Microplastic induces mitochondrial pathway mediated cellular apoptosis in mussel (Mytilus galloprovincialis) via inhibition of the AKT and ERK signaling pathway.

Microplastics (MPs) is an escalating aquatic environmental crisis that poses significant threats to marine organisms, especially mussels. Here, we compare the cumulative toxic effects of the two most abundant morphotypes of MPs in the environment, microspheres, and microfibers, on the gill and digestive gland (DG) of Mytilus galloprovincialis in a dose-dependent (1, 10, and 100 mg/L) and time-dependent (1, 4, 7, 14, 21 days exposure) manner. DNA fragmentation assessment through TUNEL assay revealed consistency in the pattern of morphological disturbance degree and cell apoptosis proportions indicated by histopathological analysis. Upon the acute phase of exposure (day 1-4), gill and DG treated with low MPs concentration exhibited preserved morphology and low proportion of TUNEL+ cells. At higher concentrations, spherical and fibrous MP-induced structural impairments and DNA breakage occurred at distinct levels. 100 mg/L microfibers was lethal to all mussels on day 21, indicating the higher toxicity of the fibrous particles. During the chronic phase, both morphological abnormalities degree and DNA fragmentation level increased over time and with increasing concentration, but the differentials between the spherical and fibrous group was gradually reduced, particularly diminished in 10 and 100 mg/L in the last 2 weeks. Furthermore, analysis of transcriptional activities of key genes for apoptosis of 100 mg/L-day 14 groups revealed the upregulation of both intrinsic and extrinsic apoptotic induction pathway and increment in gene transcripts involving genotoxic stress and energy metabolism according to MP morphotypes. Overall, microfibers exert higher genotoxic effects on mussel. In response, mussels trigger more intense apoptotic responses together with enhanced energy metabolism to tolerate the adverse effects in a way related to the accumulation of stimuli.

Control of the growth and development of murine preantral follicles in a biomimetic ovary using a decellularized porcine scaffold.

This study aimed to derive mature oocytes from murine preantral follicles cultured in a biomimetic ovary with a porcine scaffold using decellularization technology. We evaluated the DNA content and the presence of cell and extracellular matrix (ECM) components, including collagen, elastin, and glycosaminoglycans (GAGs), in decellularized (decell) porcine ovaries. The DNA content inthe decell ovarian tissues was approximately 94 % less than that in native tissues (66 ± 9.8 ng/mg vs. 1139 ± 269 ng/mg). Furthermore, the ECM component integrity was maintained in the decell ovarian tissue. The soluble collagen concentration of native ovarian tissue (native) was 195.34 ± 15.13 µg/mg (dry wt.), which was less than 878.6 ± 8.24 µg/mg for the decell ovarian tissue due to the loss of cellular mass. Hydrogels derived from decell porcine ovaries were prepared to develop an in vitro biomimetic ovary with appropriate ECM concentration (2-6 mg/mL). Scanning electron microscope (SEM) imagining revealed that the complex fiber network and porous structure were maintained in all groups treated with varying ECM concentration (2-6 mg/mL). Furthermore, rheometer analysis indicated that mechanical strength increased with ECM concentration in a dose-dependently. The preantral follicles cultured with 4 mg/mL ECM showed high rates of antral follicle (66 %) and mature oocyte (metaphase II) development (47 %). The preantral follicles cultured in a biomimetic ovary with a decell porcine scaffold showed a higher rate of antral follicle and mature oocytes than those cultured in other biomaterials such as collagen and Matrigel. In mature oocytes derived from antral follicles, meiotic spindles and nuclei were stained using a tubulin antibody and Hoechst, respectively. Two-cell embryos were developed from MII oocytes following parthenogenetic activation. Preantral follicles were cultured in a biomimetic ovary derived from the ECM of a decell porcine ovary, and embryos were generated from MII oocytes. This biomimetic ovary could contribute to restoring fertility in infertile women with reduced ovarian function, benefit mating efforts for endangered species, and maintain animals with valuable genetic traits.

Responses of multifunctional immune complement component 1q (C1q) and apoptosis-related genes in Macrophthalmus japonicus tissues and human cells following exposure to environmental pollutants.

