Podoplanin is a small membrane mucin expressed in tumors associated with malignant progression. It is enriched at cell-surface protrusions where it colocalizes with members of the ERM (ezrin, radixin, moesin) protein family. Here, we found that human podoplanin directly interacts with ezrin (and moesin) in vitro and in vivo through a cluster of basic amino acids within its cytoplasmic tail, mainly through a juxtamembrane dipeptide RK. Podoplanin induced an epithelial-mesenchymal transition in MDCK cells linked to the activation of RhoA and increased cell migration and invasiveness. Fluorescence time-lapse video observations in migrating cells indicate that podoplanin might be involved in ruffling activity as well as in retractive processes. By using mutant podoplanin constructs fused to green fluorescent protein we show that association of the cytoplasmic tail with ERM proteins is required for upregulation of RhoA activity and epithelial-mesenchymal transition. Furthermore, expression of either a dominant-negative truncated variant of ezrin or a dominant-negative mutant form of RhoA blocked podoplanin-induced RhoA activation and epithelial-mesenchymal transition. These results provide a mechanistic basis to understand the role of podoplanin in cell migration or invasiveness.

Nab-paclitaxel and gemcitabine have demonstrated a survival benefit over gemcitabine alone in advanced pancreatic cancer (PDA). This study aimed to investigate the clinical, biological, and imaging effects of the regimen in patients with operable PDA. Patients with operable PDA received two cycles of nab-paclitaxel and gemcitabine before surgical resection. FDG-PET and CA19.9 tumour marker levels were used to measure clinical activity. Effects on tumour stroma were determined by endoscopic ultrasound (EUS) elastography. The collagen content and architecture as well as density of cancer-associated fibroblasts (CAFs) were determined in the resected surgical specimen and compared with a group of untreated and treated with conventional chemoradiation therapy controls. A co-clinical study in a mouse model of PDA was conducted to differentiate between the effects of nab-paclitaxel and gemcitabine. A total of 16 patients were enrolled. Treatment resulted in significant antitumour effects with 50% of patients achieving a >75% decrease in circulating CA19.9 tumour marker and a response by FDG-PET. There was also a significant decrement in tumour stiffness as measured by EUS elastography. Seven of 12 patients who completed treatment and were operated had major pathological regressions. Analysis of residual tumours showed a marked disorganised collagen with a very low density of CAF, which was not observed in the untreated or conventionally treated control groups. The preclinical co-clinical study showed that these effects were specific of nab-paclitaxel and not gemcitabine. These data suggest that nab-paclitaxel and gemcitabine decreases CAF content inducing a marked alteration in cancer stroma that results in tumour softening. This regimen should be studied in patients with operable PDA.

MT1‐matrix metalloproteinase (MT1‐MMP) is one of the most critical factors in the invasion machinery of tumor cells. Subcellular localization to invasive structures is key for MT1‐MMP proinvasive activity. However, the mechanism driving this polarized distribution remains obscure. We now report that polarized exocytosis of MT1‐MMP occurs during MDA‐MB‐231 adenocarcinoma cell migration into collagen type I three‐dimensional matrices. Polarized trafficking of MT1‐MMP is triggered by β1 integrin‐mediated adhesion to collagen, and is required for protease localization at invasive structures. Localization of MT1‐MMP within VSV‐G/Rab8‐positive vesicles, but not in Rab11/Tf/TfRc‐positive compartment in invasive cells, suggests the involvement of the exocytic traffic pathway. Furthermore, constitutively active Rab8 mutants induce MT1‐MMP exocytic traffic, collagen degradation and invasion, whereas Rab8‐ but not Rab11‐knockdown inhibited these processes. Altogether, these data reveal a novel pathway of MT1‐MMP redistribution to invasive structures, exocytic vesicle trafficking, which is crucial for its role in tumor cell invasiveness. Mechanistically, MT1‐MMP delivery to invasive structures, and therefore its proinvasive activity, is regulated by Rab8 GTPase.