ALDH activity selectively defines an enhanced tumor-initiating cell population relative to CD133 expression in human pancreatic adenocarcinoma

Background

Multiple studies in recent years have identified highly tumorigenic populations of cells that drive tumor formation. These cancer stem cells (CSCs), or tumor-initiating cells (TICs), exhibit properties of normal stem cells and are associated with resistance to current therapies. As pancreatic adenocarcinoma is among the most resistant human cancers to chemo-radiation therapy, we sought to evaluate the presence of cell populations with tumor-initiating capacities in human pancreatic tumors. Understanding which pancreatic cancer cell populations possess tumor-initiating capabilities is critical to characterizing and understanding the biology of pancreatic CSCs towards therapeutic ends.

Methodology/Principal Findings

We have isolated populations of cells with high ALDH activity (ALDH^high) and/or CD133 cell surface expression from human xenograft tumors established from multiple patient tumors with pancreatic adenocarcinoma (direct xenograft tumors) and from the pancreatic cancer cell line L3.6pl. Through fluorescent activated cell sorting (FACs)-mediated enrichment and depletion of selected pancreatic cancer cell populations, we sought to discriminate the relative tumorigenicity of cell populations that express the pancreatic CSC markers CD133 and aldehyde dehydrogenase (ALDH). ALDH^high and ALDH^low cell populations were further examined for co-expression of CD44 and/or CD24. We demonstrate that unlike cell populations demonstrating low ALDH activity, as few as 100 cells enriched for high ALDH activity were capable of tumor formation, irrespective of CD133 expression. In direct xenograft tumors, the proportions of total tumor cells expressing ALDH and/or CD133 in xenograft tumors were unchanged through a minimum of two passages. We further demonstrate that ALDH expression among patients with pancreatic adenocarcinoma is heterogeneous, but the expression is constant in serial generations of individual direct xenograft tumors established from bulk human pancreatic tumors in NOD/SCID mice.

Conclusions/Significance

We conclude that, in contrast to some previous studies, cell populations enriched for high ALDH activity alone are sufficient for efficient tumor-initiation with enhanced tumorigenic potential relative to CD133⁺ and ALDH^low cell populations in some direct xenograft tumors. Although cell populations enriched for CD133 expression may alone possess tumorigenic potential, they are significantly less tumorigenic than ALDH^high cell populations. ALDH^high/CD44⁺/CD24⁺ or ALDH^low/CD44⁺/CD24⁺ phenotypes do not appear to significantly contribute to tumor formation at low numbers of inoculated tumor cells. ALDH expression broadly varies among patients with pancreatic adenocarcinoma and the apparent expression is recapitulated in serial generations of direct xenograft tumors in NOD/SCID. We have thus identified a distinct population of TICs that should lead to identification of novel targets for pancreatic cancer therapy.     

An inhibitor of K+ channels modulates human endometrial tumor-initiating cells

Background

Many potassium ion (K⁺) channels function as oncogenes to sustain growth of solid tumors, but their role in cancer progression is not well understood. Emerging evidence suggests that the early progenitor cancer cell subpopulation, termed tumor initiating cells (TIC), are critical to cancer progression.

Results

A non-selective antagonist of multiple types of K⁺ channels, tetraethylammonium (TEA), was found to suppress colony formation in endometrial cancer cells via inhibition of putative TIC. The data also indicated that withdrawal of TEA results in a significant enhancement of tumorigenesis. When the TIC-enriched subpopulation was isolated from the endometrial cancer cells, TEA was also found to inhibit growth in vitro.

Conclusions

These studies suggest that the activity of potassium channels significantly contributes to the progression of endometrial tumors, and the antagonists of potassium channels are candidate anti-cancer drugs to specifically target tumor initiating cells in endometrial cancer therapy.     

