TRIB2 Stimulates Cancer Stem-Like Properties through Activating the AKT-GSK3β-β-Catenin Signaling Axis

Tribbles homolog 2 (TRIB2) is implicated in tumorigenesis and drug resistance in various types of cancers. However, the role of TRIB2 in the regulation of tumorigenesis and drug resistance of cancer stem cells (CSCs) is still elusive. In the present study, we showed increased expression of TRIB2 in spheroid-forming and aldehyde dehydrogenase-positive CSC populations of A2780 epithelial ovarian cancer cells. Short hairpin RNA-mediated silencing of TRIB2 expression attenuates the spheroid-forming, migratory, tumorigenic, and drug-resistant properties of A2780 cells, whereas overexpression of TRIB2 increases the CSC-like characteristics. TRIB2 overexpression induced GSK3β inactivation by augmenting AKT-dependent phosphorylation of GSK3β at Ser9, followed by increasing β-catenin level via reducing the GSK3β-mediated phosphorylation of β-catenin. Treatment of TRIB2-ovexpressed A2780 cells with the phosphoinositide-3-kinase inhibitor LY294002 abrogated TRIB2-stimulated proliferation, migration, drug resistance of A2780 cells. These results suggest a critical role for TRIB2 in the regulation of CSC-like properties by increasing the stability of β-catenin protein via the AKT-GSK3β-dependent pathways.     

CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166⁺ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166⁺ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anti-cancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.     

Establishment of human ovarian serous carcinomas cell lines in serum free media

Ovarian cancers are the fifth leading cause of cancer death among US woman. The majority of ovarian cancers belong to a category of serous adenocarcinomas. This type of cancer is often diagnosed at a late stage of the disease. Surgical debulking, followed by chemotherapy is the current treatment. Half of all patients will die within 5 years of diagnosis of the disease. Poor survival may be due to disease progression as a consequence of development of drug resistance, cancer cell heterogeneity within the tumor, or the persistence of cancer stem cells. Cancer stem cells (CSC) are defined as a minority cell type in the tumor, which retains the capacity, through asymmetric division, for self-renewal as well as differentiation into multiple cell types. Through this process, CSC can regenerate the entire tumor phenotype and subsequent metastases. Initial in vitro work in the area of solid tumor CSC biology has focused on the isolation and propagation of cells with CSC-like properties from breast and colon tumors. Breast and colon cell lines with CSC-like properties have been isolated and maintained in vitro for extended periods of time. The in vitro maintenance of these CSC requires growth in hormone-supplemented serum-free media and the use of matrix or growth as tumor spheres (Roberts, Ricci-Vitiani et al., Cammareri et al.). Based on the pioneering work generating breast and colon CSC, our lab has begun to develop methods for the establishment cell lines with CSC-like properties from additional solid tumors. In this article, we describe methods, using defined medium, which allow for the successful establishment of continuous cell cultures from a minority cell type within serous ovarian cancers. The cell lines established using these methods grow in serum-free hormone-supplemented medium either as a monolayer on a matrix, or as tumor spheres in suspension. These cells express markers previously reported for tumor stem cells, including CD44 and CD133, and form tumors that recreate the morphology of the original patient tumor when implanted in immune deficient mice. The introduction of this method will facilitate the expansion of ovarian cancer cells for investigating cancer stem cell biology as well as providing tools to aid in the development of new treatments for this deadly disease.     

Ionizing radiation induces stemness in cancer cells

The cancer stem cell (CSC) model posits the presence of a small number of CSCs in the heterogeneous cancer cell population that are ultimately responsible for tumor initiation, as well as cancer recurrence and metastasis. CSCs have been isolated from a variety of human cancers and are able to generate a hierarchical and heterogeneous cancer cell population. CSCs are also resistant to conventional chemo- and radio-therapies. Here we report that ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. Exposure of non-stem cancer cells to ionizing radiation enhanced spherogenesis, and this was accompanied by upregulation of the pluripotency genes Sox2 and Oct3/4. Knockdown of Sox2 or Oct3/4 inhibited radiation-induced spherogenesis and increased cellular sensitivity to radiation. These data demonstrate that ionizing radiation can activate stemness pathways in heterogeneous cancer cells, resulting in the enrichment of a CSC subpopulation with higher resistance to radiotherapy.     

