Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis.

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.

Cell-matrix interface regulates dormancy in human colon cancer stem cells.

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.

Somatic inflammatory gene mutations in human ulcerative colitis epithelium.

With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations1-7. However, whether such clonal expansion exists in the non-neoplastic intestine remains unknown. Here, using whole-exome sequencing data from 76 clonal human colon organoids, we identify a unique pattern of somatic mutagenesis in the inflamed epithelium of patients with ulcerative colitis. The affected epithelium accumulates somatic mutations in multiple genes that are related to IL-17 signalling-including NFKBIZ, ZC3H12A and PIGR, which are genes that are rarely affected in colon cancer. Targeted sequencing validates the pervasive spread of mutations that are related to IL-17 signalling. Unbiased CRISPR-based knockout screening in colon organoids reveals that the mutations confer resistance to the pro-apoptotic response that is induced by IL-17A. Some of these genetic mutations are known to exacerbate experimental colitis in mice8-11, and somatic mutagenesis in human colon epithelium may be causally linked to the inflammatory process. Our findings highlight a genetic landscape that adapts to a hostile microenvironment, and demonstrate its potential contribution to the pathogenesis of ulcerative colitis.

Visualization and targeting of LGR5+ human colon cancer stem cells.

The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+ CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+ CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.

Microtubule severing by katanin p60 AAA+ ATPase requires the C-terminal acidic tails of both α- and ß-tubulins and basic amino acid residues in the AAA+ ring pore.

The microtubule (MT) network is highly dynamic and undergoes dramatic reorganizations during the cell cycle. Dimers of α- and ß-tubulins rapidly polymerize to and depolymerize from the end of MT fibrils in an intrinsic GTP-dependent manner. MT severing by ATP-driven enzymes such as katanin and spastin contributes significantly to microtubule dynamics, and it has been shown that katanin p60, a AAA+ family protein, has ATPase and MT-severing activities. The mechanism of MT severing by katanin p60 is poorly understood, and the residues in katanin p60 and tubulins important for severing activity were therefore explored in this study. MT-severing activity, but not ATPase activity, was inhibited by mutations of the conserved aromatic residue and the flanking basic residues in the pore region of the katanin p60 hexameric ring. When the acidic residue-rich C-terminal unstructured segment of either α- or ß-tubulin was removed, polymerized MTs were resistant to katanin p60 treatment. Interactions between katanin p60 and the mutant MTs, on the other hand, were unaffected. Taken together, these findings led us to propose that the interactions between the positively charged residues of katanin p60 and the acidic tails of both tubulins are essential for efficient severing of MTs.

A PDE3A-SLFN12 Molecular Glue Exhibits Significant Antitumor Activity in TKI-Resistant Gastrointestinal Stromal Tumors.

PURPOSE: Gastrointestinal stromal tumor (GIST), the most common mesenchymal tumor with KIT or PDGFRA driver mutations, is typically treated with tyrosine kinase inhibitors (TKI). However, resistance to TKIs due to secondary mutations is a common challenge in advanced GISTs. In addition, there are currently no effective therapies for several other molecular subtypes, such as succinate dehydrogenase-deficient GISTs. Therefore, novel therapeutic strategies are needed. EXPERIMENTAL DESIGN: To address this need, we tested the efficacy of a novel non-TKI compound, OPB-171775, using patient-derived xenograft models of GISTs. In parallel, we sought to elucidate the mechanism of action of the compound. RESULTS: Our study revealed that OPB-171775 exhibited significant efficacy against GISTs regardless of their KIT mutation status by inducing complex formation between phosphodiesterase 3A (PDE3A) and Schlafen family member 12 (SLFN12), which are highly expressed in GISTs, leading to SLFN12 RNase-mediated cell death. Furthermore, we identified the activation of general control non-derepressible 2 and its downstream response as an effector pathway of SLFN12 in mediating anticancer activity and revealed potential pharmacodynamic markers. CONCLUSIONS: These findings suggest that OPB-171775, with its significant efficacy, could potentially serve as a novel and effective treatment option for advanced GISTs, particularly those resistant to TKIs.

