Aurora kinase A promotes inflammation and tumorigenesis in mice and human gastric neoplasia

Abstract

Background & aims: Chronic inflammation contributes to the pathogenesis of gastric tumorigenesis. The aurora kinase A (AURKA) gene is frequently amplified and overexpressed in gastrointestinal cancers. We investigated the roles of AURKA in inflammation and gastric tumorigenesis.

Methods: We used quantitative real-time reverse transcription polymerase chain reaction, immunofluorescence, immunohistochemistry, luciferase reporter, immunoblot, co-immunoprecipitation, and in vitro kinase assays to analyze AGS and MKN28 gastric cancer cells. We also analyzed Tff1(-/-) mice, growth of tumor xenografts, and human tissues.

Results: We correlated increased expression of AURKA with increased levels of tumor necrosis factor-α and inflammation in the gastric mucosa of Tff1(-/-) mice (r = 0.62; P = .0001). MLN8237, an investigational small-molecule selective inhibitor of AURKA, reduced nuclear staining of nuclear factor-κB (NF-κB) p65 in human gastric cancer samples and mouse epithelial cells, suppressed NF-κB reporter activity, and reduced expression of NF-κB target genes that regulate inflammation and cell survival. Inhibition of AURKA also reduced growth of xenograft tumors from human gastric cancer cells in mice and reversed the development of gastric tumors in Tff1(-/-) mice. AURKA was found to regulate NF-κB activity by binding directly and phosphorylating IκBα in cells. Premalignant and malignant lesions from the gastric mucosa of patients had increased levels of AURKA protein and nuclear NF-κB, compared with healthy gastric tissue.

Conclusions: In analyses of gastric cancer cell lines, human tissue samples, and mouse models, we found AURKA to be up-regulated during chronic inflammation to promote activation of NF-κB and tumorigenesis. AURKA inhibitors might be developed as therapeutic agents for gastric cancer.

Keywords: AURKA; Aurora kinase A; Gene Regulation; Mouse Model; NF-κB; Stomach Cancer; TNF; TNF-α; mRNA; messenger RNA; nuclear factor κB; tumor necrosis factor.

Figure 1. AURKA expression levels directly correlate with inflammation in Tff1-knockout mouse model

A) Immunohistochemical staining of Tff1-knockout mouse (KO) (2 months of age) stomach tissue shows increased AURKA protein levels as compared to wild-type mice (WT). B) Real-time RT-PCR analysis of Tff1-knockout mouse gastric tissue showed significant increase in AURKA expression levels at 2 and 4 months of age relative to wild-type (p<0.001). C) AURKA mRNA expression levels are significantly associated with inflammation scores in Tff1-knockout animals (p=0.0001). D) Pro-inflammatory cytokine Tnf-α mRNA expression was significantly up-regulated in Tff1-knockout mice at 2 and 4 months of age (p<0.02) (left panel), and directly correlated with increased AURKA mRNA levels (p=0.0007) (right panel).

Figure 2. AURKA regulates NF-κBp65 nuclear translocation ex vivo and in vitro

A) Antrum epithelial cells were extracted from Tff1-knockout mice. Selectively cultured epithelial cells were treated with vehicle (ctrl) or MLN8237 (500 nmol/L) and subjected to immunofluorescence analysis of NF-κBp65. Original magnification is shown at ×100. Treatment with MLN8237 significantly reduced nuclear NF-κBp65–positive staining relative to vehicle-treated control (p<0.001). B–C) NF-κBp65 immunofluorescence in AGS (B) and MKN28 (C) cells after 30 min treatment with NF-κB inhibitor Bay 11-7085 (10 μmol/L) or AURKA inhibitor MLN8237 (500 nmol/L). Nuclear localization of NF-κBp65 is shown in green. DAPI (blue) was used as a nuclear counterstain. Graphs indicate the quantification of nuclear NF-κBp65–positive staining in at least 200 counted cells presented as a percentage. D) Western blot analysis of AURKA, p-NF-κBp65 (S536), and NF-κBp65 proteins in AGS and MKN28 cell lines suggesting a correlation between AURKA and p-NF-κBp65 (S536) protein levels.

