Stromal loss of TGFβ drives cancer growth in the epithelium via inflammation

(A) Tgfbr2 deletion in stromal fibroblasts results in inflammation and infiltration of CD45+ cells. (B) Inflammation induces DNA damage in epithelia leading to decreased p21, p15, p16. (C) The loss of cell cycle control and increased proliferation of epithelia leads to development of SCC.

(A) Tgfbr2 deletion in stromal fibroblasts results in inflammation and infiltration of CD45+ cells. (B) Inflammation induces DNA damage in epithelia leading to decreased p21, p15, p16. (C) The loss of cell cycle control and increased proliferation of epithelia leads to development of SCC.

Interactions between epithelial and stromal cells play an important role in cancer development and progression. Epithelial cancers develop when changes occur to tumor suppressor genes in stromal fibroblast cells. For example, loss of tumor suppressor, p53, in stromal fibroblasts leads to p53 inactivation in the epithelium in a prostate cancer model, and disruption of the transforming growth factor-b receptor II (TGF-βRII) in stromal fibroblasts results in intraepithelial dysplasia in prostate cancer and invasive squamous cell carcinoma (SCC) in mouse forestomach.

Previous studies suggest that the stroma is a vital part of epithelial homeostasis; however, the molecular mechanisms involved in this relationship are unknown. Li Yang, Ph.D., and her colleagues in CCR’s Laboratory of Cancer Biology and Genetics set out to identify the molecular mechanisms triggering epithelial cancer development resulting from changes in stromal cells. The researchers used an animal model in which TGF-β signaling is deleted in stromal fibroblasts (Tgfbr2fspKO). Tgfbr2fspKO die by 7 weeks with an average survival of 38 days. At 3 weeks of age, Tgfbr2fspKO mice begin to have hyperplasia in the forestomach, followed by dysplasia, carcinoma in situ, then invasive SCC.

The researchers found that Tgfbr2fspKO SCC tumors contained many CD45+ leukocytes between weeks 3 and 5 of age compared to control mice, which signifies inflammation due to loss of Tgfbr2. Inflammation has been linked to carcinogenesis and is known to cause DNA damage and histone modification in cancer. To determine whether damage to DNA occurred in the Tgfbr2fspKO forestomach, the researchers looked for a major product of DNA oxidation, which signifies DNA damage. They found that DNA damage could be detected at 3 weeks of age and became progressively worse by 5 weeks. DNA damage often results in genetic changes. The researchers analyzed epithelial cells from forestomach tumors from Tgfbr2fspKO mice and found that p15 and p16 tumor suppressor gene expression was lost in the epithelial cells. p15 and p16 have an important role in cell cycle control and are needed for suppressing tumor development.

The next step for the researchers was to examine other key molecules involved in the cell cycle that may be affected by loss of TGF-β signaling. Expression of Cyclin D1 and phospho-p53 was increased in forestomach of Tgfbr2fspKO mice compared to controls, while p21, the downstream regulator of p53 was reduced. DNA sequencing showed that there was increased methylation of the p21 promoter, which likely prevented p53-mediated p21 transcription, resulting in the decreased expression of p21 in Tgfbr2fspKO mice. Downregulation of p21, p15, and p16 may lead to increased proliferation, so cell proliferation in the forestomach was analyzed. Tissue was stained for stroma- and epithelial-specific markers, in addition to a marker that signifies proliferation. Immunofluorescence imaging showed uncontrolled proliferation in the Tgfbr2fspKO mice, which was much more prominent in the epithelium than in the stroma.

In order to characterize the role of inflammation in Tgfbr2fspKO mice, forestomach tissue was analyzed for a variety of inflammatory mediators and infiltrating immune cells were identified. There was increased expression of inducible nitrogen oxide synthatase (NOS2), cyclooxygenase-2 (COX-2), and nuclear factor (NF) kB subunit (p65) in the epithelium and stroma of the Tgfbr2fspKO mice compared to controls. The infiltrating CD45+ immune cells included myeloid derived suppressor cells (MDSCs) and TH17 cells. MDSCs have significant impact on tumor microenvironment and are well-known for their role in cancer associated immune suppression.

The researchers investigated the role of inflammation in SCC development by treating Tgfbr2fspKO mice with the COX-2 inhibitor, Celecoxib. Treatment restored p21 expression, delayed tumorigenesis, and increased survival. Celecoxib also decreased p53, COX-2, NOS2, and p65 expression to levels similar to control samples. These results support that inflammation-induced DNA damage causes genetic and epigenetic changes of cell cycle mediators, which results in the progression of SCC in the forestomachs of Tgfbr2fspKOmice.

Importantly, the researchers showed that their findings in the Tgfbr2fspKO mice are supported by what is found in human esophageal SCC. This work suggests that anti-inflammation therapeutics may be an option in treating human SCCs with low levels of TβRII in the stroma.

Summary Posted: 03/2013

Reference

Achyut BR, Bader DA, Robles AI, Wangsa D, Harris CC, Ried T, Yang L. Inflammation-Mediated Genetic and Epigenetic Alterations Drive Cancer Development in the Neighboring Epithelium upon Stromal Abrogation of TGF-β Signaling. PLoS Genetics February 2013. PubMed Link