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.     

Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substrate Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.     

Identification of a distinct side population of cancer cells in the Cal-51 human breast carcinoma cell line

“Side population” (SP) cells, which pump out the fluorescent dye H33342 via the ABCG2 transporter, define a putative stem/progenitor cell population in the mammary gland. Breast cancer SP cells recently isolated from the MCF-7 cell line possess similar properties and may represent stem cell-like cancer cells. This study extends SP cell analysis to a broad panel of human breast cancer cell lines and investigates the expression of differentiation-associated markers in isolated cancer SP cells. Expression of ABCG2 was determined in 16 breast cancer cell lines by quantitative RT-PCR, Western blotting and immunohistochemistry. Subsequently, all cell lines were screened for the presence of SP cells. Human breast cancer cell lines commonly express ABCG2. ABCG2-immunoreactivity was clearly restricted to rare cancer cells in several cell lines including Cal-51. Analysis of H33342-labeled Cal-51 cells revealed a small fraction of putative SP cells accounting for one percent of all cells. The genuine nature of Cal-51 SP cells was unambiguously verified by demonstrating a 30-fold increased ABCG2-expression in isolated Cal-51 SP cells. During in vitro expansion, Cal-51 SP cells generated heterologous non-SP (NSP) cells and ABCG2-expression declined dramatically. In contrast, NSP cells failed to sustain proliferation. Freshly isolated Cal-51 SP cells also exhibited increased expression of Muc1 and CALLA. Noteworthy, non-malignant mammary epithelial SP cells lack these differentiation markers, highlighting fundamental differences between non-malignant and breast cancer-derived SP cells. In summary, we established Cal-51 SP cells as a novel in vitro model to study differential gene expression in breast cancer-derived SP and NSP cells.     

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

Presence and role of stem cells in ovarian cancer

Ovarian cancer is the deadliest gynecological malignancy. It is typically diagnosed at advanced stages of the disease, with metastatic sites disseminated widely within the abdominal cavity. Ovarian cancer treatment is challenging due to high disease recurrence and further complicated pursuant to acquired chemoresistance. Cancer stem cell(CSC) theory proposes that both tumor development and progression are driven by undifferentiated stem cells capable of self-renewal and tumor-initiation. The most recent evidence revealed that CSCs in terms of ovarian cancer are not only responsible for primary tumor growth, metastasis and relapse of disease, but also for the development of chemoresistance. As the elimination of this cell population is critical for increasing treatment success, a deeper understanding of ovarian CSCs pathobiology, including epithelial-mesenchymal transition, signaling pathways and tumor microenvironment, is needed. Finally, before introducing new therapeutic agents for ovarian cancer, targeting CSCs, accurate identification of different ovarian stem cell subpopulations, including the very small embryoniclike stem cells suggested as progenitors, is necessary. To these ends, reliable markers of ovarian CSCs should be identified. In this review, we present the current knowledge and a critical discussion concerning ovarian CSCs and their clinical role.     

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.     

Autophagy inhibition suppresses the tumorigenic potential of cancer stem cell enriched side population in bladder cancer

The mechanisms that underlie tumor formation and progression have not been elucidated in detail in cancer biology. Recently, the identification of a tumor cell subset defined as cancer stem cells (CSCs), which is enriched for tumor initiating capacity, has engendered new perspectives towards selective targeting of tumors. In this study, we isolated the side population (SP) cells which share characteristics of CSCs from bladder cancer cell lines, T24 and UM-UC-3 by fluorescence activated cell sorting. The cells were cultured in serum free medium and expression profile of stem cell like markers (SOX-2, NANOG, KLF-4 and OCT-4), drug resistant genes (ABCG2 and MDR1) and spheroid forming capability were examined in SP, non-side population (NSP) and bulk T24 and UM-UC-3 cells. We observed that SP cells possessed a higher mRNA expression of SOX-2, NANOG, KLF-4, OCT-4, ABCG2, and MDR1 as well as a higher spheroid forming ability as compared to other bulk cells or NSP cells. The SP cells had low ROS levels and high GSH/GSSG ratio which may contribute to radio-resistance. The SP cells also showed substantial resistance to gemcitabine, mitomycin and cisplatin compared with the NSP counterpart. A high autophagic flux was observed in the SP cells. Both pharmacological and siRNA mediated inhibition of autophagy potentiated the chemotherapeutic effects of gemcitabine, mitomycin and cisplatin in these cells. We concluded that the ABCG2 expressing SP cells show autophagy associated cell survival and may be a potent target for developing more effective treatment in bladder carcinoma to enhance patient survival.     

