Paclitaxel aggravating radiation-induced pulmonary fibrosis is associated with the down-regulation of the negative regulatory function of Spry2

Paclitaxel (PTX) is capable of aggravating radiation-induced pulmonary fibrosis (RIPF), but the mechanism is unknown. Spry2 is a negative regulator of receptor tyrosine kinase-related Ras/Raf/ERK pathway. This experiment was aimed at exploring whether the aggravation of RIPF by PTX is related to Spry2. The RIPF model was established with C57BL/6 mice by thoracic irradiation, and PTX was administered concurrently. Western blot was used to detect the expression level of ERK signaling molecules and the distribution of Spry2 in the plasma membrane/cytoplasm. Co-IP and immunofluorescence were used to observe the co-localization of Spry2 with the plasma membrane and tubulin. The results showed that PTX-concurrent radiotherapy could aggravate fibrotic lesions in RIPF, down-regulate the content of membrane Spry2 and up-regulate the levels of p-c-Raf and p-ERK in lung tissue. It was found that knockdown of Spry2 in fibroblast abolished the up-regulation of p-c-Raf and p-ERK by PTX. Both Co-IP results and immunofluorescence staining showed that PTX increased the binding of Spry2 to tubulin and that microtubule depolymerizing agents could abolish PTX's inhibition of Spry2 membrane distribution and inhibit PTX's up-regulation of Raf/ERK signaling. Both Nintedanib and ERK inhibitor were effective in relieving PTX-exacerbated RIPF. Taken together, the mechanism of PTX's aggravating RIPF was related to its ability to enhance Spry2's binding to tubulin, thus attenuating Spry2's negative regulation on Raf/ERK pathway. Significance Statement The obtained mechanism of paclitaxel aggravating RIPF by restraining Spry2 provides targets such as Raf/ERK for reducing radiation-induced damage to normal tissues caused by paclitaxel combined with radiation therapy.