Dynamic imbalance between cancer cell subpopulations induced by Transforming Growth Factor Beta (TGF-β) is associated with a DNA methylome switch

Background

Distinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display pronounced ability to initiate new tumors and induce metastasis. Recent evidence suggests that signals from transforming growth factor beta (TGF-β) may increase the survival of these so called tumor initiating cells leading to poor HCC prognosis. However, how TGF-β establishes and modifies the key features of these cell subpopulations is not fully understood.

Results

In the present report we describe the differential DNA methylome of CD133-negative and CD133-expressing liver cancer cells. Next, we show that TGF-β is able to increase the proportion of CD133+ cells in liver cancer cell lines in a way that is stable and persistent across cell division. This process is associated with stable genome-wide changes in DNA methylation that persist through cell division. Differential methylation in response to TGF-β is under-represented at promoter CpG islands and enriched at gene bodies, including a locus in the body of the de novo DNA methyl-transferase DNMT3B gene. Moreover, phenotypic changes induced by TGF-β, including the induction of CD133, are impaired by siRNA silencing of de novo DNA methyl-transferases.

Conclusions

Our study reveals a self-perpetuating crosstalk between TGF-β signaling and the DNA methylation machinery, which can be relevant in the establishment of cellular phenotypes. This is the first indication of the ability of TGF-β to induce genome-wide changes in DNA methylation, resulting in a stable change in the proportion of liver cancer cell subpopulations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-435) contains supplementary material, which is available to authorized users.     

Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma

Background

This study evaluated the safety and immune responses to an autologous dendritic cell vaccine pulsed with class I peptides from tumor-associated antigens (TAA) expressed on gliomas and overexpressed in their cancer stem cell population (ICT-107).

Methods

TAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2. HLA-A1- and/or HLA-A2-positive patients with glioblastoma (GBM) were eligible. Mononuclear cells from leukapheresis were differentiated into dendritic cells, pulsed with TAA peptides, and administered intradermally three times at two-week intervals.

Results

Twenty-one patients were enrolled with 17 newly diagnosed (ND-GBM) and three recurrent GBM patients and one brainstem glioma. Immune response data on 15 newly diagnosed patients showed 33 % responders. TAA expression by qRT-PCR from fresh-frozen tumor samples showed all patient tumors expressed at least three TAA, with 75 % expressing all six. Correlations of increased PFS and OS with quantitative expression of MAGE1 and AIM-2 were observed, and a trend for longer survival was observed with gp100 and HER2 antigens. Target antigens gp100, HER1, and IL13Rα2 were downregulated in recurrent tumors from 4 HLA-A2+ patients. A decrease in or absence of CD133 expression was seen in five patients who underwent a second resection. At a median follow-up of 40.1 months, six of 16 ND-GBM patients showed no evidence of tumor recurrence. Median PFS in newly diagnosed patients was 16.9 months, and median OS was 38.4 months.

Conclusions

Expression of four ICT-107 targeted antigens in the pre-vaccine tumors correlated with prolonged overall survival and PFS in ND-GBM patients. The goal of targeting tumor antigens highly expressed on glioblastoma cancer stem cells is supported by the observation of decreased or absent CD133 expression in the recurrent areas of gadolinium-enhanced tumors.     

Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Background

Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.

Methodology/Principal Findings

Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.

Conclusions/Significance

These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.     

Analysis of indoleamine 2-3 dioxygenase (IDO1) expression in breast cancer tissue by immunohistochemistry

Introduction

The immunosuppressive enzyme, indoleamine 2,3 dioxygenase (IDO), is overexpressed in many different tumor types including breast cancer. IDO inhibitors synergize with chemotherapy in breast cancer murine models. Characterizing IDO expression in breast cancer could define which patients receive IDO inhibitors. This study analyzed IDO protein expression in 203 breast cancer cases. The relationship between IDO, overall survival (OS), disease-specific survival (DSS), clinicopathologic, molecular, and immune tumor infiltrate factors was evaluated.

