Glucose-regulated protein 94 deficiency induces squamous cell metaplasia and suppresses PTEN-null driven endometrial epithelial tumor development

Abstract

Endometrial carcinoma is the most prevalent gynecologic cancer in the United States. The tumor suppressor gene Pten (phosphatase and tensin homolog) is commonly mutated in the more common type 1 (endometrioid) subtype. The glucose-regulated protein 94 (GRP94) is emerging as a novel regulator for cancer development. Here we report that expression profiles from the Cancer Genome Atlas (TCGA) showed significantly increased Grp94 mRNA levels in endometrial tumor versus normal tissues, correlating with highly elevated GRP94 protein expression in patient samples and the requirement of GRP94 for maintaining viability of human endometrioid adenocarcinoma (EAC) cell lines. Through generation of uterus-specific knockout mouse models with deletion of Grp94 alone (c94f/f) or in combination with Pten (cPf/f94f/f), we discovered that c94f/f uteri induced squamous cell metaplasia (SCM) and reduced active nuclear β-catenin. The cPf/f94f/f uteri showed accelerated SCM and suppression of PTEN-null driven EAC, with reduced cellular proliferation, attenuated β-catenin signaling and decreased AKT/S6 activation in the SCM. In contrast to single PTEN knockout uteri (cPf/f), cPf/f94f/f uteri showed no decrease in E-cadherin level and no invasive lesion. Collectively, our study implies that GRP94 downregulation induces SCM in EAC and suppresses AKT/S6 signaling, providing a novel mechanism for suppressing EAC progression.

Keywords: PTEN; endometrial cancer; glucose-regulated protein 94 (GRP94); squamous cell metaplasia; β-catenin.

Figure 1. Increased GRP94 expression in human EAC and its effect on EAC cell viability

Grp94 mRNA expression in A. normal uterine (n=24) and EAC tissues (n=177), B. different grade EAC, and C. different stage EAC. D. Pten mRNA expression in normal uterine and EAC tissues. Student's t-test p values are indicated. E. Immunohistochemistry (IHC) analysis of GRP94 in human normal uterine and EAC tissues. Scale bar, 50 μm. F. Scatterplot of Grp94 and Pten mRNA expression of human normal uterine (green) and tumor samples (red). Pearson correlation p value is indicated. Western blot analysis of GRP94 knockdown efficiency in G. AN3CA cells and H. ECC-1 cells. The level of GRP94 reduction after normalization to GAPDH which served as loading control is shown below. WST-1 assay measuring cell viability in si-control (siCtrl) and si-Grp94 (si94) treated G. AN3CA cells at day 4 and H. ECC-1 cells at day 5. Data are presented as mean ± s.e., **p<0.01 (Student's t-test).

Figure 2. Generation of PR-Cre-mediated GRP94 knockout mouse models

A. Representative mouse tail PCR genotyping of the indicated alleles. B. Representative gross anatomy of uteri of the indicated genotypes at 4 and 8 weeks. Scale bar, 1 cm. C. The ratio of uterine weight to body weight in WT and c94^f/f mice at 4 weeks (n=16 and 5, respectively) and 8 weeks (n=4 and 11, respectively). The data are presented as mean ± standard error (s.e.), *p<0.05, **p<0.01 (LSD Method, and data was log transformed prior to analysis). D. H&E staining (upper panel) and IHC analysis of GRP94 (lower panel) of WT and c94^f/f uteri from 4 and 8 weeks. Scale bar, 50 μm. White arrows indicate glands and black and red bars denote columnar luminal epithelial cells and SCM, respectively.

Figure 3. Induction of squamous metaplasia in developing c94^f/f uteri

A. IHC of p63 and cytokeratin 14 (K14). B. IHC of active β-catenin and β-catenin in WT and c94^f/f uteri at 4 and 8 weeks. Enlarged views of the boxed regions are shown. Scale bar, 50 μm.