Reprogramming of non-genomic estrogen signaling by the stemness factor SOX2 enhances the tumor-initiating capacity of breast cancer cells

The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka’s nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E₂/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E₂/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E₂ signaling that leads to nuclear phospho-Ser118-ERα, which ultimately exacerbates genomic ER signaling in response to E₂. Because E₂ stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140, which targets SOX2, the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator, SOX2, and the local and systemic ability of E₂ to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy.     

α-Mangostin attenuates stemness and enhances cisplatin-induced cell death in cervical cancer stem-like cells through induction of mitochondrial-mediated apoptosis

Cancer stem cells (CSCs) exhibit specific characteristics including decontrolled self-renewal, tumor-initiating, promoting, and metastatic potential, abnormal stemness signaling, and chemotherapy resistance. Thus, targeting CSC is becoming an emerging cancer treatment. α-Mangostin has been shown to have potent and multiple anticancer activities. Accordingly, we hypothesized that α-mangostin may diminish the stemness and proliferation of CSC-like cervical cancer cells. In our results, comparing to the parent cells, CSC-like SiHa and HeLa cells highly expressed CSC marker Sox2, Oct4, Nanog, CK-17, and CD49f. α-Mangostin significantly reduced the cell viability, sphere-forming ability, and expression of the CSC stemness makers of CSC-like cervical cancer cells. Further investigation showed that α-mangostin induced mitochondrial depolarization and mitochondrial apoptosis signaling, including upregulation of Bax, downregulation of Mcl-1 and Bcl-2, and activation of caspase-9/3. Moreover, α-mangostin synergically enhanced the cytotoxicity of cisplatin on CSC-like SiHa cells by promoting mitochondrial apoptosis and inhibiting the expression of CSC markers. Consistent with in vitro findings, in vivo tumor growth assay revealed that α-mangostin administration significantly inhibited the growth of inoculated CSC-like SiHa cells and synergically enhanced the antitumor effect of cisplatin. Our findings indicate that α-mangostin can reduce the stemness and proliferation of CSC-like SiHa and HeLa cells and promote the cytotoxicity of cisplatin, which may attribute to the mitochondrial apoptosis activation. Thus, it suggests that α-mangostin may have clinical potential to improve chemotherapy for cervical cancer by targeting cervical CSC.     

Inhibition of DNMT suppresses the stemness of colorectal cancer cells through down-regulating Wnt signaling pathway

Cancer stem cell (CSC) theory reveals a new insight into the understanding of tumorigenesis and metastasis. Recently, DNA methylation is suggested to be a potential epigenetic mechanism for maintenance of CSCs. What's more, studies have shown that DNA methyltransferase (DNMT) is essential for CSCs and deletion of DNMT can reduce tumorigenesis by limiting CSC pool. Therefore, targeting the epigenetic modifiers especially DNA methylation offers an optional strategy for treating human cancers. In the present study we found that DNMT inhibitor 5-Aza-2′-deoxycytidine (5-AzaDC) markedly reduced colorectal CSC abundance in vitro and suppressed liver metastatic tumor growth in vivo. And 5-AzaDC inhibited the expression of active β-catenin and down-regulated the Wnt signaling pathway. The Wnt inhibitors were frequently inactivated by promoter methylation in colorectal cancer; however analysis of TCGA data base showed that only the expression of SFRP1 was significantly reduced in tumors compared to normal tissues. In addition, restoring of SFRP1 expression inhibited the stem cell-like potential of colorectal cancer cells. Our results indicated that inhibition of DNMT blocked the self-renewal of colorectal CSCs and SFRP1 was essential for the maintenance of colorectal CSCs.     

