Acquisition Of CSC-like Properties Research Articles

Continuous TNF-α exposure in mammary epithelial cells promotes cancer phenotype acquisition via EGFR/TNFR2 activation.

Tumor necrosis factor alpha (TNF-α), an abundant inflammatory cytokine in the tumor microenvironment (TME), is linked to breast cancer growth and metastasis. In this study, we established MCF10A cell lines incubated with TNF-α to investigate the effects of continuous TNF-α exposure on the phenotypic change of normal mammary epithelial cells. The established MCF10A-LE cell line, through long-term exposure to TNF-α, displayed cancer-like features, including increased proliferation, migration, and sustained survival signaling even in the absence of TNF-α stimulation. Unlike the short-term exposed cell line MCF10A-SE, MCF10A-LE exhibited elevated levels of epidermal growth factor receptor (EGFR) and subsequent TNF receptor 2 (TNFR2), and silencing of EGFR or TNFR2 suppressed the cancer-like phenotype of MCF10A-LE. Notably, we demonstrated that the elevated levels of NAD(P)H oxidase 4 (NOX4) and the resulting increase in reactive oxygen species (ROS) were associated with EGFR/TNFR2 elevation in MCF10A-LE. Furthermore, mammosphere-forming capacity and the expression of cancer stem cell (CSC) markers increased in MCF10A-LE. Silencing of EGFR reversed these effects, indicating the acquisition of CSC-like properties via EGFR signaling. In conclusion, our results reveal that continuous TNF-α exposure activates the EGFR/TNFR2 signaling pathway via the NOX4/ROS axis, promoting neoplastic changes in mammary epithelial cells within the inflammatory TME.

Selective evaluation of potentially carcinogenic polycyclic aromatics in mineral oil by the DMSO based IP346 method

Lung cancer is the leading cause of cancer-related death worldwide; tobacco smoke (TS) constitutes the main causes of lung cancer. Acquisition of cancer stem cells (CSCs)-like properties is the essential progression for the initiation of lung cancer. However, the mechanisms for tobacco smoke-induced lung carcinogenesis remain elusive. In the present study, we demonstrated that long-term exposure of human bronchial epithelial (HBE) cells to TS resulted in malignant transformation and acquisition of CSC-like properties. Moreover, Wnt/β-catenin pathway was involved in acquisition of the CSC-like phenotype during neoplastic transformation of HBE cells induced by TS. Downregulation of β-catenin reduced the tumorsphere and decreased the protein expression of lung CSCs markers in TS-transformated HBE sphere-forming cells. Furthermore, Diallyl trisulfide (DATS) inhibited the CSCs activity of TS-transformed HBE cells, as well as Wnt/β-catenin suppression. Activation of Wnt/β-catenin diminished the inhibitory effects of DATS on TS-induced stemness of HBE cells. Together, the present investigation elucidates the modulation of Wnt/β-catenin in chronic TS exposure-triggered pulmonary acquisition of CSCs properties and DATS intervention, which may provide new insights into the interventional strategies against lung CSCs.

Wnt/β-catenin modulates chronic tobacco smoke exposure-induced acquisition of pulmonary cancer stem cell properties and diallyl trisulfide intervention

Lung cancer is the leading cause of cancer-related death worldwide; tobacco smoke (TS) constitutes the main causes of lung cancer. Acquisition of cancer stem cells (CSCs)-like properties is the essential progression for the initiation of lung cancer. However, the mechanisms for tobacco smoke-induced lung carcinogenesis remain elusive. In the present study, we demonstrated that long-term exposure of human bronchial epithelial (HBE) cells to TS resulted in malignant transformation and acquisition of CSC-like properties. Moreover, Wnt/β-catenin pathway was involved in acquisition of the CSC-like phenotype during neoplastic transformation of HBE cells induced by TS. Downregulation of β-catenin reduced the tumorsphere and decreased the protein expression of lung CSCs markers in TS-transformated HBE sphere-forming cells. Furthermore, Diallyl trisulfide (DATS) inhibited the CSCs activity of TS-transformed HBE cells, as well as Wnt/β-catenin suppression. Activation of Wnt/β-catenin diminished the inhibitory effects of DATS on TS-induced stemness of HBE cells. Together, the present investigation elucidates the modulation of Wnt/β-catenin in chronic TS exposure-triggered pulmonary acquisition of CSCs properties and DATS intervention, which may provide new insights into the interventional strategies against lung CSCs.

