SummaryCells inversely adjust the plasma membrane levels of integrins and cadherins during cell migration and cell-cell adhesion but the regulatory mechanisms that coordinate these trafficking events remain unknown. Here, we demonstrate that the small GTPase Rab35 maintains cadherins at the cell surface to promote cell-cell adhesion. Simultaneously, Rab35 supresses the activity of the GTPase Arf6 to downregulate an Arf6-dependent recycling pathway for b1-integrin and EGF receptors, resulting in inhibition of cell migration and attenuation of signaling downstream of these receptors. Importantly, the phenotypes of decreased cell adhesion and increased cell migration observed following Rab35 knock down are consistent with the epithelial-mesenchymal transition, a feature of invasive cancer cells, and we show that Rab35 expression is suppressed in a subset of cancers characterized by Arf6 hyperactivity. Our data thus identify a key molecular mechanism that efficiently coordinates the inverse intracellular sorting and cell surface levels of cadherin and integrin receptors for cell migration and differentiation.

Our study highlights the biological heterogeneity of murine glioma models and illustrates that cotargeting of the VEGF and TGF-β pathways might lead to improved tumor control only in subsets of glioblastoma.

We recently demonstrated that lack of Furin-processing of the N-cadherin precursor (proNCAD) in highly invasive melanoma and brain tumor cells results in the cell-surface expression of a nonadhesive protein favoring cell migration and invasion in vitro. Quantitative polymerase chain reaction analysis of malignant human brain tumor cells revealed that of all proprotein convertases (PCs) only the levels of Furin and PC5A are modulated, being inversely (Furin) or directly (PC5A) correlated with brain tumor invasive capacity. Intriguingly, the N-terminal sequence following the Furin-activated NCAD site (RQKR↓DW(161), mouse nomenclature) reveals a second putative PC-processing site (RIRSDR↓DK(189)) located in the first extracellular domain. Cleavage at this site would abolish the adhesive functions of NCAD because of the loss of the critical Trp(161). This was confirmed upon analysis of the fate of the endogenous prosegment of proNCAD in human malignant glioma cells expressing high levels of Furin and low levels of PC5A (U343) or high levels of PC5A and negligible Furin levels (U251). Cellular analyses revealed that Furin is the best activating convertase releasing an ~17-kDa prosegment, whereas PC5A is the major inactivating enzyme resulting in the secretion of an ~20-kDa product. Like expression of proNCAD at the cell surface, cleavage of the NCAD molecule at RIRSDR↓DK(189) renders the U251 cancer cells less adhesive to one another and more migratory. Our work modifies the present view on posttranslational processing and surface expression of classic cadherins and clarifies how NCAD possesses a range of adhesive potentials and plays a critical role in tumor progression.

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e13027 Background: Epigenetic methylation of the O6-methylguanine-DNA methyltransferase (MGMT) DNA repair gene promoter in tumor tissue from glioblastoma multiforme patients is associated with improved survival after treatment with radiotherapy plus concomitant and adjuvant temozolomide (TMZ). We hypothesized that MGMT promoter methylation mosaicism exists in glial tumors and would affect response to TMZ. Methods: This is a nonrandomized, prospective, open-label, two cohort, single-center phase II study. Twenty-three patients with brain tumors detected by MRI scan and suspected to be gliomas were evaluated. All eligible patients were treatment naive and were self-selected into a TMZ group or a control group. The primary goal of the study was to evaluate the effect of TMZ 75 mg/m2 daily prior to surgery on the brain tumor MGMT expression. Secondary endpoints included safety, tolerability, and MGMT promoter methylation mosaicism in glial tumors. Samples were obtained from multiple regions of each tumor intra-operatively and were analyzed by methylation specific PCR. Results: Our results on MGMT promoter methylation demonstrate that three groups of patients can be identified: Type I: all sites assessed in the tumor demonstrate no methylation of the MGMT promoter; Type II: all sites demonstrate high levels of MGMT promoter methylation; and Type III: a mixed promoter methylation pattern is observed. Conclusions: These results suggest that 1) preoperative neoadjuvant temozolomide is not associated with increased postoperative complications; 2) glial tumors can have very heterogeneous areas of MGMT promoter methylation; and 3) three patterns of MGMT promoter methylation can be discerned. This experimental paradigm may be a useful experimental platform for the assessment of the effect of new drugs at the tumor level. [Table: see text]

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The current standard of care for glioblastoma includes surgery, radiotherapy and chemotherapeutic agents such as temozolomide (TMZ), a DNA methylating compound. The cytotoxic effects of TMZ have been linked to guanine methylation at the N7 and O6 positions. These adducts are not currently used as markers of TMZ efficacy. Using liquid chromatography/mass spectrometry (LC/MS), we have established a sensitive analytical assay to directly detect both N7- and O6-methylguanine adducts from DNA following TMZ treatment. A limit of detection below 1 fmol was observed for O6-methylguanine, while N7-methylguanine was observed below 5 fmol. O6- and N7-methylguanine were successfully detected by LC/MS in tumour and normal brain tissue samples from patients treated with a neoadjuvant TMZ regimen for 14 days (75 mg/m2). Variations in levels of both methylated guanines were detected between patients as well as within different locations of the same tumour sample. This technique provides a direct detection of the damage inflicted by TMZ. This could potentially indicate the efficacy of the drug, allowing for prompt analysis and response. It also holds potential for determining efficacy of treatment dose, schedule and possible concomitant drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B24.

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