Generation Of Cancer Stem Cells Research Articles

Hepatitis B virus PreS1 facilitates hepatocellular carcinoma development by promoting appearance and self-renewal of liver cancer stem cells

Hepatitis B virus (HBV) is a major etiologic agent of hepatocellular carcinoma (HCC). However, the molecular mechanism by which HBV infection contributes to HCC development is not fully understood. Here, we initially showed that HBV stimulates the production of cancer stem cells (CSCs)-related markers (CD133, CD117 and CD90) and CSCs-related genes (Klf4, Sox2, Nanog, c-Myc and Oct4) and facilitates the self-renewal of CSCs in human hepatoma cells. Cellular and clinical studies revealed that HBV facilitates hepatoma cell growth and migration, enhances white blood cell (WBC) production in the sera of patients, stimulates CD133 and CD117 expression in HCC tissues, and promotes the CSCs generation of human hepatoma cells and clinical cancer tissues. Detailed studies revealed that PreS1 protein of HBV is required for HBV-mediated CSCs generation. PreS1 activates CD133, CD117 and CD90 expression in normal hepatocyte derived cell line (L02) and human hepatoma cell line (HepG2 and Huh-7); facilitates L02 cells migration, growth and sphere formation; and finally enhances the abilities of L02 cells and HepG2 cells to induce tumorigeneses in nude mice. Thus, PreS1 acts as a new oncoprotein to play a key role in the appearance and self-renewal of CSCs during HCC development.

Long Non-Coding RNAs: Key Regulators of Epithelial-Mesenchymal Transition, Tumour Drug Resistance and Cancer Stem Cells.

Epithelial mesenchymal transition (EMT), the adoption by epithelial cells of a mesenchymal-like phenotype, is a process co-opted by carcinoma cells in order to initiate invasion and metastasis. In addition, it is becoming clear that is instrumental to both the development of drug resistance by tumour cells and in the generation and maintenance of cancer stem cells. EMT is thus a pivotal process during tumour progression and poses a major barrier to the successful treatment of cancer. Non-coding RNAs (ncRNA) often utilize epigenetic programs to regulate both gene expression and chromatin structure. One type of ncRNA, called long non-coding RNAs (lncRNAs), has become increasingly recognized as being both highly dysregulated in cancer and to play a variety of different roles in tumourigenesis. Indeed, over the last few years, lncRNAs have rapidly emerged as key regulators of EMT in cancer. In this review, we discuss the lncRNAs that have been associated with the EMT process in cancer and the variety of molecular mechanisms and signalling pathways through which they regulate EMT, and finally discuss how these EMT-regulating lncRNAs impact on both anti-cancer drug resistance and the cancer stem cell phenotype.

RNA editing-dependent epitranscriptome diversity in cancer stem cells.

Cancer stem cells (CSCs) can regenerate all facets of a tumour as a result of their stem cell-like capacity to self-renew, survive and become dormant in protective microenvironments. CSCs evolve during tumour progression in a manner that conforms to Charles Darwin's principle of natural selection. Although somatic DNA mutations and epigenetic alterations promote evolution, post-transcriptional RNA modifications together with RNA binding protein activity (the 'epitranscriptome') might also contribute to clonal evolution through dynamic determination of RNA function and gene expression diversity in response to environmental stimuli. Deregulation of these epitranscriptomic events contributes to CSC generation and maintenance, which governs cancer progression and drug resistance. In this Review, we discuss the role of malignant RNA processing in CSC generation and maintenance, including mechanisms of RNA methylation, RNA editing and RNA splicing, and the functional consequences of their aberrant regulation in human malignancies. Finally, we highlight the potential of these events as novel CSC biomarkers as well as therapeutic targets.

Evolution of Cancer Stem-like Cells in Endocrine-Resistant Metastatic Breast Cancers Is Mediated by Stromal Microvesicles.

