RAB27B Drives a Cancer Stem Cell Phenotype in NSCLC Cells Through Enhanced Extracellular Vesicle Secretion.

Cancer stem cells (CSC) within non-small cell lung carcinoma (NSCLC) tumors drive NSCLC progression, metastasis, relapse, and intrinsic chemoresistance. Understanding the mechanisms that support the malignant phenotypes of NSCLC CSCs may provide insights for improved NSCLC therapeutic interventions. Here, we report that expression of RAB27B, a small GTPase, is significantly upregulated in NSCLC CSCs when compared with bulk cancer cells (BCC). Short hairpin RNA-mediated knockdown of RAB27B leads to a loss of stem cell marker gene expression and reduced NSCLC spheroid growth, clonal expansion, transformed growth, invasion, and tumorigenicity. We find that NSCLC CSCs secrete significantly more extracellular vesicles (EV) than BCCs, and that this is RAB27B-dependent. Furthermore, CSC-derived EVs, but not BCC-derived EVs, induce spheroid growth, clonal expansion, and invasion in BCCs. Finally, RAB27B is required for CSC-derived EV-induced stemness in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating, invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Our findings further suggest that inhibition of RAB27B-dependent EV secretion may be a potential therapeutic strategy for NSCLC. Significance: Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.

Glioblastoma disrupts the ependymal wall and extracellular matrix structures of the subventricular zone.

BACKGROUND: Glioblastoma (GBM) is the most aggressive and common type of primary brain tumor in adults. Tumor location plays a role in patient prognosis, with tumors proximal to the lateral ventricles (LVs) presenting with worse overall survival, increased expression of stem cell genes, and increased incidence of distal tumor recurrence. This may be due in part to interaction of GBM with factors of the subventricular zone (SVZ), including those contained within the cerebrospinal fluid (CSF). However, direct interaction of GBM tumors with CSF has not been proved and would be hindered in the presence of an intact ependymal cell layer. METHODS: Here, we investigate the ependymal cell barrier and its derived extracellular matrix (ECM) fractones in the vicinity of a GBM tumor. Patient-derived GBM cells were orthotopically implanted into immunosuppressed athymic mice in locations distal and proximal to the LV. A PBS vehicle injection in the proximal location was included as a control. At four weeks post-xenograft, brain tissue was examined for alterations in ependymal cell health via immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. RESULTS: We identified local invading GBM cells within the LV wall and increased influx of CSF into the LV-proximal GBM tumor bulk compared to controls. In addition to the physical disruption of the ependymal cell barrier, we also identified increased signs of compromised ependymal cell health in LV-proximal tumor-bearing mice. These signs include increased accumulation of lipid droplets, decreased cilia length and number, and decreased expression of cell channel proteins. We additionally identified elevated numbers of small fractones in the SVZ within this group, suggesting increased indirect CSF-contained molecule signaling to tumor cells. CONCLUSIONS: Our data is the first to show that LV-proximal GBMs physically disrupt the ependymal cell barrier in animal models, resulting in disruptions in ependymal cell biology and increased CSF interaction with the tumor bulk. These findings point to ependymal cell health and CSF-contained molecules as potential axes for therapeutic targeting in the treatment of GBM.

Protein kinase Cι promotes UBF1-ECT2 binding on ribosomal DNA to drive rRNA synthesis and transformed growth of non-small-cell lung cancer cells.

Epithelial cell-transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho GTPases that is overexpressed in many cancers and involved in signal transduction pathways that promote cancer cell proliferation, invasion, and tumorigenesis. Recently, we demonstrated that a significant pool of ECT2 localizes to the nucleolus of non-small-cell lung cancer (NSCLC) cells, where it binds the transcription factor upstream binding factor 1 (UBF1) on the promoter regions of ribosomal DNA (rDNA) and activates rDNA transcription, transformed cell growth, and tumor formation. Here, we investigated the mechanism by which ECT2 engages UBF1 on rDNA promoters. Results from ECT2 mutagenesis indicated that the tandem BRCT domain of ECT2 mediates binding to UBF1. Biochemical and MS-based analyses revealed that protein kinase Cι (PKCι) directly phosphorylates UBF1 at Ser-412, thereby generating a phosphopeptide-binding epitope that binds the ECT2 BRCT domain. Lentiviral shRNA knockdown and reconstitution experiments revealed that both a functional ECT2 BRCT domain and the UBF1 Ser-412 phosphorylation site are required for UBF1-mediated ECT2 recruitment to rDNA, elevated rRNA synthesis, and transformed growth. Our findings provide critical molecular insight into ECT2-mediated regulation of rDNA transcription in cancer cells and offer a rationale for therapeutic targeting of UBF1- and ECT2-stimulated rDNA transcription for the management of NSCLC.