Spasmolytic polypeptide expressing metaplasia (SPEM) develops as a preneoplastic lesion in the stomachs of mice and humans after parietal cell loss. To identify the commonalities and differences between phenotypic SPEM lineages, we studied SPEM from three different mouse models of parietal cell loss: 1) with chronic inflammation with H. felis infection, 2) with acute inflammation with L635 treatment and 3) without inflammation following DMP-777 treatment.
RNA transcripts from laser capture microdissected normal chief cells and SPEM lineages were compared utilizing gene microarray. Alterations in transcripts were validated by qRT-PCR. Clusterin and cystic fibrosis transmembrane conductance regulator (CFTR) were selected for immunohistochemical analysis in all mouse models as well as in human SPEM, intestinal metaplasia, and gastric cancer.
Transcript expression patterns demonstrated differences among the phenotypic SPEM models. Clusterin expression was significantly upregulated in all three mouse SPEM models as well as in human SPEM. The highest clusterin expression in human gastric cancers correlated with poor survival. Conversely, CFTR expression was upregulated only in SPEM with inflammation in mice. In humans, intestinal metaplasia, but not SPEM, expressed CFTR.
While markers such as clusterin are expressed in all phenotypic SPEM lineages, distinct patterns of upregulated genes including CFTR are present in murine metaplasia associated with inflammation, indicative of progression of metaplasia towards a more intestinalized metaplastic phenotype.
Clusterin; CFTR; H. felis; DMP-777; Spasmolytic Polypeptide-Expressing Metaplasia
A tripartite association of Rab11a with both Rab11-FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11-FIP2 with MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11-FIP2 that caused loss of interaction with MYO5B in yeast 2-hybrid assays as well as loss of interaction of Rab11-FIP2(129-356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full-length Rab11-FIP2 with MYO5B tail in HeLa cells. While EGFP-Rab11-FIP2 wild type co-localized with endogenous MYO5B staining in MDCK cells, EGFP-Rab11-FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a-containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11-FIP2 association is perturbed by mutation or by Rab11-FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11-FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles.
Rab11; MYO5B; Rab11-FIP2; membrane recycling
Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea.
Epithelial cell carcinogenesis involves the loss of polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation and increased cell motility and invasion. Elements of membrane vesicle trafficking underlie all of these processes. Specific membrane trafficking regulators, including Rab small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine cell surface presentation of proteins and overall function of both differentiated and neoplastic cells. While mutations in vesicle trafficking proteins may not be direct drivers of transformation, elements of the machinery of vesicle movement play critical roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are critical mediators of the full spectrum of cell physiologies driving cancer cell biology, including initial loss of polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may provide important points for manipulation of cancer cell behaviour.
Upper gastrointestinal neoplasia in the esophagus, stomach and pancreas is associated with the formation of pre-neoplastic metaplasias. We have previously reported the up-regulation of Human epididymis protein 4 (HE4) in all metaplasias in the stomach of humans and mice. We have now sought to evaluate the expression of HE4 in metaplasias/pre-neoplastic precursors and cancers of the human stomach, pancreas and esophagus. Tissue microarrays for gastric cancers, pancreatic cancers and esophageal adenocarcinoma were stained with antibodies against HE4. Immunostaining was quantified by digital imaging and the results were evaluated to assess expression in metaplasias, expression in cancer pathological subtypes and the effects of expression on survival in cancer patients. In gastric cancer patients from Korea, HE4 was detected in 74% of intestinal and 90% of diffuse cancers, while in a gastric cancer cohort from Johns Hopkins HE4 was detected 74% of intestinal type and 92% of diffuse cancers. Nevertheless, in both cohorts there was no impact of HE4 expression on overall survival. In the esophagus, we observed expression of HE4 in scattered endocrine cells within Barrett’s esophagus samples, but Barrett’s columnar metaplasias and HE4 was detected in only 2% of esophageal adenocarcinomas. Finally, in the pancreas, HE4 expression was not observed in pancreatic intraepithelial neoplasia (PanIN) lesions, but 46.8% of pancreatic adenocarcinomas expressed HE4 expression. Still, we did not observe any influence of HE4 expression on survival. The results suggest that HE4 is up-regulated during gastric and pancreatic carcinogenesis.
