JMJD3 acts as a tumor suppressor in PDAC
JMJD3 expressions in tissues from 132 PDAC patients and 12 normal individuals were analyzed by immunohistochemical staining. JMJD3 expression levels were much lower in tumor tissues compared to the normal pancreatic tissues (Fig. 1A). The primary tumors showed higher overall JMJD3 expression than the metastatic tumors, and a statistically significant correlation between JMJD3 expression levels and tumor grades were found (Fig. 1B). Importantly, low expression of JMJD3 was correlated with shorter overall survival in PDAC patients (Fig. 1C), which was further validated by using GEO data (Fig. S1A). We examined the in vivo anti-cancer activities of JMJD3 by over-expressing wild type (wt) JMJD3, or mutant JMJD3 which lacked H3K27me3 demethylase activity, in PDAC cells. The tumors in control group showed a faster and more stable growth. The mutant JMJD3 treated group showed a significant tumor inhibition, and there was no tumor formed after transfected with wt JMJD3 (Fig. 1D, E, F). In in vitro studies also revealed a tumor suppressive role of JMJD3 in PDAC (Fig. S1B, C, D, E, F, G).
Jmjd3 Regulates Hkdc1 Expression By Histone Demethylation
We found that JMJD3 caused no changes on the p16INK4A, p19Arf and p53 expression levels in PDAC cell (Fig. S2A). To further elucidate the role of JMJD3 in PDAC, we performed the genome-wide gene expression microarray to screen differentially expressed genes in wt JMJD3/mutant JMJD3 over-expressed Panc-1 cells. AGR2 and HKDC1 were the most up-regulated genes after ectopic expression of wt JMJD3. However, AGR2 was also up-regulated upon mutant JMJD3 over-expression, indicated that AGR2 was not involved in JMJD3 demethylase activity. Then, HKDC1 was further studied in PDAC (Fig. S2B, C, D and supplementary Table 2).
Both mRNA and protein level of HKDC1 were upregulated by wt JMJD3 in PDAC cells (Fig. 2A, Fig. S3A). The expression of HKDC1 was decreased by the knockdown of JMJD3 in HPDE cells (Fig. 2B). To determine whether HKDC1 was regulated under histone modification, we firstly treated PDAC cells with DZnep which was an H3K27me3 inhibitor[21]. DZnep increased HKDC1 expression in PDAC cells (Fig. 2C), indicated that histone H3K27me3 may serve to inhibit HKDC1 transcription. By ChIP assay, a higher H3K27me3 mark at HKDC1 promoter in PDAC cells (Panc-1 and SW1990) was identified when compared to that in human pancreatic duct epithelial (HPDE) cells (Fig. 2D). JMJD3 was reported to pave the way for the RNA pol Ⅱ progression, then activated the transcription of JMJD3 downstream targets[22]. By ChIP assay with Pol Ⅱ antibody, we found a significant loss of pol Ⅱ and JMJD3 enrichment at HKDC1 promoters in PDAC cells when compared to that in HPDE cells (Fig. 2D). As a high H3K27me3 level was observed at HKDC1 promoter in PDAC cells, we speculated whether ectopic expression of JMJD3 alone would be sufficient to induce HKDC1 transcription by reducing H3K27me3 at HKDC1 loci. By ChIP assay using antibodies specific for JMJD3, we found that ectopic expression of JMJD3 increased the binding of JMJD3 to HKDC1 promoter and correlated with a decrease of H3K27me3 levels at HKDC1 locus (Fig. 2E). These results suggested that JMJD3 contributed to the transcriptional induction of HKDC1 by demethylating H3K27me3. We also performed ChIP assays using JMJD3-knockdown HPDE cells. The result showed that depletion of JMJD3 impaired the H3K27me3 reduction and repressed HKDC1 transcription (Fig. 2F). These results suggested that JMJD3 activated HKDC1 expression by demethylating H3K27me3 associated with the locus.
The Tumor Suppressive Role Of Hkdc1 In Pdac
We performed immunohistochemistry to examine HKDC1 expression in a PDAC tissue microarray. We found that HKDC1 expression was much lower in tumor tissues compared to tumor-adjacent normal tissues (Fig. 3A), and negatively correlated with tumor grades (Fig. 3B). A significant positive correlation between expression of JMJD3 and HKDC1 was proved in PDAC tissues (Fig. 3C). Low expression of HKDC1 was correlated with shorter overall survival in 25 PDAC patients (Fig. 3D). Meanwhile, In in vitro studies also proved a tumor suppressive role of HKDC1 in PDAC (Fig. S3B, C, D, E). Low expression of HKDC1 was correlated with shorter overall survival which was also validated by using GEO data (Fig. S3F).
