IL-13Rα2 expression in pancreatic cancer cell lines
Eleven pancreatic cancer cell lines and three types of normal cell lines (fibroblast, umbilical vein endothelial cells and pancreatic ductal epithelial cells) were examined for IL-13Rα2
expression. qRT-PCR analysis identified five pancreatic cancer cell lines (HS766T, MIAPaCa2, KLM, SW1990 and BxPC3), which expressed high levels of IL-13Rα2
mRNA, and six cell lines (Panc-1, ASPC-1, HPAF-II, Mpanc96, PK-1 and Capan-1) expressed low levels IL-13Rα2
mRNA (negative cell line) (Figure ). All three normal cell lines showed extremely low levels of IL-13Rα2
mRNA. We also examined IL-13Rα2 protein expression in these cell lines by flow-cytometric analysis using monoclonal antibody to IL-13Rα2. These results essentially corroborated the mRNA results (data not shown) [15
Figure 1 IL-13Rα2 expression in pancreatic cancer and normal cell lines and DNA methylation and Histone modification of IL-13Rα2 promoter. A, qRT-PCR for IL-13Rα2 expression in pancreatic cancer and normal cell lines was performed. Data (more ...)
Mutation analysis of IL-13Rα2 cDNA
We investigated whether there were gene sequence changes in the IL-13Rα2 gene by performing sequencing of IL-13Rα2 cDNA. However, no mutations were detected in any pancreatic cancer cell lines studied (data not shown).
DNA methylation in IL-13Rα2 promoter
We next examined any epigenetic changes in IL-13Rα2
gene. Since there is only one CpG site in the IL-13Rα2
promoter region, we examined DNA methylation at this site [32
]. We picked more than 10 independent clones for analysis. In at least 80% of the clones tested from all cell lines including three normal cell lines, no methylation was detected (Figure ). As a control, we also studied DNA methylation of other CpG sites located ~100 bases upstream from the IL-13Rα2
promoter region. In contrast to the CpG in the IL-13Rα2 promoter region, the distant CpG site showed methylation in all cell lines (Supplementary Figure ).
Regulation of histone acetylation and methylation in IL-13Rα2 promoter region
We also examined histone acetylation of the IL-13Rα2 promoter region using a chromatin-immunoprecipitation technique (ChIP). In all IL-13Rα2-positive pancreatic cell lines, histone H3 was highly acetylated compared to IL-13Rα2-negative and normal cell lines (Figure ). Similar acetylation results were observed for histone H4. In sharp contrast, the methylation status at the H3K9 site, which is a site for transcriptional repression, was high in IL-13Rα2-negative cell lines compared to IL-13Rα2-positive cell lines (Figure ).
Next, we examined the effect of histone acetylation inhibition by HDAC inhibitors on IL-13Rα2 expression. When pancreatic cancer lines expressing undetectable levels of IL-13Rα2 were treated with TSA, histone H3 and H4 acetylation was dramatically increased. TSA also increased acetylation in pancreatic cancer cells expressing high levels of IL-13Rα2 but this increase was less dramatic (Figure ). In contrast, TSA caused a significant decrease in H3K9 methylation in pancreatic cancer cells with undetectable levels of IL-13Rα2 expression but no change in high IL-13Rα2 expressing cell lines (Figure ).
Histone deacetylation inhibition increases IL-13Rα2 expression in pancreatic cancer cell lines
As the relationship between histone acetylation and IL-13Rα2 expression levels was observed, we tested whether HDAC inhibitors can modulate IL-13Rα2 expression in pancreatic cancer cell lines. Interestingly, similar to histone acetylation, TSA treatment resulted in increased IL-13Rα2 mRNA expression in pancreatic cancer cell lines that normally have undetectable levels of IL-13Rα2 expression, while no changes were seen in cells expressing high levels of IL-13Rα2 mRNA or normal cell lines (Figure ). Similar results were obtained with another HDAC inhibitor, sodium butyrate (NaB) (Figure ).
Figure 2 Regulation of IL-13Rα2 expression by HDAC and AP-1 inhibitors. A, Conventional RT-PCR of IL-13Rα2 mRNA after incubation with TSA. Cells were incubated with 1 or 5 μM TSA for 24 hours and total RNA was extracted. PM-RCC cells were (more ...)
