PPARD regulation in gastric progenitor cells drives gastric tumorigenesis in mice

Little is known about the cell origin of gastric cancer. Peroxisome proliferator-activated receptor-delta (PPARD) is a druggable ligand-activated nuclear receptor that impacts protumorigenic cellular events. However, PPARD’s role in tumorigenesis, especially gastric tumorigenesis, remains to be defined. We found that targeting PPARD overexpression in murine gastric progenitor cells (GPC), via a villin promoter, spontaneously induced gastric tumorigenesis that progressed to invasive adenocarcinoma. PPARD overexpression in GPC upregulated tumorigenic proinflammatory cytokine and CD44 expression, expanded GPC population in vivo, enhanced GPC self-renewal and proliferation in organoid cultures, and endowed these cells with tumorigenic properties. Our findings identify PPARD as a driver of gastric tumorigenesis via GPC transformation.

Gastric cancer (GC) is the second leading cause of cancer-related death globally with a 5-year survival rate of less than 25% 1 . Identification of preventive and therapeutic molecular targets in gastric tumorigenesis is needed to improve this poor outcome. Peroxisome proliferator-activated receptor-delta (PPARD) is a ligandactivated nuclear receptor that regulates important molecular cellular events, which strongly impact tumorigenesis (e.g., inflammation, metabolism) 2 . Although PPARD expression is upregulated in many major human cancers 2 , PPARD's role in tumorigenesis remains controversial 2, 3 . This controversy is primarily based on studies of germ-line PPARD knockout in Apc min mice showing conflicting results regarding the role of PPARD in intestinal tumorigenesis 2 . However, PPARD upregulation is common in human colorectal cancer 2,4 . To study the mechanistic role of PPARD upregulation in colonic tumorigenesis, we generated a mouse model with intestinally targeted PPARD overexpression via the villin-promoter (hereafter designated villin-PPARD mice) 5 .
We serendipitously found that villin-PPARD mice spontaneously developed invasive GC during longitudinal follow-up studies ( Figure 1A-I and Supplementary Figure 1A). Gastric tumorigenesis progressed in age-dependent fashion ( Figure 1I) from hyperplasia ( Figure 1E) and low-grade dysplasia ( Figure 1F) at 25 weeks to high-grade dysplasia ( Figure 1G) at 35 weeks to large invasive adenocarcinoma at 55 weeks ( Figure   1H). None of the wild-type (WT) littermates followed concomitantly developed gastric tumors ( Figure 1A-D, I and Supplementary Figure 1A). Lesions were initially found in the lesser gastric curvature at 25 weeks (Supplementary Figure 1A) and eventually expanded to occupy the whole gastric corpus at 55 weeks ( Figure   1A). PPARD has been reported to be required for Helicobacter pylori infection promotion of gastric epithelial proliferation in rodent and human gastric mucosa 6 . GW501516, a PPARD agonist, promoted DMBA carcinogen-induced squamous GC, a rare form of human GC 7 . These prior reports suggested that PPARD may contribute to GC. These data have remained insufficient to determine the true contribution of PPARD to gastric tumorigenesis, especially the more common adenocarcinoma type. Our new findings demonstrate for the first time that PPARD gene overexpression in villin-positive gastric epithelial cells was sufficient to induce gastric adenocarcinoma. Limited prior studies have shown a vague and questionable relation between PPARD and human GC 8 . On examining PPARD expression in human GC, we found that PPARD protein expression was higher in epithelial cells of human GC and adjacent tissues than in paired normal gastric tissues ( Figure 1J, K). Furthermore, PPARD upregulation was associated with lower GC patients' survival (Supplemental Figure 1B). These findings demonstrate the clinical relevance of our mouse data to human GC.
Chronic inflammation scores of gastric mucosa were significantly higher in villin-PPARD mice than their WT littermates, and these differences increased with age ( Figure 1L, M and Supplementary Figure 1C and expanded gastric progenitor cell compartments in gastric crypts of villin-PPARD mice ( Figure 2E). CD44 is a putative GC stem cell marker that regulates normal and metaplastic gastric epithelial progenitor cell proliferation 13,14 and significantly contributes to inflammation promotion of gastric tumorigenesis 11,[15][16][17][18] .
We therefore examined the effects of PPARD on gastric progenitor cells' self-renewal capacity and tumorigenicity. PPARD overexpression or activation by PPARD agonist GW501516 treatment markedly increased gastric epithelial crypts-derived spheroid formation in ex vivo 3-dimensional gastric organoid cultures ( Figure 2G,H and Supplementary Figure 3A-D), a well-established method to assess cancer stem cell self-renewal. The 3-dimensional cultures of organoids in this experiment were derived from gastric epithelial crypts of villin-PPARD mice at age 10 weeks, before the earliest tumorigenic changes were observed (Supplementary Figure 1A). Thus, the observed increases in spheroid formation were driven by PPARD and During the preparation of this manuscript, a report was published showing that PPARD activation enhanced the tumorigenicity of intestinal progenitor cells in APC null mice 19 , which is in agreement with our new findings. However, PPARD's effects in these cells occurred in association with APC mutation in intestinal cells. APC mutations are a major driver of intestinal tumorigenesis and regulator of stemness; thus, the distinct effects of PPARD are yet to be defined. Our new findings show for the first time in an in vivo experimental model that PPARD is sufficient not only to enhance epithelial progenitor cell self-renewal but also, more importantly, to transform these cells so they acquire tumorigenic properties to drive gastric tumorigenesis.
In summary, our results provide the first evidence that PPARD overexpression in progenitor cells is sufficient to induce invasive GC and demonstrate that PPARD can transform gastric progenitor cells. Thus, PPARD is potentially an attractive target for the development of small-molecule PPARD antagonists to prevent and treat GC.

Supplementary Materials
Methods and any associated references are available in the online version of the paper.

Conflicts of interest
The authors disclose no conflicts.