WFDC2; HE4; metaplasia; adenocarcinoma
The Rab11 GTPases and Rab11 family-interacting proteins (Rab11-FIPs) define integrated yet distinct compartments within the slow recycling pathway. The lipid content of these compartments is less well understood, although past studies have indicated phosphatidylserine (PS) is an integral component of recycling membranes. We sought to identify key differences in the presence of PS within Rab and Rab11-FIP containing membranes. We used live cell fluorescence microscopy and structured illumination microscopy to determine whether the previously published LactC2 probe for PS displays differential patterns of overlap with various Rab GTPases and Rab11-FIPs. Selective overlap was observed between the LactC2 probe and Rab GTPases when co-expressed in HeLa cells. Rab11-FIP1 proteins consistently overlapped with LactC2 along peripheral and pericentriolar compartments. The specificity of Rab11-FIP1 association with LactC2 was further confirmed by demonstrating that additional Rab11-FIPs (FIP2, FIP3, and FIP5) exhibited selective association with LactC2 containing compartments. Live cell dual expression studies of Rab11-FIPs with LactC2 indicated that PS is enriched along tubular compartments of the Rab11a-dependent recycling system. Additionally, we found that the removal of C2 domains from the Rab11-FIPs induced an accumulation of LactC2 probe in the pericentriolar region, suggesting that inhibition of trafficking through the recycling system can influence the distribution of PS within cells. Finally, we confirmed these findings using structured illumination microscopy suggesting that the overlapping fluorescent signals were on the same membranes. These results suggest distinct associations of Rab GTPases and Rab11-FIPs with PS-containing recycling system membrane domains.
Rab11-FIP; Rab11a; Rab5; Rab7; Rab8a; live cell microscopy; phophatidylserine; structured illumination
Little research has addressed the role of membrane trafficking and recycling in the regulation of the transformed phenotype of neoplastic cells. The small GTPase Rab25 is an epithelial-specific modulator of membrane recycling. Recent studies have demonstrated that Rab25 expression is up-regulated in a number of epithelial cancers and over-expression may increase the aggressive phenotype of certain cancers. We have utilized the non-transformed RIE cell line to examine the influence of Rab25 on transformation. Over-expression of Rab25 in RIE cells leads to morphological transformation as well as growth in soft agar, tumor formation in nude mice, disruption of integrin-based focal adhesions and alteration in modified microtubule subsets. Although the predominance of recent cancer research has focused on the manipulation of the actin-based cytoskeleton, recycling trafficking relies on microtubules. Transformation of RIE cells through over-expression of Rab25, but not with H-RasV12, was reversed by inhibitors of microtubule polymerization. These results suggest that up-regulation of Rab25 in RIE cells leads to microtubule-dependent transformation. Thus, depolymerization of microtubules may be a potent therapeutic target for cancer therapy through the reversal of the invasive phenotype of certain cancer cells.
Rab25; β1-integrin; paxillin; microtubules; indibulin; nocodazole
Metaplastic lineages in the oxyntic mucosa of the stomach are critical preneoplastic precursors of gastric cancer. Recent studies have demonstrated that Spasmolytic polypeptide-expressing metaplasia (SPEM) in the mouse oxyntic mucosa arises from transdifferentiation of mature gastric chief cells. Other investigations of intestinal progenitor cells have shown that cells demonstrating transcriptional activity for Lgr5 in the intestine, colon and gastric antrum function as adult stem cells. We have now investigated whether cells demonstrating Lgr5 transcriptional activity in the oxyntic mucosa of mice might be responsible for development of metaplasia.
Lgr5-EGFP-IRES-CreERT2/+;Rosa26R mice were utilized to examine the distribution of Lgr5 transcriptionally active cells in the normal oxyntic mucosa as well as after treatment with DMP-777 or L635 to induce acute SPEM. Lineage mapping was performed to determine if LGR5-expressing cells gave rise to SPEM.
