STAT3 and STAT1 mediate IL-11-dependent and inflammation-associated gastric tumorigenesis in gp130 receptor mutant mice.

Deregulated activation of STAT3 is frequently associated with many human hematological and epithelial malignancies, including gastric cancer. While exaggerated STAT3 signaling facilitates an antiapoptotic, proangiogenic, and proproliferative environment for neoplastic cells, the molecular mechanisms leading to STAT3 hyperactivation remain poorly understood. Using the gp130(Y757F/Y757F) mouse model of gastric cancer, which carries a mutated gp130 cytokine receptor signaling subunit that cannot bind the negative regulator of cytokine signaling SOCS3 and is characterized by hyperactivation of the signaling molecules STAT1 and STAT3, we have provided genetic evidence that IL-11 promotes chronic gastric inflammation and associated tumorigenesis. Expression of IL-11 was increased in gastric tumors in gp130(Y757F/Y757F) mice, when compared with unaffected gastric tissue in wild-type mice, while gp130(Y757F/Y757F) mice lacking the IL-11 ligand-binding receptor subunit (IL-11Ralpha) showed normal gastric STAT3 activation and IL-11 expression and failed to develop gastric tumors. Furthermore, reducing STAT3 activity in gp130(Y757F/Y757F) mice, either genetically or by therapeutic administration of STAT3 antisense oligonucleotides, normalized gastric IL-11 expression and alleviated gastric tumor burden. Surprisingly, the genetic reduction of STAT1 expression also reduced gastric tumorigenesis in gp130(Y757F/Y757F) mice and coincided with reduced gastric inflammation and IL-11 expression. Collectively, our data have identified IL-11 as a crucial cytokine promoting chronic gastric inflammation and associated tumorigenesis mediated by excessive activation of STAT3 and STAT1.

Elevated Dnmt3a activity promotes polyposis in Apc(Min) mice by relaxing extracellular restraints on Wnt signaling.

BACKGROUND & AIMS: Aberrant DNA methylation is a common early event in neoplasia, but it is unclear how this relates to dysregulation of DNA (cytosine-5) methyltransferases (Dnmts). Here we use knock-in transgenic mice to investigate the consequences of intestinal epithelium-specific overexpression of de novo Dnmt3a. METHODS: A novel gene targeting strategy, based on the intestinal epithelium-specific, uniform expression of the A33 glycoprotein, is employed to restrict Dnmt3a overexpression in homozygous A33(Dnmt3a) mutant mice. RESULTS: A33(Dnmt3a) mice infrequently develop spontaneous intestinal polyps. However, when genetically challenged, tumor multiplicity in A33(Dnmt3a);Apc(Min) compound mice is 3-fold higher than in Apc(Min) mice. Although we observe a requirement for spontaneous loss of heterozygosity of the adenomatous polyposis coli (Apc) gene to trigger tumorigenesis in Apc(Min) mice, lesions in A33(Dnmt3a);Apc(Min) mice frequently retain the wild-type Apc allele. However, epithelia from normal mucosa and polyps of A33(Dnmt3a);Apc(Min) mice show hypermethylation-mediated transcriptional silencing of the Wnt antagonists Sfrp5, and to a lesser extent, Sfrp1 and increased nuclear beta-catenin alongside activation of the Wnt-target gene Axin2/Conductin. Conversely, enforced Sfrp5 expression suppresses canonical Wnt-signaling more effectively in wild-type than in Apc(Min) cells. CONCLUSIONS: Aberrant activation of the canonical Wnt pathway, either by mono-allelic Apc loss or transcriptional silencing of Sfrp5 is largely insufficient to promote polyposis, but epistatic interactions between these genetic and epigenetic events enables initiation and promotion of disease. This mechanism is likely to play a role in human colorectal cancer, because we also show that elevated DNMT3A expression coincides with repressed SFRP5 and enhanced AXIN2/CONDUCTIN expression in paired patient biopsies.

gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis.

