Liposarcoma Cells with Aldefluor and CD133 Activity have a Cancer Stem Cell Potential

ABSTRACT: Aldehyde dehydrogenase (ALDH) has recently been shown to be a marker of cancer stem-like cells (CSCs) across tumour types. The primary goals of this study were to investigate whether ALDH is expressed in liposarcomas, and whether CSCs can be identified in the ALDHhigh subpopulation. We have demonstrated that ALDH is indeed expressed in 10 out of 10 liposarcoma patient samples. Using a liposarcoma xenograft model, we have identified a small population of cells with an inducible stem cell potential, expressing both ALDH and CD133 following culturing in stem cell medium. This potential CSC population, which makes up for 0,1-1,7 % of the cells, displayed increased self-renewing abilities and increased tumourigenicity, giving tumours in vivo from as few as 100 injected cells.     

Mitochondrial biogenesis is required for the anchorage-independent survival and propagation of stem-like cancer cells

Here, we show that new mitochondrial biogenesis is required for the anchorage independent survival and propagation of cancer stem-like cells (CSCs). More specifically, we used the drug XCT790 as an investigational tool, as it functions as a specific inhibitor of the ERRα-PGC1 signaling pathway, which governs mitochondrial biogenesis. Interestingly, our results directly demonstrate that XCT790 efficiently blocks both the survival and propagation of tumor initiating stem-like cells (TICs), using the MCF7 cell line as a model system. Mechanistically, we show that XCT790 suppresses the activity of several independent signaling pathways that are normally required for the survival of CSCs, such as Sonic hedgehog, TGFβ-SMAD, STAT3, and Wnt signaling. We also show that XCT790 markedly reduces oxidative mitochondrial metabolism (OXPHOS) and that XCT790-mediated inhibition of CSC propagation can be prevented or reversed by Acetyl-L-Carnitine (ALCAR), a mitochondrial fuel. Consistent with our findings, over-expression of ERRα significantly enhances the efficiency of mammosphere formation, which can be blocked by treatment with mitochondrial inhibitors. Similarly, mammosphere formation augmented by FOXM1, a downstream target of Wnt/β-catenin signaling, can also be blocked by treatment with three different classes of mitochondrial inhibitors (XCT790, oligomycin A, or doxycycline). In this context, our unbiased proteomics analysis reveals that FOXM1 drives the expression of >90 protein targets associated with mitochondrial biogenesis, glycolysis, the EMT and protein synthesis in MCF7 cells, processes which are characteristic of an anabolic CSC phenotype. Finally, doxycycline is an FDA-approved antibiotic, which is very well-tolerated in patients. As such, doxycycline could be re-purposed clinically as a ‘safe’ mitochondrial inhibitor, to target FOXM1 and mitochondrial biogenesis in CSCs, to prevent tumor recurrence and distant metastasis, thereby avoiding patient relapse.     

Cancer stem cells: distinct entities or dynamically regulated phenotypes?

The origins of tumor-propagating neoplastic stem-like cells [cancer stem cells (CSC)] and their relationship to the bulk population of tumor cells that lack stem-like tumor-propagating features (i.e., transit-amplifying cancer progenitor cells) remain unclear. Recent findings from multiple laboratories show that cancer progenitor cells have the capacity to dedifferentiate and acquire a stem-like phenotype in response to either genetic manipulation or environmental cues. These findings suggest that CSCs and relatively differentiated progenitors coexist in dynamic equilibrium and are subject to bidirectional conversion. In this review, we discuss emerging concepts regarding the stem-like phenotype, its acquisition by cancer progenitor cells, and the molecular mechanisms involved. Understanding the dynamic equilibrium between CSCs and cancer progenitor cells is critical for the development of therapeutic strategies to deplete tumors of their tumor-propagating and treatment-resistant cell subpopulations.     

Sequencing of breast cancer stem cell populations indicates a dynamic conversion between differentiation states in vivo

Introduction

The cancer stem cell model implies a hierarchical organization within breast tumors maintained by cancer stem-like cells (CSCs). Accordingly, CSCs are a subpopulation of cancer cells with capacity for self-renewal, differentiation and tumor initiation. These cells can be isolated through the phenotypic markers CD44+/CD24-, expression of ALDH1 and an ability to form nonadherent, multicellular spheres in vitro. However, controversies to describe the stem cell model exist; it is unclear whether the tumorigenicity of CSCs in vivo is solely a proxy for a certain genotype. Moreover, in vivo evidence is lacking to fully define the reversibility of CSC differentiation.

