Targeting PDGF signaling of cancer-associated fibroblasts blocks feedback activation of HIF-1α and tumor progression of clear cell ovarian cancer.

Ovarian clear cell carcinoma (OCCC) is a gynecological cancer with a dismal prognosis; however, the mechanism underlying OCCC chemoresistance is not well understood. To explore the intracellular networks associated with the chemoresistance, we analyze surgical specimens by performing integrative analyses that combine single-cell analyses and spatial transcriptomics. We find that a chemoresistant OCCC subpopulation with elevated HIF activity localizes mainly in areas populated by cancer-associated fibroblasts (CAFs) with a myofibroblastic phenotype, which is corroborated by quantitative immunostaining. CAF-enhanced chemoresistance and HIF-1α induction are recapitulated in co-culture assays, which show that cancer-derived platelet-derived growth factor (PDGF) contributes to the chemoresistance and HIF-1α induction via PDGF receptor signaling in CAFs. Ripretinib is identified as an effective receptor tyrosine kinase inhibitor against CAF survival. In the co-culture system and xenograft tumors, ripretinib prevents CAF survival and suppresses OCCC proliferation in the presence of carboplatin, indicating that combination of conventional chemotherapy and CAF-targeted agents is effective against OCCC.

FXYD3 functionally demarcates an ancestral breast cancer stem cell subpopulation with features of drug-tolerant persisters.

The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain-containing ion transport regulator 3 (FXYD3), a component of the Na+/K+ pump. Accordingly, FXYD3+ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3+ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3+ CSCs were sensitive to senolytic Na+/K+ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3+ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na+/K+ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.

Elevated stress response marks deeply quiescent reserve cells of gastric chief cells.

Gastrointestinal tract organs harbor reserve cells, which are endowed with cellular plasticity and regenerate functional units in response to tissue damage. However, whether the reserve cells in gastrointestinal tract exist as long-term quiescent cells remain incompletely understood. In the present study, we systematically examine H2b-GFP label-retaining cells and identify a long-term slow-cycling population in the gastric corpus but not in other gastrointestinal organs. The label-retaining cells, which reside near the basal layers of the corpus, comprise a subpopulation of chief cells. The identified quiescent cells exhibit induction of Atf4 and its target genes including Atf3, a marker of paligenosis, and activation of the unfolded protein response, but do not show elevated expression of Troy, Lgr5, or Mist. External damage to the gastric mucosa induced by indomethacin treatment triggers proliferation of the quiescent Atf4+ population, indicating that the gastric corpus harbors a specific cell population that is primed to facilitate stomach regeneration.

PROX1 induction by autolysosomal activity stabilizes persister-like state of colon cancer via feedback repression of the NOX1-mTORC1 pathway.

Cancer chemoresistance is often attributed to slow-cycling persister populations with cancer stem cell (CSC)-like features. However, how persister populations emerge and prevail in cancer remains obscure. We previously demonstrated that while the NOX1-mTORC1 pathway is responsible for proliferation of a fast-cycling CSC population, PROX1 expression is required for chemoresistant persisters in colon cancer. Here, we show that enhanced autolysosomal activity mediated by mTORC1 inhibition induces PROX1 expression and that PROX1 induction in turn inhibits NOX1-mTORC1 activation. CDX2, identified as a transcriptional activator of NOX1, mediates PROX1-dependent NOX1 inhibition. PROX1-positive and CDX2-positive cells are present in distinct populations, and mTOR inhibition triggers conversion of the CDX2-positive population to the PROX1-positive population. Inhibition of autophagy synergizes with mTOR inhibition to block cancer proliferation. Thus, mTORC1 inhibition-mediated induction of PROX1 stabilizes a persister-like state with high autolysosomal activity via a feedback regulation that involves a key cascade of proliferating CSCs.

NF-κB suppression synergizes with E7386, an inhibitor of CBP/ß-catenin interaction, to block proliferation of patient-derived colon cancer spheroids.

