Joseph N. Contessa

Stress and radiation-induced activation of multiple intracellular signaling pathways.

Abstract Dent, P., Yacoub, A., Contessa, J., Caron, R., Amorino, G., Valerie, K., Hagan, M. P., Grant, S. and Schmidt-Ullrich, R. Stress and Radiation-Induced Activation of Multiple Intracellular Signaling Pathways. Radiat. Res. 159, 283–300 (2003). Exposure of cells to a variety of stresses induces compensatory activations of multiple intracellular signaling pathways. These activations can play critical roles in controlling cell survival and repopulation effects in a stress-specific and cell type-dependent manner. Some stress-induced signaling pathways are those normally activated by mitogens such as the EGFR/RAS/PI3K-MAPK pathway. Other pathways activated by stresses such as ionizing radiation include those downstream of death receptors, including pro-caspases and the transcription factor NFKB. This review will attempt to describe some of the complex network of signals induced by ionizing radiation and other cellular stresses in animal cells, with particular attention to signaling by growth factor and death receptors. This includes radiation-induced signaling via the EGFR and IGFI-R to the PI3K, MAPK, JNK, and p38 pathways as well as FAS-R and TNF-R signaling to pro-caspases and NFKB. The roles of autocrine ligands in the responses of cells and bystander cells to radiation and cellular stresses will also be discussed. Based on the data currently available, it appears that radiation can simultaneously activate multiple signaling pathways in cells. Reactive oxygen and nitrogen species may play an important role in this process by inhibiting protein tyrosine phosphatase activity. The ability of radiation to activate signaling pathways may depend on the expression of growth factor receptors, autocrine factors, RAS mutation, and PTEN expression. In other words, just because pathway X is activated by radiation in one cell type does not mean that pathway X will be activated in a different cell type. Radiation-induced signaling through growth factor receptors such as the EGFR may provide radioprotective signals through multiple downstream pathways. In some cell types, enhanced basal signaling by proto-oncogenes such as RAS may provide a radioprotective signal. In many cell types, this may be through PI3K, in others potentially by NFKB or MAPK. Receptor signaling is often dependent on autocrine factors, and synthesis of autocrine factors will have an impact on the amount of radiation-induced pathway activity. For example, cells expressing TGFα and HB-EGF will generate protection primarily through EGFR. Heregulin and neuregulins will generate protective signals through ERBB4/ERBB3. The impact on radiation-induced signaling of other autocrine and paracrine ligands such as TGFβ and interleukin 6 is likely to be as complicated as described above for the ERBB receptors.

Radiation-induced proliferation of the human A431 squamous carcinoma cells is dependent on EGFR tyrosine phosphorylation

Accelerated cellular repopulation has been described as a response of tumors to fractionated irradiation in both normal tissue and tumor systems. To identify the mechanisms by which cells enhance their proliferative rate in response to clinically used doses of ionizing radiation (IR) we have studied human mammary and squamous carcinoma cells which are autocrine growth regulated by the epidermal growth factor receptor (EGFR) and its ligands, transforming growth factor-α and EGF. Both EGF and IR induced EGFR autophosphorylation, comparable levels of phospholipase Cγ activation as measured by inositol-1,4,5-triphosphate production, and as a consequence oscillations in cytosolic [Ca2+]. Activities of Raf-1 and mitogen-activated protein kinase (MAPK) were also stimulated by EGF and IR by Ca2+-dependent mechanisms. All these responses to EGF and IR were dependent upon activation of EGFR as judged by the use of the specific inhibitor of EGFR autophosphorylation, tyrphostin AG1478. Importantly, IR-induced proliferation of A431 cells was also inhibited by AG1478. This is the first report which demonstrates a link between IR-induced activation of proliferative signal transduction pathways and enhanced proliferation. We propose that accelerated repopulation of tumors whose growth is regulated by EGFR is initiated by an IR-induced EGFR activation mechanism that mimics the effects of growth factors.

Ionizing radiation activates Erb-B receptor dependent Akt and p70 S6 kinase signaling in carcinoma cells

In this study we have investigated the effects of low dose ionizing radiation (2 Gy) on p70 S6 kinase and Akt signaling with respect to Erb-B receptors in both the A431 squamous and the MDA-MB-231 mammary carcinoma cell lines. Ionizing radiation caused a 2–3-fold increase in p70 S6 kinase activity that was blocked pharmacologically using an EGFR inhibitor (AG1478) alone, or in combination with an Erb-B2 inhibitor (AG825). These results suggested that both EGFR and Erb-B2 receptors could initiate radiation-induced activation of p70 S6K. EGFR dependent Erb-B3 signaling also contributed to p70 S6 kinase activity through recruitment and activation of PI3K, which has been shown to regulate p70 S6 kinase activity. Furthermore, inhibition of the EGFR blocked IR stimulated increases in protein translation, a biologic consequence of p70 S6 kinase activation. We also report that ionizing radiation stimulated Akt activity that was partially independent of PI3K activity, but dependent on Erb-B2 function. Erb-B2 inhibition also correlated with enhanced apoptosis following IR exposure, suggesting an important role for Erb-B2 in cell survival. Together this work demonstrates that the Erb-B receptor tyrosine kinase network stimulates cytoprotective p70 S6 kinase and Akt activity in response to clinically relevant doses of ionizing radiation.

