EGFR Inhibition in Glioma Cells Modulates Rho Signaling to Inhibit Cell Motility and Invasion and Cooperates with Temozolomide to Reduce Cell Growth

Enforced EGFR activation upon gene amplification and/or mutation is a common hallmark of malignant glioma. Small molecule EGFR tyrosine kinase inhibitors, such as erlotinib (Tarceva), have shown some activity in a subset of glioma patients in recent trials, although the reported data on the cellular basis of glioma cell responsiveness to these compounds have been contradictory. Here we have used a panel of human glioma cell lines, including cells with amplified or mutant EGFR, to further characterize the cellular effects of EGFR inhibition with erlotinib. Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects. Flow cytometric analyses confirm that EGFR inhibition does not induce apoptosis in glioma cells, leading to cell cycle arrest in G1. Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth. This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect. Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27kip1 levels and pRB hypophosphorylation. Interestingly, EGFR inhibition also perturbs Rho GTPase signaling and cellular morphology, leading to Rho/ROCK-dependent formation of actin stress fibres and the inhibition of glioma cell motility and invasion

Decreased aortic growth and middle aortic syndrome in patients with neuroblastoma after radiation therapy

Background: Long-term CT follow-up studies are required in pediatric patients who have received intraoperative radiation therapy (IORT) and external beam radiation therapy (EBRT) to assess vascular toxicities and to determine the exact complication rate. Objective: To analyze with CT the effects of radiation therapy (RT) on the growth of the aorta in neuroblastoma patients.Materials and methods Abdominal CT scans of 31 patients with intraabdominal neuroblastoma (stage II–IV), treated with RT (20 IORT±EBRT, 11 EBRT alone), were analyzed retrospectively. The diameter of the abdominal aorta was measured before and after RT. These data were compared to normal and predicted normal aortic diameters of children, according to the model of Fitzgerald, Donaldson and Poznanski (aortic diameter in centimeters = 0.844 + 0.0599 × age in years), and to the diameters of a control group of children who had not undergone RT. Statistical analyses for the primary aims were performed using the chi-squared test, t-test, Mann-Whitney test, nonparametric Wilcoxon matched-pairs test and analysis of variance for repeated measures. Clinical files and imaging studies were evaluated for signs of late vascular complications of neuroblastoma patients who had received RT. Results: The mean diameter before and after RT and the growth of the aorta were significantly lower than expected in patients with neuroblastoma (P<0.05 for each) and when compared to the growth in a control group with normal and nonirradiated aortas. Among the patients who had received RT, there was no difference due to the type of RT. Seven patients from the IORT±EBRT group developed vascular complications, which included hypertension (five), middle aortic syndrome (two), death due to mesenteric ischemia (one) and critical aortic stenosis, which required aortic bypass surgery (two).Conclusion Patients with neuroblastoma who had received RT showed impaired growth of the abdominal aorta. Significant long-term vascular complications occurred in seven patients who received IORT±EBRT. Thus, CT evaluation of patients with neuroblastoma who receive RT should include not only reports of changes in tumor extension, but also documentation of perfusion, and the size and growth of the aorta and its branches over time

MMP-2 siRNA Inhibits Radiation-Enhanced Invasiveness in Glioma Cells

Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma.We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls.MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients

Gefitinib in patients with progressive high-grade gliomas: a multicentre phase II study by Gruppo Italiano Cooperativo di Neuro-Oncologia (GICNO)

To investigate the role of gefitinib in patients with high-grade gliomas (HGGs), a phase II trial (1839IL/0116) was conducted in patients with disease recurrence following surgery plus radiotherapy and first-line chemotherapy. Adult patients with histologically confirmed recurrent HGGs following surgery, radiotherapy and first-line chemotherapy, were considered eligible. Patients were treated with gefitinib (250 mgday−1) continuously until disease progression. The primary end point was progression-free survival at 6 months progression-free survival at 6 months (PFS-6). Tissue biomarkers (epidermal growth factor receptor (EGFR) gene status and expression, phosphorylated Akt (p-Akt) expression) were assessed. Twenty-eight patients (median age, 55 years; median ECOG performance status, 1) were enrolled; all were evaluable for drug activity and safety. Sixteen patients had glioblastoma, three patients had anaplastic oligodendrogliomas and nine patients had anaplastic astrocytoma. Five patients (17.9%, 95% CI 6.1–36.9%) showed disease stabilisation. The overall median time to progression was 8.4 (range 2–104+) weeks and PFS-6 was 14.3% (95% CI 4.0–32.7%). The median overall survival was 24.6 weeks (range 4–104+). No grade 3–4 gefitinib-related toxicity was found. Gefitinib showed limited activity in patients affected by HGGs. Epidermal growth factor receptor expression or gene status, and p-Akt expression do not seem to predict activity of this drug

Separation of Anti-Proliferation and Anti-Apoptotic Functions of Retinoblastoma Protein through Targeted Mutations of Its A/B Domain