Methods

Expression of IDO, estrogen receptor (ER), progesterone receptor (PR), human epithelial receptor 2, cytokeratin 5/6, epithelial growth factor receptor, phosphorylated AKT, neoangiogenesis, nitrogen oxide synthetase 2 (NOS2), cyclooxygenase 2 (COX2), FoxP3, CD8, and CD11b on archival breast cancer tissue sections was evaluated by immunohistochemistry. Associations between IDO and these markers were explored by a univariate and multivariate analysis. Survival was analyzed using Kaplan–Meier (OS) and Wilcoxon two-sample (DSS) tests.

Results

IDO expression was higher in ER+ tumors compared to ER? tumors. IDO was lower in those with higher neoangiogenesis. OS was better in ER+ patients with high IDO expression. DSS was better in node-positive patients with high IDO expression. IDO activity positively correlates with NOS2. COX2 as positively correlated with IDO on univariate but not multivariate analysis. There was a trend toward greater numbers of CD11b+ cells in IDO-low tumors.

Conclusions

IDO protein expression is lower in ER- breast tumors with greater neoangiogenesis. Future clinical trials evaluating the synergy between IDO inhibitors and chemotherapy should take this finding into account and stratify for ER status in the trial design.     

Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties

Background

The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential.

Methodology/Principal Finding

The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44⁺ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44⁺ cells consisted of mixed CD44⁺ and CD44⁻ cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44⁺ and CD44⁺ cells derived from CD44⁺-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44⁻ cells. Furthermore, freshly sorted CD44⁺ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44⁻ cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas.

Conclusion/Significance

Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment.     

MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma

Background

MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.

Methods

TaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44^high oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers.

Results

MiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44^high oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids.

Conclusions

MiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.     

Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions

Background

The tumor-initiating capacity of many cancers is considered to reside in a small subpopulation of cells (cancer stem cells). We have previously shown that rare prostate epithelial cells with a CD133⁺/α₂β₁ ^hi phenotype have the properties of prostate cancer stem cells. We have compared gene expression in these cells relative to their normal and differentiated (CD133^-/α₂β₁ ^low) counterparts, resulting in an informative cancer stem cell gene-expression signature.

Results

Cell cultures were generated from specimens of human prostate cancers (n = 12) and non-malignant control tissues (n = 7). Affymetrix gene-expression arrays were used to analyze total cell RNA from sorted cell populations, and expression changes were selectively validated by quantitative RT-PCR, flow cytometry and immunocytochemistry. Differential expression of multiple genes associated with inflammation, cellular adhesion, and metastasis was observed. Functional studies, using an inhibitor of nuclear factor κB (NF-κB), revealed preferential targeting of the cancer stem cell and progenitor population for apoptosis whilst sparing normal stem cells. NF-κB is a major factor controlling the ability of tumor cells to resist apoptosis and provides an attractive target for new chemopreventative and chemotherapeutic approaches.

Conclusion

We describe an expression signature of 581 genes whose levels are significantly different in prostate cancer stem cells. Functional annotation of this signature identified the JAK-STAT pathway and focal adhesion signaling as key processes in the biology of cancer stem cells.     

Isolation of Stem-Like Cancer Cells in Primary Endometrial Cancer Using Cell Surface Markers CD133 and CXCR4

Endometrial cancer (EC) is the most common familiar gynecologic malignant tumor identified in the female reproductive system and has been increasing yearly. In this study, we will identify the surface markers and stem cell markers related with cancer stem cells (CSCs) of EC. Tissue samples were obtained from endometrial cancer patients during surgical procedures. Single cells were isolated from the tissues for culturing, transfection into nude mice, and histopathology analysis. RT-PCR demonstrated that the cultured cells strongly expressed stemness-related genes, such as c-Myc, Sox-2, Nanog, Oct 4A, ABCG2, BMI-1, CK-18, Nestin and β-actin. The expression of surface markers CD24, CD133, CD47, CD29, CD44, CXCR4, SSEA3 and SSEA4, CD24, and CD133 and chemokine markers such as CXCR4 were measured by flow cytometry. Then the double percentage of CD133+CXCR4+ cells constituted 7.2% and 9.3% in EC cells originated from two different patients, respectively. The CD133+CXCR4+ primary endometrial cancer cells grew faster, exhibited high expression of mRNA of stemness-related genes, produced more spheres, and had higher clonogenic ability than other subpopulations. They are also more resistant to anti-cancer drugs than other subpopulations. These findings indicate that CD133+CXCR4+ cells may possess some characteristics of CSCs in primary endometrial cancer. These cell surface markers may be useful for the development of drugs against CSC molecular targets or as a predictive marker for poor prognosis in primary endometrial cancer.     