Role of AP-1 transcription factor activity in IL-13Rα2 regulation in pancreatic cancer cell lines
To determine the mechanism of the differential effect of HDAC inhibition in cells expressing undetectable levels of IL-13Rα2, we examined whether the transcription factor (AP-1) is activated in these cell lines as reported by Wu et al. [32
]. We found that pancreatic cancer cell lines that highly express IL-13Rα2
(HS766T, MIAPaCa2, and KLM), and those which express undetectable levels (Panc-1 and ASPC-1), both show high c-jun activity (Supplementary Figure ). In contrast, normal cell lines showed low c-jun activity. We did not observe any significant differences in c-Fos activity, another AP-1 member (Supplementary Figure ) between cancer and normal cell lines.
Interestingly, when high IL-13Rα2-expressing cells were treated with the c-jun N-terminal kinase inhibitor, SP600125, IL-13Rα2 expression decreased (Figure ), whereas SP600125 had no effect on cells expressing undetectable levels of IL-13Rα2. Another pan-AP-1 inhibitor, SR11302, also decreased IL-13Rα2 expression in IL-13Rα2 expressing cell lines in a concentration-dependent manner (Figure ). The effects of TSA and SP600125 on IL-13Rα2 protein expression in pancreatic cancer cells were also analyzed by IHC. IL-13Rα2 protein levels were also found to increase in the presence of TSA and decrease in the presence of SP600125. In addition, SP600125 prevented the increase of IL-13Rα2 protein by TSA (Figure ).
Figure 3 Modulation of IL-13Rα2 protein by HDAC and AP-1 inhibitors and stability of IL-13Rα2 expression. A, ICC of IL-13Rα2 after incubation with TSA and SP600125 is shown. Cells were incubated with 1 μM TSA and/or 10 μM (more ...)
Stability of upregulated IL-13Rα2 expression by HDAC inhibitor
We examined the stability of upregulated IL-13Rα2 expression in IL-13Rα2-expressing and negative pancreatic cancer cell lines when treated with HDAC inhibitor. After treatment with TSA and SP600125 for 24 hours, the drugs were removed and cell culture was continued. IL-13Rα2 expression was still elevated 3 days after TSA removal in IL-13Rα2 undetectable cell lines (Figure ). In contrast, in IL-13Rα2 positive cell lines, IL-13Rα2 expression returned to pre-treatment levels within 24 hours following SP600125 removal (Figure ).
HDAC inhibition increases IL-13 induced matrix metalloproteinases via IL-13Rα2 upregulation
As we have shown that IL-13 can upregulate Matrix metalloproteinases (MMPs) expression in IL-13Rα2 expressing pancreatic cancer cell lines [28
], we investigated the impact of IL-13Rα2 upregulation by HDAC inhibitors by examining IL-13 induced MMPs expression. TSA treatment increased mRNA expression for MMPs
through upregulation of IL-13Rα2
after treatment with IL-13 in two IL-13Rα2 negative cell lines (Figure ). Interestingly, when IL-13 signaling was blocked by an inhibitor of the AP-1 pathway (SP600125), it prevented the increase in MMPs expression by TSA. Thus, MMPs
expression showed a positive correlation with IL-13Rα2
expression in IL-13 treated cells.
Figure 4 HDAC inhibitor inhibits MMPs expression activated by IL-13 through induction of IL-13Rα2. A, Conventional RT-PCR for expression of MMPs was performed after cells were incubated with 1 μM TSA and/or 10 μM SP600125 for 24 hours. (more ...)
To confirm whether TSA increased MMPs expression as a result of IL-13Rα2 induction, we conducted a knock-down of the IL-13Rα2 gene using two different sequences of siRNA in Panc-1 and ASPC-1 cell lines. MMPs expression was suppressed in IL-13Rα2 knock-down cells treated with TSA (Figure ).
