Subversion of cellular defence mechanisms in H. pylori-induced stomach cancer

Project Overview

In nearly half of the human population, a Gram-negative bacterium called Helicobacter pylori colonises the gastric epithelium and is associated with an increased risk of developing gastric adenocarcinomas at the sites of infection. The epithelial depolarisation and hyperproliferation induced by H.pylori infection might be relevant events for cancer initiation (Bagnoli, Buti et al. 2005). However, the molecular mechanisms underlying H. Pylori-induced malignant transformation of gastric epithelial cells are still not well understood, but are probably related to the ability of the host cells to sense bacterial infection but with a failure in mounting and adequate response. Recent evidence suggests that one mechanism to maintain H. pylori infection, and consequently increase the probabililty of progression to gastric cancer, is to reduce the ability of the lysosomal machinery and autophagosomes to clear the infection (Terebiznik, Vazquez et al. 2006).

 

One of the key regulators of lysosomal biogenesis and autophagy is the MiT family of transcription factor comprising TFEB, TFE3 and MITF. These factors translocate from the cytoplasm to the nucleus in response to nutrient limitation or cellular stress, where they bind and regulate a wide range of genes involved in lysosome function, autophagy, the cell cycle and metabolism (Li, Friedrichsen et al. 2018). As such, they play a critical role in restoring homeostasis by for example, rebalancing nutrient supply and demand (Falletta, Sanchez-Del-Campo et al. 2017). Importantly in C. elegans, the TFEB homologue plays a key role in orchestrating the anti-microbial response (Visvikis, Ihuegbu et al. 2014). How TFEB or its related transcription factors TFE3 and MITF impact H. pylori infection and whether this pathogenic bacteria can  disrupt the regulation and activity of these key transcription factors is poorly understood.

 

To address this question we will use our newly developed functional, long-lived, epithelial polarised monolayers from healthy human stomachs, which fully recapitulate the different cell lineages found in situ, and accumulate mucins on the apical side. This ‘mucosoid’ culture system recapitulates the in vivo gastric epithelial layer and permits advanced studies of the bacterial sensing mechanisms and epithelial inflammation (Boccellato, Woelffling et al. 2018). This mucosoid culture system will be used to assess the bi-directional interplay between the MiT family members and H. pylori using a combination of real-time live cell imaging combined with fluorescent reporters for TFEB.

Training Opportunities

The project will provide training in a wide range of advanced 3D culture systems, flow cytometry and cell sorting as well as real-time live cell imaging, and will also provide opportunities for genome-wide transcriptomic analysis. All students at the Ludwig Institute are provided with training in all aspects of scientific development and are integrated into a highly interdisciplinary and collaborative environment.

Theme

Cancer Biology

Admissions

Project reference number: 1000

Funding and admissions information

Supervisors

Name Department Institution Country Email
Professor Colin R Goding Oxford Ludwig Institute Oxford University, Old Road Campus Research Building GBR colin.goding@ludwig.ox.ac.uk
Dr Francesco Boccellato Oxford Ludwig Institute Oxford University, Old Road Campus Research Building GBR francesco.boccellato@ludwig.ox.ac.uk

Visvikis O, Ihuegbu N, Labed SA, Luhachack LG, Alves AF, Wollenberg AC, Stuart LM, Stormo GD, Irazoqui JE. 2014. Innate host defense requires TFEB-mediated transcription of cytoprotective and antimicrobial genes. Immunity, 40 (6), pp. 896-909. Read abstract | Read more

Animal host defense against infection requires the expression of defense genes at the right place and the right time. Understanding such tight control of host defense requires the elucidation of the transcription factors involved. By using an unbiased approach in the model Caenorhabditis elegans, we discovered that HLH-30 (known as TFEB in mammals) is a key transcription factor for host defense. HLH-30 was activated shortly after Staphylococcus aureus infection, and drove the expression of close to 80% of the host response, including antimicrobial and autophagy genes that were essential for host tolerance of infection. TFEB was also rapidly activated in murine macrophages upon S. aureus infection and was required for proper transcriptional induction of several proinflammatory cytokines and chemokines. Thus, our data suggest that TFEB is a previously unappreciated, evolutionarily ancient transcription factor in the host response to infection. Hide abstract

Terebiznik MR, Vazquez CL, Torbicki K, Banks D, Wang T, Hong W, Blanke SR, Colombo MI, Jones NL. 2006. Helicobacter pylori VacA toxin promotes bacterial intracellular survival in gastric epithelial cells. Infect. Immun., 74 (12), pp. 6599-614. Read abstract | Read more

Helicobacter pylori colonizes the gastric epithelium of at least 50% of the world's human population, playing a causative role in the development of chronic gastritis, peptic ulcers, and gastric adenocarcinoma. Current evidence indicates that H. pylori can invade epithelial cells in the gastric mucosa. However, relatively little is known about the biology of H. pylori invasion and survival in host cells. Here, we analyze both the nature of and the mechanisms responsible for the formation of H. pylori's intracellular niche. We show that in AGS cells infected with H. pylori, bacterium-containing vacuoles originate through the fusion of late endocytic organelles. This process is mediated by the VacA-dependent retention of the small GTPase Rab7. In addition, functional interactions between Rab7 and its downstream effector, Rab-interacting lysosomal protein (RILP), are necessary for the formation of the bacterial compartment since expression of mutant forms of RILP or Rab7 that fail to bind each other impaired the formation of this unique bacterial niche. Moreover, the VacA-mediated sequestration of active Rab7 disrupts the full maturation of vacuoles as assessed by the lack of both colocalization with cathepsin D and degradation of internalized cargo in the H. pylori-containing vacuole. Based on these findings, we propose that the VacA-dependent isolation of the H. pylori-containing vacuole from bactericidal components of the lysosomal pathway promotes bacterial survival and contributes to the persistence of infection. Hide abstract