Positive cooperativity of the p97 AAA ATPase is critical for essential functions.

p97 is composed of two conserved AAA (ATPases associated with diverse cellular activities) domains, which form a tandem hexameric ring. We characterized the ATP hydrolysis mechanism of CDC-48.1, a p97 homolog of Caenorhabditis elegans. The ATPase activity of the N-terminal AAA domain was very low at physiological temperature, whereas the C-terminal AAA domain showed high ATPase activity in a coordinated fashion with positive cooperativity. The cooperativity and coordination are generated by different mechanisms because a noncooperative mutant still showed the coordination. Interestingly, the growth speed of yeast cells strongly related to the positive cooperativity rather than the ATPase activity itself, suggesting that the positive cooperativity is critical for the essential functions of p97.

AAA+ chaperone ClpX regulates dynamics of prokaryotic cytoskeletal protein FtsZ.

AAA(+) chaperone ClpX has been suggested to be a modulator of prokaryotic cytoskeletal protein FtsZ, but the details of recognition and remodeling of FtsZ by ClpX are largely unknown. In this study, we have extensively investigated the nature of FtsZ polymers and mechanisms of ClpX-regulated FtsZ polymer dynamics. We found that FtsZ polymerization is inhibited by ClpX in an ATP-independent manner and that the N-terminal domain of ClpX plays a crucial role for the inhibition of FtsZ polymerization. Single molecule analysis with high speed atomic force microscopy directly revealed that FtsZ polymer is in a dynamic equilibrium between polymerization and depolymerization on a time scale of several seconds. ClpX disassembles FtsZ polymers presumably by blocking reassembly of FtsZ. Furthermore, Escherichia coli cells overproducing ClpX and N-terminal domain of ClpX show filamentous morphology with abnormal localization of FtsZ. These data together suggest that ClpX modulates FtsZ polymer dynamics in an ATP-independent fashion, which is achieved by interaction between the N-terminal domain of ClpX and FtsZ monomers or oligomers.

FtsH cleavage of non-native conformations of proteins.

FtsH is a peculiar prokaryotic protease with low unfoldase activity. Different reports have proposed that FtsH substrates could be either tagged proteins or proteins of low stability. We show here that FtsH degradation of 31 point mutants of Anabaena apoflavodoxin is inversely proportional to their conformational stabilities, and that the same applies to other substrate proteins. In contrast, highly stable proteins such as GST and holoflavodoxin are not degraded at all. Attempts to identify sequence tags signaling for degradation in apoflavodoxin fragments have been unsuccessful. Apoflavodoxin adopts three conformations: native, partly unfolded and fully unfolded. It is revealing that degradation of the 31 variants is proportional to the molar fraction of fully unfolded molecules and inversely proportional to the fraction of stable apoflavodoxin molecules. This indicates that FtsH, rather than unfolding the protein, acts on the fraction that is already unfolded.

p97 Homologs from Caenorhabditis elegans, CDC-48.1 and CDC-48.2, suppress the aggregate formation of huntingtin exon1 containing expanded polyQ repeat.

Polyglutamine (polyQ)-expanded proteins are associated with cytotoxicity in some neurodegenerative disorders such as Huntington's disease. We have reported that the aggregation of the polyQ-expanded protein is partially suppressed by co-expression of either of two homologs of an AAA chaperone p97, CDC-48.1 or CDC-48.2, in Caenorhabditis elegans, but how p97 regulates the aggregation of polyQ-expanded proteins remains unclear. Here we present direct evidence that CDC-48.1 and CDC-48.2 suppress the aggregation of a huntingtin (Htt) exon1 fragment containing an expanded polyQ repeat in vitro. CDC-48.1 and CDC-48.2 bound the Htt exon1 fragment directly, and suppressed the formation of SDS-insoluble aggregates of Htt fragments containing 53 glutamine residues (HttQ53) independently of nucleotides. CDC-48.1 and CDC-48.2 also modulated the oligomeric states of HttQ53 during the aggregate formation. In the absence of CDC-48.1 and CDC-48.2, HttQ53 formed 70-150 kDa oligomers, whereas 300-500 kDa oligomers as well as 70-150 kDa oligomers accumulated in the presence of CDC-48.1 and CDC-48.2. Taken together, these results suggest that p97 plays a protective role in neurodegenerative disorders by directly suppressing the protein aggregation as a molecular chaperone.

Chelation of cadmium ions by phytochelatin synthase: role of the cysteine-rich C-terminal.