Non-invasive in vivo imaging of tumor-associated CD133/prominin

Background

Cancer stem cells are thought to play a pivotal role in tumor maintenance, metastasis, tumor therapy resistance and relapse. Hence, the development of methods for non-invasive in vivo detection of cancer stem cells is of great importance.

Methodology/Principal Findings

Here, we describe successful in vivo detection of CD133/prominin, a cancer stem cell surface marker for a variety of tumor entities. The CD133-specific monoclonal antibody AC133.1 was used for quantitative fluorescence-based optical imaging of mouse xenograft models based on isogenic pairs of CD133 positive and negative cell lines. A first set consisted of wild-type U251 glioblastoma cells, which do not express CD133, and lentivirally transduced CD133-overexpressing U251 cells. A second set made use of HCT116 colon carcinoma cells, which uniformly express CD133 at levels comparable to primary glioblastoma stem cells, and a CD133-negative HCT116 derivative. Not surprisingly, visualization and quantification of CD133 in overexpressing U251 xenografts was successful; more importantly, however, significant differences were also found in matched HCT116 xenograft pairs, despite the lower CD133 expression levels. The binding of i.v.-injected AC133.1 antibodies to CD133 positive, but not negative, tumor cells isolated from xenografts was confirmed by flow cytometry.

Conclusions/Significance

Taken together, our results show that non-invasive antibody-based in vivo imaging of tumor-associated CD133 is feasible and that CD133 antibody-based tumor targeting is efficient. This should facilitate developing clinically applicable cancer stem cell imaging methods and CD133 antibody-based therapeutics.     

Treatment with anti-CD137 mAbs causes intense accumulations of liver T cells without selective antitumor immunotherapeutic effects in this organ

Background/aims

Cancer therapy with agonist anti-CD137 mAbs has been shown to induce immune-mediated tumor rejections in mice, and equivalent agents of this kind are currently being tested in cancer patients. Previous reports indicated that CD137 stimulation induced polyclonal infiltrates of T lymphocytes in the liver. This study characterizes the liver infiltrates and the target dependency of the phenomena and addresses the question of whether tumors nested in the liver are a more favorable target for CD137-based immunotherapy.

Methods

Liver infiltrates were studied with conventional histology and multiple color flow cytometry of total liver leukocytes. CD137^?/? mice, mice with a single rearrangement of the TCR (OT-1 mice) and Rag^?/? mice were used to clarify molecular requirements. Mice implanted with MC38 colon carcinomas either subcutaneously or inside the liver were used for comparative studies under treatment with agonist anti-CD137 mAbs.

Results

CD137 treatment caused mononuclear inflammation in the portal spaces of the liver, which gave rise to moderate increases in transaminases without signs of cholestasis. Marked increases in the numbers of CD8+ T cells were observed, including CD8+ T lymphocytes co-expressing CD11c. Infiltrates were absent in CD137^?/? mice and mitigated in mice harboring a single transgenic TCR on their CD8 T cells. Despite the tumor-independent accumulation of T cells in the liver, immunotherapeutic effects were not more prominent against tumors located in this organ.

Conclusions

Target-dependent effects of CD137 stimulation lead to liver infiltration with T cells, but lymphocyte enrichment in this organ does not privilege this site for immunotherapeutic effects against transplanted tumors.     