SIRT1 coordinates with the CRL4B complex to regulate pancreatic cancer stem cells to promote tumorigenesis

Pancreatic cancer is a common malignant tumor with poor prognosis. Recently, cancer stem cells (CSCs) were identified in several solid tumors, including pancreatic cancer. Although accumulating evidence indicates that sirtuin 1 (SIRT1) exerts biological functions in various cancers, how it contributes to tumorigenesis and metastasis of pancreatic cancer, as well as its role in CSCs, is still poorly defined. Here we show that SIRT1 interacts with the Cullin 4B (CUL4B)-Ring E3 ligase (CRL4B) complex, which is responsible for H2AK119 monoubiquitination (H2AK119ub1), collaborating as a functional unit. Genome-wide analysis of SIRT1/CUL4B targets identified a cohort of genes, including GRHL3 and FOXO3, critically involved in cell differentiation, growth, and migration. Furthermore, we found that SIRT1 and CUL4B collectively promote the proliferation, autophagy, and invasion of pancreatic cancer cells. Remarkably, we demonstrate that SIRT1/CUL4B promotes CSC-like properties, including increased stemness marker expression and sphere formation. In vivo experiments implied that SIRT1 promoted established tumor xenograft growth, increased tumor-initiating capacity in NOD/SCID mice, and increased CSC frequency. Strikingly, SIRT1 and CUL4B expression is markedly upregulated in a variety of human cancers, including pancreatic cancer. Our data provide a molecular basis for the functional interplay between histone deacetylation and ubiquitination. The results also implicate the SIRT1/CRL4B complex in pancreatic cancer metastasis and stem cell properties, thus supporting SIRT1 as a promising potential target for cancer therapy development.Subject terms: Cancer stem cells, Metastasis     

Translocalization of enhanced PKM2 protein into the nucleus induced by cancer upregulated gene 2 confers cancer stem cell-like phenotypes

Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Ex-pression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of β-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound heal-ing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator.     

Interleukin-6 signaling regulates anchorage-independent growth, proliferation, adhesion and invasion in human ovarian cancer cells

It has been widely reported that Interleukin-6 (IL-6) is overexpressed in the serum and ascites of ovarian cancer (OVCA) patients, and elevated IL-6 level correlates with poor prognosis and survival. However, the exact role that IL-6 plays in this malignancy or whether IL-6 can regulate tumorigenic properties has not been established. Here we demonstrate that overexpression of IL-6 in non-IL-6-expressing A2780 cells (by transfecting with plasmid encoding for sense IL-6) increases anchorage-independent growth, proliferation, adhesion and invasion, while depletion of endogenous IL-6 expression in IL-6-overexpressing SKOV-3 cells (by transfecting with plasmid encoding for antisense IL-6) decreases. Further investigation indicates that IL-6 promotes OVCA cell proliferation by altering cell cycle distribution rather than inhibiting apoptosis and that IL-6-enhanced OVCA cell invasive may be associated with increased matrix metalloproteinase (MMP)-9 but not MMP-2 proteolytic activity. In addition, overexpressing or deleting of IL-6 in OVCA cells enhances or reduces its receptor (IL-6Rα and gp130) expression and basal phosphorylation levels of both ERK and Akt, and additional treatment with specific inhibitor of the ERK or Akt signaling pathway significantly inhibits the proliferation of IL-6-overexpressing A2780 cells. Our data suggest that the autocrine production of IL-6 by OVCA cells regulates tumorigenic properties of these cells by inducing IL-6 signaling pathways. Thus, regulation of IL-6 expression or its related signaling pathway may be a promising strategy for controlling the progression of OVCA.     