Dysregulated activation of the WNT/ß-catenin signaling pathway is essential for the initiation and development of various cancers. E7386, a small-molecule compound, attenuates WNT signaling by blocking the interaction between ß-catenin and CREB-binding protein (CBP); hence, it is regarded as a therapeutic candidate for cancers with activated WNT signaling. In the present study, we evaluated the biological characteristics associated with E7386 sensitivity by using a panel of patient-derived colon cancer spheroids. An integrative approach that combined E7386 sensitivity and gene expression profiles revealed that the resistance of the cancer spheroids to E7386 was associated with the activation of the NF-κB pathway. NF-κB pathway inhibitors acted synergistically with E7386 to block proliferation and induce cell cycle arrest in E7386-resistant spheroids. These findings suggest a possibility that a combination of E7386 and NF-κB inhibition may effectively block the proliferation of a subset of colon cancer cells.

Stemness and immune evasion conferred by the TDO2-AHR pathway are associated with liver metastasis of colon cancer.

The aryl hydrocarbon receptor (AHR) pathway modulates the immune system in response to kynurenine, an endogenous tryptophan metabolite. IDO1 and TDO2 catalyze kynurenine production, which promotes cancer progression by compromising host immunosurveillance. However, it is unclear whether the AHR activation regulates the malignant traits of cancer such as metastatic capability or cancer stemness. Here, we carried out systematic analyses of metabolites in patient-derived colorectal cancer spheroids and identified high levels of kynurenine and TDO2 that were positively associated with liver metastasis. In a mouse colon cancer model, TDO2 expression substantially enhanced liver metastasis, induced AHR-mediated PD-L1 transactivation, and dampened immune responses; these changes were all abolished by PD-L1 knockout. In patient-derived cancer spheroids, TDO2 or AHR activity was required for not only the expression of PD-L1, but also for cancer stem cell (CSC)-related characteristics and Wnt signaling. TDO2 was coexpressed with both PD-L1 and nuclear ß-catenin in colon xenograft tumors, and the coexpression of TDO2 and PD-L1 was observed in clinical colon cancer specimens. Thus, our data indicate that the activation of the TDO2-kynurenine-AHR pathway facilitates liver metastasis of colon cancer via PD-L1-mediated immune evasion and maintenance of stemness.

Single-cell DNA and RNA sequencing reveals the dynamics of intra-tumor heterogeneity in a colorectal cancer model.

BACKGROUND: Intra-tumor heterogeneity (ITH) encompasses cellular differences in tumors and is related to clinical outcomes such as drug resistance. However, little is known about the dynamics of ITH, owing to the lack of time-series analysis at the single-cell level. Mouse models that recapitulate cancer development are useful for controlled serial time sampling. RESULTS: We performed single-cell exome and transcriptome sequencing of 200 cells to investigate how ITH is generated in a mouse colorectal cancer model. In the model, a single normal intestinal cell is grown into organoids that mimic the intestinal crypt structure. Upon RNAi-mediated downregulation of a tumor suppressor gene APC, the transduced organoids were serially transplanted into mice to allow exposure to in vivo microenvironments, which play relevant roles in cancer development. The ITH of the transcriptome increased after the transplantation, while that of the exome decreased. Mutations generated during organoid culture did not greatly change at the bulk-cell level upon the transplantation. The RNA ITH increase was due to the emergence of new transcriptional subpopulations. In contrast to the initial cells expressing mesenchymal-marker genes, new subpopulations repressed these genes after the transplantation. Analyses of colorectal cancer data from The Cancer Genome Atlas revealed a high proportion of metastatic cases in human subjects with expression patterns similar to the new cell subpopulations in mouse. These results suggest that the birth of transcriptional subpopulations may be a key for adaptation to drastic micro-environmental changes when cancer cells have sufficient genetic alterations at later tumor stages. CONCLUSIONS: This study revealed an evolutionary dynamics of single-cell RNA and DNA heterogeneity in tumor progression, giving insights into the mesenchymal-epithelial transformation of tumor cells at metastasis in colorectal cancer.

Integrative analyses of gene expression and chemosensitivity of patient-derived ovarian cancer spheroids link G6PD-driven redox metabolism to cisplatin chemoresistance.

