Downregulation of protein kinase C gamma reduces epithelial property and enhances malignant phenotypes in colorectal cancer cells.

Loss of epithelial integrity is associated with colorectal cancer (CRC) aggressiveness. Protein kinase C (PKC) is frequently implicated in human cancers, but the role of PKCγ in CRC remains poorly understood. Here, we show that PKCγ, a conventional PKC, is expressed in normal colonic epithelium, but this is lower in dedifferentiated CRC. PKCγ expression was downregulated by SNAI1 overexpression, and low PKCγ expression was associated with poor prognosis in patients with CRC. Transient or stable knockdown of PKCγ reduced E-cadherin expression in CRC cells. PKCγ knockdown enhanced proliferation, anchorage-independent cell growth, resistance to anti-cancer drugs, and in vivo tumor growth of DLD-1 cells. We have also identified phosphorylation substrates for PKCγ. Among them, ARHGEF18, a RhoA activator that stabilizes cell-cell junctions, was phosphorylated and stabilized by PKCγ. Thus, these results suggest that the downregulation of PKCγ decreases the epithelial property of CRC cells and enhances its malignant phenotypes.

Cell­to­cell communication via extracellular vesicles among human pancreatic cancer cells derived from the same patient.

Despite existing multimodal therapies, pancreatic cancer exhibits high metastatic capability and poor prognosis. Extracellular vesicles (EVs) are nanoparticles comprising lipid bilayers and various other components, such as protein and nucleic acids, derived from secreted cells. Recent research has demonstrated the involvement of EVs released from cancer cells in the metastasis of cancer cells to distant organs. However, the effects of EVs released from pancreatic cancer cells on other pancreatic cancer cells in a tumor microenvironment remain unclear. The present study aimed to elucidate that EVs released from PK­45H pancreatic cancer cells are taken up by PK­45P pancreatic cancer cells derived from the same patient through dynamin­related endocytosis. Additionally, EVs released from PK­45H cells augment the phosphorylation of classical mitogen­activated protein kinase (MAPK) pathways in PK­45P cells. The uptake of EVs released from PK­45H cells by PK­45P cells stimulates cell migration through the classical MAPK­dependent pathway, suggesting that EVs released from one pancreatic cancer cell are taken up by other surrounding pancreatic cancer cells and could be critical inducers of cancer metastasis in the tumor microenvironment.

Exosomes released from pancreatic cancer cells enhance angiogenic activities via dynamin-dependent endocytosis in endothelial cells in vitro.

Pancreatic cancer has the lowest 5 year survival rate among all cancers. Several extracellular factors are involved in the development and metastasis of pancreatic cancer to distant organs. Exosomes are lipid-bilayer, membrane-enclosed nanoparticles that are recognised as important mediators of cell-to-cell communications. However, the role of exosomes released from pancreatic cancer cells in tumour micro-environment remains unknown. Here, we show that exosomes released from pancreatic cancer PK-45H cells activate various gene expressions in human umbilical vein endothelial cells (HUVECs) by in vitro analyses. In addition, these exosomes released from PK-45H cells promote phosphorylation of Akt and ERK1/2 signalling pathway molecules and tube formation via dynamin-dependent endocytosis in HUVECs. Our findings suggested that exosomes released from pancreatic cancer cells may act as a novel angiogenesis promoter.

Exosomes derived from SW480 colorectal cancer cells promote cell migration in HepG2 hepatocellular cancer cells via the mitogen-activated protein kinase pathway.

Exosomes are membrane-derived extracellular vesicles that have recently been recognized as important mediators of intercellular communication. In the present study, we investigated the effects of exosomes derived from SW480 colorectal cancer cells in recipient HepG2 hepatocellular cancer cells. We demonstrated that SW480-derived exosomes were taken up by the recipient HepG2 cells via dynamin-dependent endocytosis and were localized to the HepG2 lysosomes. In addition, SW480-derived exosomes induced the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 following their uptake into HepG2 cells. Of note, these changes occurred during the early phase after exosome treatment. Furthermore, SW480-derived exosomes promoted the migration of recipient HepG2 cells in a wound-healing assay, which was suppressed by pretreatment with U0126, an upstream inhibitor of ERK1/2. These results indicated that SW480-derived exosomes activated a classical mitogen-activated protein kinase pathway in recipient HepG2 cells via dynamin-dependent endocytosis and subsequently enhanced cell migration by ERK1/2 activation. Our results provide new insights into the regulation of cellular functions by exosomes.

Secretion of small/microRNAs including miR-638 into extracellular spaces by sphingomyelin phosphodiesterase 3.

A recent study demonstrated that intracellular small/microRNAs are released from cells, and some of these extracellular RNAs are embedded in vesicles, such as ceramide-rich exosomes, on lipid-bilayer membranes. In the present study, we examined the effects of sphingomyelin phosphodiesterase 3 (SMPD3), which generates ceramide from sphingomyelin, on the release of small/microRNAs from intracellular to extracellular spaces. In these experiments, SW480 human colorectal and HuH-7 human hepatocellular cancer cells were cultured for 48 h in serum-free media. Culture supernatants were then collected, and floating cells and debris were removed by centrifugation and filtration through a 0.22-µm filter. Extracellular small RNAs in purified culture supernatants were stable for 4 weeks at room temperature, after 20 freeze-thaw cycles and exposure to pH 2.0, and were resistant to ribonuclease A degradation. Amino acid sequence analyses of SMPD3 showed high homology between mammals, indicating evolutionary conservation. Therefore, to investigate the mechanisms of cellular small/microRNA export, SW480 and HuH-7 cells were treated with the SMPD3 inhibitor GW4869 in serum-free media. Culture supernatants were collected for microarray and/or reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments. The number of microRNAs in culture supernatants was decreased following treatment with GW4869. Among these, extracellular and intracellular miR-638 were dose-dependently decreased and increased, respectively. These data suggest that SMPD3 plays an important role in the release of microRNAs into extracellular spaces.