Gastroenterology

Gastroenterology

Volume 159, Issue 4, October 2020, Pages 1444-1458.e15
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Loss of NFKB1 Results in Expression of Tumor Necrosis Factor and Activation of Signal Transducer and Activator of Transcription 1 to Promote Gastric Tumorigenesis in Mice

https://doi.org/10.1053/j.gastro.2020.06.039Get rights and content

Background & Aims

Activity of nuclear factor κB transcription factors and signaling via signal transducer and activator of transcription (STAT) are frequently altered in gastric cancer cells. Mice lacking NFKB1 (Nfkb1–/– mice) develop invasive gastric cancer, and their gastric tissues have increased levels of cytokines, such as interleukin (IL) 6, IL22, IL11, and tumor necrosis factor (TNF), as well as increased activation of STAT1. We investigated whether these cytokines were required for STAT1 activation in gastric tissues of mice and critical for gastric tumorigenesis.

Methods

We crossed Nfkb1–/– mice with Il6–/–, Il22–/–, Il11Rα–/–, and Tnf–/– mice. Stomach tissues from compound mutant mice were analyzed by histology, immunoblotting, and RNA sequencing. Lymphoid, myeloid, and epithelial cells were isolated from stomachs, and the levels of cytokines were determined by flow cytometric analysis.

Results

Nfkb1–/– mice developed gastritis, oxyntic atrophy, gastric dysplasia, and invasive tumors, whereas Nfkb1–/–Stat1–/– mice did not, even when followed for as long as 2 years. The levels of Il6, Il11, Il22, and Tnf messenger RNA were increased in the body and antrum of the stomachs from Nfkb1–/– mice, from 3-6 months of age. However, Nfkb1–/–Il6–/–, Nfkb1–/–Il22–/–, and Nfkb1–/–Il11Rα–/– mice still developed gastric tumors, although the absence of IL11 receptor (IL11R) significantly reduced development of invasive gastric tumors. Stomachs from Nfkb1–/–Tnf–/– mice exhibited significantly less gastritis and oxyntic atrophy and fewer tumors than Nfkb1–/– mice. This correlated with reduced activation of STAT1 and STAT3 and fewer numbers of T cells and B cells infiltrating the gastric body. Loss of STAT1 or TNF significantly reduced expression of PD-L1 on epithelial and myeloid (CD11b+) cells in the gastric mucosa of Nfkb1–/– mice—indeed, to the levels observed on the corresponding cells from wild-type mice.

Conclusions

In studies of gastric tumor development in knockout mice, we found that loss of NFKB1 causes increased expression of TNF in the stomach and thereby drives activation of STAT1, resulting in an inflammatory immune response and the development of gastric cancer. IL11R appears to be required for the progression of gastric tumors to the invasive stage. These findings suggest that inhibitors of TNF, and possibly also inhibitors of IL11/IL11Rα, might be useful in the treatment of gastric cancer.

Section snippets

Mice

All animal experiments complied with and were approved by The Walter and Eliza Hall Institute of Medical Research Animals Ethics Committee. Nfkb1–/– mice were crossed with Il6–/–, Il11Rα–/–, Il22–/–, Tnf–/–, or STAT1–/– mice (see Supplementary Materials for original sources). The resulting intercrossed mice were used to generate Nfkb1–/–Il6–/–, Nfkb1–/–Il11Rα–/–, Nfkb1–/–Il22–/–, Nfkb1–/–Tnf–/–, and Nfkb1–/–STAT1–/– mice. Genotyping was performed by polymerase chain reaction using published

The Absence of Signal Transducer and Activator of Transcription 1 Affords Nfkb1–/– Mice With Long-Term Protection From Gastric Cancer

We previously reported that the loss of STAT1 prevented gastric pathology in Nfkb1–/– mice, whereas loss of 1 allele of Stat3 (complete loss of STAT3 causes embryonic lethality) had no effect.7 However, Nfkb1–/–Stat1–/– compound mutant animals were only followed for 1 year. To determine whether STAT1 loss only delayed disease progression or was sufficient to abrogate IGC development in Nfkb1–/– mice, we aged a cohort of Nfkb1–/–Stat1–/– mice to timepoints at which we would expect invasive GC

Discussion

Cytokine/cytokine receptor signaling is a key component in the overall regulation of the immune system. Accordingly, its dysregulation has been linked to many chronic inflammatory diseases, autoimmune disorders, and cancer.45,46 Pertinently, chronic inflammation has been identified as a key initiator of the development of IGC.3,45 Even though activation of the NF-κB family of transcription factors has been linked to the control of cytokine and chemokine production in several

Acknowledgments

The authors thank Professors Emad El-Omar and John Silke for discussions and critical reading of this manuscript, G. Siciliano and K. Hughes for animal care, B. Helbert for genotyping, E. Tsui and her team for preparation of histologic sections, and C. McLean for secretarial assistance.

CRediT Authorship Contributions

Jun Ting Low, PhD (Data curation: Lead; Formal analysis: Lead; Investigation: Lead; Writing – original draft: Lead); Michael Christie, PhD, Medical degree (Data curation: Supporting; Formal analysis: Supporting;

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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was supported by a Cancer Council Victoria Grant-in-Aid project grant 1161400 (to Lorraine A. O’Reilly, Andreas Strasser, Tracy Putoczki, and Matthias Ernst) a Cancer Australia and Cancer Council New South Wales project grant 1047672 (to Lorraine A. O’Reilly, Tracy Putoczki, Andreas Strasser, and Matthias Ernst) and, a Cancer Council NSW (Box Rallies) RG 20-13 (to Lorraine A. O'Reilly, Tracy Putoczki, Michael Christie, Matthias Ernst). Additional support included fellowships and grants from the National Health and Medical Research Council, Canberra, program 1016701 (Andreas Strasser), fellowship; 1020363 (Andreas Strasser) and project grants; 1046010 (Andreas Strasser); 1008614, 1080498 (Tracy Putoczki); The Walter and Eliza Hall Institute of Medical Research Dyson Bequest Centenary Fellowship (Tracy Putoczki), Victorian Cancer Agency Fellowship MCRF16009 (Tracy Putoczki), Worldwide Cancer Research project grant 14-1197 (Tracy Putoczki), Australian Postgraduate Award (Jun Ting Low), Top-Up Scholarship, Cancer Therapeutics CRC (Australia; Jun Ting Low) and an NHMRC infrastructure grant, Independent Research Institutes Infrastructure Support Scheme grant 361646, the Victorian State Government (Operational Infrastructure Support Program grant), the Leukemia and Lymphoma Society (grant 7413 and 7001-13) and the Juvenile Diabetes Research Foundation/National Health and Medical Research Council 466658 (Andreas Strasser).

    Authors share co-senior authorship.

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