Methods

In order to answer these questions, we undertook exome sequencing of CSCs from 12 breast cancer patients, along with paired primary tumor samples. As suggested by stem classical cell biology, we assumed that the number of mutations in the CSC subpopulation should be lower and distinct compared to the differentiated tumor cells with higher proliferation.

Results

Our analysis revealed that the majority of somatic mutations are shared between CSCs and bulk primary tumor, with similar frequencies in the two.

Conclusions

The data presented here exclude the possibility that CSCs are only a phenotypic consequence of certain somatic mutations, that is a distinct and non-reversible population of cells. In addition, our results imply that CSCs must be a population of cells that can dynamically switch from differentiated tumor cells, and vice versa. This finding increases our understanding of CSC function in tumor heterogeneity and the importance of identifying drugs to counter de-differentiation rather than targeting CSCs.     

Hyaluronan synthase HAS2 promotes tumor progression in bone by stimulating the interaction of breast cancer stem-like cells with macrophages and stromal cells

The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here, we report that upregulation of hyaluronan synthase 2 (HAS2) occurs in highly metastatic breast cancer stem-like cells (CSC) defined by CD44(+)/CD24(-)/ESA(+) phenotype, where it plays a critical role in the generation of a prometastatic microenvironment in breast cancer. HAS2 was critical for the interaction of CSCs with tumor-associated macrophages (TAM), leading to enhanced secretion of platelet-derived growth factor-BB from TAMs, which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone, an inhibitor of HAS, which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings show a critical role of HAS2 in the development of a prometastatic microenvironment and suggest that HAS2 inhibitors can act as antimetastatic agents that disrupt a paracrine growth factor loop within this microenvironment.     

High aldehyde dehydrogenase activity enhances stem cell features in breast cancer cells by activating hypoxia-inducible factor-2α

High aldehyde dehydrogenase (ALDH) activity has been recognized as a marker of cancer stem cells (CSCs) in breast cancer. In this study, we examined whether inhibition of ALDH activity suppresses stem-like cell properties in a 4T1 syngeneic mouse model of breast cancer. We found that ALDH-positive 4T1 cells showed stem cell-like properties in vitro and in vivo. Blockade of ALDH activity reduced the growth of CSCs in breast cancer cell lines. Treatment of mice with the ALDH inhibitor diethylaminobenzaldehyde (DEAB) significantly suppressed 4T1 cell metastasis to the lung. Recent evidence suggests that ALDH affects the response of stem cells to hypoxia; therefore, we examined a possible link between ALDH and hypoxia signaling in breast cancer. Hypoxia-inducible factor-2α (HIF-2α) was highly dysregulated in ALDH-positive 4T1 cells. We observed that ALDH was highly correlated with the HIF-2α expression in breast cancer cell lines and tissues. DEAB treatment of breast cancer cells reduced the expression of HIF-2α in vitro. In addition, reduction of HIF-2α expression suppressed in vitro self-renewal ability and in vivo tumor initiation in ALDH-positive 4T1 cells. Therefore, our findings may provide the evidence necessary for exploring a new strategy in the treatment of breast cancer.     

Cancer stem cells in squamous cell carcinoma switch between two distinct phenotypes that are preferentially migratory or proliferative

