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As demonstrated recently, ionizing radiation (IR) can mediate phosphorylation of DNA-PKcs in human tumor cells through stimulation of the PI3K/Akt pathway. It is also known that DNA-PKcs directly interacts the X-ray repair cross-complementing group 1 protein (XRCC1) involved in base excision repair (BER). Therefore, in the present study we investigated the role of PI3K/Akt activity and DNA-PKcs on XRCC1 expression/stabilization. In contrast to the DNA-PKcs-deficient glioblastoma cell line MO59J, the DNA-PKcs-proficient counterpart MO59K as well as human lung adenocarcinoma A549 cells presented a high basal level of XRCC1 expression. Radiation doses of 3-12Gy did not stimulate a further enhanced expression of XRCC1 in DNA-PKcs-proficient cells (MO59K and A549) within 180min post-irradiation. However, a marked induction of XRCC1 expression was apparent in DNA-PKcs-deficient MO59J cells. Targeting of DNA-PKcs as well as PI3K/Akt pathway by specific kinase inhibitors and/or siRNA reduced basal XRCC1 expression in un-irradiated DNA-PKcs-proficient cells to the level observed in DNA-PKcs-deficient cells. Reduction of basal expression of XRCC1 by XRCC1-siRNA, AKT-siRNA as well as DNA-PKcs inhibitor facilitated IR-induced XRCC1 expression. XRCC1 expression induced by irradiation, however, was independent of PI3K/Akt signaling, but dependent of MAPK-ERK1/2. By immuno-precipitation experiments and confocal microscopy a complex formation of XRCC1 and DNA-PKcs was shown. Applying gamma-H2AX foci analysis it was shown that basal expression of XRCC1 is important for the repair of IR-induced DNA-double strand breaks (DNA-DSBs). These data indicate that IR-induced XRCC1 expression is dependent on the expression level of DNA-PKcs and basal activity status of PI3K/Akt signaling. Likewise, potential of IR-induced XRCC1 expression depends on its basal expression level.

Over the last decade evidence has accumulated indicating that cell membrane-bound growth factor receptor of the erbB family and especially the epidermal growth factor receptor EGFR (erbB1) mediates resistance of tumor cells to both chemo- and radiotherapy when mutated or overexpressed. More recently a novel link between EGFR signaling pathways and DNA repair mechanisms, especially non-homologous end joining (NHEJ) repair could be demonstrated. The following review summarizes the current knowledge on the role of EGFR and its downstream signaling pathways in the regulation of cellular radiation response and DNA repair.The novel findings on radiation-induced EGFR-signaling and its involvement in regulating DNA-double strand break repair need further investigations of the detailed mechanisms involved. The results to be obtained may not only improve our knowledge on basic mechanisms of radiation sensitivity/resistance but also will promote translational approaches to test new strategies for clinically applicable molecular targeting.

Non-small cell lung cancer (NSCLC) cells expressing K-RAS mutation are resistant to anti-EGFR antibody cetuximab. Oncogenic Ras regulates basal effector PI3K/Akt and MAPK/ERK pathways. In this study, we investigated impact of K-RAS activity independent of its mutational status on targetibility of EGFR as well as the PI3K/Akt and MAPK/ERK pathways. A panel of five NSCLC cells (A549, H460, H661, SK-MES-1, HTB-182) and five head and neck squamous carcinoma (HNSCC) cells (SAS, FaDu, UT-SCC-5, UT-SCC-15, UT-SCC-5R (UT-SCC-5R are UT5 cells acquired resistance to cetuximab) were studied. K-Ras was constitutively activate not only in K-RAS mutated (K-RASmut) A549 and H460 cells, but also in K-RAS wild-type (K-RASwt) SAS and UT-SCC-5R cells. Enhanced K-Ras-GTP either endogenously or following overexpression of K-RAS(V12) was associated with significant shortening of population doubling time and increased clonogenic activity of tu-mor cells from both origin. Constitutive activation of K-Ras independent of K-RAS muta-tional status led to desensitization of both K-RASmut and K-RASwt cells to anti-EGFR an-tibody cetuximab and EGFR-tyrosine kinase inhibitors (TKI) erlotinib and AG1478, tested by standard clonogenic assay. Analyzing EGFR downstream pathways revealed that lack of response to the EGFR-TK inhibitors is through activation of the PI3K/Akt pathway. Thus, inhibition of PI3K was supposed to be an efficient approach to block clonogenic activity. However, PI3K inhibitor completely blocked clonogenic activity only in cells expressing low level of K-RASwt from both origins. In K-RASmut NSCLC cells or in HNSCC cells with overexpression of K-RASwt a partial response to PI-103 was observed. Analyzing pathway activity revealed that PI-103 does not inhibit Akt activity after 24 h pre-treatment while up to 4 h after post-treatment with PI-103, phosphorylation of Akt and Akt substrates (PRAS40 and GSK3αβ) are completely blocked. Lack of effect of PI103 at 24 h post-treatment was shown to be through K-RAS/MEK/ERK dependent reactivation of Akt. In line with this observation, targeting MEK by PD98059 significantly improved the anti-clonogenic activity of PI-103. Although, PD98059 blocked phosphorylation of ERK1/2, no marked effect was observed on clonogenic activity. Thus, EGFR or PI3K, but not MEK are appropriate target to inhibit clonogenic activity of tumor cells expressing low level of K-RASwt. In tumor cells with constitutive K-RAS activity, dual targeting of PI3K and MEK is an efficient approach to inhibit clonogenicity. Supported by grants from the Deutsche Forschungsgemeinschaft [Ro527/7-1 and SFB-773-TP B02] awarded to HPR, GRK 1302/2 (T11) awarded to MT/HPR. Citation Format: Mahmoud Toulany, Mohammad Saki, H. Peter Rodemann. Constitutive K-Ras activity independent of K-RAS mutational status leads to resistance to anti-EGFR molecular targeting approaches and MEK-dependent reactivation of Akt following PI3K inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A121.

