Cancer Stem-like Properties Research Articles

Abstract 1451: MicroRNAs regulated by ESE3/EHF control important mediators of epithelial cell differentiation and stemness in prostate tumors

Abstract Deregulated expression of ETS transcription factors has emerged as an important event in prostate cancer pathogenesis. We found that loss of the ETS factor ESE3/EHF induced a broad dedifferentiation program associated with epithelial-to-mesenchymal transition (EMT) and induction of metastatic and cancer stem-like cell properties. To understand the mechanism by which ESE3/EHF controls differentiation of prostate epithelial cells, we examined microRNA (miRNA) expression in a cohort of primary prostate tumors and prostate epithelial normal and cancer cells using miRNA gene arrays. A distinct set of miRNAs was specifically deregulated in cancer cells and tumors with low ESE3/EHF expression. Bioinformatic analysis indicated that the deregulated miRNAs controlled many genes involved in EMT and cell stemness. Interestingly, we found that miR-424 was at the top list of the miRNAs up-regulated in ESE3 low expressing tumors and cell lines. This finding was confirmed by qRT-PCR both in cells and human tumors. Functional assays showed that ESE3/EHF controlled directly miR-424 by binding to the pre-miRNA promoter and repressing its transcription. Inhibition of miR-424 using an antimiR reduced anchorage-independent growth and cell migration in cancer cells with low ESE3 expression and high miR-424 level. On the contrary, stable expression of pre-miR-424 in cells with low endogenous miR-424 level increased anchorage-independent growth and cell migration. Furthermore, modulation of miR-424 expression affected in vitro prostatosphere formation, a phenotype associated with cancer stem-like cell properties. Consistently, inhibition of miR-424 in DU145 prostate cancer cells reduced growth of tumor xenografts in immunodeficient mice. Integrating bioinformatic analyses of the predicted targets and gene profiling of cells with miR-424 overexpression we found that miRNA-424 controlled several factors involved in protein degradation. Collectively, these results show for the first time that ESE3/EHF controls a distinct network of miRNAs with both oncogenic and tumor suppressor functions. Loss of ESE3/EHF resulted specifically in increased expression of miR-424, which has oncogenic properties in prostate epithelial cells. This represents a novel mechanism by which deregulation of ESE3/EHF impact on prostate tumorigenesis. Thus, targeting miR-424 could be a novel therapeutic strategy for prostate cancer. Citation Format: Cecilia Dallavalle, Domenico Albino, Gianluca Civenni, Paola Ostano, Davide Genini, Ramon Garcia-Escudero, Laura Curti, Sandra Pinton, Manuela Sarti, Giovanna Chiorino, Carlo V. Catapano, Giuseppina M. R. Carbone. MicroRNAs regulated by ESE3/EHF control important mediators of epithelial cell differentiation and stemness in prostate tumors. [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 1451. doi:10.1158/1538-7445.AM2014-1451

Abstract 691: Down-regulation of Wnt10b following targeted theranostic nanoparticles in a patient derived xenograft model of triple negative breast cancer

Abstract BACKGROUND: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive disease with a poor clinical prognosis. While the primary therapeutic option for TNBC patients is pre-operative neo-adjuvant chemotherapy followed by surgical resection, about 60% of patients develop drug resistance and have a very high incidence of tumor recurrence and poorer prognosis. Given its poor survival and lack of available targeted therapeutics, the development of novel treatment options for chemo-resistant TNBC is critical. Cancer progenitor cells or stem-like cells (CSCs) are generally thought to contribute to the drug-resistant phenotype of TNBC which are typically identified as CD44+/CD24-. CSCs can also rely on activation of stem cell signaling through the Wnt/ β-catenin pathway. Components of the Wnt pathway, including Wnt ligands, have been shown to be over-expressed in human breast cancers. Our lab has developed urokinase plasminogen activator receptor (uPAR) targeted theranostic magnetic iron oxide nanoparticles (IONPs) carrying therapeutic agents that are clinically beneficial to TNBC patients such as doxorubicin (Dox). uPAR is an ideal target due to its high expression in malignant tumors including aggressive breast cancer tissues and tumor stromal cells that are enriched in TNBC tissues. METHODS: We generated patient derived xenografts of drug-resistant TNBC for testing the therapeutic efficacy of uPAR-targeted nanoparticle-drugs from tumor fragments from patients obtained from an ongoing Phase II clinical trial at Winship Cancer Institute. Histological analysis of post-chemotherapy TNBC with or without uPAR-targeted IONP-Dox was conducted using immunohistochemistry and immunofluorescence techniques. RESULTS: We have previously reported that treatment with uPAR-targeted IONP-Dox caused tumor growth inhibition following systemic delivery. We further investigated the effect of uPAR-targeted IONP on Wnt10b, a ligand of the Wnt signaling pathway. We observed that uPAR-targeted-IONP-Dox treatment decreases Wnt10b expression relative to the untreated and conventional Dox groups. We also investigated the level of CD44 expression, which is a target gene of the Wnt signaling pathway. We observed that Wnt10b and CD44 expression was co-localized and that treatment with uPAR-targeted IONP-Dox decreased the level of CD44 expression that is comparable to the level of decrease in Wnt10b expression. CONCLUSION: Our findings show that uPAR-targeted theranostic IONP can effectively target the Wnt activated CSC population. It is likely that uPAR expressing tumor cells are potential drug resistant cell populations with breast cancer stem-like cell properties. Our results support further investigation of theranostic nanoparticles as a viable therapeutic approach to overcome drug resistance in TNBC. Citation Format: Jasmine Miller-Kleinhenz, Weiping Qian, Ruth O'Regan, Toncred Styblo, Amelia Zelnak, Andrew Wang, Lily Yang. Down-regulation of Wnt10b following targeted theranostic nanoparticles in a patient derived xenograft model of triple negative breast cancer. [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 691. doi:10.1158/1538-7445.AM2014-691