Abstract 1707: Epithelial mesenchymal transition generates cancer stem cells in CD44- colorectal cancer cells

Abstract Background: EpCAMhigh CD44+colorectal cancer (CRC) cells are thought to be cancer stem cells. Recently CD44- CRC cells are also suggested to acquire a property of cancer stem cells. Epithelial-mesenchymal transition (EMT) is a possible process of acquisition of cancer stem cell (CSC)-like properties. However, it is unclear whether EMT can be induced in primary human CRC cells. Patients and Methods: We obtained surgical specimens from 51 CRC patients, and cultured isolated cancer cells on matrigel-coated dish with medium containing growth factors. For induction of EMT, TGF-beta was added into the culture medium. Otherwise, TWIST1 expression was enforced in the cells by using lentiviral transduction. Immunocytochemical analysis, flow cytometric analysis and single cell PCR analysis using 24-genes set containing embryonic stem cell (ES)-related and EMT-related genes were performed. PCR analysis was carried out by C1 single cell auto prep system. CD44- CRC cells with or without enforced expression of TWIST1 were injected into immunodeficient mice. Results: Fifteen out of 51 samples of cancer cells formed sphere (>50 μm in diameter) after one week culture. The sphere-forming ability was related with clinical stage (Stage 1 and 2;16.7%, Stage 3 and 4;41.3%). Sorted single CD44+ cell had higher sphere-forming ability, compared to CD44- cell. The higher expression of ES- and EMT-related genes was observed in short term culture than in long-term culture, suggesting that differentiation occurred in sphere cells after long-term culture. Single cell PCR analysis revealed that sphere-forming cells were classified into 2 different populations on the basis of primary component analysis. Correlation analysis showed expression of TWIST1 and ES-related genes were correlated. In addition, flow cytometric analysis revealed that sphere forming CD44+ cells gave rise to CD44- cells. These results suggest that CD44+ cells have an ability to reconstruct the heterogeneous population, and EMT is involved in acquisition of CSC-like properties. TGF-beta increased the number of CD44+ cells in CD44- cells, and enhanced sphere-forming ability of CD44- cells. TGF-beta also induced expression of ES-related genes and TWIST1. Enforced expression of TWIST1 induced sphere-forming ability and tumorigenicity in CD44- cells. Conclusions: We established the culture system to observe the differentiation of CSCs and revealed that EMT might be involved in maintenance of CSCs. We firstly demonstrated that primary CD44- CRC cells undergo EMT and become CD44+ cells by TGF-beta treatment or enforced expression of TWIST1. Citation Format: Michitaka Nakano, Mamoru Tanaka, Taichi Isobe, Kohta Miyawaki, Yoshikane Kikushige, Hitoshi Kusaba, Shigeo Takaishi, Takashi Ueki, Eishi Baba, Koichi Akashi. Epithelial mesenchymal transition generates cancer stem cells in CD44- colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1707.

5-Fluorouracil chemotherapy of gastric cancer generates residual cells with properties of cancer stem cells.

Background: 5-Fluorouracil (5Fu) chemotherapy is the first treatment of choice for advanced gastric cancer (GC), but its effectiveness is limited by drug resistance. Emerging evidence suggests that the existence of cancer stem cells (CSCs) contributes to chemoresistance. The aim of the present study was to determine whether 5Fu chemotherapy generates residual cells with CSC-like properties in GC. Methods: Human GC cell lines, SGC7901 and AGS, were exposed to increasing 5Fu concentrations. The residual cells were assessed for both chemosensitivity and CSC-like properties. B lymphoma Mo-MLV insertion region 1 (BMI1), a putative CSC protein, was analyzed by immunohistochemical staining and subjected to pairwise comparison in GC tissues treated with or without neoadjuvant 5Fu-based chemotherapy. The correlation between BMI1 expression and recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy was then examined. Results: The residual cells exhibited 5Fu chemoresistance. These 5Fu-resistant cells displayed some CSC features, such as a high percentage of quiescent cells, increased self-renewal ability and tumorigenicity. The 5Fu-resistant cells were also enriched with cells expressing cluster of differentiation (CD)133+, CD326+ and CD44+CD24-. Moreover, the BMI1 gene was overexpressed in 5Fu-resistant cells, and BMI1 knockdown effectively reversed chemoresistance. The BMI1 protein was highly expressed consistently in the remaining GC tissues after 5Fu-based neoadjuvant chemotherapy, and BMI1 levels were correlated positively with recurrence-free survival in GC patients who received 5Fu-based neoadjuvant chemotherapy. Conclusions: Our data provided molecular evidence illustrating that 5Fu chemotherapy in GC resulted in acquisition of CSC-like properties. Moreover, enhanced BMI1 expression contributed to 5Fu resistance and may serve as a potential therapeutic target to reverse chemoresistance in GC patients.

