Iqgap3-Ras axis drives stem cell proliferation in the stomach corpus during homoeostasis and repair

Abstract

Objective: Tissue stem cells are central regulators of organ homoeostasis. We looked for a protein that is exclusively expressed and functionally involved in stem cell activity in rapidly proliferating isthmus stem cells in the stomach corpus.

Design: We uncovered the specific expression of Iqgap3 in proliferating isthmus stem cells through immunofluorescence and in situ hybridisation. We performed lineage tracing and transcriptomic analysis of Iqgap3 +isthmus stem cells with the Iqgap3-2A-tdTomato mouse model. Depletion of Iqgap3 revealed its functional importance in maintenance and proliferation of stem cells. We further studied Iqgap3 expression and the associated gene expression changes during tissue repair after tamoxifen-induced damage. Immunohistochemistry revealed elevated expression of Iqgap3 in proliferating regions of gastric tumours from patient samples.

Results: Iqgap3 is a highly specific marker of proliferating isthmus stem cells during homoeostasis. Iqgap3+isthmus stem cells give rise to major cell types of the corpus unit. Iqgap3 expression is essential for the maintenance of stem potential. The Ras pathway is a critical partner of Iqgap3 in promoting strong proliferation in isthmus stem cells. The robust induction of Iqgap3 expression following tissue damage indicates an active role for Iqgap3 in tissue regeneration.

Conclusion: IQGAP3 is a major regulator of stomach epithelial tissue homoeostasis and repair. The upregulation of IQGAP3 in gastric cancer suggests that IQGAP3 plays an important role in cancer cell proliferation.

Keywords: cell proliferation; gastric cancer; gastric pre-cancer; gastric wound repair; stem cells.

Figure 1

Expression of Iqgap3 in the isthmus of corpus epithelium. (A) Schematic diagram of a gastric unit in the corpus of the mouse stomach. (B) Immunofluorescence (IF) staining for Iqgap3, Ki67 and E-cadherin (E-cad) on the corpus of wild-type (WT) mouse stomach (n=3). (C) Quantification of Iqgap3+cells in isthmus or base (n=3). Error bar represents SD from 2025 Iqgap3+cells of 3 mice. The Ki67+region was defined as the isthmus zone. (D) In situ hybridisation (ISH) for Iqgap3 (green) and Mki67 (red) on the corpus of WT mice (n=2). (E) IF staining for tdTomato and Ki67 on the corpus of Iqgap3-2A-tdTomato mice (n=3). (F) Flow cytometry to isolate tdTomato/Iqgap3 high expression epithelial cell fraction (Iqgap3^high) and tdTomato/Iqgap3 low or negative expression epithelial cell fraction (Iqgap3^low/neg) from stomach of Iqgap3-2A-tdTomato reporter mice (n=5). (G) qPCR for Cdh1, tdTomato, Iqgap3, Mki67, Stathmin1 (Stmn1), Lgr5, Bhlha15 (Mist1) and Tnfrsf19 (Troy) mRNA from isolated tdTomato^high (Iqgap3^high) and tdTomato^low/neg (Iqgap3^low/^neg) gastric epithelial cells. mRNA expression was normalised by Gapdh expression (n=3). Error bars represent SD scale bar=100 µm. qPCR, quantitative PCR.

