Pleural mesothelioma (PM) is a relatively rare and very aggressive malignancy often caused by the inhalation of asbestos fibers. Treatment options remain limited and suffer from relatively poor efficacy. PM is characterized by a lack of oncogenic driver mutations, suggesting that growth-driving signals may derive from the tumor microenvironment (TME). Indeed, mesothelioma-associated fibroblasts (Meso-CAFs) have previously been demonstrated to stimulate the growth and migration of mesothelioma cells. Additionally, Y-box binding protein 1 (YB-1), which is not mutated but often overexpressed in PM, has emerged as a key multifunctional protein involved in multiple hallmarks of cancer. Recent studies have shown that YB-1 overexpression in PM cells contributes to growth, migration, cisplatin resistance, and immune evasion. The function of YB-1 in CAFs, in contrast, has not been investigated so far. In the current study, we have investigated the role of YB-1 in Meso-CAFs and their ability to stimulate PM cell growth.

YBX-1 RNA levels, and Meso-CAF differentiation markers (ELN and CTGF) were analyzed by qRT-PCR. YB-1 protein expression and phosphorylation were analyzed by immunoblotting. YB-1 depletion was achieved by siRNA-mediated knockdown. Cell growth in 2D models was assessed using SYBR Green assays. Cell growth in 3D spheroid and co-culture models, cell migration, and cell fate were assessed by live-cell videomicroscopy, followed by automated image analysis and single-cell tracking.

Meso-CAFs showed a strong expression of YB-1 at the transcript and protein levels. Targeting YB-1 with siRNA achieved a potent reduction of YB-1 mRNA and protein levels in Meso-CAFs and resulted in decreased viability and migration capacity. In contrast, transcript levels of the Meso-CAF differentiation markers ELN and CTGF were not affected by YB-1 targeting. Co-culture with Meso-CAFs had a significant stimulatory effect on mesothelioma cell growth in 2D and in 3D spheroid cultures. Importantly, knockdown of YB-1 in Meso-CAFs resulted in a marked reduction of this growth stimulatory effect in the 2D and 3D models.

YB-1 has previously been shown to drive growth and migration in different types of cancer cells, including those of PM. In the current study, we show for the first time that YB-1 is also critically involved in the growth and migration of CAFs and, moreover, its expression in CAFs is required for their stimulation of tumor growth. Effects of YB-1 inhibition on cancer cells and the TME could, therefore, both contribute to therapeutic efficacy.