Systemic activation of K-ras rapidly induces gastric hyperplasia and metaplasia in mice

Abstract

Mouse models with conditional activation of K-ras (K-ras(G12D)) are used widely to investigate the role of oncogenic K-ras in a tissue-specific manner. However, the effect of ubiquitous activation of K-ras in adult mice has not been well studied. Herein, we report that systemic activation of K-ras in mice leads to rapid changes in gastric cellular homeostasis. Conditional activation of K-ras results in activation of the MAPK pathway and hyperproliferation of squamous epithelium in the forestomach and metaplasia in the glandular stomach. Parietal cells almost completely disappear from the upper part of the stomach adjacent to forestomach of K-ras activated mice. CDX2, a caudal-related homeobox transcription factor normally expressed in the intestine, is upregulated in parts of the stomach, following activation of K-ras in mice. Cyclooxygenase 2 (COX-2), a mediator of inflammation, is also upregulated in parts of the stomach of the K-ras activated mice with concomitant infiltration of hematopoietic cells in the hyperplastic tissue. Moreover, in K-ras activated mice, the expression of putative progenitor cell marker Dcamkl1 is upregulated in the glandular stomach. Expression of CD44, a candidate stomach cancer stem cell marker, is also increased in forestomach and the glandular stomach. These results suggest that cells of the stomach, potentially stem or progenitor cells, are highly susceptible to K-ras activation-induced initiation of gastric precancerous lesions. The histological changes in the K-ras activated mice resemble the pre-neoplastic changes that take place during gastric carcinogenesis in humans. Thus, a mouse model with systemic K-ras(G12D) activation could be useful for studying the early molecular events leading to gastric carcinogenesis.

Figure 1

Activation of K-ras leads to quick death of mice. A) Schematic of LSL-K-ras^G12D locus. K-ras^G12D with an upstream LoxP-STOP-LoxP cassette that can be excised by Cre recombinase. P1, P2 and P3, primers for genotyping. B) Genotyping products, before and 15 days after TAM treatment. 500 bp LSL cassette, 625 bp wild-type and 650 bp recombined fragments. TAM-induced activation of K-ras decreases survival of mice. C) Kaplan-Meier curve of control Ubc9 CreER mice and LSL-K-ras^G12D/⁺ Ubc9 Cre ER mice after TAM feeding. Mice with K-ras activation had D) decreased body weights and E) decreased random blood glucose levels. F) Tumor in stomach of K-ras Ubc9 Cre-ER mice.

Figure 2

Histology of tissues from mice two weeks after tamoxifen treatment. H & E staining of A) lung, B) Pancreas, C) Colon and D) Small intestine.

Figure 3

Activation of K-ras causes hyperplasia of squamous epithelium and glandular epithelium within 15 days after TAM treatment. A) Control and B) K-ras_G12D/₊ H and E staining of gastric squamous epithelium. C) Control and D) Kras_G12D/₊ H and E staining of glandular epithelium. E) Glandular stomach in Kras_G12D/₊ mice in higher magnification (100X). F) Cross section of tumor in Kras^G12D/⁺ mice.

Figure 4

A) Control and B) K-ras^G12D/⁺ BrdU incorporation of sqamous epithelium and C) Quantitation of BrdU positive cells in squamous epithelium. D) Control and E) K-ras^G12D/⁺ BrdU incorporation in glandular epithelium. F) Quantitation of BrdU positive cells in glandular epithelium. Immunofluorescence staining with H⁺/ K⁺ ATPase antibody to detect parietal cells in G) control and H) K-ras activated mice. Immunofluorescence staining with Chromogranin A antibody to detect enteroendocrine cells in I) control and J) K-ras activated mice. Scale bar 200μM.

Figure 5

K-ras activation causes intestinal metaplasia in 13-16 days after TAM treatment. Alcian blue staining for A) Control and B) K-ras activated mice. Immunohistochemistry staining for CDX2 level in C) Control and D) K-ras activated mice. Scale bar 200μM. E) Western blot for CDX2.

Figure 6

Activation of downstream targets of the MAPK pathway within 13-16 days after TAM treatment. A) Western blot for phospho MEK, phospho Erk1/2, and phosphor p38 in K-ras activated mice and control mice. Immunohistochemical staining for phospho Erk1/2 in the stomach of the B) control and C) K-ras activated mice, or for phospho p38 (D and E), respectively, 15 days after tamoxifen feeding. Scale bar 200μM.

Figure 7

Activation of K-ras induces inflammatory response within 15 days after TAM treatment. H and E staining for stomach of A) control and B) K-ras activated mice, Immunohistochemistryfor CD45 in stomach of C) control and D) K-ras activated mice. Immunohistochemistry for CD68, a macrophage marker, in E) control and F) K-ras activated mice. Immunohistochemical stainingfor COX-2 from G) control and H) K-ras activated mice. Scale bar 200μM.

Figure 8

Change in stem cell markers in K-ras activated mouse within 15 days after TAM treatment. Western blot for A) LGR5 or B) CD44 from the lysates of stomach from control or K-ras activated mice 13-16 days after TAM treatment. Immunofluorescence staining for CD44 in the forestomach of the C) control and D) K-ras activated mice, or in the glandular stomach (E and F), 15 days after TAM feeding. Immunofluorescence stainingfor Dcamkl1 in stomach of G) control and H) K-ras activated mice. Scale bar 200μM.