Statin prevents cancer development in chronic inflammation by blocking interleukin 33 expression

Abstract

Chronic inflammation is a major cause of cancer worldwide. Interleukin 33 (IL-33) is a critical initiator of cancer-prone chronic inflammation; however, its induction mechanism by the environmental causes of chronic inflammation is unknown. Herein, we demonstrate that Toll-like receptor (TLR)3/4-TBK1-IRF3 pathway activation links environmental insults to IL-33 induction in the skin and pancreas. FDA-approved drug library screen identified pitavastatin as an effective IL-33 inhibitor by blocking TBK1 membrane recruitment/activation through the mevalonate pathway inhibition. Accordingly, pitavastatin prevented chronic pancreatitis and its cancer sequela in an IL-33-dependent manner. IRF3-IL-33 axis was highly active in chronic pancreatitis and its associated pancreatic cancer in humans. Interestingly, pitavastatin use correlated with a significantly reduced risk of chronic pancreatitis and pancreatic cancer in patients. Our findings demonstrate that blocking the TBK1-IRF3 signaling pathway suppresses IL-33 expression and cancer-prone chronic inflammation. Statins present a safe and effective therapeutic strategy to prevent chronic inflammation and its cancer sequela.

Keywords: Interleukin-33; TBK1-IRF3 pathway; cancer prevention; chronic inflammation; chronic pancreatitis; pancreatic cancer; pitavastatin.

Figure 1. TBK1-IRF3 signaling induces IL-33 expression in chronic inflammation.

a, Representative images of IL-33 immunostaining on DNFB-treated skin and caerulein-treated pancreas compared with the acetone and PBS-treated controls, respectively. Note the nuclear IL-33 stains in the epithelial cells of the inflamed organs. b, The enrichment plot of TRIF mediated TLR3 signaling gene set in caerulein-treated compared with PBS-treated pancreas. c, The enrichment plot of activated TLR4 signaling gene set in caerulein-treated compared with PBS-treated pancreas. d, (Left) Immunoblot of p-TBK1, p-IRF3, p-NF-κB, TBK1, IRF3, NF-κB and GAPDH proteins in DNFB- versus acetone-treated WT skin (n=4 in each group). (Right) The ratio of p-TBK1/TBK1 and p-IRF3/IRF3 protein band intensity quantified from the immunoblot shown on the left. e, (Left) Immunoblot of p-TBK1, p-IRF3, p-NF-κB, TBK1, IRF3, NF-κB and GAPDH proteins in caerulein-versus PBS-treated WT pancreas (n=3 in caerulein and n=4 in PBS group). (Right) The ratio of p-TBK1/TBK1 and p-IRF3/IRF3 protein band intensity quantified from the immunoblot shown on the left. f, Il33 expression in Pam212 cells at 6 hours after treatment with poly(I:C), LPS and PBS (n=4 in each group). g, Time course of p-TBK1, p-IRF3, TBK1, IRF3 and GAPDH protein expression in Pam212 cells after poly(I:C) treatment. h, Il33 expression in Trif siRNA (siTrif)- versus siRNA control (siCon)-treated Pam212 cells in response to poly(I:C) versus PBS treatment (n=4 in each group). i, Chromatin Immunoprecipitation (ChIP)-qPCR assay for p-IRF3 binding to Il33 promoter region after the treatment of Pam212 cells with poly(I:C) versus PBS (n=4 in each group). Note the presence of a sequence very similar to the IRF3 binding motif in the Il33 promoter region. j, Il33 expression in siIrf3- versus siCon-treated Pam212 cells in response to poly(I:C) versus PBS treatment (n=4 in each group). k, Il33 expression levels in DNFB-treated WT versus Irf3^KO skin (n=8 in WT and n=5 in Irf3^KO group). l, IL-33 protein levels in DNFB-treated WT versus Irf3^KO skin (n=9 in WT and n=10 in Irf3^KO group). m, Epidermal thickness in DNFB-treated WT versus Irf3^KO skin. Each dot represents the average of three measurements in an HPF image. Ten random HPF images per skin sample are included (n=6 in each group). n, Mast cell counts in DNFB-treated WT versus Irf3^KO skin. Each dot represents cell counts from an HPF image. Three randomly selected HPF images are included per sample (n=10 in each group). o, Il33 expression levels in caerulein-treated WT versus Irf3^KO pancreas (n=6 in each group). p, IL-33 protein levels in caerulein-treated WT versus Irf3^KO pancreas (n=6 in WT and n=7 in Irf3^KO group). q, CD45⁺ immune cell counts in caerulein-treated WT versus Irf3^KO pancreas. Each dot represents cell counts from an HPF image. Three randomly selected HPF images are included per sample (n=3 in each group). Graphs show mean + SD, Fig. 1d, e, i, k-q: unpaired t test, Fig 1f, h, j: one-way ANOVA, scale bar: 100 μm