Side Population in Human Non-Muscle Invasive Bladder Cancer Enriches for Cancer Stem Cells That Are Maintained by MAPK Signalling

Side population (SP) and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs) and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC), multiple human cell lines were used to characterise SP and ABC transporter expression. In vitro and in vivo phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148), and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2^hi) relative to the non-SP (NSP) fraction (ABCG2^low). ABCG2^hi SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2) and a three-fold increase in colony forming efficiency (CFE) in comparison to ABCG2^low NSP cells. In vivo, ABCG2^hi SP cells enriched for tumour growth compared with ABCG2^low NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2^hi SP cells and MEK inhibition also inhibited the ABCG2^hi SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2^hi SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.     

Clinical significance of side population in ovarian cancer cells

Recently, accumulating evidence has suggested that tumors, including ovarian cancer, are composed of a heterogeneous cell population with a small subset of cancer stem cells (CSCs) that sustain tumor formation and growth. The emergence of drug resistance is one of the most difficult problems in the treatment of ovarian cancer, which has been explained recently by the potential of CSCs to have superior resistance against anti-cancer drugs than conventional cancer cells. In this study, we expanded this line of study to examine whether this phenomenon is also observed in clinical specimens of ovarian cancer cells. In total we could analyze 28 samples out of 60 obtained from ovarian cancer patients. The clinical samples were subjected to testing of the expression of side population (SP) as a CSC marker, and according to the presence of SP (SP+) or absence of SP (SP-), clinicopathological significances were analyzed. Although there was no statistical significance, there were more SP+s in recurrent cases as well as in ascitic and peritoneal dissemination than in primary tumor of the ovary. There was no correlation between SP status and FIGO staging. In 19 cases of those who could be followed more than 6 months from initial therapy, there were 8 cases of recurrence or death from disease, and all of these were SP+. On the other hand, in 11 cases of disease-free survivors, 6 were SP+. There was a significant difference in prognosis between SP+ and SP- (p = 0.017). Although this study was limited, it revealed that SP could be contained more in recurrent or metastatic tumors than in primary tumors, and also that the presence of SP could be a risk factor of recurrence in ovarian cancer. Therefore, a novel therapeutic strategy targeting SP could improve the prognosis of ovarian cancer.     

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 µM cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.     

Resveratrol potentially increased the tumoricidal effect of doxorubicin on SKOV3 cancer stem cells in vitro

Ovarian cancer is associated with a high percentage of recurrence of tumor and resistance to chemotherapy. Cancer stem cells (CSCs) form a rare population with a significant capacity to begin and expand malignant diseases. Eliminating the drug resistance of CSCs by factors that have fewer side effects to the patient is vital. To investigate the effect of resveratrol (RES) and doxorubicin (DOX) on drug resistance and apoptosis of CSCs; at the first, isolation of CSCs from SKOV3 ovarian carcinoma cells and their dosage adjustment (IC₅₀) with RES and DOX was performed. Then, isolated CSCs were treated with RES and DOX IC ₅₀ of 55 and 250 nM, respectively. Subsequently, their effects on drug resistance and cell death were evaluated using real-time polymerase chain reaction, rhodamine 123 uptakes. The results of the present study demonstrated that treatment of SKOV3 with 55 μM of RES and 250 nM of DOX simultaneously increased cell viability in CSCs to DOX after 24 and 48 hours by increasing the expression of Bcl-2-associated X protein (BAX) and caspase-3 genes, and decreased the expression and function of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1) genes indicated by intracellular the rhodamine 123 content. Treatment of RES could increase the activity of DOX cell viability in CSCs originated from SKOV3 ovarian carcinoma and decrease drug resistance capacity to DOX.     