HDAC inhibition increases the anti-cancer effect of IL-13-PE targeting IL-13Rα2 in vitro and in vivo
As HDAC inhibition increased IL-13Rα2 expression in IL-13Rα2-negative but not in normal cell lines, we examined whether HDAC inhibition enhanced the anti-cancer effect of IL-13-PE in IL-13Rα2-negative pancreatic cancer cell lines. The anti-cancer effect of IL-13-PE was evaluated using a protein synthesis inhibition assay in vitro (Figure ). IL-13-PE inhibited protein synthesis in IL-13Rα2-positive cancer cells (IC50 between 10 and 50 ng/ml) without TSA, but not in IL-13Rα2-negative cancer cells nor normal cells (IC50 > 1000 ng/ml). TSA treatment enhanced the cytotoxicity of IL-13-PE in IL-13Rα2-negative cancer cells (IC50 40-50 ng/ml with 5 μM TSA), but not in normal cells (IC50 > 1000 ng/ml with 5 μM TSA).
Figure 5 HDAC inhibitors induce anti tumor effect of IL-13Rα2 targeted immmunotoxin IL13-PE in IL-13Rα2-negative pancreatic cancer cell lines. A, Cytotoxicity assay was performed in IL-13Rα2-negative and -positive pancreatic cancer and (more ...)
We next examined the enhancement of the anti-cancer effect of IL-13-PE by HDAC inhibition in xenograft mouse models of human cancer. IL-13Rα2-negative pancreatic cancer cell lines (Panc-1 and ASPC-1) were implanted in the flanks of immunodeficient mice and treated with two different HDAC inhibitors, TSA and SAHA followed by IL-13-PE immunotoxin. Neither TSA nor IL-13-PE alone affected the tumor growth, but when combined, a dramatic inhibition of tumor growth was observed (Figure and ). In contrast, when IL-13Rα2 was knocked-down prior to TSA therapy, the anti-tumor effect of combination of TSA and IL-13-PE was completely eliminated compared to mock vector transfected tumors, which showed dramatic tumor response (Figure ).
A second HDAC inhibitor, SAHA, itself showed some anti-cancer effect in two tumor models (Figure and ). However, when mice were treated with SAHA followed by IL-13-PE, a significant decrease in tumor size was observed. In addition, 50% of mice showed complete elimination of their tumors in combination group.
Next, we evaluated anti-cancer effect of combination of SAHA and IL-13-PE in IL-13Rα2-positive pancreatic cancer model (HS766T and MIA-PaCa2). We observed that IL-13-PE could significantly decrease tumor size in both IL-13Rα2-positive tumors (Figure and ). But when combined with SAHA, IL-13-PE not only decreased tumor size but also completely eliminated tumors in 66 to 83% of mice. These data suggest that SAHA can enhance anti-cancer effect of IL-13-PE even in IL-13Rα2-positive pancreatic cancers.
We monitored the body weight of mice and their general condition throughout the experimental period and detected no adverse effects caused by the treatment (data not shown). In addition, we observed no organ toxicity in vital organs such as the liver, brain, lung, kidney, pancreas and spleen of IL-13-PE and HDAC inhibitor-treated mice evaluated by histological examination (Supplementary Figure )
HDAC inhibitor significantly increased IL-13Rα2 in the pancreatic tumors implanted in the mice but not in mice organs
After SAHA and IL-13-PE treatment, implanted tumors and mice organs (liver, brain, pancreas, kidney, spleen and lung) were harvested and IL-13Rα2 expression was examined at mRNA and protein levels. Human IL-13Rα2 mRNA was significantly increased in tumors in both SAHA treated mice (Figure ) and TSA treated mice (Supplementary Figure ). IL-13-PE treatment had no effect by itself but in combination with SAHA, a significant decrease in IL-13Rα2 expression was observed. In contrast, none of the organs except brain showed a modest increase in mouse IL-13Rα2 mRNA expression (Figure ).
Figure 6 IL-13Rα2 expression is upregulated in pancreatic tumors but not in organs of mice after treatment with HDAC inhibitor, SAHA. A, qRT-PCR of human IL-13Rα2 in implanted pancreatic tumors after SAHA and IL-13-PE treatment. Tumors were harvested (more ...)
We also examined IL-13Rα2 protein expression by IHC. Similar to mRNA results, human IL-13Rα2 was dramatically increased in tumors from SAHA treated mice and when combined with IL-13-PE, a decrease in IL-13Rα2 expression was observed (Figure ). In normal tissues, mouse IL-13Rα2 was not detected or levels were below the detection limit of the assay in all organs examined (Figure ).