Apoptosis is a key defense process for multiple immune system functions, playing a central role in maintaining homeostasis and cell development. The purpose of this study was to evaluate the effects of environmental pollutant exposure on immune-related apoptotic pathways in crab tissues and human cells. To do this, we characterized the multifunctional immune complement component 1q (C1q) gene and analyzed C1q expression in Macrophthalmus japonicus crabs after exposure to di(2-ethylhexyl) phthalate (DEHP) or hexabromocyclododecanes (HBCDs). Moreover, the responses of apoptotic signal-related genes were observed in M. japonicus tissues and human cell lines (HEK293T and HCT116). C1q gene expression was downregulated in the gills and hepatopancreas of M. japonicus after exposure to DEHP or HBCD. Pollutant exposure also increased antioxidant enzyme activities and altered transcription of 15 apoptotic signaling genes in M. japonicus. However, patterns in apoptotic signaling in response to these pollutants differed in human cells. HBCD exposure generated an apoptotic signal (cleaved caspase-3) and inhibited cell growth in both cell lines, whereas DEHP exposure did not produce such a response. These results suggest that exposure to environmental pollutants induced different levels of immune-related apoptosis depending on the cell or tissue type and that this induction of apoptotic signaling may trigger an initiation of carcinogenesis in M. japonicus and in humans as consumers.

Long range inter-chromosomal interaction of Oct4 distal enhancer loci regulates ESCs pluripotency.

Nuclear architecture underlies the transcriptional programs within the cell to establish cell identity. As previously demonstrated, long-range chromatin interactions of the Oct4 distal enhancer (DE) are correlated with active transcription in naïve state embryonic stem cells. Here, we identify and characterize extreme long-range interactions of the Oct4 DE through a novel CRISPR labeling technique we developed and chromosome conformation capture to identify lethal giant larvae 2 (Llgl2) and growth factor receptor-bound protein 7 (Grb7) as putative functional interacting target genes in different chromosomes. We show that the Oct4 DE directly regulates expression of Llgl2 and Grb7 in addition to Oct4. Expression of Llgl2 and Grb7 closely correlates with the pluripotent state, where knock down of either result in loss of pluripotency, and overexpression enhances somatic cell reprogramming. We demonstrated that biologically important interactions of the Oct4 DE can occur at extreme distances that are necessary for the maintenance of the pluripotent state.

Germ Cell Isolation and Cryopreservation from Reproductive Organs of Brown Mealworm.

This study aimed to isolate and freeze germ cells from the superior brown mealworm. Styrofoam diet changes were observed for 20 days to determine whether mealworms were useful insects for decomposing Styrofoam. The average weight of mealworms before the Styrofoam diet was 500 mg, which decreased to 336 mg at D20 after their diet. To preserve mealworms with excellent Styrofoam-degrading ability, we first isolated the reproductive organs of mealworms, testes, ovaries, sperms, and ovarioles. Morphologically, male and female adult brown mealworms were distinguished according to the presence or absence of a protrusion at the tip of the fifth segment of the abdomen. Sperms and ovarioles were observed in anatomically isolated testes and ovaries. We compared mechanical and enzymatic (collagenase I) methods to effectively isolate ovarioles from adult female brown mealworms. For the enzymatic method, most were torn and burst as the membrane of the ovarioles was damaged by collagenase I, unlike the mechanical method. To preserve the superior genetic resources of mealworms, we cryopreserved the ovaries of female brown mealworms using slow-freezing and vitrification. Histological analysis showed that the yolk sac was completely damaged in the ovaries after slow-freezing. However, only partial damage was achieved in the vitrification group compared to the control group (no freezing). The newly developed vitrification method with alginate-encapsulated ovarioles maintained the yolk sac in the ovarioles but was evenly distributed. These results provide basic data for reproductive studies of other useful insects and contribute to the biobanking and fertility preservation of superior mealworm germ cells and endangered insects.

Discovery of GSK3ß Inhibitors through In Silico Prediction-and-Experiment Cycling Strategy, and Biological Evaluation.