Downregulation of PRRX1 Confers Cancer Stem Cell-Like Properties and Predicts Poor Prognosis in Hepatocellular Carcinoma.

Downregulation of paired related homeobox 1 (PRRX1) is associated with the acquisition of cancer stem cell (CSC)-like properties and poor prognosis in cancers. The purpose of this study is to clarify the role of PRRX1 expression in predicting prognosis and mediating CSC-like properties in hepatocellular carcinoma (HCC). The association between PRRX1 expression and overall survival (OS) of patients with HCC was analyzed in three independent datasets: 62 resected primary cases, 242 cases from GSE14520, and 162 cases from The Cancer Genome Atlas (TCGA). A cell line expressing PRRX1 (HuH7) was established for the functional analyses. The ability to form spheres, the expression levels of the hepatic CSC surface markers (CD13, CD133, and EpCAM), in vitro chemosensitivity to 5-fluorouracil (FU), and radiosensitivity were evaluated. Univariate and multivariate analyses showed that the 5-year OS of the low PRRX1 expression group was significantly poorer than that of the high PRRX1 expression group (P = 0.024 and P = 0.045, respectively). Consistent with this, the low PRRX1 expression group in GSE14520 and TCGA datasets showed significantly shorter OS (P = 0.027 and P = 0.010, respectively). Gene set enrichment analysis on GSE14520 and TCGA datasets indicated that downregulation of PRRX1 was correlated with the stemness signature. The number of spheres and the expression levels of CSC markers were significantly decreased when PRRX1 was expressed. Moreover, PRRX1 impaired resistance to 5-FU and radiation. Downregulation of PRRX1 expression contributes to the poor prognosis of patients with HCC through acquisition of CSC-like properties.

Abstract 3882: Downregulation of PRRX1 confers cancer stem cell-like properties and poor prognosis in hepatocellular carcinoma

Abstract Background: Recent studies have been focused on the relationship between cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT). Unlike classical EMT inducers, PRRX1 is involved in the acquisition of CSC-like properties when its down-regulation induces mesenchymal-epithelial transition in breast cancer. Meanwhile, hepatocellular carcinoma (HCC) is one of the most intractable malignancies, and it is required to identify a bona-fide molecular target to regulate tumor progression and the treatment resistance. The purpose of this study is to investigate the role of PRRX1 expression on clinical significance and CSC-like properties in HCC. Methods: (1) PRRX1 expression was analyzed in 62 resected primary HCC cases by quantitative RT-PCR (qRT-PCR). Clinicopathological factors and overall survival (OS) according to PRRX1 expression were analyzed. (2) The gene set enrichment analysis (GSEA) on the published clinical data set of 242 HCC samples (GSE14520) was applied to investigate the association of PRRX1 expression levels and the stem cell-like properties. (3) PRRX1 stably expressing HCC cell lines (HuH7) were established using a lentiviral vector. The expression levels of the hepatic CSC surface markers (CD13, CD133 and EpCAM) were analyzed by flowcytometry and qRT-PCR. Furthermore, the ability of sphere formation and in vitro chemosensitivity to 5-FU were evaluated to assess the CSC-like properties. Results: (1) Univariate analysis showed that 5-year OS of the low PRRX1 expression group was significantly shorter than that of the high PRRX1 expression group (49.8% vs. 80.3%, P=0.014). On multivariate analysis, low PRRX1 expression was an independent prognostic factor for HCC (P=0.026). (2) GSEA analysis on the 247 HCC data set indicated that the down-regulation of PRRX1 was significantly correlated with the stem cell signature (P=0.039). Furthermore, using gene sets of polycomb repressive complex 2 (PCR2) targets and H3 lysine-27 trimethylation mediated by PCR2, HCC with low PRRX1 expression showed parallel enrichment patterns with human embryonic stem cells (P=0.002 and P<0.001, respectively). These results suggested that down-regulation of PRRX1 in HCC induces the acquisition of CSC-like properties through modification of chromatin structure. (3) The level of CSC markers (CD13, CD133 and EpCAM) and the number of spheres were significantly decreased in PRRX1-expressing HuH7 cells compared with the controls. Moreover, PRRX1 impaired resistance to 5-FU in HCC cells. Conclusions: The down-regulation of PRRX1 expression contributes to the poor prognosis through the acquisition of CSC-like properties in HCC patients. We might apply the aberrant expression of PRRX1 as a novel clinical biomarker for prediction of the malignant potential in HCC cases. Citation Format: Hidenari Hirata, Keishi Sugimachi, Ryutaro Uchi, Junji Kurashige, Tae Matsumura, Yuki Takano, Hiroki Ueo, Masami Ueda, Shotaro Sakimura, Yoshiaki Shinden, Hidetoshi Eguchi, Tomoya Sudo, Masakazu Hirakawa, Hiroshi Honda, Koshi Mimori. Downregulation of PRRX1 confers cancer stem cell-like properties and poor prognosis in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3882. doi:10.1158/1538-7445.AM2014-3882