Cells expressing transcriptional activity for Lgr5 in the oxyntic mucosa were present as scattered rare cells only along the lesser curvature of the stomach. These cells also stained for markers of chief cells (intrinsic factor and pepsinogen) but never showed any staining for proliferative markers (Ki-67). In Lgr5-EGFP-IRES-CreERT2/+;Rosa26R mice induced with tamoxifen, treatment with either DMP-777 or L-635 to induce acute oxyntic atrophy caused induction of SPEM, but no lineage mapping into SPEM from Lgr5-expressing cells was observed.
The results indicate that, while chief cells with Lgr5-transcriptional activity are present along the lesser curvature of the gastric oxyntic mucosa, they are not responsible for production of metaplasia.
SPEM; chief cell; Lgr5; TFF2; oxyntic atrophy
Eradication of Helicobacter pylori correlates with regeneration of the gastric epithelium, ulcer healing and re-expression of the gastric morphogen Sonic Hedgehog (Shh). We sought to identify the role of Shh as a regulator of gastric epithelial regeneration during wound healing. A mouse model expressing a parietal cell-specific, tamoxifen-inducible deletion of Shh (HKCreERT2;Shhflox/flox or PC-iShhKO) was developed. Stomachs were collected and compared 7 to 150 days after the final vehicle or tamoxifen injection. Ulcers were induced in both controls and PC-iShhKO mice using acetic acid and ulcer size compared 1 and 7 days post induction. 1) Re-expression of Shh correlates with decreased hyperproliferation: Compared to controls, PC-iShhKO mice developed foveolar hyperplasia. Restoration of normal gastric epithelial architecture and differentiation correlated with the re-expression of Shh in PC-iShhKO mice 150 days after the final tamoxifen injection. At the tamoxifen dose used to induce Cre recombination there was no genotoxicity reported in either HKCreERT2 or Shhflox/flox control mouse stomachs. 2) Delayed wound healing in PC-iShhKO mouse stomachs: To identify the role of Shh in gastric regeneration, an acetic acid ulcer was induced in control and PC-iShhKO mice. Ulcers began to heal in control mice by 7 days after induction. Ulcer healing was documented by decreased ulcer size, angiogenesis, macrophage infiltration and formation of granulation tissue that correlated with the re-expression of Shh within the ulcerated tissue. PC-iShhKO mice did not show evidence of ulcer healing. Re-expression of Shh contributes to gastric regeneration. Our current study may have clinical implications given that eradication of Helicobacter pylori correlates with re-expression of Shh, regeneration of the gastric epithelium and ulcer healing.
Helicobacter pylori (H. pylori); gastric ulcer; re-epithelialization; tissue repair; macrophages
Rab25 is a tumor suppressor in the colon, but the mechanisms underlying the influence of Rab25 on polarity are unknown. Findings on changes in polarity in Caco2-BBE cells with knockdown and rescue of Rab25 expression indicate that Rab25 regulates integrin gene expression mediated by ETV4.
Rab25 is a tumor suppressor for colon cancer in humans and mice. To identify elements of intestinal polarity regulated by Rab25, we developed Caco2-BBE cell lines stably expressing short hairpin RNA for Rab25 and lines rescuing Rab25 knockdown with reexpression of rabbit Rab25. Rab25 knockdown decreased α2-, α5-, and β1-integrin expression. We observed colocalization and direct association of Rab25 with α5β1-integrins. Rab25 knockdown also up-regulated claudin-1 expression, increased transepithelial resistance, and increased invasive behavior. Rab25-knockdown cells showed disorganized brush border microvilli with decreases in villin expression. All of these changes were reversed by reintroduction of rabbit Rab25. Rab25 knockdown altered the expression of 29 gene transcripts, including the loss of α5-integrin transcripts. Rab25 loss decreased expression of one transcription factor, ETV4, and overexpression of ETV4 in Rab25-knockdown cells reversed losses of α5β1-integrin. The results suggest that Rab25 controls intestinal cell polarity through the regulation of gene expression.
The Rab11-family interacting proteins (Rab11-FIPs) facilitate Rab11-dependent vesicle recycling, yet it is unknown how these effectors cooperate with each other during recycling. It is found that Rab11-FIPs exhibit selective cooperation along dynamic tubular compartments to fill distinct spatiotemporal roles during recycling.