Abstract 209: Pharmacological inhibition of protein kinase C alpha (PKCα) and all trans retinoic acid (ATRA) synergize to inhibit the proliferation, migration and cancer stem-like properties of a triple-negative mammary cancer model

Abstract To study the interaction between PKCα and retinoic acid pathways on the biology of breast cancer, we employed a murine mammary triple negative cell model (LM38-LP, composed by luminal (LEP), myoepithelial (MEP) and cancer stem cells (CSC). We proposed to: A) Study the effect of ATRA (1μM) on the expression of PKCα and Retinoic Acid Receptors (RARs) in LEP, MEP and CSC (mammospheres); B) Evaluate the combined effect of ATRA and a PKCα pharmacological inhibitor (1 uM Go6976) on cell proliferation employing a method to determine the interaction type; C) Analyze the effect of ATRA/PKCα inhibitor on migration and MMP activity on LM38-LP;. D) Analyze the effect of ATRA/PKCα inhibitor on proliferation, self-renewal and differentiation of CSC; E) Study the expression profile of pluripotent genes in CSC and their modulation by treatments. By RT-PCR we found that ATRA (48 h) induced a decrease in PKCα in LEP, MEP and CSC. The same treatment increased RARβ2 and RARγ2 in CSC, and only RARβ2 in LEP. ATRA and PKCα inhibitor interaction results from the proliferation assay were analyzed by Chou-Talalay´s method. We found that the inhibitory effect exerted by ATRA/PKCα inhibitor was synergistic with CI=0.59 (Synergistic with CI<0.7). ATRA/PKCα inhibitor treatment reduced LM38-LP migration more efficiently than each treatment alone, showing a synergic effect (% migration: Control: 80,1±6,9, ATRA: 55,4±5,6, PKCα inhibitor: 37,3±9,5, ATRA/PKCα inhibitor: 20,0±1,7; p≤0.05). Furthermore, MMP2 activity was strongly reduced by the combined treatment. Interestingly, we observed that only the combined treatment induced a decrease of RARγ2 expression in the highly migratory MEP cells. ATRA/PKCα inhibitor treatment synergized to reduce mammospheres growth (Diameter in µm at 96h: Control: 176±8, ATRA: 129±10; PKCα inhibitor: 130±11 ATRA/PKCα inhibitor: 103±5 p≤0.05). Pre-treatment with ATRA/PKCα inhibitor for 96h dramatically affected CSC self-renewal (Number of secondary mammospheres: Control: 313±19; ATRA/PKCα inhibitor: 69±21; p≤0.05). While in a 3D matrigel culture assay ATRA-pretreated CSC formed organized colonies with presence of lumen, the combined treatment led to the formation of small undifferentiated structures and evidence of cell death. By RT-PCR we determined that CSC expressed higher levels of pluripotent genes, such as Nanog, Sox2, Slug and Sox9, than the parental LM38-LP cell line. Besides, only the combined treatment for 96 h induced a decrease in the levels of Nanog and Slug in CSC. Our findings suggest that the pharmacological inhibition of PKCα and ATRA synergize to inhibit proliferation and migration possibly through the decrease of RARγ and the inhibition of MMP2 activity. Furthermore, the blockage of CSC expansion by the combined treatment, possibly occurred through the down regulation of Nanog and Slug. Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Pharmacological inhibition of protein kinase C alpha (PKCα) and all trans retinoic acid (ATRA) synergize to inhibit the proliferation, migration and cancer stem-like properties of a triple-negative mammary cancer model. [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 209. doi:10.1158/1538-7445.AM2014-209

Elevated expression level of long noncoding RNA MALAT-1 facilitates cell growth, migration and invasion in pancreatic cancer.

