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.

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.

Establishment of in vitro 3D spheroid cell cultivation from human gynecologic cancer tissues.

Advanced-stage gynecologic cancer remains a life-threatening disease. Here, we present a protocol for establishment of stable in vitro 3D spheroid cells derived from human uterine endometrial and ovarian cancer tissues. The tumor-derived spheroid cells have cancer stem cell-related characteristics, including tumorigenesis, and can be used for biological and biochemical analyses and drug efficacy assays. Because these cells possess the biological characteristics of original human tumors, spheroid cells and spheroid-derived xenografts will have applications in personalized medicine in the future. For complete details on the use and execution of this protocol, please refer to Ishiguro et al. (2016) and Mori et al. (2019).

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.

ALDH-Dependent Glycolytic Activation Mediates Stemness and Paclitaxel Resistance in Patient-Derived Spheroid Models of Uterine Endometrial Cancer.

Uterine endometrial cancer is associated with poor survival outcomes in patients with advanced-stage disease. Here, we developed a three-dimensional cell cultivation method of endometrioid cancer stem-like cells with high aldehyde dehydrogenase (ALDH) activity from clinical specimens. ALDH inhibition synergized with paclitaxel to block cancer proliferation. In the clinical setting, high ALDH1A1 expression was associated with poor survival. A high level of ALDH correlated with an increase of glucose uptake, activation of the glycolytic pathway, and elevation of glucose transporter 1 (GLUT1). Blockade of GLUT1 inhibited characteristics of cancer stem cells. Similarly to ALDH inhibition, GLUT1 inhibition synergized with paclitaxel to block endometrial cancer proliferation. Our data indicated that ALDH-dependent GLUT1 activation and the resulting glycolytic activation are of clinical importance for both prognostic evaluation and therapeutic decision-making in endometrial cancer patients. In addition, the synergistic effects of taxane compounds and ALDH or GLUT1 inhibitors may serve as a new clinical treatment option for endometrial cancer.

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.

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.

Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications.

Recently, many types of in vitro 3-D culture systems have been developed to recapitulate the in vivo growth conditions of cancer. The cancer 3-D culture methods aim to preserve the biological characteristics of original tumors better than conventional 2-D monolayer cultures, and include tumor-derived organoids, tumor-derived spheroids, organotypic multicellular spheroids, and multicellular tumor spheroids. The 3-D culture methods differ in terms of cancer cell sources, protocols for cell handling, and the required time intervals. Tumor-derived spheroids are unique because they are purposed for the enrichment of cancer stem cells (CSCs) or cells with stem cell-related characteristics. These spheroids are grown as floating spheres and have been used as surrogate systems to evaluate the CSC-related characteristics of solid tumors in vitro. Because eradication of CSCs is likely to be of clinical importance due to their association with the malignant nature of cancer cells, such as tumorigenicity or chemoresistance, the investigation of tumor-derived spheroids may provide invaluable clues to fight against cancer. Spheroid cultures have been established from cancers including glioma, breast, colon, ovary, and prostate cancers, and their biological and biochemical characteristics have been investigated by many research groups. In addition to the investigation of CSCs, tumor-derived spheroids may prove to be instrumental for a high-throughput screening platform or for the cultivation of CSC-related tumor cells found in the circulation or body fluids.

TNIK inhibition abrogates colorectal cancer stemness.

Canonical Wnt/ß-catenin signalling is essential for maintaining intestinal stem cells, and its constitutive activation has been implicated in colorectal carcinogenesis. We and others have previously identified Traf2- and Nck-interacting kinase (TNIK) as an essential regulatory component of the T-cell factor-4 and ß-catenin transcriptional complex. Consistent with this, Tnik-deficient mice are resistant to azoxymethane-induced colon tumorigenesis, and Tnik(-/-)/Apc(min/+) mutant mice develop significantly fewer intestinal tumours. Here we report the first orally available small-molecule TNIK inhibitor, NCB-0846, having anti-Wnt activity. X-ray co-crystal structure analysis reveals that NCB-0846 binds to TNIK in an inactive conformation, and this binding mode seems to be essential for Wnt inhibition. NCB-0846 suppresses Wnt-driven intestinal tumorigenesis in Apc(min/+) mice and the sphere- and tumour-forming activities of colorectal cancer cells. TNIK is required for the tumour-initiating function of colorectal cancer stem cells. Its inhibition is a promising therapeutic approach.