Patient-derived cells and xenografts retain the biological characteristics of clinical cancers and are instrumental in gaining a better understanding of the chemoresistance of cancer cells. Here, we have established a panel of patient-derived spheroids from clinical materials of ovarian cancer. Systematic evaluation using therapeutic agents indicated that sensitivity to platinum-based compounds significantly varied among the spheroids. To understand the molecular basis of drug sensitivity, we performed integrative analyses combining chemoresistance data and gene expression profiling of the ovarian cancer patient-derived spheroids. Correlation analyses revealed that cisplatin resistance was significantly associated with elevated levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione-producing redox enzymes. Accordingly, cisplatin-resistant spheroids established in vitro showed elevated levels of G6PD and active glutathione. Moreover, treatment with a G6PD inhibitor in combination with cisplatin suppressed spheroid proliferation in vitro and largely eradicated peritoneal metastasis in mouse xenograft models. Furthermore, G6PD expression was elevated during carcinogenesis and associated with poor prognosis. Thus, the combination of gene expression data and chemosensitivity revealed the essential roles of G6PD-driven redox metabolism in cisplatin resistance, underscoring the significance of an integrative approach using patient-derived cells.

Slow-Cycling Cancer Stem Cells Regulate Progression and Chemoresistance in Colon Cancer.

Cancer chemoresistance is often attributed to the presence of cancer stem cell (CSC)-like cells, but whether they are homogeneously chemoresistant remains unclear. We previously showed that in colon tumors, a subpopulation of LGR5+ CSC-like cells driven by TCF1 (TCF7), a Wnt-responsive transcription factor, were responsible for tumorigenicity. Here we demonstrate that the tumorigenic subpopulation of mouse LGR5+ cells exists in a slow-cycling state and identify a unique 22-gene signature that characterizes these slow-cycling CSC. Seven of the signature genes are specifically expressed in slow-cycling LGR5+ cells from xenografted human colon tumors and are upregulated in colon cancer clinical specimens. Among these seven, four genes (APCDD1, NOTUM, PROX1, and SP5) are known to be direct Wnt target genes, and PROX1 was expressed in the invasive fronts of colon tumors. PROX1 was activated by TCF1 to induce CDKN1C and maintain a slow-cycling state in colon cancer organoids. Strikingly, PROX1 was required for recurrent growth after chemotherapeutic treatment, suggesting that inhibition of slow-cycling CSC by targeting the TCF1-PROX1-CDKN1C pathway is an effective strategy to combat refractory colon cancer in combination with conventional chemotherapy. SIGNIFICANCE: These findings illustrate the importance of a slow-cycling CSC subpopulation in colon cancer development and chemoresistance, with potential implications for the identified slow-cycling CSC signatures and the TCF1-PROX1-CDKN1C pathway as therapeutic targets.

NOX1-Dependent mTORC1 Activation via S100A9 Oxidation in Cancer Stem-like Cells Leads to Colon Cancer Progression.

Cancer stem cells (CSCs) are associated with the refractory nature of cancer, and elucidating the targetable pathways for CSCs is crucial for devising innovative antitumor therapies. We find that the proliferation of CSC-enriched colon spheroids from clinical specimen is dependent on mTORC1 kinase, which is activated by reactive oxygen species (ROS) produced by NOX1, an NADPH oxidase. In the spheroid-derived xenograft tumors, NOX1 is preferentially expressed in LGR5-positive cells. Dependence on NOX1 expression or mTOR kinase activity is corroborated in the xenograft tumors and mouse colon cancer-derived organoids. NOX1 co-localizes with mTORC1 in VPS41-/VPS39-positive lysosomes, where mTORC1 binds to S100A9, a member of S100 calcium binding proteins, in a NOX1-produced ROS-dependent manner. S100A9 is oxidized by NOX1-produced ROS, which facilitates binding to mTORC1 and its activation. We propose that NOX1-dependent mTORC1 activation via S100A9 oxidation in VPS41-/VPS39-positive lysosomes is crucial for colon CSC proliferation and colon cancer progression.

Heterogeneity among Lgr5-positive colon stem cells.

Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is considered a representative marker of intestinal stem cells from both non-tumor and tumor tissues. However, it remains unclear whether all or only a fraction of Lgr5-positive cells behave as stem cells. Recently, we reported that Lgr5-positive cells from non-tumor and tumor tissues can be classified into overlapping yet distinct groups and that the tumor-specific groups are associated with tumorigenic capability, suggesting that these cells could represent targets for therapeutic intervention.

The Induction of Selected Wnt Target Genes by Tcf1 Mediates Generation of Tumorigenic Colon Stem Cells.

