Panoply™ Human CSF2 Knockdown Stable Cell Line

Bladder cancer (BCa) is one of the most common cancers of the urinary system. Colony-stimulating factor 2 (CSF2) is implicated in multiple cancers, but not BCa. Here, researchers investigated the effects of CSF2 on BCa and its underlying molecular mechanisms. CSF2 mRNA levels in BCa were analyzed using the Cancer Genome Atlas (TCGA) database. Western blotting was used to validate CSF2 expression in BCa tissue samples and cell lines. CCK8 and clonogenic assays were used to assess the effects of CSF2 on BCa cell growth. Transwell assays and wound healing assays were performed to determine the migration and invasion abilities of BCa cells. Next, Western blotting was used to explore the underlying mechanisms. Finally, a BCa xenograft mouse model was established to examine the effects of CSF2 on tumor formation in vivo. Results showed that CSF2 mRNA expression was upregulated in BCa samples. Knockdown of CSF2 significantly inhibited BCa cell proliferation and tumor formation in vitro and in vivo. Mechanistic analysis revealed that CSF2 knockdown inhibited BCa cell proliferation and invasion through AKT/mTOR signaling. Based on these results, CSF2 promoted the proliferation and tumor formation of BCa cells.

Using the cBioPortal database, KEGG pathway analysis was performed to explore the specific mechanisms by which CSF2 affects BCa progression. The results suggested that CSF2 may regulate BCa progression through the AKT signaling pathway (Figure 1A). The cBioPortal database also preliminarily confirmed that there was no significant correlation between CSF2 and AKT1 expression (S1), indicating that AKT's function is post-transcriptionally phosphorylated. Subsequently, the researchers performed Western blot experiments to verify changes in p-Akt and Akt protein levels. Nrf2 is regulated by Akt/mTOR signaling in tumor cells. To determine whether Nrf2 is regulated by the Akt/mTOR pathway, they examined activation of this pathway in T24 and 5637 cell lines. Finally, p-Akt and p-mTOR levels were significantly reduced in CSF2 knockdown T24 and 5637 cell lines (Figure 1B). Subsequently, the Akt agonist SC79 was used. After treatment of CSF2-knockdown T24 and 5637 cell lines with SC79, cell biological activity assays were performed. Western blot results showed that Nrf2 protein levels increased after SC79 treatment in CSF2-knockdown T24 (Figure 1C) and 5637 cell lines (Figure 1D). In other experiments, SC79 treatment significantly reversed the inhibition of CSF2-knockdown T24 and 5637 cell lines (Figures 1E, F, and G). Together, these results confirm that CSF2 regulates Nrf2 in T24 and 5637 cell lines through Akt/mTOR activation.

Figure 1. Akt/mTOR pathway is required for the regulation of Nrf2 by CSF2 in BCa cells. (Yu X, et al., 2024)