSCC15 and UTSCC14 were used. Upon ß1 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 ß1 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 ß1 integrins. CONCLUSIONS: Our data suggest that ß1 integrin critically contributes to the cellular radioresistance of HNSCC. Further studies are warranted to evaluate whether targeting ß1 integrin emerges as novel approach to improve radiotherapy patients' outcome.