Figure 4. Reduced endometrial cancer in cP^f/f94^f/f uteri

A. Representative mouse tail PCR genotyping of the indicated alleles. B. Representative gross anatomy of uteri of the indicated genotype at 4 weeks. Scale bar, 1 cm. C. The ratio of uterine weight to body weight in WT (n=16), cP^f/f (n=10) and cP^f/f94^f/f (n=9) mice at 4 weeks. The data are presented as mean ± s.e., **p<0.01 (LSD Method). D. Western blot analysis of GRP94 and PTEN levels in uteri of indicated genotypes with GAPDH serving as the loading control. E. H&E staining and IHC of GRP94 in uteri of the indicated genotype at 4 weeks and F. at 8 weeks. White and red arrows indicate normal and transformed glands, respectively, the broken lines denote EAC and red bars denote SCM. Scale bar, 200 μm.

Figure 5. Accelerated squamous metaplasia and reduced β-catenin signaling in cP^f/f94^f/f uteri

A. IHC of p63, K14 and B. IHC of active β-catenin, β-catenin, cyclin D1 in uteri of indicated genotypes at 4 weeks. The white arrows denote glands. Enlarged view of the boxed region is shown. C. IHC of K14, GRP94 in uteri of indicated genotypes at 8 weeks. Scale bar, 200 μm. D. Merged images of immunofluorescence (IF) of K8 (red) and K14 (green) with nuclei stained with DAPI (blue) in cP^f/f94^f/f uteri and PAS-diastase staining of cP^f/f94^f/f uteri at the indicated age. Scale bar, 200 μm. White arrows indicate glands, white bars denote SCM and yellow arrows indicate cells positive for both K8 and K14. Enlarged view of the boxed region is shown.

Figure 6. Attenuated AKT activation and decreased proliferation in cP^f/f94^f/f SCM

A. IHC of phospho-S6 (pS6) and S6 in uteri of the indicated genotypes at 4 weeks. White arrows indicate glands, black and red bars denote columnar luminal epithelial cells and SCM, respectively. B. IF of pAKT (Ser 473) and IHC of AKT. White arrows indicate glands, white and red bars denote columnar luminal epithelial cells and SCM, respectively. C. IHC of Ki67 and phospho-Histone H3 (p-H3) in mouse uteri. Scale bar, 100 μm. D. Quantification of the p-H3 positive epithelial cells in the uteri sections of WT (n=6), cP^f/f (n=3) and cP^f/f94^f/f (n=4) uteri at 4 weeks. The data are presented as mean ± s.e., **p<0.01 (LSD Method).

Figure 7. Characterization of cP^f/f94^f/f uteri on myometrial invasion and prolonged stage

A. IHC of pan-cytokeratin (panCK) and α-smooth muscle actin (α-SMA) on consecutive slides in uteri of the indicated genotypes at 8 weeks. The red broken lines denote the boundaries between the endometrium and the myometrium. Arrows indicate EAC invasion into the myometrium. Scale bar, 400 μm. B. Frequency of myometrial invasion in cP^f/f and cP^f/f94^f/f uteri at 8-9 weeks. Each circle represented one mouse. p=0.004 (2-sided Fisher's exact test). C. IHC of E-cadherin at 8 weeks. Scale bar, 200 μm. D. IF of E-cadherin on frozen tissue sections (red) at 8 weeks. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. E. H&E of WT, cP^f/f and cP^f/f94^f/f uteri at 12 weeks (upper panel) and 20 weeks (lower panel). Scale bar, 200 μm.

Figure 8. Summary model on the effect of GRP94 deficiency in mouse uteri in the presence or absence of PTEN-deficiency

The development of 4 and 8 week uteri of the indicated genotypes is shown. The morphological transitions of the glands, SCM replacement, EAC formation, alteration of the signaling pathways and E-cadherin expression are summarized for 4 and 8 week uteri. EAC: endometrial cancer. SCM: squamous cell metaplasia.