Figure 2. Pitavastatin inhibits IL-33 expression by blocking GGPP-dependent TBK1 activation.

a, Il33 expression in poly(I:C)-treated Pam212 cells that received pitavastatin, BX795, Zoliadic acid (Zol) or PBS (n=4 in each group). b, (Left) Immunoblot of p-TBK1, p-IRF3, TBK1, IRF3, and GAPDH proteins in whole cell lysates of poly(I:C)-treated Pam212 cells that received pitavastatin alone or in combination with GGPP. (Right) The ratio of p-TBK1/TBK1 and p-IRF3/IRF3 protein band intensity from immunoblots (n=3 in each group). c, (Left) Immunoblot of p-TBK1, TBK1, Na-K-ATPase, and GAPDH proteins in membrane and cytosol fraction of poly(I:C)-treated Pam212 cells that received pitavastatin alone or in combination with GGPP. (Right) The ratio of membrane-bound p-TBK1/Na,K-ATPase protein band intensity from the immunoblots (n=3 in each group). d, Il33 expression in poly(I:C)-treated Pam212 cells that received pitavastatin alone or in combination with GGPP (n=7 in each group). e, Schematic diagram of pitavastatin mechanism of action in blocking mevalonate pathway-GGPP mediated TBK1-IRF3 signaling activation. Graphs show mean + SD, one-way ANOVA.

Figure 3. Pitavastatin blocks chronic pancreatitis and its cancer sequela.

a, (Left) Immunoblot of p-TBK1, p-IRF3, p-NF-kB, TBK1, IRF3, NF-kB and GAPDH proteins in pitavastatin-versus PBS-treated WT pancreas at the completion of caerulein treatment protocol (n=4 in each group). (Right) The ratio of p-TBK1/TBK1 and p-IRF3/IRF3 protein band intensity quanti ed from the immunoblot shown on the left. b, Il33 expression in pitavastatin- versus PBS-treated WT pancreas at the completion of caerulein treatment protocol (n=9 in pitavastatin and n=7 in PBS group). c, IL-33 protein levels in pitavastatin- versus PBS-treated WT pancreas at the completion of caerulein treatment protocol (n=10 in pitavastatin and n=9 in PBS group). d, CD45⁺ immune cell counts in pitavastatin- versus PBS-treated WT pancreas at the completion of the caerulein treatment protocol. Each dot represents cell counts from an HPF image. Three randomly selected HPF images are included per sample (n=5 in pitavastatin and n=4 in PBS group). e, Representative images of macroscopic, hematoxylin and eosin (H&E), and Alcian blue-stained pancreatic tumors from pitavastatin- versus PBS-treated KPC mice that underwent caerulein-induced pancreatic cancer protocol. f, Ratio of pancreatic tumor per body weight in pitavastatin- versus PBS-treated KPC mice at the completion of caerulein-induced pancreatic cancer protocol (n=6 in each group). g, Percent mucin-positive cells in pitavastatin- versus PBS-treated KPC tumors at the completion of caerulein-induced pancreatic cancer protocol. Each dot represents % mucin-positive cells in an HPF image. Two to three randomly selected HPF images are included per sample (n=6 in each group). Graphs show mean + SD, unpaired t test, scale bars: 1cm (macroscopic images), 100 μm (microscopic images).

Figure 4. IRF3-IL-33 signaling axis is highly active in human chronic pancreatitis-associated pancreatic cancer, and pitavastatin reduces the risk of chronic pancreatitis and pancreatic cancer in patients.

a, Representative images of IL-33 and IRF3 immunostaining on adjacent sections of matched normal pancreas, chronic pancreatitis, and PDAC collected from pancreatic cancer patients. b, IL-33⁺ epithelial cell counts per HPF in the matched samples from human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=15 patients, paired t-test). c, IRF3⁺ epithelial cell counts per HPF in the matched samples from human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=15 patients, paired t-test). d, The correlation between IL-33⁺ and IRF3⁺ cell counts across normal pancreas, chronic pancreatitis, and PDAC samples (n=45 matched samples from 15 patients, t-test for the Pearson correlation coefficient). e, Box plot of IL33 expression in pancreatic cancer versus normal pancreas across TCGA/GTEx datasets (* P < 0.01, one-way ANOVA, Gene Expression Pro ling Interactive Analysis database). f, A retrospective cohort analysis of chronic pancreatitis and pancreatic cancer risk in matched cohorts of patients treated with pitavastatin (test) versus ezetimibe (control). Graphs show mean + SD, scale bar: 100 μm