Direct inhibitors of glycogen synthase kinase 3ß (GSK3ß) have been investigated and reported for the past 20 years. In the search for novel scaffold inhibitors, 3000 compounds were selected through structure-based virtual screening (SBVS), and then high-throughput enzyme screening was performed. Among the active hit compounds, pyrazolo [1,5-a]pyrimidin-7-amine derivatives showed strong inhibitory potencies on the GSK3ß enzyme and markedly activated Wnt signaling. The result of the molecular dynamics (MD) simulation, enhanced by the upper-wall restraint, was used as an advanced structural query for the SBVS. In this study, strong inhibitors designed to inhibit the GSK3ß enzyme were discovered through SBVS. Our study provides structural insights into the binding mode of the inhibitors for further lead optimization.

A Small Molecule Promoting Neural Differentiation Suppresses Cancer Stem Cells in Colorectal Cancer.

Cancer stem cells (CSCs) are a tumor cell subpopulation that drives tumor progression and metastasis, leading to a poor overall survival of patients. In colorectal cancer (CRC), the hyper-activation of Wnt/ß-catenin signaling by a mutation of both adenomatous polyposis coli (APC) and K-Ras increases the size of the CSC population. We previously showed that CPD0857 inactivates Wnt/ß-catenin signaling by promoting the ubiquitin-dependent proteasomal degradation of ß-catenin and Ras proteins, thereby decreasing proliferation and increasing the apoptosis of CRC lines. CPD0857 also decreased the growth and invasiveness of CRC cells harboring mutant K-Ras resistant to EGFR mAb therapy. Here, we show that CPD0857 treatment decreases proliferation and increases the neuronal differentiation of neural progenitor cells (NPCs). CDP0857 effectively reduced the expression of CSC markers and suppressed self-renewal capacity. CPD0857 treatment also inhibited the proliferation and expression of CSC markers in D-K-Ras MT cells carrying K-Ras, APC and PI3K mutations, indicating the inhibition of PI3K/AKT signaling. Moreover, CPD0857-treated xenograft mice showed a regression of tumor growth and decreased numbers of CSCs in tumors. We conclude that CPD0857 could serve as the basis of a drug development strategy targeting CSCs activated through Wnt/ß-catenin-Ras MAPK-PI3K/AKT signaling in CRCs.

Azathioprine antagonizes aberrantly elevated lipid metabolism and induces apoptosis in glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor with poor survival rate. Temozolomide (TMZ) is used as standard chemotherapy to treat GBM, but a large number of patients either respond poorly and/or develop resistance after long-term use, emphasizing the need to develop potent drugs with novel mechanisms of action. Here, using high-throughput compound screening (HTS), we found that azathioprine, an immunosuppressant, is a promising therapeutic agent to treat TMZ-resistant GBM. Through integrative genome-wide analysis and global proteomic analysis, we found that elevated lipid metabolism likely due to hyperactive EGFR/AKT/SREBP-1 signaling was inhibited by azathioprine. Azathioprine also promoted ER stress-induced apoptosis. Analysis of orthotopic xenograft models injected with patient-derived GBM cells revealed reduced tumor volume and increased apoptosis after azathioprine and TMZ co-treatment. These data indicate that azathioprine could be a powerful therapeutic option for TMZ-resistant GBM patients.

Small Molecule Destabilizer of ß-Catenin and Ras Proteins Antagonizes Growth of K-Ras Mutation-Driven Colorectal Cancers Resistant to EGFR Inhibitors.

BACKGROUND: Oncogenic K-Ras mutations in colorectal cancer (CRC) combined with APC mutations worsen CRC prognosis and lower drug effectiveness. Thus, inhibition of both Wnt/ß-catenin and Ras-MAPK signaling may be a rational strategy to improve the treatment of this cancer. OBJECTIVE: To identify a novel compound inhibiting both Wnt/ß-catenin and Ras-MAPK signaling in CRC. METHODS AND PATIENTS: We developed a two-part screening system consisting of analysis of TOP flash reporter cells and then potential toxicity effects on primary neural stem cells (NSCs). We then screened 2000 chemical compounds and tested efficacy of candidates against isogenic colon cancer cells harboring wild-type or mutant K-Ras. We employed immunohistochemistry and immunocytochemistry to determine marker signatures associated with development of disease phenotypes. RESULTS: We identified CPD0857, a compound that inactivates Wnt/ß-catenin signaling and promotes ubiquitin-dependent proteasomal degradation of ß-catenin and Ras proteins. CPD0857 effectively decreased proliferation and increased apoptosis of CRC cell lines, and overcame resistance of CRC harboring APC and K-Ras mutations to treatment with an EGFR monoclonal antibody (mAb). Moreover, CPD0857 attenuated invasiveness of highly migratory CRC cells in vitro. Accordingly, xenograft mice treated with CPD0857 showed slower tumor growth and significant decreases in both ß-catenin and Ras protein expression. CONCLUSIONS: CPD0857 may be a potential drug for treating aggressive CRC carrying mutations that aberrantly activate Wnt/ß-catenin and Ras-ERK pathways.

