Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease.

Parkinson's disease (PD) is a fatal neurodegenerative disorder that is primarily caused by the degeneration and loss of dopaminergic neurons of the substantia nigra in the ventral midbrain. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset PD identified to date, with G2019S being the most frequent LRRK2 mutation, which is responsible for up to 1-2% of sporadic PD and up to 6% of familial PD cases. As no treatment is available for this devastating disease, developing new therapeutic strategies is of foremost importance. Cellular models are commonly used for testing novel potential neuroprotective compounds. However, current cellular PD models either lack physiological relevance to dopaminergic neurons or are too complex and costly for scaling up the production process and for screening purposes. In order to combine biological relevance and throughput, we have developed a PD model in Lund human mesencephalic (LUHMES) cell-derived dopaminergic neurons by overexpressing wild-type (WT) and G2019S LRRK2 proteins. We show that these cells can differentiate into dopaminergic-like neurons and that expression of mutant LRRK2 causes a range of different phenotypes, including reduced nuclear eccentricity, altered mitochondrial and lysosomal morphologies, and increased dopaminergic cell death. This model could be used to elucidate G2019S LRRK2-mediated dopaminergic neural dysfunction and to identify novel molecular targets for disease intervention. In addition, our model could be applied to high-throughput and phenotypic screenings for the identification of novel PD therapeutics.

Oncogenic dependency on ß-catenin in liver cancer cell lines correlates with pathway activation.

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/ß-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (ß-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of ß-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on ß-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on ß-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) ß-catenin were significantly more sensitive to ß-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active ß-catenin. Finally, significant therapeutic benefit of ß-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. ß-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of ß-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant ß-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells.

Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.