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ERlo/Notchhi feed-forward loop responsible for the generation of CD133hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6-pStat3 pathway promoted the biogenesis of onco-miR-221hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSCs. Conversely, in models of CD133neg, HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927-41. ©2017 AACR.

Dual inhibiting OCT4 and AKT potently suppresses the propagation of human cancer cells

AKT serves as an epigenetic modulator that links epigenetic regulation to cell survival and proliferation while the epigenetic mediator OCT4 critically controls stem cell pluripotency and self-renewal. Emerging evidence indicated their complicated interplays in cancer cells and cancer stem cells (CSCs), and inhibiting either one may activate the other. Thus, in this study, we propose a strategy to targeting both factors simultaneously. Firstly, a combination of an OCT4-specific shRNA and the specific AKT inhibitor Akti-1/2 potently suppressed the propagation of human embryonal carcinoma cells, adherent cancer cells and stem-like cancer cells, establishing the proof-of-concept that dual inhibiting OCT4 and AKT can effectively target various cancer cells. Next, we combined Akti-1/2 with metformin, a widely-prescribed drug for treating type 2 diabetes, which was reported to down-regulate OCT4 expression. The metformin + Akti-1/2 combo significantly altered multiple signaling and epigenetic pathways, induced growth arrest and cell death of adherent and stem-like glioblastoma U87 cells, and attenuated their tumorigenicity in vivo. Taken together, we demonstrate here that simultaneously targeting an epigenetic mediator and an epigenetic modulator, by dual inhibiting OCT4 and AKT, can have significantly improved efficacies over single treatment in suppressing the propagation of CSCs as well as the entire bulk of differentiated cancer cells.

MP99-03 RIOK2 IS A MEDIATOR OF OBESITY ENHANCED PROSTATE CANCER GROWTH

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology IV1 Apr 2017MP99-03 RIOK2 IS A MEDIATOR OF OBESITY ENHANCED PROSTATE CANCER GROWTH Everardo Macias, David Corcoran, Jen-Tsan Chi, and Stephen Freedland Everardo MaciasEverardo Macias More articles by this author , David CorcoranDavid Corcoran More articles by this author , Jen-Tsan ChiJen-Tsan Chi More articles by this author , and Stephen FreedlandStephen Freedland More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3089AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Obesity is a growing global and U.S. health problem. For 2012, an estimated 117,000 cancer cases in the U.S. were deemed preventable by achieving and maintaining a healthy weight, including 11% of all advanced prostate cancers (PC). Obesity is associated with greater risk of high-grade PC, recurrence after therapy, metastases, and PC specific death. We exploited this link to identify actionable targets by performing a shRNA genomic screen in obese and lean mice targeting the entire kinome. Our functional screen identified multiple kinases, which appear to be essential for obesity-driven PC growth including kinases previously implicated in PC and others not previously studied such as Right Open Reading Frame Kinase 2 (RIOK2). METHODS LAPC-4 cells were inoculated with an shRNA library of ~5000 lentivirus targeting 513 kinases. 5x106 cells (~1,000 cells per shRNA) were grafted to chronically obese mice. Tumors were established to ~200 mm3 and a portion collected for reference. Remaining mice were randomized to continue on ad lib WD or 25% CR diet. Genome-integrated shRNA inserts were amplified using nested barcoded primers and sequenced using Illumina Hi-Seq 2000 and quantified. A virtual screen based on a RIOK2 homology model generated using MODELLER based on two RIOK2 crystal structures. Global gene expression analysis of RNA from scramble control and RIOK2 knockdown with two shRNAs in 22RV1 cells was conducted with Affymetrix U133A Plus Array. RESULTS RIOK2 expression correlates with Gleason grade in radical prostatectomy tissue and RIOK2 kinase activity is elevated in metastatic vs localized PCs. ENCODE ChIP-seq data shows Androgen Receptor and Myc bind to the RIOK2 promoter and regulate expression. Targeting RIOK2, via newly identified small molecule inhibitors reduces cell viability and soft agar colony growth. Gene set enrichment analysis of RIOK2 depleted PC cells showed reduction of cell cycle, adipogenesis, EMT and cancer stem cell genes. RIOK2 also regulates Neuropeptide Y2 Receptor (NYP2R), which is part of the NPY obesogenic signaling axis that correlates with obesity and worse PC outcomes. CONCLUSIONS Our in vivo screen highlighted RIOK2 as an actionable PC target in obese hosts. Targeting RIOK2, pharmacologically with our lead compounds or genetically, drastically reduces PC cell viability. RIOK2 may regulate NPY2R expression, which when coupled with elevated NPY in both obese hosts and PCs can amplify NPY pro-tumorigenic signaling. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1320-e1321 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Everardo Macias More articles by this author David Corcoran More articles by this author Jen-Tsan Chi More articles by this author Stephen Freedland More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cancer Stem Cell Gene Variants in CD44 Predict Outcome in Stage II and Stage III Colon Cancer Patients.