Figure 3. AURKA activates NF-κB through regulation of IκBα

A–B) Left panels show NF-κB Luciferase reporter assay in AGS (A) and MKN28 (B) cells infected with control or AURKA adenoviruses with or without TNF-α treatment (50 ng/ml). Right panels show NF-κB Luciferase reporter assay in AGS (A) and MKN28 (B) cells treated with either Bay κ11-7085 (10 μmol/L) or MLN8237 (500 nmol/L). The % relative luciferase units (RLU) results indicate that AURKA can enhance NF-κB luciferase reporter activity. C) Western blot analysis of the indicated proteins in AGS cells infected with control or AURKA adenoviruses and treated with vehicle or MLN8237 (5 μmol/L). D) Western blot analysis of the indicated proteins in AGS cells after knocking down AURKA with siRNA. E) Western blotting for the indicated proteins in MKN28 cells treated with the selected doses of MLN8237 or Bay 11-7085 (10 μmol/L). The results indicate that NF-κB activation by AURKA is mediated by IκBα.

Figure 4. AURKA directly binds and phosphorylates IκBα in vitro

A) The in vitro kinase assay with IκBα and increasing concentrations of AURKA recombinant proteins was performed. Assay products were analyzed by Western blotting for p-IκBα (S32), IκBα, and AURKA proteins. GPX7 protein was used as a negative control. B) The in vitro kinase assay for AURKA, IκBα, and increasing concentrations of MLN8237 was carried out, and the proteins were subjected to Western blot analysis. C) In vitro kinase assay for AURKA and IκBα with or without MLN8237 (500 nmol/L) was performed and followed by Western blotting for p-AURKA (T288), AURKA, p-IκBα (S32), and IκBα. D) Following the in vitro kinase assay (panel C), AURKA or IκBα pull-down products were subjected to Western blot analysis. The data indicate that AURKA directly binds and phosphorylates IκBα protein. E) Endogenous AURKA or IκBα pull-down proteins were subjected to Western blot analysis of AURKA in MKN28 cells. *, IgG heavy chain.

Figure 5. AURKA inhibition induces anti-inflammatory and anti-survival signaling in vivo

A) Proteins extracted from 8-day MLN8237-treated Tff1-knockout mice or control animals were analyzed by Western Blot for p-NF-κBp65 (S536) and NF-κBp65 proteins. B) Real-time RT-PCR analysis demonstrated the expression levels of pro-inflammatory and pro-survival NF-κB target genes in the Tff1-knockout animals in control or in 8 days MLN8237 treated animals. AURKA inhibition significantly attenuated mRNA expression of these genes.

Figure 6. MLN8237 treatment reduces tumor growth in vivo

A) A representative microPET coronal view of a mouse injected with [18F]FLT and imaged before and after the MLN8237 treatment. The tumors are invisible after 12 weeks post treatment. Because of the high uptake, kidneys showed typical [18F]FLT staining. B) Representative images of the antrum and H&E staining of gastric mucosa of Tff1-knockout mice. While tumors were visibly large in control (upper left panel), they became undetectable or small after 3 months of treatment (lower left panel). H&E staining indicated adenocarcinoma in control animals (upper middle and right panels) whereas MLN8237-treated animals exhibited low grade dysplasia (lower middle and right panels). C) AGS tumor xenografts were treated with MLN8237 (30 mg/kg) for 28 days. Tumor volume was dramatically reduced in the treated group as illustrated in the graph (p<0.01) (left panel). After collection of tumors at the end of experiment, representative tumors from the control or MLN8237 treated groups are shown (right panel). D) Real-time RT-PCR analysis of XIAP in xenograft tumors in control or ML8237- treated animals. E) Representative images of immunohistochemical analysis of Ki-67 protein expression in MLN8237-treated or control xenografts. F) Representative images of Immunohistochemical analysis of cleaved caspase-3 protein expression in MLN8237-treated or control xenografts.

Figure 7. Expression of AURKA and NF-κB is associated with human gastric cancer development and progression

A) IHC staining for AURKA and NF-κB on consecutive replicates of the same tissue sample. Normal gastric glands with negative AURKA and NF-κB immunostaining are indicated by arrow-heads whereas glands with intestinal metaplasia showing positive AURKA and NF-κB staining are indicated with arrows. B) IHC staining for AURKA and NF-κB on consecutive replicates of moderately differentiated gastric adenocarcinoma showing strong immunostaining for AURKA and NF-κB. C) The same as in panel B showing a case of poorly differentiated gastric adenocarcinoma. D) The graph summarizes the AURKA immunohistochemical staining results on gastric tissue microarrays. Horizontal bars indicate the median whereas red dots depict the mean. Normal, normal gastric glands; GAS, gastritis; IM, intestinal metaplasia; Dys, dysplasia; AdCa, adenocarcinoma. AURKA was significantly overexpressed in all stages of gastric tumorigenesis (p<0.001).