Epithelial-to-mesenchymal transition (EMT) is an important driver of tumor invasion and metastasis, which causes many cancer deaths. Cancer stem cells (CSC) that maintain and initiate tumors have also been implicated in invasion and metastasis, but whether EMT is an important contributor to CSC function is unclear. In this study, we investigated whether a population of CSCs that have undergone EMT (EMT CSCs) exists in squamous cell carcinoma (SCC). We also determined whether a separate population of CSCs that retain epithelial characteristics (non-EMT CSCs) is also present. Our studies revealed that self-renewing CSCs in SCC include two biologically-distinct phenotypes. One phenotype, termed CD44(high)ESA(high), was proliferative and retained epithelial characteristics (non-EMT CSCs), whereas the other phenotype, termed CD44(high)ESA(low), was migratory and had mesenchymal traits characteristic of EMT CSCs. We found that non-EMT and EMT CSCs could switch their epithelial or mesenchymal traits to reconstitute the cellular heterogeneity which was characteristic of CSCs. However, the ability of EMT CSCs to switch to non-EMT character was restricted to cells that were also ALDH1(+), implying that only ALDH1(+) EMT cells had the ability to seed a new epithelial tumor. Taken together, our findings highlight the identification of two distinct CSC phenotypes and suggest a need to define therapeutic targets that can eradicate both of these variants to achieve effective SCC treatment.     

An increase in cancer stem cell population after primary systemic therapy is a poor prognostic factor in breast cancer

Background:

The cancer stem cell (CSC) hypothesis has important clinical implications for cancer therapeutics because of the proposed role of CSCs in chemoresistance. The aim of this study was to investigate changes in the CSC populations before and after primary systemic therapy (PST) and their prognostic role in human breast cancer.

Methods:

Paired samples (before and after PST) of breast cancer tissue were obtained from clinical stage II or III patients (n=92) undergoing PST with the regimen of doxorubicin plus docetaxel (AD) (n=50) or doxorubicin plus cyclophosphamide (AC) (n=42) and subsequent breast resection. The proportions of putative CSCs with CD44+/CD24− or aldehyde dehydrogenase 1+ (ALDH1+) phenotypes were determined by immunohistochemistry.

Results:

A higher proportion of CD44+/CD24− tumour cells and ALDH1 positivity in pre-chemotherapy tissue was correlated with higher histologic grade, oestrogen receptor (ER) negativity, high Ki-67 proliferation index and basal-like subtype of breast cancer. Aldehyde dehydrogenase 1 positivity in pre-chemotherapy biopsy was also associated with a higher rate of pathologic complete response following PST. In comparisons of putative CSC populations before and after PST, the proportions of CD44+/CD24− and ALDH1+ tumour cells were significantly increased after PST. The cases with increased CD44+/CD24− tumour cell populations after PST showed high Ki-67 proliferation index in post-chemotherapy specimens and those with increased ALDH1+ tumour cell population after PST were associated with ER negativity and p53 overexpression. Furthermore, cases showing such an increase had significantly shorter disease-free survival time than those with no change or a reduced number of CSCs, and the survival difference was most notable with regard to the changes of ALDH1+ tumour cell population in the patients who received AC regimen.

Conclusion:

The present study provides the clinical evidence that the putative CSCs in breast cancer are chemoresistant and are associated with tumour progression, emphasising the need for targeting of CSCs in the breast cancer therapeutics.     

HSP DNAJB8 controls tumor-initiating ability in renal cancer stem-like cells

Cancer stem-like cells (CSC) are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. In this study, we show that the cancer-testis antigen and HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma (RCC). DNAJB8 overexpression increased the percentage of side population (SP) cells representing CSCs in RCC cells, enhancing their tumor-initiating ability. Conversely, attenuation of DNAJB8 decreased SP cells and reduced tumor-initiating ability. The utility of DNAJB8 as an immunologic target was established in DNA vaccination experiments. Compared with immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC, immunization with Dnajb8 expression plasmids yielded stronger antitumor effects. Together, our findings suggest that DNAJB8 plays a role in CSC maintenance and that it offers a candidate for CSC-targeting immunotherapy in RCC.     

Combination therapy targeting both cancer stem-like cells and bulk tumor cells for improved efficacy of breast cancer treatment