Growing evidence suggests that human cancers are stem cell diseases and recent data support the existence of cancer stem cells (CSCs) in a variety of malignancies, including colon cancer. These CSCs were shown to be capable of initiating tumor development and progression. Several studies have suggested CD133, CD26 and CD44 as markers of tumor-initiating cells of colon cancer. The purpose of the present study was to assess the impact of single-nucleotide polymorphisms (SNPs) in stem cell-related genes on clinical outcome in a large cohort of colon cancer patients with clinical stage II and III. Data from 599 consecutive patients with colon cancer stage II and III, treated between 1995 and 2011 at a single centre, were retrospectively evaluated. Genomic DNA was extracted from paraffin-embedded normal tissue distant from the tumor to obtain germline DNA. Allelic distribution of polymorphisms was tested for deviation from Hardy-Weinberg equilibrium using χ2-test. The association of polymorphisms with time to recurrence (TTR) and overall survival (OS) was analyzed using Kaplan-Meier curves and compared by the log-rank test. Case-wise deletion for missing polymorphisms was used in univariable and multivariable analyses. CD44 rs187115 showed a statistically significant association with TTR; patients carrying at least one G allele had a significant reduced risk of recurrence compared to patients with the homozygous A/A variant (hazard ratio (HR)=0.67, 95% confidence interval (CI)=0.48-0.94, p=0.019). CD44 rs13347 showed a statistically significant association with OS. Patients carrying at least one T allele in rs13347 had a significantly reduced risk of death compared to patients with the homozygous C/C variant (HR=0.61, 95% CI=0.41-0.92, p=0.019). None of the other investigated polymorphisms (CD44 rs187116, CD44 rs7116432, CD44 rs353639, DPP4 rs2268889, DPP4 rs3788979, DPP4 rs7608798 and CD133 rs2240688) were associated with either TTR or OS. Germline variants rs13347 and rs187115 in the stem cell gene CD44 are prognostically relevant in stage II and III colon cancer patients.

STROBE-compliant integrin through focal adhesion involve in cancer stem cell and multidrug resistance of ovarian cancer.

Cancer stem cells (CSCs) are considered to be the root of carcinoma relapse and drug resistance in ovarian cancer. Hunting for the potential CSC genes and explain their functions would be a feasible strategy to meet the challenge of the drug resistance in ovarian cancer. In this study, we performed bioinformatic approaches such as biochip data extraction and pathway enrichment analyses to elucidate the mechanism of the CSC genes in regulation of drug resistance. Potential key genes, integrins, were identified to be related to CSC in addition to their associations with drug resistance and prognosis in ovarian cancer. A total of 36 ovarian CSC genes involved in regulation of drug resistance were summarized, and potential drug resistance-related CSC genes were identified based on 3 independent microarrays retrieved from the Gene Expression Omnibus (GEO) Profiles. Pathway enrichment of CSC genes associated with drug resistance in ovarian cancer indicated that focal adhesion signaling might play important roles in CSC genes-mediated drug resistance. Integrins are members of the adhesion molecules family, and integrin subunit alpha 1, integrin subunit alpha 5, and integrin subunit alpha 6 (ITGA6) were identified as central CSC genes and their expression in side population cells, cisplatin-resistant SKOV3 (SKOV3/DDP2) cells, and cisplatin-resistant A2780 (A2780/DDP) cells were dysregulated as measured by real-time quantitative polymerase chain reaction. The high expression of ITGA6 in 287 ovarian cancer patients of TCGA cohort was significantly associated with poorer progression-free survival. This study provide the basis for further understanding of CSC genes in regulation of drug resistance in ovarian cancer, and integrins could be a potential biomarker for prognosis of ovarian cancer.