CD133/Src axis mediates tumor initiating property and epithelial-mesenchymal transition of head and neck cancer

Background

Head and Neck squamous cell carcinoma (HNSCC) is a human lethal cancer with clinical, pathological, phenotypical and biological heterogeneity. Caner initiating cells (CICs), which are responsible for tumor growth and coupled with gain of epithelial-mesenchymal transition (EMT), have been identified. Previously, we enriched a subpopulation of head and neck cancer initiating cells (HN-CICs) with up-regulation of CD133 and enhancement of EMT. Others demonstrate that Src kinase interacts with and phosphorylates the cytoplasmic domain of CD133. However, the physiological function of CD133/Src signaling in HNSCCs has not been uncovered.

Methodology/Principal Finding

Herein, we determined the critical role of CD133/Src axis modulating stemness, EMT and tumorigenicity of HNSCC and HN-CICs. Initially, down-regulation of CD133 significantly reduced the self-renewal ability and expression of stemness genes, and promoted the differentiation and apoptotic capability of HN-CICs. Additionally, knockdown of CD133 in HN-CICs also lessened both in vitro malignant properties including cell migration/cell invasiveness/anchorage independent growth, and in vivo tumor growth by nude mice xenotransplantation assay. In opposite, overexpression of CD133 enhanced the stemness properties and tumorigenic ability of HNSCCs. Lastly, up-regulation of CD133 increased phosphorylation of Src coupled with EMT transformation in HNSCCs, on the contrary, silence of CD133 or treatment of Src inhibitor inversely abrogated above phenotypic effects, which were induced by CD133 up-regulation in HNSCCs or HN-CICs.

Conclusion/Significance

Our results suggested that CD133/Src signaling is a regulatory switch to gain of EMT and of stemness properties in HNSCC. Finally, CD133/Src axis might be a potential therapeutic target for HNSCC by eliminating HN-CICs.     

Plasticity of DNA methylation in mouse T cell activation and differentiation

Background

Circulating CD4⁺ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4⁺ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.

Results

Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.

Conclusion

We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.     

Survival of Cancer Stem Cells under Hypoxia and Serum Depletion via Decrease in PP2A Activity and Activation of p38-MAPKAPK2-Hsp27

Hypoxia and serum depletion are common features of solid tumors that occur upon antiangiogenesis, irradiation and chemotherapy across a wide variety of malignancies. Here we show that tumor cells expressing CD133, a marker for colorectal cancer initiating or stem cells, are enriched and survive under hypoxia and serum depletion conditions, whereas CD133− cells undergo apoptosis. CD133+ tumor cells increase cancer stem cell and epithelial-mesenchymal transition properties. Moreover, via screening a panel of tyrosine and serine/threonine kinase pathways, we identified Hsp27 is constitutively activated in CD133+ cells rather than CD133− cell under hypoxia and serum depletion conditions. However, there was no difference in Hsp27 activation between CD133+ and CD133− cells under normal growth condition. Hsp27 activation, which was mediated by the p38MAPK-MAPKAPK2-Hsp27 pathway, is required for CD133+ cells to inhibit caspase 9 and 3 cleavage. In addition, inhibition of Hsp27 signaling sensitizes CD133+ cells to hypoxia and serum depletion -induced apoptosis. Moreover, the antiapoptotic pathway is also activated in spheroid culture-enriched CD133+ cancer stem cells from a variety of solid tumor cells including lung, brain and oral cancer, suggesting it is a common pathway activated in cancer stem cells from multiple tumor types. Thus, activation of PP2A or inactivation of the p38MAPK-MAPKAPK2-Hsp27 pathway may develop new strategies for cancer therapy by suppression of their TIC population.     

c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

Background

Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells.

Methodology/Principal Findings

Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G₀/G₁ phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice.

Conclusions/Significance

These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.     