Proteasome inhibitors sensitize ovarian cancer cells to TRAIL induced apoptosis

In the present study we have explored the sensitivity of ovarian cancer cells to TRAIL and proteasome inhibitors. Particularly, we have explored the capacity of proteasome inhibitors to bypass TRAIL resistance of ovarian cancer cells. For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/DDP and A2780/ADR, providing evidence that: (i) the three cell lines are either scarcely sensitive (A2780 and A2780/ADR) or moderately sensitive (A2780/DDP) to the cytotoxic effects of TRAIL; (ii) the elevated c-FLIP expression observed in ovarian cancer cells is a major determinant of TRAIL resistance of these cells; (iii) proteasome inhibitors (PS-341 or MG132) are able to exert a significant pro-apoptotic effect and to greatly enhance the sensitivity of both chemosensitive and chemoresistant A2780 cells to TRAIL; (iv) proteasome inhibitors damage mitochondria through stabilization of BH3-only proteins, Bax and caspase activation and significantly enhance TRAIL-R2 expression; (v) TRAIL-R2, but not TRAIL-R1, mediates the apoptotic effects of TRAIL on ovarian cancer cells. Importantly, studies on primary ovarian cancer cells have shown that these cells are completely resistant to TRAIL and proteasome inhibitors markedly enhance the sensitivity of these cells to TRAIL. Given the high susceptibility of ovarian cancer cells to proteasome inhibitors, our results further support the experimental use of these compounds in the treatment of ovarian cancer.     

Metabolic regulation of cancer cell side population by glucose through activation of the Akt pathway

Side population (SP) cells within tumors are a small fraction of cancer cells with stem-like properties that can be identified by flow cytometry analysis based on their high ability to export certain compounds such as Hoechst 33342 and chemotherapeutic agents. The existence of stem-like SP cells in tumors is considered as a key factor contributing to drug resistance, and presents a major challenge in cancer treatment. Although it has been recognized for some time that tumor tissue niches may significantly affect cancer stem cells (CSCs), the role of key nutrients such as glucose in the microenvironment in affecting stem-like cancer cells and their metabolism largely remains elusive. Here we report that SP cells isolated from human cancer cells exhibit higher glycolytic activity compared to non-SP cells. Glucose in the culture environment exerts a profound effect on SP cells as evidenced by its ability to induce a significant increase in the percentage of SP cells in the overall cancer cell population, and glucose starvation causes a rapid depletion of SP cells. Mechanistically, glucose upregulates the SP fraction through ATP-mediated suppression of AMPK and activation of the Akt pathway, leading to elevated expression of the ATP-dependent efflux pump ABCG2. Importantly, inhibition of glycolysis by 3-BrOP significantly reduces SP cells in vitro and impairs their ability to form tumors in vivo. Our data suggest that glucose is an essential regulator of SP cells mediated by the Akt pathway, and targeting glycolysis may eliminate the drug-resistant SP cells with potentially significant benefits in cancer treatment.     

Characterization of a stem cell population in lung cancer A549 cells

We isolated a stem cell subpopulation from human lung cancer A549 cells using FACS/Hoechst 33342. This side population (SP), which comprised 24% of the total cell population, totally disappeared after treatment with the selective ABCG 2 inhibitor fumitremorgin C. In a repopulation study, isolated SP and non-SP cells were each able to generate a heterogeneous population of SP and non-SP cells, but this repopulation occurred more rapidly in SP cells than non-SP. An MTT assay and cell cycle distribution analysis reveal a similar profile between SP and non-SP groups. However, in the presence of doxorubicin (DOX) and methotrexate (MTX), SP cells showed significantly lower Annexin V staining when compared to non-SP cells. Taken together, these results demonstrate that SP cells have an active regeneration capacity and high anti-apoptotic activity compared with non-SP cells. Furthermore, our GeneChip^® data revealed a heightened mRNA expression of ABCG2 and ABCC2 in SP cells. Overall these data explain why the SP of A549 has a unique ability to resist DOX and MTX treatments. Therefore, we suggest that the expression of the ABCG2 transporter plays an important role in the multidrug resistance phenotype of A549 SP cells.     

Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G₂/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced when combined with GSI. Interestingly, this effect was especially significant in CD133+ cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer.     

Lumiflavin increases the sensitivity of ovarian cancer stem‐like cells to cisplatin by interfering with riboflavin

Here, we used lumiflavin, an inhibitor of riboflavin, as a new potential therapeutic chemosensitizer to ovarian cancer stem‐like cells (CSCs). This study demonstrates that the enrichment of riboflavin in CSCs is an important cause of its resistance to chemotherapy. Lumiflavin can effectively reduce the riboflavin enrichment in CSCs and sensitize the effect of cisplatin Diamminedichloroplatinum (DDP) on CSCs. In this study, CSCs of human ovarian cancer cell lines HO8910 were separated using a magnetic bead (CD133+). We also show the overexpression of the mRNA and protein of riboflavin transporter 2 and the high content of riboflavin in CSCs compared to non‐CSCs (NON‐CSCs). Moreover, CSCs were less sensitive to DDP than NON‐CSCs, whereas, the synergistic effect of lumiflavin and DDP on CSCs was more sensitive than NON‐CSCs. Further research showed that lumiflavin had synergistic effects with DDP on CSCs in increasing mitochondrial function damage and apoptosis rates and decreasing clonic function. In addition, we found that the combination of DDP and lumiflavin therapy in vivo has a synergistic cytotoxic effect on an ovarian cancer nude mice model by enhancing the DNA‐damage response and increasing the apoptotic protein expression. Notably, the effect of lumiflavin is associated with reduced riboflavin concentration, and riboflavin could reverse the effect of DDP in vitro and in vivo. Accordingly, we conclude that lumiflavin interfered with the riboflavin metabolic pathways, resulting in a significant increase in tumour sensitivity to DDP therapy. Our study suggests that lumiflavin may be a novel treatment alternative for ovarian cancer and its recurrence.     

肿瘤干细胞与肿瘤转移

近年来,肿瘤干细胞(cancer stem cell,CSC)学说研究认为CSC与肿瘤发生、发展、转移和复发关系极为密切。研究还发现CSC具有明显的异质性,即CSC可分为增生、耐药、侵袭和转移等行为不同的亚群细胞,其中具有转移生物学特性的CSC亚群细胞称为肿瘤转移干细胞(migrating cancer stem cell,MCSC)。目前认为,上皮-间质转变、趋化因子和靶器官微环境可能在肿瘤转移过程中起着重要作用。针对MCSC及其相关机制的靶向治疗有望能更有效地遏制肿瘤的转移。     

Whole Genome Expression Profiling Reveals a Significant Role for the Cell Junction and Apoptosis Pathways in Breast Cancer Stem Cells

Side population (SP) cells in primary tumors and cell lines are a small cell population, but they are known to enrich cancer stem cells (CSCs). In this study, we isolated SP cells from the human breast cancer cell line MCF7 as a model for studying CSCs. Compared with non-SP cells, MCF7 SP cells had higher mammosphere-formation efficiency (MFE) in vitro and greater tumorigenicity in vivo, suggesting that MCF7 SP cells enrich CSCs. We first directly compared the gene expression profile of SP and non-SP cells from MCF7 cell line. Comparing the expression signature of SP to non-SP cells, we identified 753 differentially expressed genes (DEGs). Using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified multiple pathways that were aberrantly regulated in SP compared with non-SP cells. Several pathways, including cell junction and apoptosis, play important roles in breast CSC function. This study demonstrates that combining global gene expression analysis with detailed annotated pathway resources can enhance our understanding of the critical pathways that regulate breast CSCs.