IL-33 facilitates endocrine resistance of breast cancer by inducing cancer stem cell properties

Breast cancers with estrogen receptor (ER) expressions account for the majority of all clinical cases. Due to hormone therapy with tamoxifen, prognoses of patients with ER-positive breast cancer are significantly improved. However, endocrine resistance to tamoxifen is common and inevitable, leading to compromised efficacy of hormone therapy. Herein, we identify a crucial role of IL-33 in inducing endocrine resistance of breast cancer. IL-33 overexpression in breast cancer cells results in resistance to tamoxifen-induced tumor growth inhibition, while IL-33 knockdown corrects this problem. Mechanistically, IL-33 induces breast cancer stem cell properties evidenced by mammosphere formation and xenograft tumorigenesis, as well as expression of cancer stem cell genes including ALDH1A3, OCT4, NANOG and SOX2. In breast cancer patients, higher serum IL-33 levels portend advanced clinical stages, poorly differentiated cancer cells and tumor recurrence. IL-33 expression levels in patients' freshly isolated breast cancer cells predicts tamoxifen resistance and cancer stem cell features in individual patient. Collectively, IL-33 induces endocrine resistance of breast cancer by promoting cancer stem cell properties. These findings provide novel mechanisms connecting IL-33 with cancer pathogenesis and pinpoint IL-33 as a promising target for optimizing hormone therapy in clinical practice.

Abstract P5-07-07: DYRK2 contributes to the generation of breast cancer stem cells through KLF4

Abstract <Introduction> Cancer stem cells (CSCs) have been defined by the potential to self-renew and to differentiate. CSCs pose a major hurdle in the treatment of cancer. However, the mechanisms by which cells acquire CSC properties such as drug resistance remain unclear. Dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a protein kinase that phosphorylates its substrates on serine/threonine. Initially, we found that DYRK2 phosphorylates p53 at Ser 46 to regulate apoptotic cell death in response to DNA damage. Recently, we have shown that DYRK2 controls Snail degradation in breast cancer and ovarian serous adenocarcinoma. We also found that knockdown of DYRK2 in luminal-type breast cancer MCF-7 cells increased the cancer stem cell population. Kruppel-like factor 4 (KLF4) is one of the Yamanaka factors. It has been reported that pluripotent stem cells from mouse embryonic or adult fibroblasts are induced by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4. This finding led us to determine if KLF4 is indispensable for the maintenance of CSCs. The aim of this study is to clarify whether DYRK2 regulates CSCs through KLF4 in breast cancer. <Methods> Cell lines: MCF-7 (human mammary carcinoma: ATCC) cells were grown according to standard protocols. We established stable DYRK2-depleted cells. MCF-7 cells were transfected with pSuper-puro vector (pSuper control) or pSuper-puro DYRK2 shRNAs (shDYRK2) with puromycin to isolate stable cell lines. In turn, we established both stable DYRK2- and KLF4-depleted cells. shDYRK2 cells were transfected with pSuper-neo vector (pSuper-neo control) or pSuper-neo KLF4 shRNAs (shKLF4) with puromycin and G418. Knockdown of DYRK2 or KLF4 was confirmed by real-time RT-PCR and immunoblotting. The depleted cells were compared with the control cells using real-time RT-PCR, immunoblotting, flow cytometric analysis, mammosphere assay, xenograft models and immunohistological staining. <Results> We analyzed the population of breast cancer stem cells by flow cytometric analysis and in vitro mammosphere assay. The results showed that knockdown of DYRK2 was associated with the increase of CD44+/CD24- cells. While pSuper control cells formed mammospheres, they did in a lesser extent compared to shDYRK2 cells. In real-time RT-PCR and immunoblotting analysis, stable DYRK2 depletion in MCF-7 cells induced KLF4 accumulation. We then investigated the effect of KLF4 on stemness by flow cytometric analysis and in vitro mammosphere assay. The results showed that knockdown of KLF4 in shDYRK2 cells reduced the proportion of CD44+/CD24- cells. Whereas shDYRK2/shKLF4 cells formed mammospheres, they did in a lesser extent compared to shDYRK2/pSuper-neo control cells. Moreover, the scale of the mammospheres formed in shDYRK2/shKLF4 cells was significantly smaller, as compared with that in shDYRK2/pSuper-neo control cells. In xenograft models, the loss of KLF4 protein expression significantly decreased tumor formation. Immunohistological staining of fifty-nine samples from surgically treated breast cancer patients showed an inverse correlation between DYRK2 and KLF4 expression. <Conclusion> These findings revealed that DYRK2 contributes to the generation of breast cancer stem cells through KLF4. Citation Format: Imawari Y, Mimoto RK, Yamaguchi N, Kamio M, Kato K, Nogi H, Toriumi Y, Uchida K, Takeyama H, Yoshida K. DYRK2 contributes to the generation of breast cancer stem cells through KLF4 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-07.