Although gastrointestinal cancers are frequently associated with chronic inflammation, the underlying molecular links have not been comprehensively deciphered. Using loss- and gain-of-function mice in a colitis-associated cancer model, we establish here a link comprising the gp130/Stat3 transcription factor signaling axis. Mutagen-induced tumor growth and multiplicity are reduced following intestinal epithelial cell (IEC)-specific Stat3 ablation, while its hyperactivation promotes tumor incidence and growth. Conversely, IEC-specific Stat3 deficiency enhances susceptibility to chemically induced epithelial damage and subsequent mucosal inflammation, while excessive Stat3 activation confers resistance to colitis. Stat3 has the capacity to mediate IL-6- and IL-11-dependent IEC survival and to promote proliferation through G1 and G2/M cell-cycle progression as the common tumor cell-autonomous mechanism that bridges chronic inflammation to tumor promotion.

Identification of putative targets of DNA (cytosine-5) methylation-mediated transcriptional silencing using a novel conditionally active form of DNA methyltransferase 3a.

Aberrant DNA methylation of gene promoters is a recurrent finding associated with diseases such as cancer and inflammation, and is thought to contribute to disease through its role in transcriptional repression. Indeed, recent evidence suggests that DNA (cytosine-5) methyltransferases (DNMTs) may mediate the activity of factors promoting cell growth. Here, we utilise a novel experimental system for the conditional and reversible activation of a de novo DNMT by constructing a steroid-hormone analogue activated version, Dnmt3a-mERtrade mark. Following treatment with the oestrogen analogue 4-hydroxy tamoxifen of murine embryonic stem cells expressing this protein, we have identified by microarray analysis, several potential targets of Dnmt3a mediated transcriptional repression including the cancer associated genes Ssx2ip, Hmga1 and Wrnip. These results were validated using quantitative reverse transcriptase PCR and we confirm the biological significance of these in vitro observations by demonstrating a reduction in mRNA transcripts of the same genes within the intestinal epithelium of cancer-prone transgenic knock-in mutant mice over-expressing Dnmt3a throughout the intestinal epithelium.

Pathologic consequences of STAT3 hyperactivation by IL-6 and IL-11 during hematopoiesis and lymphopoiesis.

We have previously demonstrated that STAT3 hyperactivation via the interleukin 6 (IL-6) cytokine family receptor gp130 in gp130 (Y757F/Y757F) mice leads to numerous hematopoietic and lymphoid pathologies, including neutrophilia, thrombocytosis, splenomegaly, and lymphadenopathy. Because IL-6 and IL-11 both signal via a gp130 homodimer, we report here a genetic approach to dissect their individual roles in these pathologies. Neutrophilia and thrombocytosis were absent in gp130 (Y757F/Y757F) mice lacking either IL-6 (gp130 (Y757F/Y757F): IL-6 (-/-)) or the IL-11 receptor alpha subunit (gp130 (Y757F/Y757F): IL-11Ralpha1 (-/-)), and this was associated with a normalized bone marrow compartment. The elevated myelopoiesis and megakaryopoiesis in bone marrow of gp130 (Y757F/Y757F) mice was attributable to an increase by either IL-6 or IL-11 in the STAT3-driven impairment of transforming growth factor beta (TGF-beta) signaling, which is a suppressor of these lineages. In contrast, the absence of IL-6, but not IL-11 signaling, prevented the splenomegaly, abnormal lymphopoiesis, and STAT3 hyperactivation in lymphoid organs of gp130 (Y757F/Y757F) mice. Furthermore, hyperactivation of STAT3 in lymphoid organs was associated with increased expression of IL-6Ralpha, and IL-6Ralpha expression was reduced in gp130 (Y757F/Y757F): Stat3 (+/-) mice displaying normal levels of STAT3 activity. Collectively, these data genetically define distinct roles of IL-6 and IL-11 in driving pathologic hematopoietic and lymphoid responses mediated by STAT3 hyperactivation.