Abstract 200: DDX3X induces signal switching to stem cell-specific Wnt/β-catenin signaling, resulting in EGFR-TKI resistance in lung cancer cells harboring EGFR activating mutation

Abstract [Background] The treatment targeting signal addiction caused by oncogenic driver mutation has led to unprecedented results in clinical setting; however, it is still difficult to achieve cure. Tumor diversity based on both genetic and non-genetic influences give rise of survivors as treatment-resistant cells. Most of the acquired resistance reflects the selection of the cancer cells harboring stochastic resistance-conferring genetic alterations. However, the mechanisms how the cancer cells survive until acquisition of additional mutations are unclear. We identified DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked (DDX3X), an ATP-dependent RNA helicase, was preferentially expressed in a cancer stem cell (CSC)-like population of murine melanoma. DDX3X is believed to be involved in epigenetic regulation of gene expression based on metabolism of mRNA and miRNA. [Objectives] In the current study, we examine that DDX3X affects phenotype and signal status of PC9 cells, a lung adenocarcinoma harboring EGFR exon19 deletion as an oncogenic driver mutation. [Results] DDX3X plays a role in acquisition of CSC-like properties. PC9 A-1 cells that were engineered to overexpress DDX3X acquired ability to proliferate as tumor spheres and showed up-regulated Sox2 and ALDH expression. Epithelial mesenchymal transition with cadherin switching from E-cadherin to N-cadherin accompanied with vimentin expression was detected in non-adherent A-1 population. E-cadherin+ non-adherent cells exhibited strong CD44 expression, a putative CSC marker. Surprisingly, A-1 cells exhibited almost no EGFR phosphorylation even in the presence of EGF. Knocking-down of DDX3X restored EGFR phosphorylation. On the other hand, activated nuclear translocation of β-catenin was observed. Loss of EGFR signal addiction resulted in resistance to EGFR-TKI. On contrast, PC9-A1 cells were sensitive to ICG-001, a β-catenin signal inhibitor. Further, we identified a minor non-adherent subpopulation of parental PC9 cells strongly expressed DDX3X and that they were resistant to EGFR-TKI because they did not addict to EGFR signaling. Survivor cells of parental PC9 after long term-exposure to EGFR-TKI strongly expressed DDX3X. [Conclusion] DDX3X plays a critical role in a novel EGFR-TKI resistance mechanism, signal switching, correlating with CSC transformation. DDX3X and β-catenin are likely promising target molecules to overcome EGFR-TKI resistance. Citation Format: Satoshi Shoji, Hiroshi Kagamu, Koichiro Nozaki, Natsue Igarashi, Masaaki Okajima, Satoru Miura, Satoshi Watanabe, Hrohisa Yoshizawa, Ichiei Narita. DDX3X induces signal switching to stem cell-specific Wnt/β-catenin signaling, resulting in EGFR-TKI resistance in lung cancer cells harboring EGFR activating mutation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 200. doi:10.1158/1538-7445.AM2014-200

Nuclear reprogramming of luminal-like breast cancer cells generates Sox2-overexpressing cancer stem-like cellular states harboring transcriptional activation of the mTOR pathway