Abstract P4-04-02: Bromodomain and extra-terminal proteins regulate the epithelial-mesenchymal transition in breast cancer

Abstract P4-04-02: Bromodomain and extra-terminal proteins regulate the epithelial-mesenchymal transition in breast cancer

Contribution of nuclear events in generation and maintenance of cancer stem cells: revisiting chemo-resistance

While chemotherapy is often capable of reducing the tumor bulk, disease-free survival of patients is curtailed by recurrence because of chemoresistance. In fact, the cardinal reason of treatment failure can be attributed to chemoresistance, which involves a complex mechanism. In the present scenario, cancer stem cells (CSCs) have been identified to play a pivotal role in chemoresistance. A few reviews have discussed numerous mechanisms of chemoresistance in CSCs and the therapeutic strategies targeting stem cell signaling pathways. References are also there elaborating the applications of therapeutic nanoparticles and epigenetic drugs in targeting CSCs. However, there is a paucity of information in this area that should be explored and addressed. It is a well known fact that the nucleus harbors the entire genome of an organism and is the master regulator of all the genetic and epigenetic programs. In fact, majority of the features of CSCs evolve from cell nucleus. Here we review, the prevailing and emanating concepts of how the key nuclear events like (1) genetic regulation, (2) epigenetic regulation, (3) regulation by microRNAs (miRNA), and (4) DNA repair, can influence CSC properties like (a) induction of EMT (epithelial-mesenchymal transition), (b) self renewal, (c) drug resistance, (d) cellular plasticity, and (e) pluripotency, thereby finally aiding in chemoresistance and cancer recurrence. Information obtained from the discussion will help in revisiting chemoresistance and provide approaches for treating CSCs by targeting nuclear events, thereby improving the prognosis and survival rate of cancer patients in near future.