Pancreatic cancer is one of the most aggressive solid malignancies with a dismal survival rate. Recent studies have shown that high expression levels of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) correlate with several solid tumors. However, the underlying molecular mechanisms and its clinical significance in pancreatic cancer remain to be elucidated. In the present study, our results showed that MALAT-1 expression levels were upregulated in pancreatic cancer tissues compared with adjacent noncancerous controls. Consistently, higher expression level of MALAT-1 was found in all seven pancreatic cancer cell lines relative to the human pancreatic ductal epithelial cell. Further function analysis revealed that downregulation of MALAT-1 could inhibit tumor cell proliferation and decrease cell migration and invasion in vitro. The underlying mechanisms are possibly involved in inducing G2/M cell cycle arrest, promoting cell apoptosis, suppressing epithelial-mesenchymal transition and reducing cancer stem-like properties. In conclusion, this study indicated that MALAT-1 may serve as an oncogenic lncRNA that is involved in malignancy phenotypes of pancreatic cancer. Therefore, it may be used as a potential therapeutic target.

The forkhead transcription factor FOXM1 promotes endocrine resistance and invasiveness in estrogen receptor-positive breast cancer by expansion of stem-like cancer cells

IntroductionThe forkhead transcription factor FOXM1 coordinates expression of cell cycle-related genes and plays a pivotal role in tumorigenesis and cancer progression. We previously showed that FOXM1 acts downstream of 14-3-3ζ signaling, the elevation of which correlates with a more aggressive tumor phenotype. However, the role that FOXM1 might play in engendering resistance to endocrine treatments in estrogen receptor-positive (ER+) patients when tumor FOXM1 is high has not been clearly defined yet.MethodsWe analyzed FOXM1 protein expression by immunohistochemistry in 501 ER-positive breast cancers. We also mapped genome-wide FOXM1, extracellular signal-regulated kinase 2 and ERα binding events by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in hormone-sensitive and resistant breast cancer cells after tamoxifen treatment. These binding profiles were integrated with gene expression data derived from cells before and after FOXM1 knockdown to highlight specific FOXM1 transcriptional networks. We also modulated the levels of FOXM1 and newly discovered FOXM1-regulated genes and examined their impact on the cancer stem-like cell population and on cell invasiveness and resistance to endocrine treatments.ResultsFOXM1 protein expression was high in 20% of the tumors, which correlated with significantly reduced survival in these patients (P = 0.003 by logrank Mantel-Cox test). ChIP-seq analyses revealed that FOXM1 binding sites were enriched at the transcription start site of genes involved in cell-cycle progression, maintenance of stem cell properties, and invasion and metastasis, all of which are associated with a poor prognosis in ERα-positive patients treated with tamoxifen. Integration of binding profiles with gene expression highlighted FOXM1 transcriptional networks controlling cell proliferation, stem cell properties, invasion and metastasis. Increased expression of FOXM1 was associated with an expansion of the cancer stem-like cell population and with increased cell invasiveness and resistance to endocrine treatments. Use of a selective FOXM1 inhibitor proved very effective in restoring endocrine therapy sensitivity and decreasing breast cancer aggressiveness.ConclusionsCollectively, our findings uncover novel roles for FOXM1 and FOXM1-regulated genes in promoting cancer stem-like cell properties and therapy resistance. They highlight the relevance of FOXM1 as a therapeutic target to be considered for reducing invasiveness and enhancing breast cancer response to endocrine treatments.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0436-4) contains supplementary material, which is available to authorized users.

IL-6 enriched lung cancer stem-like cell population by inhibition of cell cycle regulators via DNMT1 upregulation.

