Cholangiocarcinoma Malignant Traits Are Promoted by Schwann Cells through TGFß Signaling in a Model of Perineural Invasion.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFß receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFß signaling in regulating this process.

Autophagy impairment in human bile duct carcinoma cells.

Bile duct epithelial cells, named cholangiocytes, may undergo a neoplastic transformation leading to cholangiocarcinoma. The role autophagy plays in cancer is still debated and few information are available in cholangiocarcinoma. We report in vitro data, at least in part validated in vivo,i ndicating that autophagy is impaired in intrahepatic cholangiocarcinoma cells, as compared to healthy cholangiocytes, evaluated through LC3II and p62 Western blot analyses. Autophagy impairment was found to be associated with low expression of TFEB protein and high expression of three proteins i.e., c-FLIP, caspase-10 and cleaved BCLAF-1, as compared to healthy cholangiocytes. We highlight biological effects of autophagy impairment in cholangiocarcinoma showing that autophagy induction, via rapamycin, as well as caspase inhibition, via Q-VD-OPh, are able to reduce proliferation marker PCNA level, colony size and protein content of cultured cholangiocarcinoma cells. The increased protein expression of p62, c-FLIP, caspase-10 observed in vitro in cholangiocarcinoma cells was paralleled by significant increase at gene expression levels in vivo; in fact, significant increase of transcript levels of p62, c-FLIP and caspase-10 was observed in 34 biopsies from human cholangiocarcinoma patients compared to 9 biopsies from 9 healthy controls, as reported in the GEPIA2 public database. The significant increase of p62 level in cholangiocarcinoma was found as a relatively uncommon finding in solid cancers, since it was also found in only 7 cancer types out of 31 cancer types investigated, including melanoma and hepatocarcinoma. In conclusion, we present data suggesting a molecular machinery controlling autophagy in cholangiocytes and autophagy impairment in cholangiocarcinoma.

Autophagy Activation Associates with Suppression of Prion Protein and Improved Mitochondrial Status in Glioblastoma Cells.

Cells from glioblastoma multiforme (GBM) feature up-regulation of the mechanistic Target of Rapamycin (mTOR), which brings deleterious effects on malignancy and disease course. At the cellular level, up-regulation of mTOR affects a number of downstream pathways and suppresses autophagy, which is relevant for the neurobiology of GBM. In fact, autophagy acts on several targets, such as protein clearance and mitochondrial status, which are key in promoting the malignancy GBM. A defective protein clearance extends to cellular prion protein (PrPc). Recent evidence indicates that PrPc promotes stemness and alters mitochondrial turnover. Therefore, the present study measures whether in GBM cells abnormal amount of PrPc and mitochondrial alterations are concomitant in baseline conditions and whether they are reverted by mTOR inhibition. Proteins related to mitochondrial turnover were concomitantly assessed. High amounts of PrPc and altered mitochondria were both mitigated dose-dependently by the mTOR inhibitor rapamycin, which produced a persistent activation of the autophagy flux and shifted proliferating cells from S to G1 cell cycle phase. Similarly, mTOR suppression produces a long-lasting increase of proteins promoting mitochondrial turnover, including Pink1/Parkin. These findings provide novel evidence about the role of autophagy in the neurobiology of GBM.

Protease Activated Receptor 1 and Its Ligands as Main Regulators of the Regeneration of Peripheral Nerves.

In contrast with the brain and spinal cord, peripheral nerves possess a striking ability to regenerate after damage. This characteristic of the peripheral nervous system is mainly due to a specific population of glial cells, the Schwann cells. Schwann cells promptly activate after nerve injury, dedifferentiate assuming a repair phenotype, and assist axon regrowth. In general, tissue injury determines the release of a variety of proteases which, in parallel with the degradation of their specific targets, also activate plasma membrane receptors known as protease-activated receptors (PARs). PAR1, the prototypical member of the PAR family, is also known as thrombin receptor and is present at the Schwann cell plasma membrane. This receptor is emerging as a possible regulator of the pro-regenerative capacity of Schwann cells. Here, we summarize the most recent literature data describing the possible contribution of PAR1 and PAR1-activating proteases in regulating the regeneration of peripheral nerves.

Anti-tumor Effect of Oleic Acid in Hepatocellular Carcinoma Cell Lines via Autophagy Reduction.

Oleic acid (OA) is a component of the olive oil. Beneficial health effects of olive oil are well-known, such as protection against liver steatosis and against some cancer types. In the present study, we focused on OA effects in hepatocellular carcinoma (HCC), investigating responses to OA treatment (50-300 µM) in HCC cell lines (Hep3B and Huh7.5) and in a healthy liver-derived human cell line (THLE-2). Upon OA administration higher lipid accumulation, perilipin-2 increase, and autophagy reduction were observed in HCC cells as compared to healthy cells. OA in the presence of 10% FBS significantly reduced viability of HCC cell lines at 300 µM through Alamar Blue staining evaluation, and reduced cyclin D1 expression in a dose-dependent manner while it was ineffective on healthy hepatocytes. Furthermore, OA increased cell death by about 30%, inducing apoptosis and necrosis in HCC cells but not in healthy hepatocytes at 300 µM dosage. Moreover, OA induced senescence in Hep3B, reduced P-ERK in both HCC cell lines and significantly inhibited the antiapoptotic proteins c-Flip and Bcl-2 in HCC cells but not in healthy hepatocytes. All these results led us to conclude that different cell death processes occur in these two HCC cell lines upon OA treatment. Furthermore, 300 µM OA significantly reduced the migration and invasion of both HCC cell lines, while it has no effects on healthy cells. Finally, we investigated autophagy role in OA-dependent effects by using the autophagy inducer torin-1. Combined OA/torin-1 treatment reduced lipid accumulation and cell death as compared to single OA treatment. We therefore concluded that OA effects in HCC cells lines are, at least, in part dependent on OA-induced autophagy reduction. In conclusion, we report for the first time an autophagy dependent relevant anti-cancer effect of OA in human hepatocellular carcinoma cell lines.

Thrombin regulates the ability of Schwann cells to support neuritogenesis and to maintain the integrity of the nodes of Ranvier.

Schwann cells (SC) are characterized by a remarkable plasticity that enables them to promptly respond to nerve injury promoting axonal regeneration. In peripheral nerves after damage SC convert to a repair-promoting phenotype activating a sequence of supportive functions that drive myelin clearance, prevent neuronal death, and help axon growth and guidance. Regeneration of peripheral nerves after damage correlates inversely with thrombin levels. Thrombin is not only the key regulator of the coagulation cascade but also a protease with hormone-like activities that affects various cells of the central and peripheral nervous system mainly through the protease-activated receptor 1 (PAR1). Aim of the present study was to investigate if and how thrombin could affect the axon supportive functions of SC. In particular, our results show that the activation of PAR1 in rat SC cultures with low levels of thrombin or PAR1 agonist peptides induces the release of molecules, which favor neuronal survival and neurite elongation. Conversely, the stimulation of SC with high levels of thrombin or PAR1 agonist peptides drives an opposite effect inducing SC to release factors that inhibit the extension of neurites. Moreover, high levels of thrombin administered to sciatic nerve ex vivo explants induce a dramatic change in SC morphology causing disappearance of the Cajal bands, enlargement of the Schmidt-Lanterman incisures and calcium-mediated demyelination of the paranodes. Our results indicate thrombin as a novel modulator of SC plasticity potentially able to favor or inhibit SC pro-regenerative properties according to its level at the site of lesion.