Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the five most lethal malignancies in the world and has a 5-year relative overall survival rate of less than 5%. Thus, there is a great need for molecular-targeting strategies. As cell-matrix adhesion is essential for the survival, invasion and therapy resistance, we sought to identify the function of 117 focal adhesion proteins (FAP) in PDAC cell radioresistance. Intriguingly 8 integrin turned out to be one of the most potential novel targets in PDAC.
Material and methods: For FAP detection, we performed a 3D endoribonuclease-prepared siRNA (esiRNA)-based screening (3DHTesiS) in PDAC cell culture (established and primary) grown in laminin-rich extracellular matrix (IrECM). After esiRNA-mediated knockdown and X-ray irradiation (2-6 Gy single dose), clonogenic survival assay and sphere formation were determined. Beta 8 integrin expression level and distribution were detected by using Western blot and immunofluorescence staining. Beta 8 integrin staining was also combined with vesicle trafficking proteins (Caveolin-1, APPL2) and the cis-Golgi matrix protein GM130. Fiji software was used to analyze vesicle distribution after irradiation and Peason’s correlation coefficients were calculated.
Results: We identified a series of novel targets with radiosensitizing potential including beta 8 integrin. Without cytotoxicity, beta 8 integrin knockdown conferred a significant radiosensitizing effect in established patient-derived PDAC cell cultures. Moreover, beta 8 integrin depletion reduced invasion and sphere forming ability. Intriguingly, we found beta 8 integrin located in the perinuclear area colocalized with GM130 but neither in the cell membrane nor colocalized with Caveolin-1 and APPL2. Further, we observed an increased beta 8 integrin expression after irradiation associated with enhanced beta 8 integrin-positive vesicle formation in both cytoplasm and nucleus. This suggests that beta 8 integrin may contribute to intracellular vesicle trafficking under stress conditions.
Summary: We successfully designed a high-throughput radiosensitivity screening method for cell growing in a physiological 3D matrix-based environment. Interestingly, beta 8 integrin has, although not found in the cell membrane to facilitate cell adhesion, a critical role in the radiation response of pancreatic cancer cell. Ongoing work will unravel the underlying mechanisms how beta 8 integrin is controlling cytoplasmic and nuclear survival pathways.