The Rab11-family interacting proteins (Rab11-FIPs) facilitate Rab11-dependent vesicle recycling. We hypothesized that Rab11-FIPs define discrete subdomains and carry out temporally distinct roles within the recycling system. We used live-cell deconvolution microscopy of HeLa cells expressing chimeric fluorescent Rab11-FIPs to examine Rab11-FIP localization, transferrin passage through Rab11-FIP–containing compartments, and overlap among Rab11-FIPs within the recycling system. FIP1A, FIP2, and FIP5 occupy widely distributed mobile tubules and vesicles, whereas FIP1B, FIP1C, and FIP3 localize to perinuclear tubules. Internalized transferrin entered Rab11-FIP–containing compartments within 5 min, reaching maximum colocalization with FIP1B and FIP2 early in the time course, whereas localization with FIP1A, FIP1C, FIP3, and FIP5 was delayed until 10 min or later. Whereas direct interactions with FIP1A were only observed for FIP1B and FIP1C, FIP1A also associated with membranes containing FIP3. Live-cell dual-expression studies of Rab11-FIPs revealed the tubular dynamics of Rab11-FIP–containing compartments and demonstrated a series of selective associations among Rab11-FIPs in real time. These findings suggest that Rab11-FIP1 proteins participate in spatially and temporally distinct steps of the recycling process along a complex and dynamic tubular network in which Rab11-FIPs occupy discrete domains.
SMAD proteins are downstream effectors of the TGF-β signaling pathway. Smad3 null mice develop colorectal cancer by 6 months of age. In this study, we have examined whether the loss of Smad3 promotes gastric neoplasia in mice.
The stomachs of Smad3−/− mice were compared with age-matched Smad3 heterozygous and wild type mice. E-cadherin, Ki-67, phosphoSTAT3, and TFF2/SP expression was analyzed by immunohistochemisty. The shRNA-mediated knockdown of Smad3 in AGS and MKN28 cells was also performed. In addition, we examined alterations in DCLK1-expressing cells.
Smad3−/− mouse stomachs at 6 months of age revealed the presence of exophytic growths along the lesser curvature in the proximal fundus. Six-month-old Smad3−− mouse stomachs showed metaplastic columnar glands initiating from the transition zone junction between the forestomach and the glandular epithelium along the lesser curvature. Ten month-old Smad3−/− mice all exhibited invasive gastric neoplastic changes with increased Ki-67, phosphoSTAT3 expression, and aberrant cytosolic E-cadherin staining in papillary glands within the invading submucosal gland. The shRNA-mediated knockdown of Smad3 in AGS and MKN28 cells promoted the expression of phosphoSTAT3. DCLK1-expressing cells, which also stained for the tuft cell marker acetylated-α-tubulin, were observed in 10-month-old Smad3−/− mice within tumors and in fundic invasive lesions.
Smad3 null mice develop gastric tumors in the fundus, which arise from the junction between the forestomach and the glandular epithelium and progress to prominent invasive tumors over time. Smad3 null mice represent a novel model of fundic gastric tumor initiated from forestomach/glandular transition zone along the lesser curvature.
Animal model; Forestomach; DCLK1; HE4; Gastric tumor development; Smad3; SPEM; Tuft cell
The digestive-enzyme secreting, gastric epithelial chief (zymogenic) cell is remarkable and underappreciated. Here, we discuss how all available evidence suggests that mature chief cells in the adult, mammalian stomach are postmitotic, slowly turning over cells that arise via a relatively long-lived progenitor, the mucous neck cell, The differentiation of chief cells from neck cells does not involve cell division, and the neck cell has its own distinct pattern of gene expression and putative physiological function. Thus, the ontogeny of the normal chief cell lineage exemplifies transdifferentiation. Furthermore, under pathophysiogical loss of acid-secreting parietal cell, the chief cell lineage can itself trasndifferentiate into a mucous cell metaplasia designated Spasmolytic Polypeptide Expressing Metaplasia (SPEM). Especially in the presence of inflammation, this metaplastic lineage can regain proliferative capacity and, in humans may also further differentiate into intestinal metaplasia. The results indicate that gastric fundic lineages display remarkable plasticity in both physiological ontogeny and pathophysiological pre-neoplastic metaplasia.