Constitutive K-RAS activity in K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells leads to resistance to EGFR targeting strategies. Analyzing the K-RAS activity status revealed that a constitutive high K-RAS activity is observed not only in K-RASmut NSCLC cells but also in head and neck squamous cell carcinoma (HNSCC) cells presenting K-RAS wild-type (K-RASwt) overexpression. Similar to K-RAS-mutated NSCLC cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Overexpression of mutated K-RAS in HNSCC cells expressing low level of K-RASwt stimulated Akt1 phosphorylation and increased clonogenic activity. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in both EGFR-dependent and EGFR-independent Akt1 activity. Short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in repression of Akt1 activation, whereas long-term treatment (24 h) with inhibitors led to reactivation of Akt1 and stimulated clonogenicity. Akt1 re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, inhibition of clonogenicity in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by the combination of PI3K and MAPK inhibitors. Supported by grants from the Deutsche Forschungsgemeinschaft [Ro527/5-1 and SFB-773-TP B02] and the Federal Ministry of Research and Education(BMBF grants 0258416, 03NUK006D) awarded to HPR as well as GRK 1302/2 (T11) awarded to MT/HPR. Citation Format: Mahmoud Toulany, H. Peter Rodemann. Crosstalk between ERK2 and Akt1 in tumor cells with constitutive K-RAS activity leads to the limited response to PI3K inhibition. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A48.

Purpose: It is known that blockage of epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K) activity enhances radiation sensitivity of human tumor cells presenting a K-RAS mutation. In the present study, we investigated whether impaired repair of DNA double-strand breaks (DSB) is responsible for the radiosensitizing effect of EGFR and PI3K inhibition in K-RAS mutated (K-RASmt) cells. Experimental Design: The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS (BIBX) on cellular radiosensitivity was determined in K-RASmt (A549) and K-RASwt (FaDu) cell lines by clonogenic survival assay. Radiation-induced phosphorylation of H2AX (Ser139), ATM (Ser1981), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr2609) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of γH2AX foci and frequency of micronuclei. Results: BIBX reduced clonogenic survival of K-RASmt-A549 cells, but not of K-RASwt-FaDu cells, after single-dose irradiation. Analysis of the radiation-induced H2AX phosphorylation revealed that BIBX, as well as the PI3K inhibitor LY294002, leads to a marked reduction of P-H2AX in K-RASmt-A549 and MDA-MB-231 cells, but not in K-RASwt-FaDu and HH4ded cells. Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr2609 was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection. However, neither in K-RASmt nor in K-RASwt cells the inhibitors did affect radiation-induced ATM phosphorylation. As a consequence of inhibitor treatment, a significant enhancement of both residual DSBs and frequency of micronuclei was apparent only in A549 but not in FaDu cells following radiation. Conclusion: Targeting of the EGFR-dependent PI3K-AKT pathway in K-RAS-mutated A549 cells significantly affects postradiation survival by affecting the activation of DNA-PKcs, resulting in a decreased DSB repair capacity.