Tumors are influenced by a microenvironment rich in inflammatory cytokines, growth factors and chemokines, which may promote tumor growth. Interleukin-6 (IL-6) is a multifunctional cytokine and known as a regulator of immune and inflammation responses. IL-6 has also been reported to be associated with tumor progression and chemoresistance in different types of cancers. In our study, we demonstrated that IL-6 enriches the properties of lung cancer stem-like cells in A549 lung cancer cells cultured in spheroid medium. IL-6 also promotes sphere formation and stem-like properties of A549 cells by enhancing cell proliferation. Methylation-specific polymerase chain reaction (PCR) was performed and revealed that IL-6 increased methylation of p53 and p21 in A549 cancer cells. Western blot analysis and quantitative real-time PCR demonstrated that IL-6 increased the expression of DNA methyltransferase 1 (DNMT1) in A549 cells cultured in spheroid medium, but not the expression of DNMT3a or DNMT3b. Knockdown of DNMT1 eliminated IL-6-mediated hypermethylation of cell cycle regulators and enrichment of lung cancer stem-like properties. In conclusion, our study, for the first time, shows that the IL-6/JAK2/STAT3 pathway upregulates DNMT1 and enhances cancer initiation and lung cancer stem cell (CSC) proliferation by downregulation of p53 and p21 resulting from DNA hypermethylation. Upon blockage of the IL-6/JAK2/STAT3 pathway and inhibition of DNMT1, the proliferation of lung CSCs was reduced and their formation of spheres and ability to initiate tumor growth were decreased. These data suggest that targeting of the IL-6/JAK2/STAT3 signaling pathway and DNMT1 may become important strategies for treating lung cancer.

MiR-145 regulates cancer stem-like properties and epithelial-to-mesenchymal transition in lung adenocarcinoma-initiating cells.

MicroRNA-145 (MiR-145) is an important regulator of tumorigenesis. Our previous work indicated that miR-145 reduced the proliferation and invasion as well as the tumorosphere growth capacity in lung adenocarcinoma cells. However, the underlying molecular mechanisms remain elusive. Here, we reported that the expression level of miR-145 was downregulated in lung adenocarcinoma tissues and negatively correlated with the expression level of Oct4. MiR-145 inhibited the proliferation of lung cancer-initiating cells (LCICs), partially by regulating Oct4 expression. Furthermore, we found that miR-145 exerted repressive effect on cancer stem cell properties and inhibited epithelial-mesenchymal transition (EMT) in vitro, also partially by regulating Oct4. Finally, we confirmed the repressive effect of miR-145 on cancer stem cell properties and EMT in vivo. Taken together, these evidences suggest that miR-145 serves as a tumor suppressor which downregulates LCICs' cancer stem cell properties and EMT process by targeting Oct4, leading to the inhibition of tumor growth and metastasis.

ALDH1A1 maintains ovarian cancer stem-like cells' properties by regulating KLF4/p21-mediated signaling cascade

Objectives: The cancer stem cell (CSC) theory of chemoresistance proposes that the proportion of CSCs correlate to enhanced chemoresistance and early disease recurrence. Therefore, novel therapeutics aimed at the innate molecular pathways responsible for this resistance would be paramount to overcoming platinum resistance in ovarian cancer.

MicroRNA142-3p promotes tumor-initiating and radioresistant properties in malignant pediatric brain tumors.

Primary central nervous system (CNS) atypical teratoid/rhabdoid tumor (ATRT) is an extremely malignant pediatric brain tumor observed in infancy and childhood. It has been reported that a subpopulation of CD133(+) cells isolated from ATRT tumors present with cancer stem-like and radioresistant properties. However, the exact biomolecular mechanisms of ATRT or CD133-positive ATRT (ATRT-CD133(+)) cells are still unclear. We have previously shown that ATRT-CD133(+) cells have pluripotent differentiation ability and the capability of malignant cells to be highly resistant to ionizing radiation (IR). By using microRNA array and quantitative RT-PCR in this study, we showed that expression of miR142-3p was lower in ATRT-CD133(+) cells than in ATRT-CD133(-) cells. miR142-3p overexpression significantly inhibited the self-renewal and tumorigenicity of ATRT-CD133(+) cells. On the contrary, silencing of endogenous miR142-3p dramatically increased the tumor-initiating and stem-like cell capacities in ATRT cells or ATRT-CD133(-) cells and further promoted the mesenchymal transitional and radioresistant properties of ATRT cells. Most importantly, therapeutic delivery of miR142-3p in ATRT cells effectively reduced its lethality by blocking tumor growth, repressing invasiveness, increasing radiosensitivity, and prolonging survival time in orthotropic-transplanted immunocompromised mice. These results demonstrate the prospect of developing novel miRNA-based strategies to block the stem-like and radioresistant properties of malignant pediatric brain cancer stem cells.

New insights into the role of ID proteins in breast cancer metastasis: a MET affair.