Gastric adenocarcinoma; metaplasia; SPEM; intestinal metaplasia; transdifferentiation; chief cell; zymogenic cell
Ser-227 phosphorylation of Rab11-FIP2 by Par1b/MARK2 regulates the establishment of polarized epithelial monolayers in three-dimensional MDCK cell cultures and has an ongoing influence on the composition of both adherens and tight junctions in polarized epithelial cells.
The Rab11 effector Rab11-family interacting protein 2 (Rab11-FIP2) regulates transcytosis through its interactions with Rab11a and myosin Vb. Previous studies implicated Rab11-FIP2 in the establishment of polarity in Madin–Darby canine kidney (MDCK) cells through phosphorylation of Ser-227 by MARK2. Here we examine the dynamic role of Rab11-FIP2 phosphorylation on MDCK cell polarity. Endogenous Rab11-FIP2 phosphorylated on Ser-227 coalesces on vesicular plaques during the reestablishment of polarity after either monolayer wounding or calcium switch. Whereas expression of the nonphosphorylatable Rab11-FIP2(S227A) elicits a loss in lumen formation in MDCK cell cysts grown in Matrigel, the putative pseudophosphorylated Rab11-FIP2(S227E) mutant induces the formation of cysts with multiple lumens. On permeable filters, Rab11-FIP2(S227E)–expressing cells exhibit alterations in the composition of both the adherens and tight junctions. At the adherens junction, p120 catenin and K-cadherin are retained, whereas the majority of the E-cadherin is lost. Although ZO-1 is retained at the tight junction, occludin is lost and the claudin composition is altered. Of interest, the effects of Rab11-FIP2 on cellular polarity did not involve myosin Vb or Rab11a. These results indicate that Ser-227 phosphorylation of Rab11-FIP2 regulates the composition of both adherens and tight junctions and is intimately involved in the regulation of polarity in epithelial cells.
Background & Aims
Intestinal metaplasia (IM) and spasmolytic polypeptide-expressing metaplasia (SPEM) are precursors to gastric carcinogenesis. We sought to identify molecular biomarkers of gastric metaplasias and gastric cancer by gene expression profiling of metaplastic lesions from patients.
cDNA microarray analysis was performed on IM and SPEM cells isolated from patient samples using laser capture microdissection. Up-regulated transcripts in metaplstic lesions were confirmed by immunostaining analysis in IM, SPEM, and gastric cancer tissues. Proteins that were highly expressed specifically in gastric cancer tissues were analyzed for their association with survival in a test set (n=450) and a validation set (n=502) of samples from gastric cancer patients.
Compared to normal chief cells, 858 genes were differentially expressed in IM or SPEM samples. Immunostaining was detected for 12 proteins, including 3 new markers of IM (ACE2, LGALS4, AKR1B10) and 3 of SPEM (OLFM4, LYZ, DPCR1). Of 13 proteins expressed in IM or SPEM, 8 were expressed by 17%–50% of human gastric cancer tissues (MUC13, OLFM4, CDH17, KRT20, MUC5AC, LGALS4, AKR1B10, REG4). Expression of CDH17 or MUC13 correlated with patient survival in the test and a validation sets. Multivariate analysis showed that CDH17 was an independent prognostic factor in patients with stage I or node-negative disease.
We identified several novel biomarkers for IM, SPEM, and gastric cancer using gene expression profiling of human metaplastic lesions. Expression of CDH17 and MUC13 was upregulated in gastric cancer tissues. CDH17 is a promising prognostic marker for early-stage gastric cancer.