Many types of tumors are organized in a hierarchy of heterogeneous cell populations. The cancer stem-like cells (CSCs) hypothesis suggests that tumor development and metastasis are driven by a minority population of cells, which are responsible for tumor initiation, growth and recurrences. The inability to efficiently eliminate CSCs during chemotherapy, together with CSCs being highly tumorigenic and invasive, may result in treatment failure due to cancer relapse and metastases. CSCs are emerging as a promising target for the development of translational cancer therapies. Ideal panacea for cancer would kill all malignant cells, including CSCs and bulk tumor cells. Since both chemotherapy and CSCs-specific therapy are insufficient to cure cancer, we propose combination therapy with CSCs-targeted agents and chemotherapeutics for improved breast cancer treatment. We generated in vitro mammosphere of 2 breast cancer cell lines, and demonstrated ability of mammospheres to grow and enrich cancer cells with stem-like properties, including self-renewal, multilineage differentiation and enrichment of cells expressing breast cancer stem-like cell biomarkers CD44⁺/CD24^?/low. The formation of mammospheres was significantly inhibited by salinomycin, validating its pharmacological role against the cancer stem-like cells. In contrast, paclitaxel showed a minimal effect on the proliferation and growth of breast cancer stem-like cells. While combination therapies of salinomycin with conventional chemotherapy (paclitaxel or lipodox) showed a potential to improve tumor cell killing, different subtypes of breast cancer cells showed different patterns in response to the combination therapies. While optimization of combination therapy is warranted, the design of combination therapy should consider phenotypic attributes of breast cancer types.     

染色质重塑蛋白MORC2通过调节ALDH1A3表达促进乳腺癌细胞的干性

背景与目的:染色质重塑蛋白MORC家族CW型锌指结构蛋白2(microrchidia family CW-type zinc finger 2,MORC2)在DNA介导的基因转录及DNA损伤修复等基本生物学过程中发挥重要作用,但其对乳腺癌细胞生物学行为的影响目前尚无相关报道.乙醛脱氢酶(aldehyde dehydrogenase,ALDH)家族成员ALDH1A3(alde-hyde dehydrogenase family 1 member A3,ALDH1A3)是一种公认的乳腺癌干细胞的标志物,但其在乳腺癌细胞中的调控机制目前尚不清楚.该研究拟探讨敲低MORC2基因对ALDH1A3表达以及对乳腺癌细胞干性的影响.方法:利用短发夹RNA(short hairpin RNA,shRNA)介导的基因沉默方法构建稳定敲低MORC2基因的乳腺癌细胞系;利用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)分析敲低MORC2基因对ALDH1A3表达的影响;利用微球体形成实验和流式细胞荧光分选技术(fluorescence activated cell sorting,FACS)分析敲低MORC2对乳腺癌干细胞亚群的影响.结果:Western blot和RTFQ-PCR分析结果显示,敲低MORC2基因显著下调ALDH1A3蛋白及mRNA水平;微球体形成实验显示敲低MORC2基因显著抑制MCF-7细胞的成球能力;FACS分析显示敲低MORC2基因显著下调ALDH1A3阳性乳腺癌干细胞亚群,而对CD44+CD24-乳腺癌干细胞亚群没有明显影响.结论:MORC2通过调节ALDH1A3的表达促进乳腺癌干细胞表型.     

Combinatorial TGF-β attenuation with paclitaxel inhibits the epithelial-to-mesenchymal transition and breast cancer stem-like cells

Distant relapse after chemotherapy is an important clinical issue for treating breast cancer patients and results from the development of cancer stem-like cells (CSCs) during chemotherapy. Here we report that blocking epithelial-to-mesenchymal transition (EMT) suppresses paclitaxel-induced CSCs properties by using a MDA-MB-231-xenografted mice model (in vivo), and breast cancer cell lines (in vitro). Paclitaxel, one of the cytotoxic taxane-drugs such as docetaxel, increases mesenchymal markers (Vimentin and Fibronectin) and decreases an epithelial marker (Zo-1). Blocking TGF-β signaling with the TGF-β type I receptor kinase (ALK5) inhibitor, EW-7197, suppresses paclitaxel-induced EMT and CSC properties such as mammosphere-forming efficiency (MSFE), aldehyde dehydrogenase (ALDH) activity, CD44⁺/CD24⁻ ratio, and pluripotency regulators (Oct4, Nanog, Klf4, Myc, and Sox2). The combinatorial treatment of EW-7197 improves the therapeutic effect of paclitaxel by decreasing the lung metastasis and increasing the survival time in vivo. We confirmed that Snail is increased by paclitaxel-induced intracellular reactive oxygen species (ROS) and EW-7197 suppresses the paclitaxel-induced Snail and EMT by attenuating paclitaxel-induced intracellular ROS. Knock-down of SNAI1 suppresses paclitaxel-induced EMT and CSC properties. These data together suggest that blocking the Snail-induced EMT with the ALK5 inhibitor attenuates metastasis after paclitaxel-therapy and that this combinatorial approach could prove useful in treating breast cancer.     

