Panoply™ Human PDGFB Over-expressing Stable Cell Line

Clear cell renal cell carcinoma (ccRCC) is a highly vascularized tumor characterized by biallelic inactivation of the tumor suppressor gene VHL and hyperactivation of the mTOR signaling pathway. The pro-angiogenic factor PDGFB is a transcriptional target of KLF6-a transcription factor driven by super-enhancers-and is capable of activating the mTORC1 signaling pathway in ccRCC. Here, researchers investigated whether ccRCC cells secrete PDGFB into the extracellular environment to stimulate mTORC1 signaling activity. They found that ccRCC cells secrete PDGFB; using ccRCC cells genetically engineered for KLF6 and PDGFB, they demonstrated a positive correlation between PDGFB secretion levels and the intracellular expression levels of KLF6 and PDGFB. Furthermore, in KLF6-knockdown or -knockout ccRCC cells, the reintroduction of either KLF6 or PDGFB sustained mTORC1 signaling activity. The researchers further confirmed that conditioned medium from PDGFB-overexpressing ccRCC cells could reactivate mTORC1 activity in KLF6-knockdown or -knockout cells. In summary, cancer cell-derived PDGFB mediates the activation of the mTORC1 signaling pathway in ccRCC, thereby further establishing the link between the KLF6-PDGFB axis and mTORC1 signaling activity in this cancer type.

Here, the researchers examined the levels of phosphorylated S6 protein-a readout of mTORC1 activity-in KLF6- and PDGFB-expressing 786-M1A iKLF6_2 cells that had undergone overnight serum starvation. The results showed that cells expressing KLF6 and PDGFB exhibited higher mTORC1 signaling activity compared to 786-M1A iKLF6_2 control cells (Figure 1a). Next, they tested whether secreted PDGFB could stimulate mTORC1 activity in a paracrine manner. The researchers subjected 786-M1A iKLF6_2 cells (in which mTORC1 activity was suppressed) to overnight serum starvation and subsequently cultured them for one hour under the following conditions: (i) serum-free medium, (ii) conditioned medium from parental 786-M1A cells, or (iii) conditioned medium from PDGFB-overexpressing 786-M1A cells. Prior to the collection of conditioned media, both the parental cells and the PDGFB-overexpressing cells had been cultured in serum-free medium overnight. Addition of conditioned medium from PDGFB-overexpressing cells to the KLF6-suppressed cells resulted in the reactivation of mTORC1 activity (Figure 1b). Conditioned medium from parental 786-M1A cells also stimulated mTORC1 activity, although the magnitude of induction was far lower than that observed with conditioned medium from PDGFB-overexpressing cells (Figure 1b).

Figure 1. Secreted PDGFB stimulates mTORC1 signalling pathway activation. (Abuhamad A Y, et al., 2023)