Low-folate stress reprograms cancer stem cell-like potentials and bioenergetics metabolism through activation of mTOR signaling pathway to promote in vitro invasion and in vivo tumorigenicity of lung cancers

Low-folate (LF) nutritional status is associated with increased risks of lung cancer. It has unexplored effects on lung cancer malignancy, a cancer stem cell (CSC) disease. We hypothesized that LF may reprogram CSC-like potential and bioenergetics metabolism to increase metastasis potential of lung cancers. Cultivation of human non-small-cell lung cancer cells (H23) in an LF medium enhanced CSC-like properties signified by increased expressions of the CSC surface marker CD44 and pluripotency markers Sox2, Oct4 and ALDH1A1, and promoted self-renewal ability of anchorage-independent oncospheroid formation. The CSC-like phenotype of LF-treated H23 cells coupled with the metabolic reprogramming to aerobic glycolysis evident by elevated lactate release and medium acidification suppressed expressions of pyruvate dehydrogenase E1-α, and elevated redox status of the NADH/NAD+ and NADPH/NADP+ ratios. The LF-induced metabostemness phenotype of H23 cells was modified by DNA methylation inhibitor 5-AdC and histone acetylation inhibitor EX. Treatment of H23 cells with mTOR siRNA or the mTOR inhibitor rapamycin abrogated LF-activated Akt-mTOR-Hif1-Foxo signaling and stemness-associated sonic hedgehog pathway, reversed Warburg metabolic switch and diminished invasion of H23 cells. Intrapleural injection of LF-induced lung oncospheres into the LF recipient mice, but not the control recipient mice, caused metastasis xenograft lung tumors. The in vitro and in vivo data corroboratively demonstrate that LF stress reprograms metabostemness signatures through activated mTOR signaling pathway to promote metastasis tumorigenicity of lung cancers.     

DGCR5 promotes cancer stem cell-like properties of radioresistant laryngeal carcinoma cells by sponging miR-506 via Wnt pathway

Cancer stem cells (CSCs) have been recognized as the significant cause of tumor recurrence. Long noncoding RNAs (lncRNAs) are involved in various cancers, including human laryngeal cancer. So far the correlation between lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) and CSC-like properties in human laryngeal cancer remains barely known. In our current study, two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R) with different radio sensitivities were cultured. Interestingly, CSC-like phenotypes were much more enriched in Hep-2R cells. We found that DGCR5 was upregulated and microRNA-506 (miR-506) was downregulated in Hep-2R cells. In addition, silence of DGCR5 could inhibit the stemness and enhance the radiosensitivity of Hep-2R cells. Meanwhile, overexpression of miR-506 also suppressed the CSC-like traits and the radiosensitivity was increased significantly. In addition, miR-506 was predicted as target of DGCR5 and the correlation between them was validated in our study. Finally, we observed that Wnt pathway exerted a significant role in human laryngeal CSCs and DGCR5 inhibition could repress Wnt signaling activity by sponging miR-506. In vivo assays were performed and we found that DCGR5 depressed stemness of human laryngeal cancer cells through modulating miR-506 and Wnt signaling pathway. Taken these together, we reported that DGCR5 induced CSC-like properties by sponging miR-506 through activating Wnt in human laryngeal carcinoma cells.     

Methylation of cancer-stem-cell-associated Wnt target genes predicts poor prognosis in colorectal cancer patients

Gene signatures derived from cancer stem cells (CSCs) predict tumor recurrence for many forms of cancer. Here, we derived a gene signature for colorectal CSCs defined by high Wnt signaling activity, which in agreement with previous observations predicts poor prognosis. Surprisingly, however, we found that elevated expression of Wnt targets was actually associated with good prognosis, while patient tumors with low expression of Wnt target genes segregated with immature stem cell signatures. We discovered that several Wnt target genes, including ASCL2 and LGR5, become silenced by CpG island methylation during progression of tumorigenesis, and that their re-expression was associated with reduced tumor growth. Taken together, our data show that promoter methylation of Wnt target genes is a strong predictor for recurrence of colorectal cancer, and suggest that CSC gene signatures, rather than reflecting CSC numbers, may reflect differentiation status of the malignant tissue.     