We next studied the effect of HKDC1 knockdown to the growth inhibition induced by JMJD3. It showed that the expression of HKDC1 decreased significantly in JMJD3 over-expressed PDAC cells upon knockdown of HKDC1 (Fig. 3E). By MTT assay, we found that JMJD3-induced growth inhibition could be reversed by knockdown of HKDC1 (Fig. 3F). The colony formation assay also showed that knockdown of HKDC1 increased the number of colony formation by PDAC cells when compared to cells over-expressing JMJD3 only (Fig. 3G).
HKDC1 bond to Spectrin beta Ⅱ to suppress PDAC cell growth
Bioinformatics prediction suggested that HKDC1 contain a spectrin binding domain (SBD) (Fig. S4A). We found that HKDC1 co-localized with spectrin in HPDE cells (Fig. 4A). We then constructed a series of plasmids which prokaryotically expressed truncated or full length HKDC1, Spectrin alpha Ⅱ and Spectrin beta Ⅱ recombinant proteins (Fig. S5A, B, C, D). By binding assay, the interaction between full length HKDC1 and C-terminal domain of Spectrin beta Ⅱ were observed (Fig. 4B). To further prove whether HKDC1 bound to Spectrin beta Ⅱ through SBD, two other HKDC1 recombinant proteins contain SBD only or the SBD-deleted (HKDC1△SBD) were used in binding assay. The SBD only protein bond to spectrin beta Ⅱ fragments (Fig. 4C). These results indicated that HKDC1 bond to C-terminal domain of spectrin beta Ⅱ through SBD.
We next examined the interaction between HKDC1 and Spectrin beta Ⅱ in vitro. Firstly, we found that HKDC1 bound to Spectrin beta Ⅱ (Fig. 4D). When HKDC1 was inhibited, the interaction between HKDC1 and Spectrin beta Ⅱ was diminished (Fig. 4D). By reverse-immunoprecipitation, knockdown of Spectrin beta Ⅱ also diminished the interaction between these two proteins (Fig. 4E). Moreover, after ectopic expression of HKDC1 and Spectrin beta Ⅱ with specific tags in Panc-1 cells, the binding between HKDC1 and Spectrin beta Ⅱ was further confirmed (Fig. 4F). We then identified that SBD domain was needed while HKDC1 exhibited its anti-cancer activities in PDAC cells (Fig. S4B, C).
HKDC1 disrupted the Spectrin beta Ⅱ-Spectrin alpha Ⅱ tetramerization by competing with Spectrin alpha Ⅱ for binding to Spectrin beta Ⅱ in PDAC cells
Previous study reported that Spectrin alpha Ⅱ and Spectrin beta Ⅱ formed a stable tetramer[23]. Our study showed that HKDC1 bound to Spectrin beta Ⅱ and ectopic expression of JMJD3 caused no expression changes of Spectrin beta Ⅱ in PDAC cells (Fig. S6A). We found that Spectrin beta Ⅱ bound to Spectrin alpha Ⅱ in PDAC cells (Fig. 5A). In turn, we showed that there was a decreased binding between these two proteins in PDAC cells with JMJD3 ovexpression (Fig. 5A). From the extracellular binding assay, we found the binding between spectrin beta Ⅱ and spectrin alpha Ⅱ was diminished by presenting HKDC1 recombinant protein (Fig. 5B). These results showed that HKDC1 competed with Spectrin alpha Ⅱ for binding with Spectrin beta Ⅱ in PDAC cells.
Spectrin beta Ⅱ-Spectrin alpha Ⅱ tetramerization was one of the important components of cytoskeleton. We further asked whether ectopic expression of HKDC1 disrupted the cytoskeleton in PDAC cells. We found that co-localization of HKDC1 and spectrin beta Ⅱ (orange color) in JMJD3 over-expressed cells but not in vehicle control (Fig. 5C). Spectrin beta Ⅱ and Spectrin alpha Ⅱ co-localization was diminished in JMJD3 over-expressed cells (Fig. 5D). Finally, the binding between HKDC1 and Spectrin beta Ⅱ was found to disrupt the cytoskeleton in PDAC cells which may be involved in PDAC progression ((Fig. S6A, B, C, D).