Establishment and Characterization of an In Vitro Model of Ovarian Cancer Stem-like Cells with an Enhanced Proliferative Capacity.

The establishment of cancer stem-like cell (CSC) culture systems may be instrumental in devising strategies to fight refractory cancers. Inhibition of the Rho kinase ROCK has been shown to favorably affect CSC spheroid cultures. In this study, we show how ROCK inhibition in human serous ovarian cancer (SOC) cells can help establish a CSC system, which illuminates cancer pathophysiology and its treatment in this setting. In the presence of a ROCK kinase inhibitor, spheroid cultures of SOC cells expressed characteristic CSC markers including ALDH1A1, CD133, and SOX2, along with differentiation and tumorigenic capabilities in mouse xenograft models of human SOC. High expression levels of ALDH, but not CD133, correlated with spheroid formation CSC marker expression and tumor forming capability. In clinical specimens of SOC, high levels of ALDH1A1 correlated with advanced stage and poor prognosis. Pharmacologic or genetic blockade of ALDH blocked cell proliferation and reduced expression of SOX2, the genetic ablation of which abolished spheroid formation, whereas SOX2 overexpression inhibited ALDH1A1 expression and blocked spheroid proliferation. Taken together, our findings illustrated a new method to culture human ovarian CSC, and they defined a reciprocal regulatory relationship between ALDH1A1 and SOX2, which impacts ovarian CSC proliferation and malignant progression.

APOLLON protein promotes early mitotic CYCLIN A degradation independent of the spindle assembly checkpoint.

In the mammalian cell cycle, both CYCLIN A and CYCLIN B are required for entry into mitosis, and their elimination is also essential to complete the process. During mitosis, CYCLIN A and CYCLIN B are ubiquitylated by the anaphase-promoting complex/cyclosome (APC/C) and then subjected to proteasomal degradation. However, CYCLIN A, but not CYCLIN B, begins to be degraded in the prometaphase when APC/C is inactivated by the spindle assembly checkpoint (SAC). Here, we show that APOLLON (also known as BRUCE or BIRC6) plays a role in SAC-independent degradation of CYCLIN A in early mitosis. APPOLON interacts with CYCLIN A that is not associated with cyclin-dependent kinases. APPOLON also interacts with APC/C, and it facilitates CYCLIN A ubiquitylation. In APPOLON-deficient cells, mitotic degradation of CYCLIN A is delayed, and the total, but not the cyclin-dependent kinase-bound, CYCLIN A level was increased. We propose APPOLON to be a novel regulator of mitotic CYCLIN A degradation independent of SAC.

NuMA is required for the selective induction of p53 target genes.

The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription.

Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system.

Haploid embryonic stem cells (ESCs) are useful for studying mammalian genes because disruption of only one allele can cause loss-of-function phenotypes. Here, we report the use of haploid ESCs and the CRISPR RNA-guided Cas9 nuclease gene-targeting system to manipulate mammalian genes. Co-transfection of haploid ESCs with vectors expressing Cas9 nuclease and single-guide RNAs (sgRNAs) targeting Tet1, Tet2, and Tet3 resulted in the complete disruption of all three genes and caused a loss-of-function phenotype with high efficiency (50%). Co-transfection of cells with vectors expressing Cas9 and sgRNAs targeting two loci on the same chromosome resulted in the creation of a large chromosomal deletion and a large inversion. Thus, the use of the CRISPR system in combination with haploid ESCs provides a powerful platform to manipulate the mammalian genome.

Induction of the stem-like cell regulator CD44 by Rho kinase inhibition contributes to the maintenance of colon cancer-initiating cells.