Roles of Wnt/β-catenin signaling in the gastric cancer stem cells proliferation and salinomycin treatment

The Wnt1 protein, a secreted ligand that activates Wnt signaling pathways, contributes to the self-renewal of cancer stem cells (CSCs) and thus may be a major determinant of tumor progression and chemoresistance. In a series of gastric cancer specimens, we found strong correlations among Wnt1 expression, CD44 expression, and the grade of gastric cancer. Stable overexpression of Wnt1 increased AGS gastric cancer cells'' proliferation rate and spheroids formation, which expressed CSC surface markers Oct4 and CD44. Subcutaneous injection of nude mice with Wnt1-overexpressing AGS cells resulted in larger tumors than injection of control AGS cells. Salinomycin, an antitumor agent, significantly reduced the volume of tumor caused by Wnt1-overexpressing AGS cells in vivo. This is achieved by inhibiting the proliferation of CD44+Oct4+ CSC subpopulation, at least partly through the suppression of Wnt1 and β-catenin expression. Taken together, activation of Wnt1 signaling accelerates the proliferation of gastric CSCs, whereas salinomycin acts to inhibit gastric tumor growth by suppressing Wnt signaling in CSCs. These results suggest that Wnt signaling might have a critical role in the self-renewal of gastric CSCs, and salinomycin targeting Wnt signaling may have important clinical applications in gastric cancer therapy.     

Interleukin-17 programs liver progenitor cell transformation into cancer stem cells through miR-122 downregulation with increased risk of primary liver cancer initiation

Chronic inflammation is a key component in the development of virtually all types of primary liver cancers. However, how chronic inflammation potentiates or even may initiate liver parenchymal cell transformation remains unclear. Cancer stem cells (CSCs) represent an exciting target for novel anticancer therapeutic strategies in several types of cancers and were also described in primary liver cancers as tumor initiating cells. Recently, we reported a key role of Interleukin (IL)-17 in Liver Progenitor Cell (LPC) accumulation in preneoplastic cirrhotic livers. In this study, we evidenced in vitro, that long-term stimulation of LPCs with IL-17 led to their transformation into CSCs. Indeed, they acquired CSC-marker expression, and self-renewal properties, showed by their increased capacity to form spheroids. The miRNome analysis revealed that long-term IL-17 treatment of LPCs led to a 90% decrease in miR-122 expression. In a model using immunodeficient mice, ectopic engraftment of LPCs in an IL-17-enriched environment led to tumor occurrence with an aggressive phenotype. Contrastingly, in a murine model of hepatocellular carcinoma induced by a unique injection of diethyl-nitrosamine associated with chronic administration of carbon tetrachloride, IL-17-deficiency or anti-IL-17 therapy protected mice from liver tumor growth. In conclusion, we showed that a chronic exposure of LPCs to IL-17 cytokine promotes their transformation into CSCs. In addition, we demonstrated that IL-17-neutralizing strategies limit CSC occurrence and liver tumor progression through miR-122 restored-expression.     

Oral epithelial stem cells—Implications in normal development and cancer metastasis

Oral mucosa is continuously exposed to environmental forces and has to be constantly renewed. Accordingly, the oral mucosa epithelium contains a large reservoir of epithelial stem cells necessary for tissue homeostasis. Despite considerable scientific advances in stem cell behavior in a number of tissues, fewer studies have been devoted to the stem cells in the oral epithelium. Most of oral mucosa stem cells studies are focused on identifying cancer stem cells (CSC) in oral squamous cell carcinomas (OSCCs) among other head and neck cancers. OSCCs are the most prevalent epithelial tumors of the head and neck region, marked by their aggressiveness and invasiveness. Due to their highly tumorigenic properties, it has been suggested that CSC may be the critical population of cancer cells in the development of OSCC metastasis. This review presents a brief overview of epithelium stem cells with implications in oral health, and the clinical implications of the CSC concept in OSCC metastatic dissemination.     

In vitro derivation and characterization of a colorectal cancer stem cell subpopulation

In the present publication we describe for the first time the derivation of cancer stem cells from a weakly metastatic human colorectal carcinoma cell line MIP101 via selecting from the native population the cells that express intensively an embryonic stem cell marker, POU5F1 (Oct4). We provide the evidence that these cells possess an elevated clonogenic and tumorigenic potential when compared to the native population, and this correlates to the hypothesis of cancer stem cells’ primary role in the development of malignant neoplasms.