A novel spontaneous model of epithelial-mesenchymal transition (EMT) using a primary prostate cancer derived cell line demonstrating distinct stem-like characteristics

Cells acquire the invasive and migratory properties necessary for the invasion-metastasis cascade and the establishment of aggressive, metastatic disease by reactivating a latent embryonic programme: epithelial-to-mesenchymal transition (EMT). Herein, we report the development of a new, spontaneous model of EMT which involves four phenotypically distinct clones derived from a primary tumour-derived human prostate cancer cell line (OPCT-1), and its use to explore relationships between EMT and the generation of cancer stem cells (CSCs) in prostate cancer. Expression of epithelial (E-cadherin) and mesenchymal markers (vimentin, fibronectin) revealed that two of the four clones were incapable of spontaneously activating EMT, whereas the others contained large populations of EMT-derived, vimentin-positive cells having spindle-like morphology. One of the two EMT-positive clones exhibited aggressive and stem cell-like characteristics, whereas the other was non-aggressive and showed no stem cell phenotype. One of the two EMT-negative clones exhibited aggressive stem cell-like properties, whereas the other was the least aggressive of all clones. These findings demonstrate the existence of distinct, aggressive CSC-like populations in prostate cancer, but, importantly, that not all cells having a potential for EMT exhibit stem cell-like properties. This unique model can be used to further interrogate the biology of EMT in prostate cancer.

Epithelial plasticity and cancer stem cells: Major mechanisms of cancer pathogenesis and therapy resistance.

Epithelial-mesenchymal transition (EMT) has been linked with aggressive tumor biology and therapy resistance. It plays central role not only in the generation of cancer stem cells (CSCs) but also direct them across the multiple organ systems to promote tumor recurrence and metastasis. CSCs are reported to express stem cell genes as well as specific cell surface markers and allow aberrant differentiation of progenies. It facilitates cancer cells to leave primary tumor, acquire migratory characteristics, grow into new environment and develop radio-chemo-resistance. Based on the current information, present review discusses and summarizes the recent advancements on the molecular mechanisms that derive epithelial plasticity and its major role in generating a subset of tumor cells with stemness properties and pathophysiological spread of tumor. This paper further highlights the critical need to examine the regulation of EMT and CSC pathways in identifying the novel probable therapeutic targets. These improved therapeutic strategies based on the co-administration of inhibitors of EMT, CSCs as well as differentiated tumor cells may provide improved anti-neoplastic response with no tumor relapse.

Cancer Stem Cell Gene Variants Predict Disease Recurrence in Patients Treated with Radical Prostatectomy for Prostate Cancer.

Background: Cancer stem cells (CSCs) are involved in tumor progression and drug resistance. We hypothesized that variants in CSC marker genes influence treatment outcomes in prostate cancer.Methods: Ten potentially functional single nucleotide polymorphisms (SNPs) in seven prostate CSC marker genes, TACSTD2, PROM1, ITGA2, POU5F1, EZH2, PSCA, and CD44, were selected for analysis of their association with disease recurrence by Kaplan-Meier analysis and Cox regression in a cohort of 320 patients with localized prostate cancer receiving radical prostatectomy.Results: We identified one independent SNP, rs2394882, in POU5F1 that was associated with prostate cancer recurrence (hazard ratio 0.32, 95% confidence interval 0.14-0.71, P = 0.005) after adjustment for known clinical predictors. Further in silico functional analyses revealed that rs2394882 affects POU5F1 expression, which in turn is significantly correlated with prostate cancer aggressiveness and patient prognosis.Conclusion: Our results suggest that rs2394882 is prognostically relevant in prostate cancer, possibly by modulating the expression of the CSC gene POU5F1.

Targeting cancer stem cell propagation with palbociclib, a CDK4/6 inhibitor: Telomerase drives tumor cell heterogeneity.