The establishment of lethal metastases depends on the capacity of a small number of cancer cells to regenerate a tumor after entering a target organ. Stankic and colleagues have identified a role for the inhibitor of differentiation protein, ID1, as a critical regulator of breast cancer stem-like properties and metastatic colonization. Under the control of tumor growth factor-beta signaling, ID1 induces mesenchymal-epithelial transition at the metastatic site by antagonizing the activity of the basic helix-loop-helix transcription factor Twist1. This study sheds light on mechanisms that initiate metastatic outgrowth, and strengthens the concept that epithelial-mesenchymal plasticity is crucial at different stages of metastasis.

MP34-12 TARGETING IGF-BP2 AND 5 WITH THE BISPECIFIC ANTISENSE OLIGODEOXYNUCLEOTIDE OGX-225 IN CHEMORESISTANT BLADDER CANCER

observed in chemo-resistant patients (**p< 0.01), which also showed correlation to prognosis. Similar results obtain by qPCR array. Furthermore, Oct4 and Nanog expression were also upregulated in multidrug resistant (253J-ADM) cell compared with parental cell, the higher tumorigenesis ability were also observed by in vitro and in vivo tumorigenesis assay(*p<0.05). Knocking down of Oct4 or Nanog in multidrug resistant BCa cell line dramatically suppressed cell renew ability (*p<0.05) and enhanced chemosensitivity to chemo-drugs (mitomycin/ pirarubicin) (*p<0.05). CONCLUSIONS: These data indicate that cancer stem-like properties were expanded during the acquisition of multidrug resistance in BCa. Clinically, BCa stemness markers (Oct4 and nanog) overexpression may promote the BCa recurrence to resist multidrugs, which could be a potential therapeutic target for chemoresistant BCa treatment.

MP34-11 MARKEDLY INCREASED OCT-4 AND NANOG EXPRESSION CORRELATES WITH MULTIDURG RESISTANCE (MDR) PROCESS IN BLADDER CARCINOMA

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2014MP34-11 MARKEDLY INCREASED OCT-4 AND NANOG EXPRESSION CORRELATES WITH MULTIDURG RESISTANCE (MDR) PROCESS IN BLADDER CARCINOMA Yi Sun, Liang Liang, Yule Chen, Yongyi Cheng, Yonggang Xu, and Dalin He Yi SunYi Sun More articles by this author , Liang LiangLiang Liang More articles by this author , Yule ChenYule Chen More articles by this author , Yongyi ChengYongyi Cheng More articles by this author , Yonggang XuYonggang Xu More articles by this author , and Dalin HeDalin He More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1024AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer (BCa) is the most prevalent and easy recurrent cancer worldwide, high propensity for recurrence and progression are still serious problems leading to high costs and life-treatment. Multidrug resistant always induced metastasis and treatment failure. Cancer stem cells (CSC) model theoretically contribute to BCa metastasis and mulitidrug resistantce. In this study, we screened BCa stemness specific marker and further detected the potential roles during multidrug resistant process. METHODS 65 pairs of primary and relapsed chemo-resistant tumors were collected during past 10 years from shaanxi provincial people¡¯s hospital, differential expression profile of stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2) were screened by IHC staining and qPCR assay. The specific stemness markers were further confirmed with newly constructed multidrug resistant (253J-ADM) and their parental cell by WB, qPCR ,in vitro and in vivo tumorigenesis assay. We finally evaluated the self-renewal ability and chemosensitivity change after knocking down specific markers by siRNA in multidrug resistant BCa cell line. RESULTS Highly expressed the stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2) was detected in relapsed chemo-resistant tumors vs primary tumor (*p<0.05 for Bmi1, CD117, CD133, and ABCG2). Oct4 and Nanog expression was significantly observed in chemo-resistant patients (**p< 0.01), which also showed correlation to prognosis. Similar results obtain by qPCR array. Furthermore, Oct4 and Nanog expression were also upregulated in multidrug resistant (253J-ADM) cell compared with parental cell, the higher tumorigenesis ability were also observed by in vitro and in vivo tumorigenesis assay(*p<0.05). Knocking down of Oct4 or Nanog in multidrug resistant BCa cell line dramatically suppressed cell renew ability (*p<0.05) and enhanced chemosensitivity to chemo-drugs (mitomycin/ pirarubicin) (*p<0.05). CONCLUSIONS These data indicate that cancer stem-like properties were expanded during the acquisition of multidrug resistance in BCa. Clinically, BCa stemness markers (Oct4 and nanog) overexpression may promote the BCa recurrence to resist multidrugs, which could be a potential therapeutic target for chemoresistant BCa treatment. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e365-e366 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yi Sun More articles by this author Liang Liang More articles by this author Yule Chen More articles by this author Yongyi Cheng More articles by this author Yonggang Xu More articles by this author Dalin He More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Hypoxia promotes stem-like properties of laryngeal cancer cell lines by increasing the CD133+ stem cell fraction