The difficulty in expanding cancer-initiating cells in vitro is one of major obstacles for their biochemical characterization. We found that Rho kinase (ROCK) inhibitors as well as blebbistatin, a myosin II inhibitor, greatly facilitated the establishment of spheroids from primary colon cancer. The spheroid cells expressed cancer stem cell markers, showed the ability to differentiate, and induced tumors in mice. The spheroids were composed of cells that express various levels of CD44, whereas CD44(high) cells were associated with increased sphere-forming ability, expression of the activating form of ß-catenin, and elevated levels of glycolytic genes, CD44(-/low) cells showed increased levels of differentiation markers and apoptotic cells. The spheroid cells expressed variant forms of CD44 including v6, and the induction of the variants was associated with the activating phosphorylation of c-Met. As expected from the predicted hierarchy, CD44(high) cells differentiated into CD44(-/low) cells. Unexpectedly, a fraction of CD44(-/low) cells generated CD44(high) cells, and the ROCK inhibitor or blebbistatin primed the transition by inducing CD44 expression. We propose that the transition from CD44(-/low) to CD44(high) state helps to maintain a CD44(high) fraction and the tumorigenic diversity in colon cancer.

miR-493 induction during carcinogenesis blocks metastatic settlement of colon cancer cells in liver.

Liver metastasis is a major lethal complication associated with colon cancer, and post-intravasation steps of the metastasis are important for its clinical intervention. In order to identify inhibitory microRNAs (miRNAs) for these steps, we performed 'dropout' screens of a miRNA library in a mouse model of liver metastasis. Functional analyses showed that miR-493 and to a lesser extent miR-493(*) were capable of inhibiting liver metastasis. miR-493 inhibited retention of metastasized cells in liver parenchyma and induced their cell death. IGF1R was identified as a direct target of miR-493, and its inhibition partially phenocopied the anti-metastatic effects. High levels of miR-493 and miR-493(*), but not pri-miR-493, in primary colon cancer were inversely related to the presence of liver metastasis, and attributed to an increase of miR-493 expression during carcinogenesis. We propose that, in a subset of colon cancer, upregulation of miR-493 during carcinogenesis prevents liver metastasis via the induction of cell death of metastasized cells.

Induction of ZEB proteins by inactivation of RB protein is key determinant of mesenchymal phenotype of breast cancer.

We previously showed that depletion of the retinoblastoma protein (RB) induces down-regulation of the adhesion molecule E-cadherin and thereby triggers the epithelial-mesenchymal transition. To further characterize the effect of RB inactivation on the phenotype of cancer cells, we have now examined RB expression in human breast cancer cell lines and clinical specimens. We found that RB-inactive cells exhibit a mesenchymal-like morphology and are highly invasive. We also found that ZEB proteins, transcriptional repressors of the E-cadherin gene, are markedly up-regulated in these cells in a manner sensitive to the miR-200 family of microRNAs. Moreover, depletion of ZEB in RB-inactive cells suppressed cell invasiveness and proliferation and induced epithelial marker expression. These results implicate ZEB in induction of the epithelial-mesenchymal transition, as well as in maintenance of the mesenchymal phenotype in RB-inactive cells. We also developed a screening program for inhibitors of ZEB1 expression and thereby identified several cyclin-dependent kinase inhibitors that blocked both ZEB1 expression and RB phosphorylation. Together, our findings suggest that RB inactivation contributes to tumor progression not only through loss of cell cycle control but also through up-regulation of ZEB expression and induction of an invasive phenotype.

Differential expression of nanog1 and nanogp8 in colon cancer cells.

Nanog, a homeodomain transcription factor, is an essential regulator for promotion of self-renewal of embryonic stem cells and inhibition of their differentiation. It has been demonstrated that nanog1 as well as nanogp8, a retrogene of nanog1, is preferentially expressed in advanced stages of several types of cancer, suggesting their involvement during cancer progression. Here, we investigated the expression of Nanog in well-characterized colon cancer cell lines. Expression of Nanog was detectable in 5 (HCT116, HT29, RKO, SW48, SW620) out of seven cell lines examined. RNA expression analyses of nanog1 and nanogp8 indicated that, while nanog1 was a major form in SW620 as well as in teratoma cells Tera-2, nanogp8 was preferentially expressed in HT29 and HCT116. In accordance with this, shRNA-mediated knockdown of nanog1 caused the reduction of Nanog in SW620 but not in HT29. Inhibition of Nanog in SW620 cells negatively affected cell proliferation and tumor formation in mouse xenograft. Biochemical subcellular fractionation and immunostaining analyses revealed predominant localization of Nanog in cytoplasm in SW620 and HT29, while it was mainly localized in nucleus in Tera-2. Our data indicate that nanog1 and nanogp8 are differentially expressed in colon cancer cells, and suggest that their expression contributes to proliferation of colon cancer cells.