Laboratory Investigation (2012) 92, 1670–1673 & 2012 USCAP, Inc All rights reserved 0023-6837/12 $32.00 EDITORIAL MDR-1, Bcl-xL, H. pylori, and Wnt/b-catenin signalling in the adult stomach: how much is too much? R John MacLeod Laboratory Investigation (2012) 92, 1670–1673; doi:10.1038/labinvest.2012.151 ultiple drug resistance (MDR) is a interaction between the antiapoptotic protein major cause of failure of che- Bcl-xL and MDR-1. Knockdown of MDR-1 motherapy in cancer treatment. increases the apoptotic index of these cells exposed The membrane transporter P-gly- to oxidative stress consistent with a role for MDR- coprotein (MDR-1, Pgp) encoded 1 in apoptosis. Several questions emerge from Mby the adenosine triphosphate-binding cassette, these findings. The first is why is MDR-1 increased subfamily B, member 1 is the main mechanism for in some HP-positive mucosa but in 100% of the decreased intracellular drug accumulation in MDR intestinal metaplasia samples? A likely causative cancer.1 Increases in Mdr-1 expression prevent effector of the increase in MDR-1 is the activation tumor cells from a variety of induced apoptosis, of canonical or Wnt/b-catenin signaling. It has but how this occurs is poorly understood. It is been known for a dozen years that the MDR-1 essential to understand how this occurs to be able gene may be stimulated by Tcf4.4 Yamada et al4 to design effective therapeutic interventions. The clearly demonstrated the presence of Tcf4 sites on study by Rocco et al2 (this issue) clearly shows that the MDR-1 promoter and showed that MDR-1 in the mitochondria of gastric cancer cell lines protein had substantially increased in adenomas MDR1 physically interacts with Bcl-xL, a well- and colon cancer. defined antiapoptotic effector. Furthermore, this Wnts are palmitolated glycoproteins that have elegant study compares MDR-1 expression in the key effects in development, inflammation, stem gastric mucosa of spontaneously aborted human cell maintenance, and cancer.5,6 There are two fetuses compared with normal adult mucosa, and types of Wnt signaling. Canonical or Wnt/b- as well reports on the presence of MDR-1 in catenin signaling regulates the concentration of Helicobacter pylori (HP) negative, positive chronic the effector b-catenin. Noncanonical Wnt gastritis and those with intestinal metaplasia and signaling in general will inhibit b-catenin signaling 2 þ 7 intestinal type gastritis. Together their interesting and/or stimulate JNK, PKC and [Ca ]i release. findings support the idea that in the stomach, In the absence of Wnt signaling, the amounts of MDR-1 behaves as an oncofetal protein, which the effector b-catenin are tightly regulated by a during HP-related gastric carcinogenesis may complex of scaffold proteins, Auxin and the tumor cross-talk with Bcl-xL to work in an antiapoptotic suppressor adenomatous polyposis coli (APC) Department of Bio- manner. together with GSK-3b. When a Wnt protein binds medical and Molecular The critical findings of Rocco et al2 are its two co-receptors Frizzled (Fzd) and Science and Medicine, illustrated in Figure 1a. MDR-1 is increased and is lipoprotein-related peptide 6 (LRP6), the auxin- Canada Research Chair in GI Cell Physiology, localized to the mitochondria and presumably the APC scaffold releases b-catenin. The Wnt- Queen’s University, plasma membrane of AGS and MKN-28 cell lines. stimulated Fzd recruits dishevelled (Dvl) to Kingston, ON, Canada These lines each contain well-defined mutations the trimeric Wnt-Fzd-LRP6 receptor complex. Correspondence should be addressed to: and characterized responses to Akt and MAPK This activated Dvl then interacts with auxin and [email protected] inhibitors.3 Importantly, there is physical GSK-3b to form a LRP6-associated ‘signalsome’ 1670 Laboratory Investigation | Volume 92 December 2012 | www.laboratoryinvestigation.org EDITORIAL where GSK-3b phosphorylation of b-catenin is Cre induction generated a progressive loss of prevented. Recent experiments demonstrate that parietal cells with fundic gland polyposis and Wnt induces the association of the intact scaffold adenomas comparable with those observed in the complex, even when APC is truncated, with antrum. phosphorylated LRP6.8 The released b-catenin Thus in both the antrum and corpus, activation migrates to the nucleus to stimulate Lef/Tcf of Wnt/b-catenin signaling alone was not suffi- transcription factors to activate a large number of cient to drive malignancy. We know from the Wnt target genes such as C-Myc, CylcinD1, and elegant work of the Oshima group that Wnt1 Sox9. How GSK-3b activity is inhibited by Wnt (which is upregulated in later stage gastric can- 11 stimulation has been extensively studied and cer) when present together with PGE2 will requires sequestration in multi-vesicular bodies.9 generate an invasive gastric adenocarcinoma In sporadic colorectal cancers mutations in APC formation by 1 year.12 Consistent with this work, are found