Epigenetic modulator inhibition overcomes temozolomide chemoresistance and antagonizes tumor recurrence of glioblastoma.

Glioblastoma multiforme (GBM) heterogeneity causes a greater number of deaths than any other brain tumor, despite the availability of alkylating chemotherapy. GBM stem-like cells (GSCs) contribute to GBM complexity and chemoresistance, but it remains challenging to identify and target GSCs or factors that control their activity. Here, we identified a specific GSC subset and show that activity of these cells is positively regulated by stabilization of methyl CpG binding domain 3 (MBD3) protein. MBD3 binds to CK1A and to BTRCP E3 ubiquitin ligase, triggering MBD3 degradation, suggesting that modulating this circuit could antagonize GBM recurrence. Accordingly, xenograft mice treated with the CK1A activator pyrvinium pamoate (Pyr-Pam) showed enhanced MBD3 degradation in cells expressing high levels of O6-methylguanine-DNA methyltransferase (MGMT) and in GSCs, overcoming temozolomide chemoresistance. Pyr-Pam blocked recruitment of MBD3 and the repressive nucleosome remodeling and deacetylase (NuRD) complex to neurogenesis-associated gene loci and increased acetyl-histone H3 activity and GSC differentiation. We conclude that CK1A/BTRCP/MBD3/NuRD signaling modulates GSC activation and malignancy, and that targeting this signaling could suppress GSC proliferation and GBM recurrence.

Circulating Tumor Cells Exhibit Metastatic Tropism and Reveal Brain Metastasis Drivers.

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood-brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis. SIGNIFICANCE: Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo-cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain.This article is highlighted in the In This Issue feature, p. 1.

The Presence of Neural Stem Cells and Changes in Stem Cell-Like Activity With Age in Mouse Spiral Ganglion Cells In Vivo and In Vitro.

OBJECTIVES: Spiral ganglion neurons (SGNs) include potential endogenous progenitor populations for the regeneration of the peripheral auditory system. However, whether these populations are present in adult mice is largely unknown. We examined the presence and characteristics of SGN-neural stem cells (NSCs) in mice as a function of age. METHODS: The expression of Nestin and Ki67 was examined in sequentially dissected cochlear modiolar tissues from mice of different ages (from postnatal day to 24 weeks) and the sphere-forming populations from the SGNs were isolated and differentiated into different cell types. RESULTS: There were significant decreases in Nestin and Ki67 double-positive mitotic progenitor cells in vivo with increasing mouse age. The SGNs formed spheres exhibiting self-renewing activity and multipotent capacity, which were seen in NSCs and were capable of differentiating into neuron and glial cell types. The SGN spheres derived from mice at an early age (postnatal day or 2 weeks) contained more mitotic stem cells than those from mice at a late age. CONCLUSION: Our findings showed the presence of self-renewing and proliferative subtypes of SGN-NSCs which might serve as a promising source for the regeneration of auditory neurons even in adult mice.

Valproic acid promotes the neuronal differentiation of spiral ganglion neural stem cells with robust axonal growth.