Li L, Friedrichsen HJ, Andrews S, Picaud S, Volpon L, Ngeow K, Berridge G, Fischer R, Borden KLB, Filippakopoulos P, Goding CR. 2018. A TFEB nuclear export signal integrates amino acid supply and glucose availability. Nat Commun, 9 (1), pp. 2685. Read abstract | Read more

How cells coordinate the response to fluctuating carbon and nitrogen availability required to maintain effective homeostasis is a key issue. Amino acid limitation that inactivates mTORC1 promotes de-phosphorylation and nuclear translocation of Transcription Factor EB (TFEB), a key transcriptional regulator of lysosome biogenesis and autophagy that is deregulated in cancer and neurodegeneration. Beyond its cytoplasmic sequestration, how TFEB phosphorylation regulates its nuclear-cytoplasmic shuttling, and whether TFEB can coordinate amino acid supply with glucose availability is poorly understood. Here we show that TFEB phosphorylation on S142 primes for GSK3β phosphorylation on S138, and that phosphorylation of both sites but not either alone activates a previously unrecognized nuclear export signal (NES). Importantly, GSK3β is inactivated by AKT in response to mTORC2 signaling triggered by glucose limitation. Remarkably therefore, the TFEB NES integrates carbon (glucose) and nitrogen (amino acid) availability by controlling TFEB flux through a nuclear import-export cycle. Hide abstract

Falletta P, Sanchez-Del-Campo L, Chauhan J, Effern M, Kenyon A, Kershaw CJ, Siddaway R, Lisle R, Freter R, Daniels MJ, Lu X, Tüting T, Middleton M, Buffa FM, Willis AE, Pavitt G, Ronai ZA, Sauka-Spengler T, Hölzel M, Goding CR. 2017. Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma. Genes Dev., 31 (1), pp. 18-33. Read abstract | Read more

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome. Hide abstract

Boccellato F, Woelffling S, Imai-Matsushima A, Sanchez G, Goosmann C, Schmid M, Berger H, Morey P, Denecke C, Ordemann J, Meyer TF. 2018. Polarised epithelial monolayers of the gastric mucosa reveal insights into mucosal homeostasis and defence against infection. Gut, Read abstract | Read more

OBJECTIVE: causes life-long colonisation of the gastric mucosa, leading to chronic inflammation with increased risk of gastric cancer. Research on the pathogenesis of this infection would strongly benefit from an authentic human in vitro model. DESIGN: Antrum-derived gastric glands from surgery specimens served to establish polarised epithelial monolayers via a transient air-liquid interface culture stage to study cross-talk with and the adjacent stroma. RESULTS: The resulting 'mucosoid cultures', so named because they recapitulate key characteristics of the gastric mucosa, represent normal stem cell-driven cultures that can be passaged for months. These highly polarised columnar epithelial layers encompass the various gastric antral cell types and secrete mucus at the apical surface. By default, they differentiate towards a foveolar, MUC5AC-producing phenotype, whereas Wnt signalling stimulates proliferation of MUC6-producing cells and preserves stemness-reminiscent of the gland base. Stromal cells from the lamina propria secrete Wnt inhibitors, antagonising stem-cell niche signalling and inducing differentiation. On infection with , a strong inflammatory response is induced preferentially in the undifferentiated basal cell phenotype. Infection of cultures for several weeks produces foci of viable bacteria and a persistent inflammatory condition, while the secreted mucus establishes a barrier that only few bacteria manage to overcome. CONCLUSION: Gastric mucosoid cultures faithfully reproduce the features of normal human gastric epithelium, enabling new approaches for investigating the interaction of with the epithelial surface and the cross-talk with the basolateral stromal compartment. Our observations provide striking insights in the regulatory circuits of inflammation and defence. Hide abstract

Bagnoli F, Buti L, Tompkins L, Covacci A, Amieva MR. 2005. Helicobacter pylori CagA induces a transition from polarized to invasive phenotypes in MDCK cells. Proc. Natl. Acad. Sci. U.S.A., 102 (45), pp. 16339-44. Read abstract | Read more

CagA is a bacterial effector protein of Helicobacter pylori that is translocated via a type IV secretion system into gastric epithelial cells. We previously described that H. pylori require CagA to disrupt the organization and assembly of apical junctions in polarized epithelial cells. In this study, we provide evidence that CagA expression is not only sufficient to disrupt the apical junctions but also perturbs epithelial differentiation. CagA-expressing cells lose apicobasal polarity and cell-cell adhesion, extend migratory pseudopodia, and degrade basement membranes, acquiring an invasive phenotype. Expression of the CagA C-terminal domain, which contains the tyrosine phosphorylated EPIYA motifs, induces pseudopodial activity but is not sufficient to induce cell migration. Conversely, the N terminus targets CagA to the cell-cell junctions. Neither domain is sufficient to disrupt cell adhesion or cell polarity, but coexpressed in trans, the N terminus determines the localization of both polypeptides. We show that CagA induces a morphogenetic program in polarized Madin-Darby canine kidney cells resembling an epithelial-to-mesenchymal transition. We propose that altered cell-cell and cell matrix interactions may serve as an early event in H. pylori-induced carcinogenesis. Hide abstract