| Popis: |
Background: Pancreatic ductal adenocarcinoma (PDAC), one of the fourth most lethal malig-nancies in the world, has a less than 5% five-year relative overall survival rate. Thus, there is a great need for novel therapies. PDAC is characterized as a stroma rich malignancy, composed of a large amount of extracellular matrix and pancreatic stellate cells. Accordingly, cell-matrix adhesion is crucial for cancer cell survival, invasion, and therapy resistance. We embarked on a high-throughput assay to identify the function of 117 focal adhesion proteins (FAP) in PDAC cell radiochemoresistance. Material and methods: We generated and performed a 3D tumoroid high-throughput esiRNA-based screening assay (3DHT-esiRNAs) in PDAC cell cultures (established and PDC) grown in laminin-rich extracellular matrix (IrECM). In addition to characterizing the β8 integrin expres-sion, distribution, and co-localization with other cellular organelles, such as Golgi apparatus, we also performed 3D tumoroid formation assay, sphere formation assay, type I collagen-based 3D invasion assay, and 2D clonogenic survival assay following esiRNA-mediated knockdown, 6 Gy x-ray irradiation and gemcitabine treatment. Image analysis was performed to determine Pearson's correlation coefficient, vesicle distribution and expression patterns upon irradiation or gemcitabine treatment by Fiji software (NIH). Immunoprecipitation-mass spectrometry (IP-MS) was performed to investigate the interactome of β8 integrin in the normal versus 6 Gy x-ray irradiation group. Inhibitor screen was conducted following 6 Gy x-ray irradi-ation or gemcitabine treatment to identify pathways involved in changes of β8 integrin localiza-tion upon treatment Autophagy flux was detected by monitoring LC3B puncta. Results: We identified a series of novel targets, including β8 integrin and PINCH1. Intriguingly, depletion of either β8 integrin or PINCH1 both showed radiosensitizing effect, with β8 integrin knockdown exerting a more profound radiosensitizing effect in a panel of PDAC cell lines. Without cytotoxicity, β8 integrin depletion evoked radiochemosensitization in PDAC, PDCs cell lines, and reduced sphere formation and 3D invasion into collagen-I. Intriguingly, β8 integrin was found to be located in the perinuclear region where it co-localized with the cis-Golgi matrix protein, GM130. Upon irradiation and gemcitabine treatment, β8 integrin was translocated from the perinuclear region to the cytosol, showing a slightly increased compart-mentalization in the exosome; a process that was abrogated by treatment with cytoskeletal inhibitors (paclitaxel, latrunculin B, and colchicine) and ATP synthase inhibitors (antimycin A and oligomycin). Depletion of β8 integrin influenced the autophagy via decreasing the LC3B puncta in a microtubule independent manner. Conclusion: Our results generated in 3D lrECM PDAC cell cultures, propose that β8 integrin, but not PINCH1, is a novel determinant of PDAC radiochemoresistance. Moreover, β8 integrin, although not localized to the cell membrane to facilitate cell adhesion, has a critical role in regulating intracellular vesicle trafficking under stress conditions and autophagy flux. |