The interactions between Cd(2+) and the C-terminal region of phytochelatin (PC) synthase using recombinant wild-type and mutant PC synthase were studied. We show that site-directed mutagenesis of Cys residues at C(358)C(359)XXXC(363)XXC(366) motif decreases the number of Cd(2+) and other heavy metal ions interacting with the enzyme, and that the motif binds the metals discriminatingly. The optimum binding ratio of PC synthase to Cd(2+) was also determined. The findings indicate that Cys exists as a free SH residue and that it is involved in the regulation of PC enzyme activity by transferring the metals into closer proximity with the catalytic domain. These results are important in understanding heavy metal detoxification mechanisms in higher plants, a step towards phytoremediated-applications.

Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition.

Cellular diversity that shapes tissue architecture and function is governed by multiple niche signals. Nonetheless, maintaining cellular diversity in human intestinal organoids has been challenging. Based on niche ligands present in the natural stem cell milieu, we establish a refined organoid culture condition for intestinal epithelia that allows human intestinal organoids to concurrently undergo multi-differentiation and self-renewal. High-throughput screening reveals that the combination of insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) enhances the clonogenic capacity and CRISPR-genome engineering efficiency of human intestinal stem cells. The combination equally enables long-term culture of a range of intestinal organoids, including rat small intestinal organoids. Droplet-based single-cell RNA sequencing further illustrates the conservation of the native cellular diversity in human small intestinal organoids cultured with the refined condition. The modified culture protocol outperforms the conventional method and offers a viable strategy for modeling human intestinal tissues and diseases in an in vivo relevant context.

Reconstruction of the Human Colon Epithelium In Vivo.

Genetic lineage tracing has revealed that Lgr5+ murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs in vivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium in vivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer an LGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5+ CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5+ CoSCs, human LGR5+ CoSCs were slow-cycling in vivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs in vivo, with potential therapeutic applications in regenerative medicine.

Unfolding mechanism of a hyperthermophilic protein O(6)-methylguanine-DNA methyltransferase.

Unfolding intermediates have been found only rarely in earlier studies, and how a protein unfolds is therefore poorly understood. In this paper, we show experimental evidence for multiple pathways and multiple intermediates during unfolding reaction of O(6)-methylguanine-DNA methyltransferase from hyperthermophile Thermococcus kodakaraensis (Tk-MGMT). The unfolding profiles monitored by far-UV CD and tryptophan fluorescence were both biphasic, and unfolding monitored by fluorescence was faster than that monitored by CD. GdnHCl-induced titration curves indicate that the intermediates with significant alpha-helical structure accumulate during unfolding. Dependence of kinetic phases on initial GdnHCl concentrations and cysteine reactivity of Tk-MGMT were investigated, suggesting that the heterogeneity of native conformations and parallel unfolding pathways.

Diamines prevent thermal aggregation and inactivation of lysozyme.

Protein aggregation is a major obstacle in both biological applications and biomedical fields involving proteins. In this study, we investigated the essential structure of small additives that function as chemical chaperones. Aggregation-suppressing competent additives were 1,3-diaminopropane, 1,4-diaminobutane, and 1,5-diaminopentane, which suppressed aggregation in the given order; whereas no diols or monoamines prevented the thermal aggregation and the inactivation of lysozyme. The heat-inactivation rate of lysozyme with 1,3-diaminopropane was almost identical to that of lysozyme with spermine and arginine ethylester, which are the most prominent additives reported yet.

Equilibrium and kinetic stability of a hyperthermophilic protein, O6-methylguanine-DNA methyltransferase under various extreme conditions.

In this work we have studied the equilibrium and kinetic stability of a hyperthermophilic protein, O(6)-methylguanine-DNA methyltransferase (Tk-MGMT), and its mesophilic counterpart AdaC, in various chemical solutions. In an unfolding experiment using guanidine hydrochloride (GdnHCl), the unfolding free-energy change of Tk-MGMT at 30 degrees C was 42.0 kJ mol(-1), and the half time for unfolding was 4.5 x 10(6) s, which is much slower than that of AdaC and representative mesophilic proteins. In unfolding experiments using methanol, ethanol, 2-propanol, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS), Tk-MGMT retained its native structure at high concentrations, despite the fact that these chemical solutions affect protein conformations in a number of different ways. Kinetic studies using TFE and SDS indicate that the unfolding rates of Tk-MGMT in these solutions are slow as in GdnHCl. Further, the results of a mutational experiment suggest that an ion-pair network plays a key role in this slow unfolding. This slow rate of unfolding under extreme conditions is a significant property that distinguishes Tk-MGMT from mesophilic proteins.