In this report, we systematically examined the role of telomerase activity in lung and ovarian cancer stem cell (CSC) propagation. For this purpose, we indirectly gauged telomerase activity, by linking the hTERT-promoter to eGFP. Using lung (A549) and ovarian (SKOV3) cancer cells, transduced with the hTERT-GFP reporter, we then employed GFP-expression levels to fractionate these cell lines into GFP-high and GFP-low populations. We functionally compared the phenotype of these GFP-high and GFP-low populations. More specifically, we now show that the cancer cells with higher telomerase activity (GFP-high) are more energetically activated, with increased mitochondrial mass and function, as well as increased glycolytic activity. This was further validated and confirmed by unbiased proteomics analysis. Cells with high telomerase activity also showed an increased capacity for stem cell activity (as measured using the 3D-spheroid assay) and cell migration (as measured using a Boyden chamber approach). These enhanced biological phenotypes were effectively inhibited by classical modulators of energy metabolism, which target either i) mitochondrial metabolism (i.e., oligomycin) or ii) glycolysis (i.e., 2-deoxy-glucose), or iii) by using the FDA-approved antibiotic doxycycline, which inhibits mitochondrial biogenesis. Finally, the level of telomerase activity also determined the ability of hTERT-high cells to proliferate, as assessed by measuring DNA synthesis via EdU incorporation. Consistent with these observations, treatment with an FDA-approved CDK4/6 inhibitor (PD-0332991/palbociclib) specifically blocked the propagation of both lung and ovarian CSCs. Virtually identical results were obtained with breast CSCs, which were also highly sensitive to palbociclib at concentrations in the nanomolar range. In summary, CSCs with high telomerase activity are among the most energetically activated, migratory and proliferative cell sub-populations. These observations may provide a mechanistic explanation for tumor metabolic heterogeneity, based on telomerase activity. FDA-approved drugs, such as doxycycline and palbociclib, were both effective at curtailing CSC propagation. Thus, these FDA-approved drugs could be used to target telomerase-high proliferative CSCs, in multiple cancer types. Finally, our experiments also allowed us to distinguish two different cellular populations of hTERT-high cells, one that was proliferative (i.e., replicative immortality) and the other that was non-proliferative (i.e., quiescent). We speculate that the non-proliferative population of hTERT-high cells that we identified could be mechanistically involved in tumor dormancy.

Roles of GSK-3 and microRNAs on epithelial mesenchymal transition and cancer stem cells.

Various signaling pathways exert critical roles in the epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs). The Wnt/beta-catenin, PI3K/PTEN/Akt/mTORC, Ras/Raf/MEK/ERK, hedgehog (Hh), Notch and TP53 pathways elicit essential regulatory influences on cancer initiation, EMT and progression. A common kinase involved in all these pathways is moon-lighting kinase glycogen synthase kinase-3 (GSK-3). These pathways are also regulated by micro-RNAs (miRs). TP53 and components of these pathways can regulate the expression of miRs. Targeting members of these pathways may improve cancer therapy in those malignancies that display their abnormal regulation. This review will discuss the interactions of the multi-functional GSK-3 enzyme in the Wnt/beta-catenin, PI3K/PTEN/Akt/mTORC, Ras/Raf/MEK/ERK, Hh, Notch and TP53 pathways. The regulation of these pathways by miRs and their effects on CSC generation, EMT, invasion and metastasis will be discussed.

Bile acid: a potential inducer of colon cancer stem cells.

BackgroundAlthough the unconjugated secondary bile acids, specifically deoxycholic acid (DCA) and lithocholic acid (LCA), are considered to be risk factors for colorectal cancer, the precise mechanism(s) by which they regulate carcinogenesis is poorly understood. We hypothesize that the cytotoxic bile acids may promote stemness in colonic epithelial cells leading to generation of cancer stem cells (CSCs) that play a role in the development and progression of colon cancer.MethodsNormal human colonic epithelial cells (HCoEpiC) were used to study bile acid DCA/LCA-mediated induction of CSCs. The expression of CSC markers was measured by real-time qPCR. Flow cytometry was used to isolate CSCs. T-cell factor/lymphoid-enhancing factor (TCF/LEF) luciferase assay was employed to examine the transcriptional activity of β-catenin. Downregulation of muscarinic 3 receptor (M3R) was achieved through transfection of corresponding siRNA.ResultsWe found DCA/LCA to induce CSCs in normal human colonic epithelial cells, as evidenced by the increased proportion of CSCs, elevated levels of several CSC markers, as well as a number of epithelial–mesenchymal transition markers together with increased colonosphere formation, drug exclusion, ABCB1 and ABCG2 expression, and induction of M3R, p-EGFR, matrix metallopeptidases, and c-Myc. Inhibition of M3R signaling greatly suppressed DCA/LCA induction of the CSC marker ALDHA1 and also c-Myc mRNA expression as well as transcriptional activation of TCF/LEF.ConclusionsOur results suggest that bile acids, specifically DCA and LCA, induce cancer stemness in colonic epithelial cells by modulating M3R and Wnt/β-catenin signaling and thus could be considered promoters of colon cancer.

