Abstract
Chemotherapy activates a novel cytoplasmic DNA damage response resulting in Golgi apparatus fragmentation and cancer cell survival. This mechanism is regulated by Golgi phosphoprotein-3 (GOLPH3)/Myo18A/F-actin axis. Analyzing the functions of miR-3135b, a small non-coding RNA with unknown functions, we found that its forced overexpression attenuates the Golgi apparatus fragmentation induced by chemotherapeutic drugs in colorectal cancer (CRC) cells. First, we found that miR-3135b is downregulated in CRC cell lines and clinical tumors. Bioinformatic predictions showed that miR-3135b could be regulating protein-encoding genes involved in cell survival, resistance to chemotherapy, and Golgi dynamics. In agreement, ectopic transfection of miR-3135b in HCT-15 cancer cells significantly inhibited cell proliferation, sensitized cells to 5-fluoruracil (5-FU), and promoted late apoptosis and necrosis. Also, miR-3135b overexpression impaired the cell cycle progression in HCT-15 and SW-480 cancer cells. Because GOLPH3, a gene involved in maintenance of Golgi structure, was predicted as a potential target of miR-3135b, we studied their functional relationships in response to DNA damage induced by chemotherapy. Immunofluorescence and cellular ultrastructure experiments using antibodies against TGN38 protein, a trans-Golgi network marker, showed that 5-FU and doxorubicin treatments result in an apoptosis-independent stacks dispersal of the Golgi ribbon structure in both HCT-15 and SW-480 cells. Remarkably, these cellular effects were dramatically hindered by transfection of miR-3135b mimics. In addition, our functional studies confirmed that miR-3135b binds to the 3′-UTR of GOLPH3 proto-oncogene, and also reduces the levels of p-AKT1 (Ser473) and p-mTOR (Ser2448) signaling transducers, which are key in cell survival and autophagy activation. Moreover, we found that after treatment with 5-FU, TGN38 factor coimmunolocalizes with beclin-1 autophagic protein in discrete structures associated with the fragmented Golgi, suggesting that the activation of pro-survival autophagy is linked to loss of Golgi integrity. These cellular effects in autophagy and Golgi dispersal were reversed by miR-3135b. In summary, we provided experimental evidence suggesting for the first time a novel role for miR-3135b in the protection of chemotherapy-induced Golgi fragmentation via GOLPH3/AKT1/mTOR axis and protective autophagy in colorectal cancer cells.
Highlights
Factor coimmunolocalizes with beclin-1 autophagic protein in discrete structures associated with the fragmented Golgi, suggesting that the activation of pro-survival autophagy is linked to loss of Golgi integrity
A report of Farber-Katz et al, demonstrated that Golgi fragmentation occurs after DNA damage caused by chemotherapeutic drugs as a mechanisms based on the activation of DNA-PK/Golgi phosphoprotein-3 (GOLPH3)/MYO18a pathway to allow cancer cells survival, which is largely unrelated to a poptosis[5]
Here we investigated the functional relationships between miR-3135b and the Golgi apparatus dispersal in response to therapy with DNA-damaging drugs
Summary
Factor coimmunolocalizes with beclin-1 autophagic protein in discrete structures associated with the fragmented Golgi, suggesting that the activation of pro-survival autophagy is linked to loss of Golgi integrity. Farber-Katz and c oworkers[5] demonstrated that the nuclear kinase DNA-PK is activated, which in turn phosphorylates the Golgi phosphoprotein-3 (GOLPH3) leading to strong interactions of GOLPH3 with unconventional myosin XVIIIa (MYO18a) that increases the tensile forces with F-actin cytoskeleton resulting in Golgi apparatus fragmentation and dispersal This aberrant Golgi-dispersion was necessary to support cell survival and conferred resistance to DNA-damaging agents[7]. Recent experimental findings suggested that Golgi membranes are essential sites for the assemblage and production of autophagic-vesicles that nucleate the membranous structures shaping the phagophore formation during the early stages of autophagy[10] In this scenario, GOLPH3 functions are relevant for the activation of the cytoplasmic DNA damage response and Golgi apparatus integrity. Our data suggest that miR-3135b is a novel tumor suppressor with a potential role in the regulation of the GOLPH3/AKT1/mTOR axis to maintain Golgi integrity, which adds a piece in the puzzle of the novel cytoplasmic DNA damage response to DNA-damaging agents in CRC cells
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