No abstract

Glioblastoma (GBM) is a highly invasive brain neoplasia with an elevated recurrence rate after surgical resection. The cyclin D1 (Ccnd1)/Cdk4-retinoblastoma 1 (RB1) axis is frequently altered in GBM, leading to overproliferation by RB1 deletion or by Ccnd1-Cdk4 overactivation. High levels of Ccnd1-Cdk4 also promote GBM cell invasion by mechanisms that are not so well understood. The purpose of this work is to elucidate the in vivo role of cytoplasmic Ccnd1-Cdk4 activity in the dissemination of GBM. We show that Ccnd1 activates the invasion of primary human GBM cells through cytoplasmic RB1-independent mechanisms. By using GBM mouse models, we observed that evaded GBM cells showed cytoplasmic Ccnd1 colocalizing with regulators of cell invasion such as RalA and paxillin. Our genetic data strongly suggest that, in GBM cells, the Ccnd1-Cdk4 complex is acting upstream of those regulators. Accordingly, expression of Ccnd1 induces focal adhesion kinase, RalA and Rac1 activities. Finally, in vivo experiments demonstrated increased GBM dissemination after expression of membrane-targeted Ccnd1. We conclude that Ccnd1-Cdk4 activity promotes GBM dissemination through cytoplasmic and RB1-independent mechanisms. Therefore, inhibition of Ccnd1-Cdk4 activity may be useful to hinder the dissemination of recurrent GBM.

The analysis of Tumor Interstitial Fluid (TIF) composition is a valuable procedure to identify anti-metastatic targets, and different laboratories have set up techniques for TIF isolation and proteomic analyses. However, those methods had never been compared in samples from the same tumor and patient. In this work, we compared the two most used methods, elution and centrifugation, in pieces of the same biopsy samples of cutaneous squamous cell carcinoma (cSCC). First, we established that high G-force (10000g) was required to obtain TIF from cSCC by centrifugation. Secondly, we compared the centrifugation method with the elution method in pieces of three different cSCC tumors. We found that the mean protein intensities based in the number of peptide spectrum matches was significantly higher in the centrifuged samples than in the eluted samples. Regarding the robustness of the methods, we observed higher overlapping between both methods (77-80%) than among samples (50%). These results suggest that exists an elevated consistence of TIF composition independently of the method used. However, we observed a three-fold increase of extracellular proteins in non-overlapped proteome obtained by centrifugation. We therefore conclude that centrifugation is the method of choice to study the proteome of TIF from cutaneous biopsies.