Dominant negative EGFR-CD533 and inhibition of MAPK modify JNK1 activation and enhance radiation toxicity of human mammary carcinoma cells.

Exposure of MDA-MB-231 human mammary carcinoma cells to an ionizing radiation dose of 2 Gy results in immediate activation and Tyr phosphorylation of the epidermal growth factor receptor (EGFR). Doxycycline induced expression of a dominant negative EGFR-CD533 mutant, lacking the COOH-terminal 533 amino acids, in MDA-TR15-EGFR-CD533 cells was used to characterize intracellular signaling responses following irradiation. Within 10 min, radiation exposure caused an immediate, transient activation of mitogen activated protein kinase (MAPK) which was completely blocked by expression of EGFR-CD533. The same radiation treatment also induced an immediate activation of the c-Jun-NH2-terminal kinase 1 (JNK1) pathway that was followed by an extended rise in kinase activity after 30 min. Expression of EGFR-CD533 did not block the immediate JNK1 response but completely inhibited the later activation. Treatment of MDA-TR15-EGFR-CD533 cells with the MEK1/2 inhibitor, PD98059, resulted in ∼70% inhibition of radiation-induced MAPK activity, and potentiated the radiation-induced increase of immediate JNK1 activation twofold. Inhibition of Ras farnesylation with a concomitant inhibition of Ras function completely blocked radiation-induced MAPK and JNK1 activation. Modulation of EGFR and MAPK functions also altered overall cellular responses of growth and apoptosis. Induction of EGFR-CD533 or treatment with PD98059 caused a 3 – 5-fold increase in radiation toxicity in a novel repeated radiation exposure growth assay by interfering with cell proliferation and potentiating apoptosis. In summary, this data demonstrates that both MAPK and JNK1 activation in response to radiation occur through EGFR-dependent and -independent mechanisms, and are mediated by signaling through Ras. Furthermore, we have demonstrated that radiation-induced activation of EGFR results in downstream activation of MAPK which may affect the radiosensitivity of carcinoma cells.

ERBB receptor tyrosine kinases and cellular radiation responses

Ionizing radiation induces in autocrine growth-regulated carcinoma and malignant glioma cells powerful cytoprotective responses that confer relative resistance to consecutive radiation exposures. Understanding the mechanisms of these responses should provide new molecular targets for tumor radiosensitization. ERBB and other receptor Tyr kinases have been identified as immediate early response gene products that are activated by radiation within minutes, as by their physiological growth factor ligands, and induce secondary stimulation of cytoplasmic protein kinase cascades. The simultaneous activation of all receptor Tyr kinases and nonreceptor Tyr kinases leads to complex cytoprotective responses including increased cell proliferation, reduced apoptosis and enhanced DNA repair. Since these responses contribute to cellular radioresistance, ERBB1, the most extensively studied ERBB receptor, is examined as a target for tumor cell radiosensitization. The three methods of ERBB1 inhibition include blockade of growth factor binding by monoclonal antibody against the ligand-binding domain, inhibition of the receptor Tyr kinase-mediating receptor activation, and overexpression of a dominant-negative epidermal growth factor receptor-CD533 that lacks the COOH-terminal 533 amino acids and forms nonfunctional heterodimeric complexes with wild-type receptors. All the three approaches enhance radiation toxicity in vitro and in vivo. The different mechanisms of inhibition have contributed to the understanding of cellular responses to radiation, vary in relative effectiveness and pose different challenges for translation.

