Background and Aim:
Multiple studies have identified the constitutive or hyperactivation of the transcription factor STAT3 as a leading driver for a variety of cancers including gastric cancer. Regulating genes involve in cell proliferation, cell survival, angiogenesis and invasion, STAT3 and its related pathway therefore is an attractive target for therapeutic inhibitors.
Aim: To genetically study the effect of specific STAT3 inhibition, we exploited a novel mouse strain in which a short hairpin (sh) RNA directed against Stat3, alongside the GFP reporter gene, can be reversibly induced by administration of Doxycycline. As a proof of principle, the resulting shStat3 mice were crossed with gp130F/F which develop gastric tumour in response to excessive STAT3 activation [Putoczki et al., Cancer Cell 2013].
Methods:
CAG-rtTA3;shSTAT3 compound mutant mice were treated with doxycycline for various amounts of time and stomach, spleen and other organs were collected. Flow cytometric analysis (FACS) was used to estimate the extent of shSTAT3 expression based on the amount of GFP reporter activity. In parallel, tissue samples were also subjected to Western blot, q-PCR and immunohistochemical analysis to monitor the expression of STAT3 on the level of RNA and protein, respectively.
Results:
The FACS analysis indicated broad expression and reversibility of the short hairpin. This was supported by a decrease in STAT3 protein and RNA and re-establishment of STAT3 expression was documented by Western blot and q-PCR analysis after removal of Doxycycline from the food. Importantly, these result were confirmed on the level of STAT3 activity using the extent of phospho-STAT3 on Western Blot and quantification of STAT3 target genes by q-PCR. Collectively, these observations are highly likely to underpin the mechanism accounting for the reduced tumour weight in Doxycycline-treated mice.
Conclusion:
Our data provide the first compelling evidence that transient reduction of STAT3 confers a therapeutic benefit in a mouse model of STAT3-driven (gastric) tumourigenesis.