Mutational effects on O(6)-methylguanine-DNA methyltransferase from hyperthermophile: contribution of ion-pair network to protein thermostability.

Ion pairs have been considered to be general stabilizing factors in hyperthermophilic proteins, but the present experimental data cannot fully explain how ion pairs and ion-pair networks contribute to the stability. In this paper, we show experimental evidence that not all of the internal ion pairs contribute to the thermal and thermodynamic stability, using O(6)-methylguanine-DNA methyltransferase from Thermococcus kodakaraensis KOD1 (Tk-MGMT) as a model protein. Of three mutants in which an inter-helical ion pair was disrupted, only one mutant (E93A) was shown to be destabilized. Delta G of E93A was lower by approximately 4 kJ mol(-1) than that of the wild type, and E93A unfolded one order of magnitude faster than did the wild type and other variants. Glu 93 has unique properties in forming an ion-pair network that bridges the N- and C-terminal domains and connects three helices in the protein interior.

High temperature increases the refolding yield of reduced lysozyme: implication for the productive process for folding.

Misfolding poses a serious problem in the biotechnological field in obtaining the active protein from inclusion bodies. Here we show that high temperature increases the refolding yield of reduced lyosyzme by a simple dilution method. The refolding yields at 98 degrees C were three times higher than those at 20 degrees C in the solutions tested, which is related to the fact that the thermally unfolded state of lysozyme is a more productive form for folding than the denaturant-induced fully unfolded state. The thermal-assisted refolding could be used for various reduced and denatured proteins as a result of its simplicity and low cost.

Contribution of protein-surface ion pairs of a hyperthermophilic protein on thermal and thermodynamic stability.

Hyperthermophilic proteins possess many ion pairs on their surface. To reveal the role of the ion pairs, O6-methylguanine-DNA methyltransferase from Thermococcus kodakaraensis KOD1 (Tk-MGMT) was studied as a model protein. The maximum free-energy changes of the protein in 0.1 and 0.5 M NaCl at pH 7.0 were 61.7 kJ mol(-1) at 31.5 degrees C and 77.4 kJ mol(-1) at 39.7 degrees C, respectively. On the other hand, mid points of the thermal unfolding temperatures in 0.1 and 0.5 M NaCl at pH 7.0 were 94.8 degrees C and 90.1 degrees C, respectively. The results suggest that the protein-surface ion pairs contribute to thermal stability (Tm), rather than thermodynamic stability (DeltaG).

Biophysical analysis of heat-induced structural maturation of glutamate dehydrogenase from a hyperthermophilic archaeon.

Tertiary structure of the recombinant glutamate dehydrogenase from Thermococcus kodakaraensis KOD1 (Tk-rGDH) converts into an intact form induced by the heat treatment. This phenomenon, heat-induced structural maturation, means that high temperature plays an important role in the proper folding and oligomerization of Tk-rGDH. In this work, we analyzed the heat-induced structural maturation of Tk-rGDH by differential scanning microcalorimetry (DSC), circular dichroism (CD), and activity measurements. In DSC measurements, the peak of adsorption of non-heated Tk-rGDH (nh-Tk-rGDH) was two times smaller than that of Tk-rGDH heated at 70 degrees C for 30 min (h-Tk-rGDH). The transition temperature (T(m)) of h-Tk-rGDH was 115 degrees C, which was about 3 degrees C higher than that of nh-Tk-rGDH. In the presence of 0.5 M NaCl, the nh-Tk-rGDH showed two peaks at 107 degrees C and 114 degrees C, while the h-Tk-rGDH showed a single peak at 115.7 degrees C. The heat-induced conformational change process was monitored by changes in CD intensity at 222 nm, and the result showed that heat-induced structural maturation is irreversible. The heat treatment at 70 degrees C showed the highest enhancement in activity, which was 15% larger than that of heat-treated Tk-rGDH at 40 degrees C. The results indicate that heat-induced structural maturation involves an irreversible process, transforming the non-heated form to the stable and active form.