Extended Survival and Prognostic Factors for Patients With ALK-Rearranged Non–Small-Cell Lung Cancer and Brain Metastasis

PURPOSE We performed a multi-institutional study to identify prognostic factors and determine outcomes for patients with ALK-rearranged non-small-cell lung cancer (NSCLC) and brain metastasis. PATIENTS AND METHODS A total of 90 patients with brain metastases from ALK-rearranged NSCLC were identified from six institutions; 84 of 90 patients received radiotherapy to the brain (stereotactic radiosurgery [SRS] or whole-brain radiotherapy [WBRT]), and 86 of 90 received tyrosine kinase inhibitor (TKI) therapy. Estimates for overall (OS) and intracranial progression-free survival were determined and clinical prognostic factors were identified by Cox proportional hazards modeling. RESULTS Median OS after development of brain metastases was 49.5 months (95% CI, 29.0 months to not reached), and median intracranial progression-free survival was 11.9 months (95% CI, 10.1 to 18.2 months). Forty-five percent of patients with follow-up had progressive brain metastases at death, and repeated interventions for brain metastases were common. Absence of extracranial metastases, Karnofsky performance score ≥ 90, and no history of TKIs before development of brain metastases were associated with improved survival (P = .003, < .001, and < .001, respectively), whereas a single brain metastasis or initial treatment with SRS versus WBRT were not (P = .633 and .666, respectively). Prognostic factors significant by multivariable analysis were used to describe four patient groups with 2-year OS estimates of 33%, 59%, 76%, and 100%, respectively (P < .001). CONCLUSION Patients with brain metastases from ALK-rearranged NSCLC treated with radiotherapy (SRS and/or WBRT) and TKIs have prolonged survival, suggesting that interventions to control intracranial disease are critical. The refinement of prognosis for this molecular subtype of NSCLC identifies a population of patients likely to benefit from first-line SRS, close CNS observation, and treatment of emergent CNS disease.

Inhibition of N-linked glycosylation disrupts receptor tyrosine kinase signaling in tumor cells.

Receptor tyrosine kinases (RTK) are therapeutic targets for the treatment of malignancy. However, tumor cells develop resistance to targeted therapies through the activation of parallel signaling cascades. Recent evidence has shown that redundant or compensatory survival signals responsible for resistance are initiated by nontargeted glycoprotein RTKs coexpressed by the cell. We hypothesized that disrupting specific functions of the posttranslational machinery of the secretory pathway would be an effective strategy to target both primary and redundant RTK signaling. Using the N-linked glycosylation inhibitor, tunicamycin, we show that expression levels of several RTKS (EGFR, ErbB2, ErbB3, and IGF-IR) are exquisitely sensitive to inhibition of N-linked glycosylation. Disrupting this synthetic process reduces both cellular protein levels and receptor activity in tumor cells through retention of the receptors in the endoplasmic reticulum/Golgi compartments. Using U251 glioma and BXPC3 pancreatic adenocarcinoma cell lines, two cell lines resistant to epidermal growth factor receptor-targeted therapies, we show that inhibiting N-linked glycosylation markedly reduces RTK signaling through Akt and radiosensitizes tumor cells. In comparison, experiments in nontransformed cells showed neither a reduction in RTK-dependent signaling nor an enhancement in radiosensitivity, suggesting the potential for a therapeutic ratio between tumors and normal tissues. This study provides evidence that enzymatic steps regulating N-linked glycosylation are novel targets for developing approaches to sensitize tumor cells to cytotoxic therapies.

EGFRvIII-mediated radioresistance through a strong cytoprotective response.

The constitutively active, truncated epidermal growth factor receptor EGFRvIII lacks the ability of EGF binding due to a deletion of the NH2-terminal domain. EGFRvIII confers increased tumorigenicity, is coexpressed with EGFR wild type (wt) in human carcinoma and malignant glioma cells when grown as xenografts, but is not expressed in vitro. The effects of EGFRvIII expression on cellular radiation responses were studied in Chinese hamster ovary (CHO) cells transfected with plasmids expressing EGFRvIII (CHO.EGFRvIII) or EGFRwt (CHO.EGFRwt). CHO cells expressing similar levels of either receptor were employed to define their roles in response to EGF and ionizing radiation. EGF activated EGFRwt with no effect on EGFRvIII. In contrast, a single radiation exposure of 2 Gy resulted in a 2.8- and 4.3-fold increase in Tyr phosphorylation of EGFRwt and EGFRvIII, respectively. Downstream consequences of this radiation-induced activation were examined by inhibiting EGFRwt and EGFRvIII with AG1478 (kinase inhibitor). The radiation-induced 8.5-fold activation of the pro-proliferative mitogen-activated protein kinase and the 3.2-fold stimulation of the antiapoptotic AKT/phosphatidylinositol-3-kinase pathways by EGFRvIII far exceeded that in CHO.EGFR wt cells. Thus, based on colony formation and apoptosis assays, EGFRvIII expression conferred a stronger cytoprotective response to radiation than EGFRwt, resulting in relative radioresistance. Therefore, disabling EGFRvIII in addition to EGFRwt needs to be considered in any therapeutic approach aimed at targeting EGFR for tumor cell radiosensitization.

Estimating Survival in Patients With Lung Cancer and Brain Metastases: An Update of the Graded Prognostic Assessment for Lung Cancer Using Molecular Markers (Lung-molGPA).