BACKGROUND: The human retinoblastoma susceptibility gene encodes a nuclear phosphoprotein RB, which is a negative regulator of cell proliferation. The growth suppression function of RB requires an evolutionarily conserved A/B domain that contains two distinct peptide-binding pockets. At the A/B interface is a binding site for the C-terminal trans-activation domain of E2F. Within the B-domain is a binding site for proteins containing the LxCxE peptide motif. METHODOLOGY/PRINCIPLE FINDINGS: Based on the crystal structure of the A/B domain, we have constructed an RB-K530A/N757F (KN) mutant to disrupt the E2F- and LxCxE-binding pockets. The RB-K530A (K) mutant is sufficient to inactivate the E2F-binding pocket, whereas the RB-N757F (N) mutant is sufficient to inactivate the LxCxE-binding pocket. Each single mutant inhibits cell proliferation, but the RB-KN double mutant is defective in growth suppression. Nevertheless, the RB-KN mutant is capable of reducing etoposide-induced apoptosis. CONCLUSION/SIGNIFICANCE: Previous studies have established that RB-dependent G1-arrest can confer resistance to DNA damage-induced apoptosis. Results from this study demonstrate that RB can also inhibit apoptosis independent of growth suppression

Incidence, mechanism and prognostic value of activated AKT in pancreas cancer

When activated, the serine/threonine kinase AKT mediates an antiapoptotic signal implicated in chemoresistance of various cancers. The mechanism(s) of AKT activation are unknown, though overexpression of HER-2/neu has been implicated in breast cancer. Therefore, we determined the incidence of activated AKT in human pancreatic cancer, whether HER-2/neu is involved in AKT activation, and if AKT activation is associated with biologic behaviour. HER-2/neu expression and AKT activation were examined in seven pancreatic cancer cell lines by Western blotting. The in vitro effect of HER-2/neu inhibition on AKT activation was similarly determined. Finally, 78 pancreatic cancer specimens were examined for AKT activation and HER-2/neu overexpression, and correlated with the clinical prognostic variable of histologic grade. HER-2/neu was overexpressed in two of seven cell lines; these two cell lines demonstrated the highest level of AKT activation. Inhibition of HER-2/neu reduced AKT activation in vitro. AKT was activated in 46 out of 78 (59%) of the pancreatic cancers; HER-2/neu overexpression correlated with AKT activation (P=0.015). Furthermore, AKT activation was correlated with higher histologic tumour grade (P=0.047). Thus, it is concluded that AKT is frequently activated in pancreatic cancer; this antiapoptotic signal may be mediated by HER-2/neu overexpression. AKT activation is associated with tumour grade, an important prognostic factor

Pediatric high-grade glioma: biologically and clinically in need of new thinking.

High-grade gliomas in children are different from those that arise in adults. Recent collaborative molecular analyses of these rare cancers have revealed previously unappreciated connections among chromatin regulation, developmental signaling, and tumorigenesis. As we begin to unravel the unique developmental origins and distinct biological drivers of this heterogeneous group of tumors, clinical trials need to keep pace. It is important to avoid therapeutic strategies developed purely using data obtained from studies on adult glioblastoma. This approach has resulted in repetitive trials and ineffective treatments being applied to these children, with limited improvement in clinical outcome. The authors of this perspective, comprising biology and clinical expertise in the disease, recently convened to discuss the most effective ways to translate the emerging molecular insights into patient benefit. This article reviews our current understanding of pediatric high-grade glioma and suggests approaches for innovative clinical management

Stage 4 neuroblastoma: sequential hemi-body irradiation or high-dose chemotherapy plus autologous haemopoietic stem cell transplantation to consolidate primary treatment

The aim of the present study was to evaluate the effectiveness of two consecutive nonrandomised treatment programs applied between 1989 and 1999 at the Istituto Nazionale Tumori of Milan in an unselected cohort of 59 children over the age of one with stage 4 neuroblastoma. Both treatment programs consisted of two phases, the induction of the remission phase and the consolidation phase. The induction of the remission phase consisted of intensive chemotherapy, and remained the same throughout the study period. The consolidation phase consisted of sequential hemi-body irradiation (HBI) (10 Gy per session, 6 weeks apart) in the first period (1988–June 1994) and sequential high-dose cyclophosphamide, etoposide, mitoxantrone+L-PAM and autologous haemopoietic stem cell transplantation in the second (July 1994–1999). Intention-to-treat analysis revealed a significantly better outcome for patients treated with the second program, the 5-year event-free survival probability being 0.12 for program 1 and 0.31 for program 2 (P=0.03). This finding led us to conclude that sequential HBI is useless as consolidation treatment. The high-dose chemotherapy adopted in the second program enabled a proportion of patients to obtain long-term survival but, since the clinical results remain unsatisfactory, new treatment strategies are warranted

Observation of associated near-side and away-side long-range correlations in √sNN=5.02 TeV proton-lead collisions with the ATLAS detector

Two-particle correlations in relative azimuthal angle (Δϕ) and pseudorapidity (Δη) are measured in √sNN=5.02  TeV p+Pb collisions using the ATLAS detector at the LHC. The measurements are performed using approximately 1  μb-1 of data as a function of transverse momentum (pT) and the transverse energy (ΣETPb) summed over 3.1<η<4.9 in the direction of the Pb beam. The correlation function, constructed from charged particles, exhibits a long-range (2<|Δη|<5) “near-side” (Δϕ∼0) correlation that grows rapidly with increasing ΣETPb. A long-range “away-side” (Δϕ∼π) correlation, obtained by subtracting the expected contributions from recoiling dijets and other sources estimated using events with small ΣETPb, is found to match the near-side correlation in magnitude, shape (in Δη and Δϕ) and ΣETPb dependence. The resultant Δϕ correlation is approximately symmetric about π/2, and is consistent with a dominant cos⁡2Δϕ modulation for all ΣETPb ranges and particle pT