Stem cells in breast tumours: Are they ready for the clinic?

The concept of stem-like cells in cancer has been gaining currency over the last decade or so since evidence for stem cell activity in human leukaemia and solid tumours, including breast cancer, was first published. The evidence established that sub-populations of cells identified by antibodies to cell surface markers behaved like developmental stem cells in their capacity to re-grow the human tumour for several generations in experimental immune-deficient hosts. The experiments established that cells with tumourigenic capacity expressed ‘cancer stem cell’ (CSC) markers and that activity could also be measured by self-renewal of tumour sphere colonies in culture. In breast and other cancers, there is good evidence that CSCs are relatively resistant to radio- and chemotherapy indicating that novel CSC-targeted therapies are needed. Several pathways are promising targets in breast CSCs. There are several ways of combating CSC activity including inducing their apoptosis, inhibiting stem cell self-renewal to either stop their division or to promote their differentiation, or targeting the CSC niche that supports them. The first challenge for developing novel CSC therapies is to ascertain which of these CSC properties is being targeted. The second challenge is to determine suitable CSC biomarkers to measure the efficacy of the novel CSC therapies. We propose using biomarkers as a means to identify and assess CSC activity in clinical trials. This is likely to be demanding but feasible in the near future. Thus, we asked if CSCs are ready for the clinic, however, the emerging question becomes: is the clinic ready for cancer stem cells?     

Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells

Breast cancer stem-like cells (CSCs) are an important therapeutic target as they are purported to be responsible for tumor initiation, maintenance, metastases, and disease recurrence. Interleukin-8 (IL-8) is upregulated in breast cancer compared with normal breast tissue and is associated with poor prognosis. IL-8 is reported to promote breast cancer progression by increasing cell invasion, angiogenesis, and metastases and is upregulated in HER2-positive cancers. Recently, we and others have established that IL-8 via its cognate receptors, CXCR1 and CXCR2, is also involved in regulating breast CSC activity. Our work demonstrates that in metastatic breast CSCs, CXCR1/2 signals via transactivation of HER2. Given the importance of HER2 in breast cancer and in regulating CSC activity, a pathway driving the activation of these receptors would have important biological and clinical consequences, especially in tumors that express high levels of IL-8 and other CXCR1/2-activating ligands. Here, we review the IL-8 signaling pathway and the role of HER2 in maintaining an IL-8 inflammatory loop and discuss the potential of combining CXCR1/2 inhibitors with other treatments such as HER2-targeted therapy as a novel approach to eliminate CSCs and improve patient survival.     

Breast-cancer stem cells-beyond semantics

Intratumoral heterogeneity in breast cancer is well documented. Although the mechanisms leading to this heterogeneity are not understood, a subpopulation of cancer cells, cancer stem cells (CSCs), that have some phenotypic similarities with adult tissue stem cells, has been suggested to contribute to tumour heterogeneity. It has been postulated that these CSCs are dormant, and by virtue of their low proliferative activity and ability to exclude intracellular toxins, are resistant to chemotherapy and radiation therapy. These cells were initially isolated based on the presence of markers such as CD44, CD24, and ALDH1, with further characterisation using mammosphere assay and transplantation into immunodeficient mice. The CSC hypothesis raises several theoretical and practical questions. Does cancer arise in normal mammary stem cells or do some malignant cells acquire a CSC phenotype through clonal evolution? Are CSCs in different molecular (intrinsic) subtypes of breast cancer similar, or do they have distinct properties based on the subtype? Does the CSC phenotype reflect plasticity or the dynamic nature of a few cancer cells? How do these cells acquire invasive behaviour, as they go through epithelial-to-mesenchymal transition and then revert to epithelial phenotype at sites of metastasis in response to tumour microenvironmental and metastasis site-specific cues? It is increasingly recognised that the methods and assays used for identifying CSCs have substantial limitations; does this negate the entire concept? In this Personal View, we argue that the CSC phenotype represents an aggressive clone that survives in an adverse environment through constant evolution and integration of various hallmarks of cancer. This evolution could involve acquiring mutations that permit asymmetric and symmetric division, converting the host immune attack to its own advantage, and plasticity to adapt to sites of metastasis through reversible change in adhesion molecules. We also argue that the cell-type origin of cancer could affect the rate at which CSCs develop in a tumour, with an eventual effect on disease outcome.     

Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer

The CD44⁺/CD24^−/low and ALDH1⁺ cell phenotypes are associated with stemness and enhanced tumorigenic potential in breast cancer. We assessed the expression of CD44, CD24 and ALDH1 on tumor cells circulating in the peripheral blood (CTCs) of patients with metastatic breast cancer using triple-marker immunofluorescence microscopy. Among a total of 1439 CTCs identified in 20 (66.7%) out of 30 patients, 35.2% had the stem-like/tumorigenic phenotype CD44⁺/CD24^−/low, whereas 17.7% of the CTCs analyzed in seven patients, were ALDH1^high/CD24^−/low. In conclusion, we report the existence of a subpopulation of CTCs with putative stem cell progenitor phenotypes in patients with metastatic breast cancer.     

BRCA1 and EZH2 cooperate in regulation of prostate cancer stem cell phenotype

Prostate cancer (PCa) is the second most common malignancy and the sixth leading cause of cancer-related death among men worldwide. Prostate carcinogenesis is driven by the accumulation of genetic and epigenetic aberrations, which regulate cancer cell transition between a stem- and nonstem-cell state and accelerate tumor evolution. Elevated expression of enhancer of zeste homolog 2 (EZH2) histone methyltransferase, a core member of the polycomb repressive complex 2 (PRC2), results in cancer progression through histone methylation-driven tumor cells dedifferentiation. Previous studies demonstrated that tumor suppressor breast cancer 1 (BRCA1) is a negative regulator of PRC2-dependent H3K27 methylation. Our recent studies revealed that inhibition of EZH2-mediated histone methylation radiosensitizes prostate cancer stem cells (CSCs) population. However, the link between BRCA1 and EZH2 in regulation of prostate CSCs remains elusive. Present study demonstrated that BRCA1 and EZH2 are coregulated in patients’ tumors and PCa cell lines, and cooperate in regulation of CSC phenotype and properties. Knockdown of BRCA1 expression significantly increases the number and the size of tumor spheres. Inhibition of BRCA1 and EZH2 expression leads to an increase of aldehyde dehydrogenase (ALDH)-positive cell population that is, at least partially, attributed to the upregulation of ALDH1A3 protein. Treatment with a global histone methylation inhibitor 3-Deazaneplanocin A abrogates this regulation, downregulates BRCA1 and EZH2 expression and has an inhibitory effect on the tumorigenic properties of radioresistant PCa cells in vivo. We found that EZH2/BRCA1 signaling mechanisms play an important role in the maintenance of prostate CSC properties and may be a promising target for tumor treatment.     

Active glycolytic metabolism in CD133(+) hepatocellular cancer stem cells: regulation by MIR-122

Although altered metabolic pathway is an important diagnostic maker and therapeutic target in cancer, it is poorly understood in cancer stem cells (CSCs). Here we show that the CD133 (+) hepatocellular CSCs have distinct metabolic properties, characterized by more active glycolysis over oxidative phosphorylation, compared to the CD133 (−) cells. Inhibition of PDK4 and LDHA markedly suppresses CD133 (+) stemness characteristics and overcome resistance to sorafenib (current chemotherapeutic agent for hepatocellular cancer). Addition of glucose or lactate to CD133 (−) cells promotes CSC phenotypes, as evidenced by increased CD133 (+) cell population, elevated stemness gene expression and enhanced spheroid formation. Furthermore, the liver-specific miRNA, miR-122, inhibits CSC phenotypes by regulating glycolysis through targeting PDK4. Our findings suggest that enhanced glycolysis is associated with CD133 (+) stem-like characteristics and that metabolic reprogramming through miR-122 or PDK4 may represent a novel therapeutic approach for the treatment of hepatocellular cancer.