metaplasia; cadherin-17; MUC13; gastric cancer; olfactomedin 4; SPEM; intestinal metaplasia
The Rab11 Family Interacting Proteins (Rab11-FIPs) are hypothesized to regulate sequential steps in the apical recycling and transcytotic pathways of polarized epithelial cells. Previous studies have suggested that Rab11-FIP proteins assemble into multi-protein complexes regulating plasma membrane recycling. Rab11-FIP2 interacts with both myosin Vb and Rab11. Recent investigations have noted that that Rab11-FIP2 mutants [Rab11-FIP2(129–512), also designated Rab11-FIP2(ΔC2) and Rab11-FIP2(S229A, R413G), also designated Rab11-FIP2(SARG)], are potent inhibitors of transcytosis in polarized MDCK cells. Interestingly, Rab11-FIP2(ΔC2), but not Rab11-FIP2(SARG), also altered the morphology of the EEA-1 positive early endosomal compartment. These findings suggested that Rab11-FIP2 mutants could differentiate different points along the recycling pathway. We therefore sought to investigate whether Rab11-FIP2 is a general regulator of the early endosomal system. Both Rab11-FIP2 mutants altered the localization and co-localized with dynein heavy chain. In contrast, both clathrin heavy chain and AP-1 accumulated with membranes containing Rab11-FIP2(SARG), but not with Rab11-FIP2(ΔC2). Expression of Rab11-FIP2(ΔC2), but not Rab11-FIP2(SARG), caused clustering of early endosomal markers Rab5b, Epsin 4 and IQGAP1, around a collapsed Rab11-FIP2 containing membranous cisternum. Interestingly, neither Rab11-FIP2 mutant had any effect on the distribution of Rab5a, a classical early endosome marker. The results support the view that Rab11-FIP2 may influence microtubule-dependent centripetal movement of subsets of early endosomes as well as processing through the common and recycling endosomal systems.
Rab11-FIP2; Rab11; trafficking; apical recycling; endosome; MDCK cells; clathrin; dynein; Rab5; epsin
BACKGROUND & AIMS
Gastric cancer evolves in the setting of a pathologic mucosal milieu characterized by both loss of acid-secreting parietal cells and mucous cell metaplasias. Indeed, mucous cell metaplasia is considered the critical preneoplastic lesion for gastric cancer. Previous investigations have shown that infection of mice with Helicobacter felis or induction of acute parietal cell loss with the drug DMP-777 leads to the emergence of a type of metaplasia designated spasmolytic polypeptide-expressing metaplasia (SPEM). We have hypothesized that SPEM arises from proliferating cells in gland bases, either from a cryptic progenitor cell or by transdifferentiation of mature chief cells.
Taking advantage of the chief cell-restricted expression of Mist1-Cre-ERT2, we used lineage mapping to examine whether SPEM lineages were derived from chief cells in 3 independent models of induction by DMP-777 treatment, L-635 treatment, or H felis infection.
Treatment of mice with L-635 for 3 days led to rapid parietal cell loss, induction of a prominent inflammatory infiltrate, and emergence of SPEM. In all 3 models, SPEM developed, at least in part, from transdifferentiation of chief cells. We further found that acute parietal cell loss in the setting of inflammation (L-635 treatment) led to more rapid induction and expansion of SPEM derived from transdifferentiation of chief cells.
These studies provide direct evidence by lineage tracing that SPEM evolves from differentiated chief cells. Thus, mature gastric chief cells have the ability to act as cryptic progenitors and reacquire proliferative capacity within the context of mucosal injury and inflammation.
SPEM; Chief Cell; Transdifferentiation; Metaplasia
Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical “canalicular” membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.
AKAP350; PKA; bile canaliculus; actin cytoskeleton; CLASP
Background & Aims
Loss of gastric parietal cells is a critical precursor to gastric metaplasia and neoplasia. However, the origin of metaplasia remains obscure. Acute parietal cell loss in gastrin-deficient mice treated with DMP-777 leads to the rapid emergence of Spasmolytic polypeptide/TFF2-expressing metaplasia (SPEM) from the bases of fundic glands. We have now sought to characterize more definitively the pathway for emergence of SPEM.
Emerging SPEM lineages in gastrin deficient mice treated with DMP-777 were examined for immunolocalization of TFF2, intrinsic factor and Mist1 and with electron microscopy. Emerging SPEM was isolated with laser capture microdissection and RNA was analyzed using gene microarrays. Immunohistochemistry in mouse and human samples was used to confirm up-regulated transcripts.