Importance Lung cancer is the leading cause of cancer-related mortality in the United States and worldwide. As systemic therapies improve, patients with lung cancer live longer and thus are at increased risk for brain metastases. Understanding how prognosis varies across this heterogeneous patient population is essential to individualize care and design future clinical trials. Objective To update the current Diagnosis-Specific Graded Prognostic Assessment (DS-GPA) for patients with non–small-cell lung cancer (NSCLC) and brain metastases. The DS-GPA is based on data from patients diagnosed between 1985 and 2005, and we set out to update it by incorporating more recently reported gene and molecular alteration data for patients with NSCLC and brain metastases. This new index is called the Lung-molGPA. Design, Setting, and Participants This is a multi-institutional retrospective database analysis of 2186 patients diagnosed between 2006 and 2014 with NSCLC and newly diagnosed brain metastases. The multivariable analyses took place between December 2015 and May 2016, and all prognostic factors were weighted for significance by hazard ratios. Significant factors were included in the updated Lung-molGPA prognostic index. Main Outcomes and Measures The main outcome was survival. Multiple Cox regression was used to select and weight prognostic factors in proportion to their hazard ratios. Log rank tests were used to compare adjacent classes and to compare overall survival for adenocarcinoma vs nonadenocarcinoma groups. Results The original DS-GPA was based on 4 factors found in 1833 patients with NSCLC and brain metastases diagnosed between 1985 and 2005: patient age, Karnofsky Performance Status, extracranial metastases, and number of brain metastases. The patients studied for the creation of the DS-GPA had a median survival of 7 months from the time of initial treatment of brain metastases. To design the updated Lung-molGPA, we analyzed data from 2186 patients from 2006 through 2014 with NSCLC and newly diagnosed brain metastases (1521 adenocarcinoma and 665 nonadenocarcinoma). Significant prognostic factors included the original 4 factors used in the DS-GPA index plus 2 new factors: EGFR and ALK alterations in patients with adenocarcinoma (mutation status was not routinely tested for nonadenocarcinoma). The overall median survival for the cohort in the present study was 12 months, and those with NSCLC-adenocarcinoma and Lung-molGPA scores of 3.5 to 4.0 had a median survival of nearly 4 years. Conclusions and Relevance In recent years, patient survival and physicians’ ability to predict survival in NSCLC with brain metastases has improved significantly. The updated Lung-molGPA incorporating gene alteration data into the DS-GPA is a user-friendly tool that may facilitate clinical decision making and appropriate stratification of future clinical trials.

Management of Brain Metastases in Tyrosine Kinase Inhibitor–Naïve Epidermal Growth Factor Receptor–Mutant Non–Small-Cell Lung Cancer: A Retrospective Multi-Institutional Analysis

Purpose Stereotactic radiosurgery (SRS), whole-brain radiotherapy (WBRT), and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are treatment options for brain metastases in patients with EGFR-mutant non-small-cell lung cancer (NSCLC). This multi-institutional analysis sought to determine the optimal management of patients with EGFR-mutant NSCLC who develop brain metastases and have not received EGFR-TKI. Materials and Methods A total of 351 patients from six institutions with EGFR-mutant NSCLC developed brain metastases and met inclusion criteria for the study. Exclusion criteria included prior EGFR-TKI use, EGFR-TKI resistance mutation, failure to receive EGFR-TKI after WBRT/SRS, or insufficient follow-up. Patients were treated with SRS followed by EGFR-TKI, WBRT followed by EGFR-TKI, or EGFR-TKI followed by SRS or WBRT at intracranial progression. Overall survival (OS) and intracranial progression-free survival were measured from the date of brain metastases. Results The median OS for the SRS (n = 100), WBRT (n = 120), and EGFR-TKI (n = 131) cohorts was 46, 30, and 25 months, respectively ( P < .001). On multivariable analysis, SRS versus EGFR-TKI, WBRT versus EGFR-TKI, age, performance status, EGFR exon 19 mutation, and absence of extracranial metastases were associated with improved OS. Although the SRS and EGFR-TKI cohorts shared similar prognostic features, the WBRT cohort was more likely to have a less favorable prognosis ( P = .001). Conclusion This multi-institutional analysis demonstrated that the use of upfront EGFR-TKI, and deferral of radiotherapy, is associated with inferior OS in patients with EGFR-mutant NSCLC who develop brain metastases. SRS followed by EGFR-TKI resulted in the longest OS and allowed patients to avoid the potential neurocognitive sequelae of WBRT. A prospective, multi-institutional randomized trial of SRS followed by EGFR-TKI versus EGFR-TKI followed by SRS at intracranial progression is urgently needed.