Hearing loss occurs with the loss of hair cells of the cochlea and subsequent degeneration of spiral ganglion neurons (SGNs). Regeneration of SGNs is a potentially promising therapeutic approach to hearing loss in addition to the use of a cochlear implant (CI), because this device stimulates SGNs directly to restore hearing bypassing the missing hair cells. The presence of SGN-neural stem cells (NSCs) has been reported in adult human and mice. These cells have the potential to become SGNs and thus represent a cellular foundation for regeneration therapies for hearing loss. Valproic acid (VPA) has been shown to influence the neural differentiation of NSCs through multiple signaling pathways involving glycogen synthase kinase3ß (GSK3ß). Our present study therefore aimed to modulate the neural differentiation potential of SGN-NSCs by treatment with VPA. We here report that a clinically relevant concentration of 1 mM VPA induced the differentiation of basic fibroblast growth factor (bFGF)-treated P1- and P14-SGN-NSCs into neuronal and glial cells, confirmed by neuronal marker (Tuj1 and MAP2) and glial cell marker (GFAP and S100ß) detection. VPA-treated cells also promoted much longer neurite outgrowth compared to differentiated cells cultured without bFGF. The effects of VPA on the regulation of differentiation may be related to the activation of the Wnt/ß-catenin signaling pathway, but not the inhibition of histone deacetylases (HDACs). We propose that VPA has the potential to convert SGN-NSCs into SGNs and thereby restore hearing when combined with a CI.

Kruppel-like factor 4-dependent Staufen1-mediated mRNA decay regulates cortical neurogenesis.

Kruppel-like factor 4 (Klf4) is a zinc-finger-containing protein that plays a critical role in diverse cellular physiology. While most of these functions attribute to its role as a transcription factor, it is postulated that Klf4 may play a role other than transcriptional regulation. Here we demonstrate that Klf4 loss in neural progenitor cells (NPCs) leads to increased neurogenesis and reduced self-renewal in mice. In addition, Klf4 interacts with RNA-binding protein Staufen1 (Stau1) and RNA helicase Ddx5/17. They function together as a complex to maintain NPC self-renewal. We report that Klf4 promotes Stau1 recruitment to the 3'-untranslated region of neurogenesis-associated mRNAs, increasing Stau1-mediated mRNA decay (SMD) of these transcripts. Stau1 depletion abrogated SMD of target mRNAs and rescued neurogenesis defects in Klf4-overexpressing NPCs. Furthermore, Ddx5/17 knockdown significantly blocked Klf4-mediated mRNA degradation. Our results highlight a novel molecular mechanism underlying stability of neurogenesis-associated mRNAs controlled by the Klf4/Ddx5/17/Stau1 axis during mammalian corticogenesis.

Smek1/2 is a nuclear chaperone and cofactor for cleaved Wnt receptor Ryk, regulating cortical neurogenesis.

The receptor-like tyrosine kinase (Ryk), a Wnt receptor, is important for cell fate determination during corticogenesis. During neuronal differentiation, the Ryk intracellular domain (ICD) is cleaved. Cleavage of Ryk and nuclear translocation of Ryk-ICD are required for neuronal differentiation. However, the mechanism of translocation and how it regulates neuronal differentiation remain unclear. Here, we identified Smek1 and Smek2 as Ryk-ICD partners that regulate its nuclear localization and function together with Ryk-ICD in the nucleus through chromatin recruitment and gene transcription regulation. Smek1/2 double knockout mice displayed pronounced defects in the production of cortical neurons, especially interneurons, while the neural stem cell population increased. In addition, both Smek and Ryk-ICD bound to the Dlx1/2 intergenic regulator element and were involved in its transcriptional regulation. These findings demonstrate a mechanism of the Ryk signaling pathway in which Smek1/2 and Ryk-ICD work together to mediate neural cell fate during corticogenesis.

Smek promotes corticogenesis through regulating Mbd3's stability and Mbd3/NuRD complex recruitment to genes associated with neurogenesis.

The fate of neural progenitor cells (NPCs) during corticogenesis is determined by a complex interplay of genetic or epigenetic components, but the underlying mechanism is incompletely understood. Here, we demonstrate that Suppressor of Mek null (Smek) interact with methyl-CpG-binding domain 3 (Mbd3) and the complex plays a critical role in self-renewal and neuronal differentiation of NPCs. We found that Smek promotes Mbd3 polyubiquitylation and degradation, blocking recruitment of the repressive Mbd3/nucleosome remodeling and deacetylase (NuRD) complex at the neurogenesis-associated gene loci, and, as a consequence, increasing acetyl histone H3 activity and cortical neurogenesis. Furthermore, overexpression of Mbd3 significantly blocked neuronal differentiation of NPCs, and Mbd3 depletion rescued neurogenesis defects seen in Smek1/2 knockout mice. These results reveal a novel molecular mechanism underlying Smek/Mbd3/NuRD axis-mediated control of NPCs' self-renewal and neuronal differentiation during mammalian corticogenesis.

