K. M. Riera1, E. Choi1, J. R. Goldenring1 1Vanderbilt University Medical Center,Surgery,Nashville, TN, USA
Introduction: Gastric adenocarcinoma develops following parietal cell loss leading to chief cell transdifferentiation into Spasmolytic Polypeptide (TFF2) Expressing Metaplasia (SPEM) and progressing to pre-neoplastic intestinal metaplasia (IM). Ras/Raf/MEK/ERK pathway is important in gastric cancer progression, and targeting downstream mediators of Ras pathway using a MEK inhibitor, selumetinib, has been shown to reverse intestinal metaplasia to normal gastric mucosa in a transgenic mouse model. Trop2 is a transmembrane glycoprotein upregulated in adult mouse stomach after damage and overexpression predicts worse prognosis in human gastric cancer. However, the mechanistic role of Trop2 in gastric pre-neoplasia progression is not well understood.
Methods: We studied Mist1-CreERT2;LSL-K-Ras(G12D) (Mist1-Kras) mice, which express the constitutively active form of Kras (Kras(G12D)) in gastric chief cells when treated with tamoxifen. Mist1-Kras mice developed SPEM, IM, and invasive IM at 1, 3, and 4 months post-tamoxifen treatment, respectively. Mist1-Kras mice were sacrificed at 2 month wild type (control), and 1, 2, 3, and 4 months after tamoxifen induction. To evaluate downstream Ras pathway inhibition with a MEK inhibitor, Mist1-Kras mice were treated with selumetinib for 2 weeks at 3 months post-tamoxifen treatment, and sacrificed 2 weeks following selumetinib withdrawal at 4 months. Stomachs from all mice were prepared for histology. Gastric tissue was stained for Ki67 (proliferation marker), Trop2, and Cd44v9 (SPEM/IM marker) using immunofluorescence and imaged using a digital scanner.
Results: There was no significant Trop2 staining in control, 1 (SPEM phenotype), or 2 month Mist1-Kras mice (not shown). In 3 month Mist1-Kras mice (IM phenotype), scattered Trop2 expression appeared along the gastric glands above a proliferative zone of cells staining for Ki67, and was not present in SPEM cells positive for Cd44v9 at the base of the glands (Figure 1A). In 4 month Mist1-Kras mice (invasive IM phenotype), Trop2 was markedly upregulated at and above the proliferative zone, and overexpression appeared patchy within the corpus mucosa (Figure 1B,D). Trop2 positive cells did not co-stain with Cd44v9. In 4 month selumetinib-treated Mist1-Kras mice, there was no significant Trop2 or Cd44v9 staining, while a normal proliferative zone was present (Figure 1C).
Conclusion: Trop2 is upregulated during IM progression to invasive IM in Mist1-Kras mouse stomach. Chronic Ras pathway activation may contribute to Trop2 regulation, and MEK inhibition with selumetinib shows loss of Trop2 and SPEM/IM. These results suggest that Trop2 may be upregulated at a critical transition point from benign gastric metaplasia to pre-neoplastic/dysplastic lineages.