The generation of tumor-initiating cells during colon carcinogenesis is associated with the dysregulation of Wnt signaling, which is known to act on Lgr5-positive intestinal stem cells. Here, using single-cell qPCR analysis, we identified a subset of Lgr5-positive stem cells that emerged during tumorigenesis in a mouse model of colon cancer. These tumor-specific Lgr5-positive cells expressed low levels of Ceacam1 and increased levels of a specific subset of Wnt targets and showed enhanced tumorigenicity. Among the Wnt targets that were specifically expressed, the long isoform of Tcf1 was required for the proliferation of tumor organoids and drove a unique Wnt target gene expression profile. Tcf1 expression increased at an early stage of colon carcinogenesis and was associated with the nuclear accumulation of ß-catenin, underscoring the importance of the induction of Tcf1 expression in generating tumorigenic colon stem cells.

DNase γ is the effector endonuclease for internucleosomal DNA fragmentation in necrosis.

Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg(2+)/Ca(2+)-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase γ, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis.

DNase gamma-dependent and -independent apoptotic DNA fragmentations in Ramos Burkitt's lymphoma cell line.

DNA fragmentation is a biochemical hallmark of apoptosis. Several endonucleases, including CAD/DFF40 and endonuclease G, are implicated in DNA fragmentation. DNase gamma has also been considered to be one of the enzymes involved, but its role in relation to CAD/DFF40 in apoptosis has not been fully elucidated. Here, we distinguished between DNase gamma-dependent and CAD/DFF40-dependent DNA fragmentations. We found that DNase gamma activities appeared in the late apoptotic phase and accelerated DNA fragmentation. Thus, even if the apoptotic DNA fragmentation is initiated by CAD/DFF40, DNase gamma is required for the more complete digestion of the genomic DNA in dying cells.

Cytoplasmic tethering is involved in synergistic inhibition of p53 by Mdmx and Mdm2.

The mdm2 and mdmx oncogenes play essential yet nonredundant roles in synergistic inactivatiosn of p53. However, the biochemical mechanism by which Mdmx synergizes with Mdm2 to inhibit p53 function remains obscure. Here we demonstrate that, using nonphosphorylatable mutants of Mdmx, the cooperative inhibition of p53 by Mdmx and Mdm2 was associated with cytoplasmic localization of p53, and with an increase of the interaction of Mdmx to p53 and Mdm2 in the cytoplasm. In addition, the Mdmx mutant cooperates with Mdm2 to induce ubiquitination of p53 at C-terminal lysine residues, and the integrity of the C-terminal lysines was partly required for the cooperative inhibition. The expression of subcellular localization mutants of Mdmx revealed that subcellular localization of Mdmx dictated p53 localization, and that cytoplasmic Mdmx tethered p53 in the cytoplasm and efficiently inhibited p53 activity. RNAi-mediated inhibition of Mdmx or introduction of the nuclear localization mutant of Mdmx reduced cytoplasmic retention of p53 in neuroblastoma cells, in which cytoplasmic sequestration of p53 is involved in its inactivation. Our data indicate that cytoplasmic tethering of p53 mediated by Mdmx contributes to p53 inactivation in some types of cancer cells.

DNase X is a glycosylphosphatidylinositol-anchored membrane enzyme that provides a barrier to endocytosis-mediated transfer of a foreign gene.

DNase X is the first mammalian DNase to be isolated that is homologous to DNase I. In this study, we have examined its function using a novel monoclonal antibody and showed it to be expressed on the cell surface as a glycosylphosphatidylinositolanchored membrane protein. High level expression was observed in human muscular tissues and in myotubes obtained in vitro from RD rhabdomyosarcoma cells. We observed that RD myotubes incorporated a foreign gene, lacZ, by endocytosis but that expression of the encoded coding product, beta-galactosidase, was strongly inhibited. Overexpression of DNase X inhibited endocytosis-mediated gene transfer, whereas knockdown of DNase X with small interfering RNA had the opposite effect. These results reveal that DNase X provides a cell surface barrier to endocytosis-mediated gene transfer.

Action of apoptotic endonuclease DNase gamma on naked DNA and chromatin substrates.