Figure 2

The Iqgap3-expressing cells in the isthmus are multipotent stem cells. (A) Iqgap3-2A-CreERT2;Rosa-tdTomato mouse model. (B) Experimental strategy for lineage tracing time course. (C) IF staining for Ki67 and tdTomato on the corpus of Iqgap3-2A-CreERT2;Rosa-tdTomato mice at 1 day (1 d), 3 months (3 m), 6 months (6 m) and 1 year (1 y) post-tamoxifen induction (p.i.) (n=3). Lineage tracing, LT. (D, E) IF staining for tdTomato and markers of major stomach differentiated cells (Muc5ac, H, K-ATPase, GS-II and Gif) on the corpus of Iqgap3-2A-CreERT2;Rosa-tdTomato mice at 1 year post-tamoxifen induction (n=3). (F) Quantification of lineage tracing on the corpus of Iqgap3-2A-CreERT2;Rosa-tdTomato mice at 6 months and 1 year post-tamoxifen induction. (n=3). The tdTomato+lineage tracing glands were categorised into pit to neck (LT #1: Pit - Neck), pit to mucus-neck/chief cell transition (LT #2: Pit-Transition) and pit to base (LT #3: Pit – Base). A total of 167 tdTomato+glands from three mice (6 months) or 172 tdTomato+glands from three mice (1 year) were counted. Error bars represent SD data sets were analysed by one-way ANOVA. ***P<0.001. (G) tdTomato expression in the isolated corpus gastric units from Iqgap3-2A-CreERT2;Rosa-tdTomato mice at 20–24 hours post-tamoxifen administration (top). Corpus organoids were generated from tdTomato +cells (n=2). (H) qPCR for IQGAP3, NANOG, OCT4, KLF4, cMYC, SOX2, CD44v9 and GFAP mRNA from IQGAP3 knockdown embryonic stem cell line NTERA-2. mRNA expression was normalised by GAPDH expression (n=3). Error bars represent SD data sets were analysed by Student’s t-test. *P<0.05, **p<0.01. (I) Immunoblot for IQGAP3, Nanog, Oct4, KLF4, CD44v9, GFAP and GAPDH from knockdown embryonic stem cell line NTERA-2 (n=3). Scale bar=100 µm. ANOVA, analysis of variance; GFAP, glial fibrillar acidic protein; IF, immunofluorescence; qPCR, quantitative PCR.

Figure 3

Iqgap3 regulates stem cell proliferation via Ras-ERK pathway. (A) Average of RNA expressions in sorted Iqgap3^high and Iqgap3^low/neg cell fractions are shown. (B) Gene set enrichment analysis (GSEA) showing enrichment of Lgr5-negative (LGR5neg) and—high (LGR5pos) corpus epithelial cell gene signature from public datasets (GSE86603) in Iqgap3^high and Iqgap3^low/neg cell fractions. P values determined by a weighted Kolmogorov–Smirnov-like statistic and adjusted for multiple hypothesis testing. (C) GSEA showing enrichment of Myc target gene signature, E2F targets gene signature, Ras pathway gene signature in Iqgap3^high and Iqgap3^low/neg cell fractions. (D) Heat MAP showing expression of representative Ras-ERK pathway genes in Iqgap3^high and Iqgap3^low/neg cell fractions. Gene expression levels are shown in Z-score of CPM of RNA-sequencing. (E–G) IF staining for HRAS, Ki67, E-cad, HER2 and phosphorylated ERK (p-Erk) on the corpus of wild-type (WT) mice (n=3). (H) Immunoprecipitation for the interaction of Venus-tagged Iqgap3 (Venus-Iqgap3) with Myc-tagged HRAS (Myc-Hras) or Myc-tagged Hras^G12V (Myc-Hras^G12V) and immunoblot for cell lysate from Venus-Iqgap3/Myc-Hras/Myc-Hras^G12V expressed 293 T cells. (I) Experimental strategy to suppress Iqgap3 expression by shRNA in organoids from WT mice. (J) Quantification of size of Iqgap3 knockdown organoids from 0 passage (6 day post doxycycline (Dox) treatment, before passage), first passage (8 days postpassage) and second passage (7 days postpassage) (n=2). Error bars represent SD from each population. Data sets were analysed by one-way ANOVA. Scale bar=100 µm. ANOVA, analysis of variance; CPM, counts per million; ERK, extracellular signal-regulated kinase; NES, normalised enrichment score.

Figure 4

Iqgap3-expressing cells drive corpus epithelial regeneration post-tissue damage. (A) H&E staining on untreated and high dose tamoxifen (HDT) treated WT corpus at 48 hours post-tamoxifen administration. (B) IF staining for Ki67 and E-cad on untreated and 48 hours post-HDT treated WT corpus. (C–E) ISH for Iqgap3(green) and Lgr5(red) on untreated, 24 hours and 48 hours post-HDT treated WT corpus. Boxes indicate enlarged regions. (F) IF staining for Muc5ac and Ki67 on 48 hours post-HDT treated WT corpus. (G, H) IF staining for tdTomato, Ki67 and GIF on 48 hours post-HDT treated corpus from Iqgap3-2A-tdTomato mice. (I) qPCR for Mki67, Iqgap3 and Lgr5 from isolated WT corpus tissue of untreated and 48 hours post-HDT treated mice (n=3). Error bars represent SE of mean. Data were analysed by Student’s t-test. (J) H&E staining on HDT treated corpus at 14 days post-tamoxifen administration. (K) IF staining of Ki67, tdTomato and H, K-ATPase on 14 days post-HDT treated corpus of Iqgap3-2A-CreERT2;Rosa-tdTomato mice. Scale bars=50 µm. IF, immunofluorescence; ISH, in situ hybridisation; LT, lineage tracing; qPCR, quantitative PCR; WT, wild-type.