CD166/ALCAM Expression Is Characteristic of Tumorigenicity and Invasive and Migratory Activities of Pancreatic Cancer Cells

Background

CD166, also known as activated leukocyte cell adhesion molecule (ALCAM), is expressed by various cells in several tissues including cancer. However, the role of CD166 in malignant tumors is controversial, especially in pancreatic cancer. This study aimed to clarify the role and significance of CD166 expression in pancreatic cancer.

Methods

We performed immunohistochemistry and flow cytometry to analyze the expression of CD166 in surgical pancreatic tissues and pancreatic cancer cell lines. The differences between isolated CD166+ and CD166- pancreatic cancer cells were analyzed by invasion and migration assays, and in mouse xenograft models. We also performed quantitative RT-PCR and microarray analyses to evaluate the expression levels of CD166 and related genes in cultured cells.

Results

Immunohistochemistry revealed high expression of CD166 in pancreatic cancer tissues (12.2%; 12/98) compared with that in normal pancreas controls (0%; 0/17) (p = 0.0435). Flow cytometry indicated that CD166 was expressed in 33.8–70.2% of cells in surgical pancreatic tissues and 0–99.5% of pancreatic cancer cell lines. Invasion and migration assays demonstrated that CD166- pancreatic cancer cells showed stronger invasive and migratory activities than those of CD166+ cancer cells (p<0.05). On the other hand, CD166+ Panc-1 cells showed a significantly stronger colony formation activity than that of CD166- Panc-1 cells (p<0.05). In vivo analysis revealed that CD166+ cells elicited significantly greater tumor growth than that of CD166- cells (p<0.05) in both subcutaneous and orthotopic mouse tumor models. mRNA expression of the epithelial-mesenchymal transition activator Zeb1 was over-expressed in CD166- cells (p<0.001). Microarray analysis showed that TSPAN8 and BST2 were over-expressed in CD166+ cells, while BMP7 and Col6A1 were over-expressed in CD166- cells.

Conclusions

CD166+ pancreatic cancer cells are strongly tumorigenic, while CD166- pancreatic cancer cells exhibit comparatively stronger invasive and migratory activities. These findings suggest that CD166 expression is related to different functions in pancreatic cancer cells.     

CD133+ Anaplastic Thyroid Cancer Cells Initiate Tumors in Immunodeficient Mice and Are Regulated by Thyrotropin

Background

Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies. Its rapid onset and resistance to conventional therapeutics contribute to a mean survival of six months after diagnosis and make the identification of thyroid-cancer-initiating cells increasingly important.

Methodology/Principal Findings

In prior studies of ATC cell lines, CD133⁺ cells exhibited stem-cell-like features such as high proliferation, self-renewal and colony-forming ability in vitro. Here we show that transplantation of CD133⁺ cells, but not CD133⁻ cells, into immunodeficient NOD/SCID mice is sufficient to induce growth of tumors in vivo. We also describe how the proportion of ATC cells that are CD133⁺ increases dramatically over three months of culture, from 7% to more than 80% of the total. This CD133⁺ cell pool can be further separated by flow cytometry into two distinct populations: CD133^+/high and CD133^+/low. Although both subsets are capable of long-term tumorigenesis, the rapidly proliferating CD133^+/high cells are by far the most efficient. They also express high levels of the stem cell antigen Oct4 and the receptor for thyroid stimulating hormone, TSHR. Treating ATC cells with TSH causes a three-fold increase in the numbers of CD133⁺ cells and elicits a dose-dependent up-regulation of the expression of TSHR and Oct4 in these cells. More importantly, immunohistochemical analysis of tissue specimens from ATC patients indicates that CD133 is highly expressed on tumor cells but not on neighboring normal thyroid cells.

Conclusions/Significance

To our knowledge, this is the first report indicating that CD133⁺ ATC cells are solely responsible for tumor growth in immunodeficient mice. Our data also give a unique insight into the regulation of CD133 by TSH. These highly tumorigenic CD133⁺ cells and the activated TSH signaling pathway may be useful targets for future ATC therapies.     

OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation

Background

Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction.

Methodology/Principal Findings

Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing.

Conclusions/Significance

Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.