The internucleosomal cleavage of genomic DNA is a biochemical hallmark of apoptosis. DNase gamma, a Mg2+/Ca2+-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases, but its biochemical characteristic has not been fully elucidated. Here, using recombinant DNase gamma, we showed that DNase gamma is a Mg2+/Ca2+-dependent single-stranded DNA nickase and has a high activity at low ionic strength. Under higher ionic strength, such as physiological buffer conditions, the endonuclease activity of DNase gamma is restricted, but its activity is enhanced in the presence of linker histone H1, which explains DNA cleavage at linker regions of apoptotic nuclei.

Structure basis for the inhibitory mechanism of a novel DNase gamma-specific inhibitor, DR396.

DNase gamma, a member of the DNase I family, has been suggested to cause DNA fragmentation during apoptosis. We recently identified 4-(4,6-dichloro-[1,3,5]-triazine-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396) as a novel specific inhibitor for human DNase gamma [Sunaga, S.; Kobayashi, T.; Yoshimori, A.; Shiokawa, D.; Tanuma, S. Biochem. Biophys. Res. Commun.2004, 325, 1292]. However, the binding mode (coordinate) of DR396 to DNase gamma has not yet been defined. Here, we examined the molecular basis for the inhibitory activity of DR396 to DNase gamma by structure-based computational docking studies. In the blind-docking study using a human DNase gamma homology model, a unique binding site of DR396 was predicted, which is tentatively named the 'DNA trapping site' because of the binding domain of the unhydrolyzed DNA strand, but not the active site. Targeting the DNA trapping site as a hot spot, new human DNase gamma inhibitors were obtained from our diverse chemical library in silico. These inhibitors showed high correlations between their predicted binding-free energies (DeltaGs) and observed IC50 values in the DNA trapping site but not the active site. The IC50 of a regioisomer of DR396, 5-(4,6-dichloro-[1,3,5]-triazine-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DF365), was 73 microM (DeltaG=-9.75 kcal/mol), a 20-fold weaker inhibitory ability than that of DR396 (IC50=3.2 microM, DeltaG=-11.22 kcal/mol). Fluorescein and triazine derivatives, partial structures of DR396, had little inhibitory activity for DNase gamma. Docking analyses of the interaction between DR396 and DNase gamma revealed that DR396 binds tightly to three subsites (S1, S2, and S3) in the trapping site of DNase gamma by forming six hydrogen bonds, whereas DF365 and the partial structures are unable to form hydrogen bonds at all three subsites. These findings suggest that the specificity and potency of the inhibitory activity of DR396 for DNase gamma is due to the specific interaction of DR396 with three subsites in the DNA trapping site of DNase gamma.

Physical and biochemical properties of mammalian DNase X proteins: non-AUG translation initiation of porcine and bovine mRNAs for DNase X.

DNase X is the first human DNase protein identified as being homologous with DNase I. In the present study we describe the isolation of several mammalian DNase X cDNAs and the molecular characterization of their coding proteins. A sequence comparison reveals some conserved characteristics: all the mammalian DNase X proteins have an N-terminal signal peptide, a potential N-linked glycosylation site and a C-terminal hydrophobic domain. Human DNase X, ectopically expressed in HeLa S3 cells, is located in the ER (endoplasmic reticulum) and is modified by an N-linked glycosylation at Asn-243. Gene expression analyses show that the high expression level in muscular tissues, a known feature of human DNASE X, is also observed in mouse DNase X. Interestingly, the translation of porcine and bovine DNase X proteins occurs in the absence of an in-frame AUG initiation codon. We show that their mRNAs utilize a conserved CUG triplet for translation initiation.

Involvement of DNase gamma in the resected double-strand DNA breaks in immunoglobulin genes.

Somatic hypermutation (SHM) of immunoglobulin variable (V) region genes occurs in the germinal center (GC) B cells during immune responses, depending on activation-induced cytidine deaminase (AID). SHM is associated with resected double-strand DNA breaks (DSBs) which were shown to occur specifically in rearranged V regions in the GC B cells and CD40-stimulated B cells expressing AID. So far, endonucleases responsible for the DSBs have not been identified. Here we show that DNase gamma, a member of DNase I family of endonucleases, is expressed in GC B cells and CD40-stimulated B cells. Overexpression of DNase gamma in the mutation-competent Ramos B-cell line resulted in a marked increase in the resected but not blunt DSBs in the V region. Conversely, a selective DNase gamma inhibitor, DR396, suppressed the generation of the resected DSBs. These results suggest that DNase gamma is involved in the generation of resected DSBs associated with SHM.