Evidence indicates that a hypoxic micro-environment plays an essential role in the regulation of cancer stem cells (CSCs). However, whether hypoxia is able to regulate the stem-like biological properties of laryngeal cancer cells remains unknown. In this study, we investigated the influence of hypoxia on the stemness of two laryngeal cancer cell lines, Hep-2 and AMC-HN-8. We cultured the two cell lines under hypoxia and normoxia and examined the influence of hypoxia on the expression of hypoxia-inducible factors (HIFs) and the cancer stem-like properties of these cells, including cell cycle distribution, expression of stem cell genes (OCT4, SOX2 and NANOG) and laryngeal CSC surface marker (CD133), proliferation, invasion, colony formation and sphere formation capacity. We determined that both of these cell lines, when maintained under hypoxic conditions, showed expanded cells in the G0/G1 phase, exhibited preferential expression of stem cell genes and CD133, and manifested upregulation of HIFs. When treated with hypoxia followed by normoxia exposure, the two cell lines exhibited enhanced capacities for proliferation, invasion, and sphere and colony formation compared with cells maintained consistently under normoxia. Our findings indicate that a hypoxic microenvironment may upgrade the stem-like biological properties of laryngeal cancer cell lines by the expansion of the CD133(+) stem cell fraction.

Sorting and identification of side population cells in the human cervical cancer cell line HeLa

BackgroundSeveral reports have revealed that cancer stem cells (CSCs) exist in many types of solid tumors. Some studies have demonstrated that side population (SP) cells isolated from diverse cancer lines harbor cancer stem-like properties, but there are few reports examining the characteristic of SP cells in human cervical cancer. The aim of this study is 1) to find out a feasible way to detect the tumor stem-like cells in cervical cancer, and 2) to analyze the properties of the SP cells being sorted.MethodsIsolated SP and non-SP cells from human cervical cancer cell line Hela by Hoechst 33342 dying method and flow cytometry analysis. Observing morphology of SP and non-SP cells. The expression of various biomarkers putatively related to cancer stem cells were investigated by immucytochemistry of SP and non-SP cells. We also analyzed cell cycle and cell apoptosis for sorted cells. The oncogenicity of the SP and non-SP cells were analyzed by tumor formation in nonobesediabeti- c/severe combined immune- deficient (NOD/SCID) mice. The drug-resistant and radiation-resistant index between SP, non-SP and Hela cells was estimated by MTS assay.ResultsThe fraction of SP cells in Hela was approximately 1.07 ± 0.32%. SP cells were smaller and rounder in shape than non-SP cells, and mostly showed colony-like growth. Immunocytochemistry showed that stem cell makers (Oct3/4, CD133, BCRP) were highly expressed in SP cells. Moreover, the number of apoptotic cells among non-SP cells (17.6 ± 3.7%) was significantly higher compared with that among SP cells (4.4 ± 1.2%). The HE staining of in vivo grown tumors result from SP cells showed more poor differentiation, though no significant differences were shown between SP and non-SP cells in NOD/SCID mice tumorigenicity. Furthermore, SP cells demonstrated a higher degree of drug resistance against trichostatin A (TSA) compared with that of non-SP and Hela cells. SP cells were also found to be more resistant against radiotherapy.ConclusionsSP cells possess some characteristics of CSCs, namely high proliferation ability, chemoresistance and radioresistance, which may be helpful to elucidate novel targets for effective clinical treatments of cervical cancer in the future.

Accumulation and biological effects of cobalt ferrite nanoparticles in human pancreatic and ovarian cancer cells