Long non-coding RNAs and genes contributing to the generation of cancer stem cells in hepatocellular carcinoma identified by RNA sequencing analysis.

Cancer stem cells(CSCs) play important roles in cancer initiation, progression and metastasis. The aim of the present study was to identify the potential targets that may contribute to the generation of hepatocellular carcinoma stem cells(HCSCs) from hepatocellular carcinoma(HCC) cells. The RNA sequencing(RNA‑Seq) dataset GSE70537 was downloaded from the Gene Expression Omnibus(GEO) database. Raw RNA sequences were mapped to the GRCh37/hg19 genome based on TopHat and assembled through Cufflinks. Cuffdiff of Cufflinks was used for the screening of differentially expressed genes(DEGs) in the two types of HCSCs (Hep3B‑C and Huh7‑C) compared with the two types of HCC cells (Hep3B and Huh7) which were satisfied by the criteria of |log2(RPKMHCSC/RPKMHCC)| >1 and p<0.05. In addition, based on the Database for Annotation, Visualization, and Integrated Discovery(DAVID), we screened the Gene Ontology(GO) terms and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways which were enriched in the DEGs. For the DEGs with consistent differential expression in the two lists of DEGs, the LncRNA2Target database was used for the identification of long non‑coding RNA(lncRNA)‑gene pairs. A total of 218and 591DEGs were identified for the Hep3B‑C and Huh7‑C samples, respectively, and 22overlaps were obtained. Biological processes and pathways related to steroid biosynthesis/metabolism or other substance transport were found to be enriched in the two lists of DEGs. Among the 22overlaps, 16were found to be consistently differentially expressed in the two HCSC samples, and the lncRNA‑gene regulatory network of these genes was constructed. Moreover, several potential biomarkers that may play important roles in the transformation of HCSCs were identified in the regulation network. Through the bioinformatics analysis of the RNA‑Seq dataset, several novel targets that were associated with the progression of HCC were obtained, and these targets may be valuable for the treatment and prognosis of HCC.

Serum depletion induced cancer stem cell-like phenotype due to nitric oxide synthesis in oncogenic HRas transformed cells.

Cancer cells rewire their metabolism and mitochondrial oxidative phosphorylation (OXPHOS) to promote proliferation and maintenance. Cancer cells use multiple adaptive mechanisms in response to a hypo-nutrient environment. However, little is known about how cancer mitochondria are involved in the ability of these cells to adapt to a hypo-nutrient environment. Oncogenic HRas leads to suppression of the mitochondrial oxygen consumption rate (OCR), but oxygen consumption is essential for tumorigenesis. We found that in oncogenic HRas transformed cells, serum depletion reversibly increased the OCR and membrane potential. Serum depletion promoted a cancer stem cell (CSC)-like phenotype, indicated by an increase in CSC markers expression and resistance to anticancer agents. We also found that nitric oxide (NO) synthesis was significantly induced after serum depletion and that NO donors modified the OCR. An NOS inhibitor, SEITU, inhibited the OCR and CSC gene expression. It also reduced anchorage-independent growth by promoting apoptosis. In summary, our data provide new molecular findings that serum depletion induces NO synthesis and promotes mitochondrial OXPHOS, leading to tumor progression and a CSC phenotype. These results suggest that mitochondrial OCR inhibitors can be used as therapy against CSC.