Background and purpose To investigate the cellular and molecular consequences of antagonizing radiation-induced EGFR-activation in vitro. Patients and methods The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS on radiation sensitivity was determined after single- and fractionated-dose irradiation in human cell lines of bronchial carcinoma (A549), breast adeno-carcinoma (MDA-MB-231), pharyngeal squamous-cell carcinoma (FaDu), squamous-cell carcinoma of cervix (HTB-35) as well as normal (HSF-7) and transformed (HH4-DED) human skin fibroblasts. Applying immuno-precipitation and western blotting pattern of radiation-dependent activation of different components of EGFR-signaling after pre-treatment with and without BIBX1382BS or other tyrosine kinase inhibitors was analyzed. Results Autophosphorylation of EGFR which occurred 1–5 min after irradiation (IR, 2 Gy) or treatment with EGF (100 ng/ml) could be inhibited in all cells tested by pre-treatment with BIBX1382BS for 30 min. Combination of drug treatment with fractionated irradiation (4×2 Gy) led to a strong radiosensitizing effect in Ras-mutated A549 and MDA-MB-231 cells, but not in normal Ras presenting cell lines FaDu and HTB-35 or normal and transformed human skin fibroblasts. Both BIBX1382BS as well as the PI3 kinase inhibitor LY294002 led to a blockage (for A549 cells) or reduction (for FaDu cells) of radiation-induced P-AKT. In contrast to FaDu cells, treatment of A549 cells with LY294002 resulted in a significant decrease of post-irradiation survival of A549 cells. Furthermore, only in Ras-mutated cells, but not in normal Ras cells clonogenic survival and phosphorylation of AKT was sensitive to pre-treatment with TGF-α-neutralizing antibody indicating an important role of TGF-α in regulating radiation-induced EGFR signaling. Conclusions Enhancement of radiation sensitivity by the specific EGFR-tyrosine kinase inhibitor BIBX1382BS is not generally achieved in human tumor cells, but depends most likely on the Ras genotype of the cell lines tested.

Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that has shown efficacy in metastatic colorectal cancer, head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC). Clinical data suggests that patients whom initially respond to cetuximab eventually acquire resistance, highlighting the importance of understanding resistant mechanisms for the development of better treatment regimes for cancer patients. Recent studies have identified a role for the TAM family of receptor tyrosine kinases (Tyro, Axl, and Mer) in tumor biology, and more specifically the Axl receptor as playing a key role in promoting tumor growth and metastasis. In the current study, we utilized NSCLC and HNSCC models of acquired resistance to cetuximab and found that Axl was overexpressed, activated, and highly associated with EGFR as compared parental controls. Further, using siRNA technology and novel Axl targeting agents (antibody and tyrosine kinase inhibitor), Axl was found to regulate proliferation, EGFR activation, and MAPK signaling in cetuximab resistant clones (CtxR). Additionally, Axl mRNA expression was regulated by EGFR induced MAPK activation in resistant cells. To investigate the role of Axl in in vivo models of acquired resistance to cetuximab, de novo acquired resistance was created by prolonged treatment of cetuximab sensitive (CtxS) tumor xenografts with cetuximab. Analysis of resultant CtxR tumors demonstrated that Axl was hyperactivated and highly associated with EGFR as compared to vehicle treated tumors. In addition, CtxR tumors expressing the highest levels of phosphorylated Axl also expressed hyperactivated EGFR, supporting the role of Axl in EGFR activation. Finally, to expand these findings to other members of the TAM family, Tyro, Axl, or Mer were stably overexpressed in CtxS parental NSCLC cells and tested for cetuximab response. The results of this experimentation indicated that stable overexpression of each TAM receptor rendered CtxS cells resistant to cetuximab therapy. Collectively, the studies presented herein identify the TAM family of receptors as key players in acquired resistance to cetuximab and provide rationale for the clinical evaluation of anti-TAM receptor therapeutics in the resistant setting. Citation Format: Toni Michel Brand, Mari Iida, Kelsey L. Corrigan, Neha Luthar, Megan Hornung, Mahmoud Toulany, Parkash Gill, Ravi Salgia, Deric L. Wheeler. The TAM family of receptor tyrosine kinases play a role in acquired resistance to cetuximab. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-215. doi:10.1158/1538-7445.AM2014-LB-215