in B80% of the cancers. While most of the CK19 Cre-Wnt1 mice show higher levels of the steps leading to colorectal cancer have been nuclear b-catenin accumulation and pre-neplastic determined,5 knowledge of the same sequence of lesions but not tumorigenesis. So, stimulation of APC and b-catenin mutations in stomach cancer Wnt/b-catenin signaling could lead to is just beginning to be understood. microadenomas in the antrum and Strong evidence that altered Wnt/b-catenin hyperproliferation in the corpus with increased signaling in the stomach epithelia rapidly gen- expression of MDR-1 (Figure 1b). The cell type erates an initiating step in gastric tumourigenesis responsible for Wnt1 secretion in the stomach is has come from recent studies deleting APC or not known in detail but IHH has shown Wnt5a at GSK-3b, as well as expression of a constitutively the base of the normal gastric corpus mucosa.13 active b-catenin protein in inducible mouse However, later stages of gastric cancer (in- models.10 This elegant study found that activating testinal type and diffuse type) correlate with an Wnt/b-catenin signaling (using floxed truncated increase in Wnt5a protein expression. In gastric APC, floxed inactivated GSK-3b, or constitutively cancer, Wnt5a has been shown to activate the activated b-catenin mice) after Cre induction, small GTP-binding protein Rac1, which has an generated in the antral glands a rapid development established role in migration and metastasis.14 of small hyperproliferative micoradenomas. With Recent reports document that polyclonal age, these small lesions developed into large antibodies against Wnt5a inhibit invasion and adenomas, and IHH demonstrated high levels of metastasis of a variety of gastric cancer cell lines.15 c-MYC and Sox9. In the corpus region, the same While the gastric cancer cell lines secrete Wnt5a to Figure 1 (a) Multiple drug resistance (MDR)-1 physically interacts with the antiapoptotic Bcl-2 family member Bcl-xL on the mitochondria of gastric cancer cell lines and is present on mucosa of fetal stomach, then lost, but increased with intestinal type gastritis. (b) Increased Wnt/b-catenin signaling, initiated by either Wnt1 or Wnt5a, could increase MDR-1.4 The degradation complex composed of Auxin1 and adenomatous polyposis coli is linked to the Wnt/Fzd (frizzled)/LRP6 while b-catenin is released.8 H.pylori can stimulate LRP6 phosphorylation, and TNFa from macrophages can increase Wnt/b- catenin signaling19 in mouse stomach. Normal stomach turnover requires some Wnt/b-catenin signaling;24,25 but how much is too much? www.laboratoryinvestigation.org | Laboratory Investigation | Volume 92 December 2012 1671 EDITORIAL various degrees, the source of Wnt5a in the late The design of small molecules that block Bcl-xL stage of gastric cancer appears to be epithelial; interaction with these and other proteins is a both intestinal type and diffuse-adherent and - rapidly growing endeavor22 in order to scattered demonstrated increased Wnt5a by overcome resistance to conventional IHH.13 The determinants of Wnt5a secretion from chemotherapy. Analyses of mitochondria these types of cells are unknown. However, work to determine the relative amounts BH3 with colon adenocarcinoma cells has shown a proteins or antiapoptotic multidomain truncated APC was necessary for Wnt5a homologs (such as Bcl-xL) are present can guide secretion.16 therapy. An example of such mitochondrial The H. pylori samples in the Rocco study2 profiling, which predicted the activity of Bcl-2 showed some expression of MDR-1 while the antagonists allowing a more designed therapy has intestinal metaplasia samples showed 100%. Is it been demonstrated in neuroblastoma.23 Is it likely that a threshold limit of Wnt/b-catenin possible to intervene earlier in the development signaling is has been surpassed in the latter, while of gastric cancer? the canonical Wnt signaling activated by If the continuum of gastric cancer is initiated H. pylori17 is insufficient to increase MDR-1? with altered Wnt/b-catenin signaling leading to H. pylori is well known to stimulate LRP6 increased MDR-1 expression (Figure 1b), then phosphorylation, a gold-standard for Wnt/b- would inhibit either Wnt secretion or events distal catenin signaling.18 Indeed there is evidence that to Wnt signalsome formation attenuate Wnt/b- CagA can also disperse b-catenin from catenin signaling to a level, which prevents MDR-1 E-cadherin-b-catenin complexes to further increases but still allows some Wnt/b-catenin increase the concentration of free b-catenin signaling to occur? Some Wnt/b-catenin signaling capable of migrating to the nucleus. Other is required for normal stomach turnover—there H. pylori constituents such as VacA, OlpA, and are Lrg5 þ stem cells present in mouse pylorus. peptidyloglycan can activate PI3K, which will When Lgr5 þ cells are isolated they will grow stimulate Akt to then cause ser9 phosphorylation expanding gastric organoids.