Energy metabolism plasticity enables stemness programs during the reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state. This relationship may introduce a new era in the understanding of Warburg’s theory on the metabolic origin of cancer at the level of cancer stem cells (CSCs). Here, we used Yamanaka’s stem cell technology in an attempt to create stable CSC research lines in which to dissect the transcriptional control of mTOR—the master switch of cellular catabolism and anabolism—in CSC-like states. The rare colonies with iPSC-like morphology, obtained following the viral transduction of the Oct4, Sox2, Klf4, and c-Myc (OSKM) stemness factors into MCF-7 luminal-like breast cancer cells (MCF-7/Rep), demonstrated an intermediate state between cancer cells and bona fide iPSCs. MCF-7/Rep cells notably overexpressed SOX2 and stage-specific embryonic antigen (SSEA)-4 proteins; however, other stemness-related markers (OCT4, NANOG, SSEA-1, TRA-1–60, and TRA-1–81) were found at low to moderate levels. The transcriptional analyses of OSKM factors confirmed the strong but unique reactivation of the endogenous Sox2 stemness gene accompanied by the silencing of the exogenous Sox2 transgene in MCF-7/Rep cells. Some but not all MCF-7/Rep cells acquired strong alkaline phosphatase (AP) activity compared with MCF-7 parental cells. SOX2-overexpressing MCF-7/Rep cells contained drastically higher percentages of CD44+ and ALDEFLUOR-stained ALDHbright cells than MCF-7 parental cells. The overlap between differentially expressed mTOR signaling-related genes in 3 different SOX2-overexpressing CSC-like cell lines revealed a notable downregulation of 3 genes, PRKAA1 (which codes for the catalytic α 1 subunit of AMPK), DDIT4/REDD1 (a stress response gene that operates as a negative regulator of mTOR), and DEPTOR (a naturally occurring endogenous inhibitor of mTOR activity). The insulin-receptor gene (INSR) was differentially upregulated in MCF-7/Rep cells. Consistent with the downregulation of AMPK expression, immunoblotting procedures confirmed upregulation of p70S6K and increased phosphorylation of mTOR in Sox2-overexpressing CSC-like cell populations. Using an in vitro model of the de novo generation of CSC-like states through the nuclear reprogramming of an established breast cancer cell line, we reveal that the transcriptional suppression of mTOR repressors is an intrinsic process occurring during the acquisition of CSC-like properties by differentiated populations of luminal-like breast cancer cells. This approach may provide a new path for obtaining information about preventing the appearance of CSCs through the modulation of the AMPK/mTOR pathway.

Membrane Type 1 Matrix Metalloproteinase induces an epithelial to mesenchymal transition and cancer stem cell-like properties in SCC9 cells

BackgroundTissue invasion and metastasis are acquired abilities of cancer and related to the death in oral squamous cell carcinoma (OSCC). Emerging observations indicate that the epithelial-to-mesenchymal transition (EMT) is associated with tumor progression and the generation of cells with cancer stem cells (CSCs) properties. Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) is a cell surface proteinase, which is involved in degrading extracellular matrix components that can promote tumor invasion and cell migration.MethodsIn the current study, we utilized SCC9 cells stably transfected with an empty vector (SCC9-N) or a vector encoding human MT1-MMP (SCC9-M) to study the role of MT1-MMP in EMT development.ResultsUpon up-regulation of MT1-MMP, SCC9-M cells underwent EMT, in which they presented a fibroblast-like phenotype and had a decreased expression of epithelial markers (E-cadherin, cytokeratin18 and β-catenin) and an increased expression of mesenchymal markers (vimentin and fibronectin). We further demonstrated that MT1-MMP-induced morphologic changes increased the level of Twist and ZEB, and were dependent on repressing the transcription of E-cadherin. These activities resulted in low adhesive, high invasive abilities of the SCC9-M cells. Furthermore, MT1-MMP-induced transformed cells exhibited cancer stem cell (CSC)-like characteristics, such as low proliferation, self-renewal ability, resistance to chemotherapeutic drugs and apoptosis, and expression of CSCs surface markers.ConclusionsIn conclusion, our study indicates that overexpression of MT1-MMP induces EMT and results in the acquisition of CSC-like properties in SCC9 cells. Our growing understanding of the mechanism regulating EMT may provide new targets against invasion and metastasis in OSCC.