Background and objectiveSuperparamagnetic iron oxide nanoparticles (SPIONs) emerge as a promising tool for early cancer diagnostics and targeted therapy. However, both toxicity and biological activity of SPIONs should be evaluated in detail. The aim of this study was to synthesize superparamagnetic cobalt ferrite nanoparticles (Co-SPIONs), and to investigate their uptake, toxicity and effects on cancer stem-like properties in human pancreatic cancer cell line MiaPaCa2 and human ovarian cancer cell line A2780. Materials and methodsCo-SPIONs were produced by Massart's co-precipitation method. The cells were treated with Co-SPIONs at three different concentrations (0.095, 0.48, and 0.95μg/mL) for 24 and 48h. Cell viability and proliferation were analyzed after treatment. The stem-like properties of cells were assessed by investigating the cell clonogenicity and expression of cancer stem cell-associated markers, including CD24/ESA in A2780 cell line and CD44/ALDH1 in MiaPaCa2 cell line. Magnetically activated cell sorting was used for the separation of magnetically labeled and unlabeled cells. ResultsBoth cancer cell lines accumulated Co-SPIONs, however differences in response to nanoparticles were observed between MiaPaCa2 and A2780 cell. In particular, A2780 cells were more sensitive to exposition to Co-SPIONs than MiaPaCa2 cells, indicating that a safe concentration of nanoparticles must be estimated individually for a particular cell type. Higher doses of Co-SPIONs decreased both the clonogenicity and ESA marker expression in A2780 cells. ConclusionsCo-SPIONs are not cytotoxic to cancer cells, at least when used at a concentration of up to 0.95μg/mL. Co-SPIONs have a dose-dependent effect on the clonogenic potential and ESA marker expression in A2780 cells. Magnetic detection of low concentrations of Co-SPIONS in cancer cells is a promising tool for further applications of these nanoparticles in cancer diagnosis and treatment; however, extensive research in this field is needed.

Genistein attenuates cancer stem cell characteristics in gastric cancer through the downregulation of Gli1

Genistein is an isoflavone from soy with multiple action targets in cellular processes. Hedgehog signaling and its activator Gli1 are involved not only in oncogenesis, but also in cancer stemness and overexpression of CD44, a typical cancer stem cell surface marker. It has been shown that levels of Gli1 and CD44 expression are downregulated by genistein. Genistein may modulate distinctive cellular characteristics in cancer stem cells by inhibiting Gli1-related signaling pathways. In the present study, we sorted cells from MKN45, a human gastric cancer cell line, according to CD44 expression. CD44(+) cells showed properties of cancer stem-like cells and formed sphere colonies. In addition, sonic hedgehog (Shh) signaling genes were upregulated in CD44(+) cells when compared with these levels in CD44(-) cells. When CD44(+) cancer stem-like cells were treated with genistein, Gli1 and CD44 mRNA and protein expression was significantly reduced. Moreover, other stem cell markers were downregulated by genistein. Gli1 siRNA was used to confirm the action of genistein in inhibiting Gli1 expression. The high cell migration capacity of CD44(+) cells was blocked by genistein. in conclusion, genistein inhibits Gli1 gene expression, resulting in the attenuation of cancer stem-like properties in gastric cancer cells. In addition, genistein suppresses the cell invasive capacity that is required for tumor growth and metastasis. Our data showed that genistein can be an effective agent for gastric cancer therapy by targeting cancer stem-like characteristics.

TGF-β-Id1 Signaling Opposes Twist1 and Promotes Metastatic Colonization via a Mesenchymal-to-Epithelial Transition

ID genes are required for breast cancer colonization of the lungs, but the mechanism remains poorly understood. Here, we show that Id1 expression induces a stem-like phenotype in breast cancer cells while retaining epithelial properties, contrary to the notion that cancer stem-like properties are inextricably linked to the mesenchymal state. During metastatic colonization, Id1 induces a mesenchymal-to-epithelial transition (MET), specifically in cells whose mesenchymal state is dependent on the Id1 target protein Twist1, but not at the primary site, where this state is controlled by the zinc finger protein Snail1. Knockdown of Id expression in metastasizing cells prevents MET and dramatically reduces lung colonization. Furthermore, Id1 is induced by transforming growth factor (TGF)-β only in cells that have first undergone epithelial-to-mesenchymal transition (EMT), demonstrating that EMT is a prerequisite for subsequent Id1-induced MET during lung colonization. Collectively, these studies underscore the importance of Id-mediated phenotypic switching during distinct stages of breast cancer metastasis.

PKCι Maintains a Tumor-initiating Cell Phenotype That Is Required for Ovarian Tumorigenesis