DMP-777-induced SPEM was immunoreactive for TFF2 and the differentiated chief cell markers, Mist1 and intrinsic factor, suggesting that SPEM derived from transdifferentiation of chief cells. Microarray analysis of microdissected SPEM lineages induced by DMP-777 showed up-regulation of transcripts associated with G1/S cell cycle transition including MCM proteins, as well as a number of secreted factors, including HE4. HE4, which was absent in the normal stomach, was expressed in SPEM of human and mouse and in intestinal metaplasia and gastric cancer in humans.
While traditionally metaplasia was thought to originate from normal mucosal progenitor cells, these studies indicate that SPEM evolves through either transdifferentiation of chief cells or activation of a basal cryptic progenitor. Additionally, induction of metaplasia elicits the expression of secreted factors, such as HE-4, relevant to gastric pre-neoplasia.
Our previous study indicated that gene expression profiling of intestinal metaplasia (IM) or spasmolytic polypeptide-expressing metaplasia (SPEM) can identify useful prognostic markers of early stage gastric cancer, and seven metaplasia biomarkers (MUC13, CDH17, OLFM4, KRT20, LGALS4, MUC5AC, and REG4) were selectively expressed in 17-50% of gastric cancer tissues. We investigated whether the combined expression of these metaplasia biomarkers could predict the prognosis of advanced stage gastric cancer.
The expression of seven metaplasia biomarkers was evaluated immunohistochemically using tissue microarrays comprised of 450 gastric cancer patients. The clinicopathologic correlations and the prognostic impact were analyzed according to the expression of multiple biomarkers.
MUC13, CDH17, LGALS4 and REG4 were significant prognostic biomarkers in univariate analysis. No expression of four markers was found in 56 cases (14.2%); 1 marker was seen in 67 cases (17.0%), 2 in 106 cases (27.0%), 3 in 101 cases (25.7%), and 4 in 63 cases (16.0%). Patients in which ≤ 2 proteins were expressed (Group B) showed younger age, undifferentiated or diffuse type cancer, a larger tumor size, a larger number of metastatic lymph nodes and more advanced stage than those in which ≥ 3 proteins were expressed (Group A). In undifferentiated or stage II/III gastric cancer, the prognosis of Group B was significantly poorer than that of Group A by multivariate analysis.
The combined loss of expression of multiple metaplasia biomarkers is considered as an independent prognostic indicator in undifferentiated or stage II/III gastric cancer.
Background & Aims
The loss of parietal cells from the fundic mucosa leads to the emergence of metaplastic lineages associated with an increased susceptibility to neoplastic transformation. Both intestinal metaplasia (IM) and spasmolytic polypeptide (TFF2/SP) expressing metaplasia (SPEM) have been identified in human stomach, but only SPEM is present in most mouse models of gastric metaplasia. We previously determined that loss of amphiregulin (AR) promotes SPEM induced by acute oxyntic atrophy. We have now examined whether SPEM in the AR−/− mouse predisposes the stomach to gastric neoplasia.
Gross pathology of 18-month-old wild-type, AR−/−, and TGF-α −/− mice were examined. Ki-67, β-catenin, Pdx-1, TFF3, and TFF2/SP expression was analyzed by immunohistochemistry. Metaplastic gastric mucosa was analyzed by dual immunostaining for TFF2/SP with MUC2 or TFF3.
By 18 months of age, more than 70% of AR−/− mice developed SPEM while 42% showed goblet cell IM labeled with MUC2, TFF3, and Pdx-1. A total of 28% had invasive gastric lesions in the fundus. No antral abnormalities were observed in AR−/− mice. Metaplastic cell lineages in AR−/− mice showed increases in cell proliferation and cytosolic β-catenin expression. Dual staining for TFF2/SP with MUC2 or TFF3 showed glands containing both SPEM and IM with intervening cells expressing both TFF2/SP and MUC2 or TFF2/SP and TFF3.
AR−/− mice develop SPEM, which gives rise to goblet cell IM and invasive fundic dysplastic lesions. The AR−/− mouse represents the first mouse model for spontaneous development of fundic SPEM with progression to IM.
