Panoply™ Human JAK1 Knockdown Stable Cell Line

Loss-of-function (LOF) mutations in JAK1, a member of the JAK kinase family, frequently occur in endometrial cancer (EC), suggesting that JAK1 may play a tumor suppressor role, at least in EC. However, the mechanisms by which JAK1 regulates tumorigenesis remain unclear. Here, researchers found that JAK1 is frequently mutated and downregulated in EC. JAK1 knockdown promoted EC cell proliferation and migration, while JAK1 overexpression inhibited EC cell proliferation and migration. Transcriptional profiling of JAK1-deficient EC cells unexpectedly revealed activation of the hypoxia-inducible factor (HIF) pathway. Mechanistically, JAK1 interacts with HIF-1/2α and reduces HIF1/2-α protein expression under hypoxic conditions. HIF-1/2α knockdown reversed the growth and migration of EC cells induced by JAK1 knockdown under hypoxic conditions. Both JAK1 knockdown and inhibition of JAK1 kinase activity with Ruxolitinib upregulated the transcription of HIF target genes under hypoxic conditions. JAK1 overexpression downregulated the transcription of HIF target genes under hypoxic conditions. These findings provide new insights into the functional link between JAK1 LOF mutations and aberrant activation of the HIF pathway in EC, and suggest that pharmacological inhibition of HIF1/2 may be an effective therapeutic strategy for JAK1-mutant EC.

Here, researchers found that under hypoxic conditions, the expression levels of HIF1α and HIF2α mRNA did not change significantly in parental cells and JAK1 knockdown cells (KLE and SPEC-2 cells) (Figure 1A and B). HIF-1α and HIF2α proteins are rapidly stabilized under hypoxic conditions but are degraded under normoxic conditions. As expected, hypoxic treatment resulted in a significant time-dependent increase in HIF1α and HIF2α protein levels. Notably, in JAK1 knockdown KLE and SPEC-2 cells, the hypoxia-induced HIF-1/2α protein expression levels were higher than in control KLE and SPEC-2 cells (Figure 1C and D). JAK1 is a protein kinase. Therefore, the researchers evaluated whether inhibiting JAK1 kinase activity would alter HIF-1/2α protein levels. They found that treatment with the potent JAK1/2 inhibitor Ruxolitinib increased HIF-1/2α protein levels in KLE and SPEC-2 cells (Figure 1E and F). Furthermore, exogenous overexpression of Myc-JAK1 reduced Flag-HIF-1/2α protein levels in a dose-dependent manner (Figure 1G). Under hypoxic conditions, endogenous HIF-1/2α physically interacted with JAK1 in KLE cells (Figure 1H). Finally, the researchers demonstrated that exogenous overexpression of Myc-JAK1 interacted with Flag-HIF-1/2α (Figure 1I). These results suggest that JAK1 may reduce the expression of HIF-1/2α protein by directly interacting with HIF-1/2α.

Figure 1. JAK1 interacts with HIF-1/2α and reduces HIF-1/2α protein levels. (Lin Q, et al., 2022)