Protein kinase Cι (PKCι) has oncogenic potential and is an attractive therapeutic target for treatment of lung cancer, particularly those tumors that express elevated PKCι. However, whether PKCι is a viable target in ovarian cancer is unknown, and virtually nothing is known about the mechanism by which PKCι drives ovarian tumorigenesis. Here, it is demonstrated that PKCι maintains a tumor-initiating cell (TIC) phenotype that drives ovarian tumorigenesis. A highly tumorigenic population of cells from human ovarian cancer cell lines exhibit cancer stem-like TIC properties, including self-renewal, clonal expansion, expression of stem-related genes, enhanced transformed growth in vitro, and aggressive tumor-initiating activity in vivo. Genetic disruption of PKCι inhibits the proliferation, clonal expansion, anchorage-independent growth, and enhanced tumorigenic properties of ovarian TICs. Biochemical analysis demonstrates that PKCι acts through its oncogenic partner Ect2 to activate a MEK/ERK signaling axis that drives the ovarian TIC phenotype. Genomic analysis reveals that PKCι and Ect2 are coordinately amplified and overexpressed in the majority of primary ovarian serous tumors, and these tumors exhibit evidence of an active PKCι-Ect2 signaling axis in vivo. Finally, this study reveals that auranofin, a potent and selective inhibitor of oncogenic PKCι signaling, inhibits the tumorigenic properties of ovarian TIC cells in vitro and in vivo. These data demonstrate that PKCι is required for a TIC phenotype in ovarian cancer, and that auranofin is an attractive therapeutic option to target deadly ovarian TICs in ovarian cancer patients. PKCι drives a tumor-initiating cell phenotype in ovarian cancer cells that can be therapeutically targeted with auranofin, a small molecule inhibitor of PKCι signaling.

ITRAQ-Based Proteomic Analysis of Polyploid Giant Cancer Cells and Budding Progeny Cells Reveals Several Distinct Pathways for Ovarian Cancer Development

Polyploid giant cancer cells (PGCCs) are a morphologically distinct subgroup of human tumor cells with increased nuclear size or multiple nuclei, but they are generally considered unimportant because they are presumed to be nondividing and thus nonviable. We have recently shown that these large cancer cells are not only viable but also can divide asymmetrically and yield progeny cancer cells with cancer stem-like properties via budding division. To further understand the molecular events involved in the regulation of PGCCs and the generation of their progeny cancer cells, we comparatively analyzed the proteomic profiles of PGCCs, PGCCs with budding daughter cells, and regular control cancer cells from the HEY and SKOv3 human ovarian cancer cell lines with and without CoCl2. We used a high-throughput iTRAQ-based proteomic methodology coupled with liquid chromatography-electrospray ionization tandem mass spectroscopy to determine the differentiated regulated proteins. We performed Western blotting and immunohistochemical analyses to validate the differences in the expression patterns of a variety of proteins between PGCCs or budding PGCCs and regular cancer cells identified by iTRAQ approach and also a selected group of proteins from the literature. The differentially regulated proteins included proteins involved in response to hypoxia, stem cell generation, chromatin remodeling, cell-cycle regulation, and invasion and metastasis. In particular, we found that HIF-1alpha and its known target STC1 are upregulated in PGCCs. In addition, we found that a panel of stem cell-regulating factors and epithelial-to-mesenchymal transition regulatory transcription factors were upregulated in budding PGCCs, whereas expression of the histone 1 family of nucleosomal linker proteins was consistently lower in PGCCs than in control cells. Thus, proteomic expression patterns provide valuable insight into the underlying mechanisms of PGCC formation and the relationship between PGCCs and cancer stem cells in patients with ovarian cancers.

Leptin receptor maintains cancer stem-like properties in triple negative breast cancer cells

Despite new therapies, breast cancer continues to be the second leading cause of cancer mortality in women, a consequence of recurrence and metastasis. In recent years, a population of cancer cells has been identified, called cancer stem cells (CSCs) with self-renewal capacity, proposed to underlie tumor recurrence and metastasis. We previously showed that the adipose tissue cytokine LEPTIN, increased in obesity, promotes the survival of CSCs in vivo. Here, we tested the hypothesis that the leptin receptor (LEPR), expressed in mammary cancer cells, is necessary for maintaining CSC-like and metastatic properties. We silenced LEPR via shRNA lentivirus transduction and determined that the expression of stem cell self-renewal transcription factors NANOG, SOX2, and OCT4 (POU5F1) is inhibited. LEPR-NANOG signaling pathway is conserved between species because we can rescue NANOG expression in human LEPR-silenced cells with the mouse LepR. Using a NANOG promoter GFP reporter, we showed that LEPR is enriched in NANOG promoter active (GFP+) cells. In lineage tracing studies, we showed that the GFP+ cells divide in a symmetric and asymmetric manner. LEPR-silenced MDA-MB-231 cells exhibit a mesenchymal to epithelial transition morphologically, increased E-CADHERIN and decreased VIMENTIN expression compared with control cells. Finally, LEPR-silenced cells exhibit reduced cell proliferation, self-renewal in tumor sphere assays, and tumor outgrowth in xenotransplant studies. Given the emergence of NANOG as a pro-carcinogenic protein in multiple cancers, these studies suggest that inhibition of LEPR may be a promising therapeutic approach to inhibit NANOG and thereby neutralize CSC functions.