Transformation of epithelial cells is associated with loss of cell polarity, which includes alterations in cell morphology as well as changes in the complement of plasma membrane proteins. Rab proteins regulate polarized trafficking to the cell membrane and therefore represent potential regulators of this neoplastic transition. Here we have demonstrated a tumor suppressor function for Rab25 in intestinal neoplasia in both mice and humans. Human colorectal adenocarcinomas exhibited reductions in Rab25 expression independent of stage, with lower Rab25 expression levels correlating with substantially shorter patient survival. In wild-type mice, Rab25 was strongly expressed in cells luminal to the proliferating cells of intestinal crypts. While Rab25-deficient mice did not exhibit gross pathology, ApcMin/+ mice crossed onto a Rab25-deficient background showed a 4-fold increase in intestinal polyps and a 2-fold increase in colonic tumors compared with parental ApcMin/+ mice. Rab25-deficient mice had decreased β1 integrin staining in the lateral membranes of villus cells, and this pattern was accentuated in Rab25-deficient mice crossed onto the ApcMin/+ background. Additionally, Smad3+/– mice crossed onto a Rab25-deficient background demonstrated a marked increase in colonic tumor formation. Taken together, these results suggest that Rab25 may function as a tumor suppressor in intestinal epithelial cells through regulation of protein trafficking to the cell surface.
Recent investigations have highlighted the importance of subcellular localization of mRNAs to cell function. While AKAP350A, a multifunctional scaffolding protein, localizes to the Golgi apparatus and centrosomes, we have now identified a cytosolic pool of AKAP350A. Analysis of AKAP350A scaffolded complexes revealed two novel interacting proteins, CCAR1 and caprin-1. CCAR1, caprin-1 and AKAP350A along with G3BP, a stress granule marker, relocate to RNA stress granules after arsenite treatment. Stress also caused loss of AKAP350 from the Golgi and fragmentation of the Golgi apparatus. Disruption of microtubules with nocodazole altered stress granule formation and changed their morphology by preventing fusion of stress granules. In the presence of nocodazole, arsenite induced smaller granules with the vast majority of AKAP350A and CCAR1 separated from G3BP-containing granules. Similar to nocodazole treatment, reduction of AKAP350A or CCAR1 expression also altered the size and number of G3BP-containing stress granules induced by arsenite treatment. A limited set of 69 mRNA transcripts was immunisolated with AKAP350A even in the absence of stress, suggesting the association of AKAP350A with mRNA transcripts. These results provide the first evidence for the microtubule dependent association of AKAP350A and CCAR1 with RNA stress granules.
AKAP350 (AKAP450/AKAP9/CG-NAP) is an A-kinase anchoring protein, which recruits multiple signaling proteins to the Golgi apparatus and the centrosomes. Several proteins recruited to the centrosomes by this scaffold participate in the regulation of the cell cycle. Previous studies indicated that AKAP350 participates in centrosome duplication. In the present study we specifically assessed the role of AKAP350 in the progression of the cell cycle. Our results showed that interference with AKAP350 expression inhibits G1/S transition, decreasing the initiation of both DNA synthesis and centrosome duplication. We identified an AKAP350 carboxyl-terminal domain (AKAP350CTD), which contained the centrosomal targeting domain of AKAP350 and induced the initiation of DNA synthesis. Nevertheless, AKAP350CTD expression did not induce centrosomal duplication. AKAP350CTD partially delocalized endogenous AKAP350 from the centrosomes, but increased the centrosomal levels of the cyclin-dependent kinase 2 (Cdk2). Accordingly, the expression of this AKAP350 domain increased the endogenous phosphorylation of nucleophosmin by Cdk2, which occurs at the G1/S transition and is a marker of the centrosomal activity of the cyclin E-Cdk2 complex. Cdk2 recruitment to the centrosomes is a necessary event for the development of the G1/S transition. Altogether, our results indicate that AKAP350 facilitates the initiation of DNA synthesis by scaffolding Cdk2 to the centrosomes, and enabling its specific activity at this organelle. Although this mechanism could also be involved in AKAP350-dependent modulation of centrosomal duplication, it is not sufficient to account for this process.
AKAP350; AKAP450; CG-NAP; Cdk2; centrosome; G1/S transition