Small-molecule binding of the axin RGS domain promotes ß-catenin and Ras degradation.

Both the Wnt/ß-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both ß-catenin and Ras, via targeting the Wnt/ß-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating ß-catenin and Ras degradation through enhancement of the ß-catenin destruction complex activating GSK3ß. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both ß-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/ß-catenin and Ras pathways.

Circular RNA profile in gliomas revealed by identification tool UROBORUS.

Recent evidence suggests that many endogenous circular RNAs (circRNAs) may play roles in biological processes. However, the expression patterns and functions of circRNAs in human diseases are not well understood. Computationally identifying circRNAs from total RNA-seq data is a primary step in studying their expression pattern and biological roles. In this work, we have developed a computational pipeline named UROBORUS to detect circRNAs in total RNA-seq data. By applying UROBORUS to RNA-seq data from 46 gliomas and normal brain samples, we detected thousands of circRNAs supported by at least two read counts, followed by successful experimental validation on 24 circRNAs from the randomly selected 27 circRNAs. UROBORUS is an efficient tool that can detect circRNAs with low expression levels in total RNA-seq without RNase R treatment. The circRNAs expression profiling revealed more than 476 circular RNAs differentially expressed in control brain tissues and gliomas. Together with parental gene expression, we found that circRNA and its parental gene have diversified expression patterns in gliomas and control brain tissues. This study establishes an efficient and sensitive approach for predicting circRNAs using total RNA-seq data. The UROBORUS pipeline can be accessed freely for non-commercial purposes at http://uroborus.openbioinformatics.org/.

Role of oncogenic K-Ras in cancer stem cell activation by aberrant Wnt/ß-catenin signaling.

BACKGROUND: Adenomatous polyposis coli (APC) loss-of-function mutations and K-Ras gain-of-function mutations are common abnormalities that occur during the initiation and intermediate adenoma stages of colorectal tumorigenesis, respectively. However, little is known about the role these mutations play in cancer stem cells (CSCs) associated with colorectal cancer (CRC) tumorigenesis. METHODS: We analyzed tissue from CRC patients (n = 49) to determine whether K-Ras mutations contributed to CSC activation during colorectal tumorigenesis. DLD-1-K-Ras-WT and DLD-1-K-Ras-MT cells were cultured and evaluated for their ability to differentiate, form spheroids in vitro, and form tumors in vivo. Interaction between APC and K-Ras mutations in colorectal tumorigenesis was evaluated using APC (Min/+)/K-Ras (LA2) mice and DLD-1-K-Ras-WT and DLD-1-K-Ras-MT cell xenografts. (n = 4) Group differences were determined by Student t test. All statistical tests were two-sided. RESULTS: The sphere-forming capability of DLD-1-K-Ras-MT cells was statistically significantly higher than that of DLD-1-K-Ras-WT cells (DLD-1-K-Ras-MT mean = 86.661 pixel, 95% confidence interval [CI] = 81.701 to 91.621 pixel; DLD-1-K-Ras-WT mean = 42.367 pixel, 95% CI = 36.467 to 48.267 pixel; P = .003). Moreover, both the size and weight of tumors from DLD-1-K-Ras-MT xenografts were markedly increased compared with tumors from DLD-1-K-Ras-WT cells. Expression of the CSC markers CD44, CD133, and CD166 was induced in intestinal tumors from APC (Min/+)/K-Ras (LA2)mice, but not K-Ras (LA2) mice, indicating that APC mutation is required for CSC activation by oncogenic K-Ras mutation. CONCLUSIONS: K-Ras mutation activates CSCs, contributing to colorectal tumorigenesis and metastasis in CRC cells harboring APC mutations. Initial activation of ß-catenin by APC loss and further enhancement through K-Ras mutation induces CD44, CD133, and CD166 expression.