Figure 5

HDT-induced tissue damage promotes stem cell activity and neoplastic characteristics. (A) IF staining for PGC, Ki67 and GS-II on untreated and 48 hours post-HDT treated WT corpus. (B) qPCR for Atp4b, Pgc, Gif, Muc6 and Chga from isolated corpus tissue of untreated or 48 hours post-HDT treated WT mice (n=4, expressed as Log₂ scale). Data were analysed by Student’s t-test. ^∗P<0.05. NS, not significant. (C) GSEA showing enrichment of embryonic stem cell gene signature, early gastric cancer gene signature and neoplastic transformation KRAS gene signature in 48 hours post-HDT treated WT corpus (n=2). P values determined by a weighted Kolmogorov-Smirnov-like statistic and adjusted for multiple hypothesis testing. (D) Heat MAP showing top 20 genes upregulated in (C) based on RNA-sequencing data from untreated and 48 hours post-HDT corpus tissue. (E) IF staining for p-Erk, Ki67 and E-cad on 48 hours post-HDT treated WT corpus. (F) Experimental strategy to generate corpus organoids from HDT treated mice. (G) Microscopic image of corpus organoids derived from untreated and 48 hours post-HDT-treated WT mice. (H, I) Organoid growth efficiency and diameter of corpus organoids derived from untreated or 48 hours post-HDT treated WT mice at 7 days of organoid culture (n=3). Data were analysed by Student’s t-test. Scale bars=50 µm (A), 100 µm (E), 500 µm (G). Error bars represent SEM. GSEA, gene set enrichment analysis; HDT, high dose of tamoxifen; IF, immunofluorescence; p-Erk, phosphorylated extracellular signal-regulated kinase; qPCR, quantitative PCR; SEM, SE of mean; WT, wild-type.

Figure 6

Iqgap3-2A-CreERT2;Kras^G12D/+ mice present pseudopyloric metaplasia. (A) Schematic representation of the genetic construct used to establish the Iqgap3-2A-CreERT2;Kras^G12D/+ mouse model. (B) Experimental strategy for inducing Iqgap3-driven active Kras^G12D expression. (C) H&E staining of the lesser and greater curvature on the corpus of Iqgap3-2A-CreERT2;Kras^G12D/+ mouse. (D, E) IF staining for Muc5ac and H, K-ATPase on the corpus of control and Iqgap3-2A-CreERT2;Kras^G12D/+ mouse. (F–I) IF staining for Muc5ac/Ki67 (F), CD44v10 (G), Pdx1/E-cad (H) and Tff2/Ki67/E-cad (I) on the corpus of Iqgap3-2A-CreERT2;Kras^G12D/+ mice (n=3). Box indicates enlarged region. Scale bars=50 µm. IF, immunofluorescence; p.i, post-tamoxifen induction.

Figure 7

IQGAP3 is coexpressed with Ki67 in human gastric cancer. (A) IF staining for IQGAP3, Ki67 and E-cad on normal corpus in the human stomach (n=3). (B) Microarray and RNA-sequencing analysis for IQGAP3 expression in gastric tumour and normal stomach tissue (Singapore cohort). Error bars for microarray (tumour=185, normal=89) and RNA-sequencing (tumour=27, normal=18) represent SEM from each population. Data were analysed by two-tailed Wilcoxon RANK sum test. (C) IQGAP3 tissue microarray (TMA) of gastric cancer and paired-adjacent normal stomach tissue (n=237). (D) Box plot for comparing IQGAP3 TMA score in adjacent normal stomach, intestinal type gastric cancer, diffuse type gastric cancer and mixed type gastric cancer. Error bars represent SEM from each population. Data sets were analysed by one-way ANOVA. (E) H&E staining on human gastric tumour (n=3). (F) IF staining for IQGAP3, Ki67 and E-cad on the human gastric tumour (n=5). (G) IF staining for IQGAP3, Ki67 and CD44v9 on the human gastric tumour (n=7). Scale bar=100 µm. ANOVA, analysis of variance; IF, immunofluorescence; SEM, SE of mean.