FDI-6, a FOXM1 inhibitor, activates the aryl hydrocarbon receptor and suppresses tumorsphere formation.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is activated by environmental contaminants such as dioxins and polycyclic aromatic hydrocarbons. Following ligand binding, AhR binds to xenobiotic responsive elements and modulates the transcription of AhR target genes. Multiple studies have shown that AhR plays important roles in a range of cancer cells and is attracting attention as a therapeutic target for cancer treatment. We have previously reported that AhR agonists inhibit tumorsphere formation in an AhR-dependent manner in the MCF-7 breast cancer cell line. In the present study, we found that FDI-6, an inhibitor of the transcription factor Forkhead Box M1 (FOXM1) induced the mRNA expression of AhR target genes, nuclear translocation of AhR, and transcriptional activity of AhR. In addition, FDI-6 dose-dependently reduced the mRNA expression of FOXM1-regulated genes in AhR-expressing MCF-7 cells, although not in AhR-deficient MCF-7 cells. Furthermore, FDI-6 was found to suppress tumorsphere formation via the AhR in MCF-7 cells and HepG2 human liver cancer cell line. On the basis of the findings of this study, we show that FDI-6, a FOXM1 inhibitor, functions as an AhR agonist, and suppresses tumorsphere formation via the AhR.

Quantification of tumorsphere migration with a physics-based machine learning method.

Current analysis techniques available for migration assays only provide quantitative measurements for overall migration. However, the potential of regional migration analyses can open further insight into migration patterns and more avenues of experimentation with the same assays. Previously, we developed an analysis pipeline utilizing the finite element (FE) method to show its potential in analyzing glioblastoma (GBM) tumorsphere migration, especially in characterizing regional changes in the migration pattern. This study aims to streamline and further automate the analysis system by integrating the machine-learning-based U-Net segmentation with the FE method. Our U-Net-based segmentation achieved a 98% accuracy in segmenting our tumorspheres. From the segmentations, FE models made up of 3D hexahedral elements were generated, and the migration patterns of the tumorspheres were analyzed under treatments B and C (under non-disclosure agreements). Our results show that our overall migration analysis correlated very strongly (R2 of 0.9611 and 0.9986 for treatments B and C, respectively) with ImageJ's method of migration area analysis, which is the most common method of tumorsphere migration analysis. Additionally, we were able to quantitatively represent the regional migration patterns in our FE models, which the methods purely based on segmentations could not do. Moreover, the new pipeline improved the efficiency and accessibility of the initial pipeline by implementing machine learning-based automated segmentation onto a mainly open-sourced FE analysis platform. In conclusion, our algorithm enables the development of a high-content and high-throughput in vitro screening platform to elucidate anti-migratory molecules that may reduce the invasiveness of these malignant tumors.

The lectin DrfL inhibits cell migration, adhesion and triggers autophagy-dependent cell death in glioma cells.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma, displaying atypical glycosylation pattern that may modulate signaling pathways involved in tumorigenesis. Lectins are glycan binding proteins with antitumor properties. The present study was designed to evaluate the antitumor capacity of the Dioclea reflexa lectin (DrfL) on glioma cell cultures. Our results demonstrated that DrfL induced morphological changes and cytotoxic effects in glioma cell cultures of C6, U-87MG and GBM1 cell lines. The action of DrfL was dependent upon interaction with glycans, and required a carbohydrate recognition domain (CRD), and the cytotoxic effect was apparently selective for tumor cells, not altering viability and morphology of primary astrocytes. DrfL inhibited tumor cell migration, adhesion, proliferation and survival, and these effects were accompanied by activation of p38MAPK and JNK (p46/54), along with inhibition of Akt and ERK1/2. DrfL also upregulated pro-apoptotic (BNIP3 and PUMA) and autophagic proteins (Atg5 and LC3 cleavage) in GBM cells. Noteworthy, inhibition of autophagy and caspase-8 were both able to attenuate cell death in GBM cells treated with DrfL. Our results indicate that DrfL cytotoxicity against GBM involves modulation of cell pathways, including MAPKs and Akt, which are associated with autophagy and caspase-8 dependent cell death.

Effects of a differentiating therapy on cancer-stem-cell-driven tumors.

The growth of many solid tumors has been found to be driven by chemo- and radiotherapy-resistant cancer stem cells (CSCs). A suitable therapeutic avenue in these cases may involve the use of a differentiating agent (DA) to force the differentiation of the CSCs and of conventional therapies to eliminate the remaining differentiated cancer cells (DCCs). To describe the effects of a DA that reprograms CSCs into DCCs, we adapt a differential equation model developed to investigate tumorspheres, which are assumed to consist of jointly evolving CSC and DCC populations. We analyze the mathematical properties of the model, finding the equilibria and their stability. We also present numerical solutions and phase diagrams to describe the system evolution and the therapy effects, denoting the DA strength by a parameter adif. To obtain realistic predictions, we choose the other model parameters to be those determined previously from fits to various experimental datasets. These datasets characterize the progression of the tumor under various culture conditions. Typically, for small values of adif the tumor evolves towards a final state that contains a CSC fraction, but a strong therapy leads to the suppression of this phenotype. Nonetheless, different external conditions lead to very diverse behaviors. For microchamber-grown tumorspheres, there is a threshold in therapy strength below which both subpopulations survive, while high values of adif lead to the complete elimination of the CSC phenotype. For tumorspheres grown on hard and soft agar and in the presence of growth factors, the model predicts a threshold not only in the therapy strength, but also in its starting time, an early beginning being potentially crucial. In summary, our model shows how the effects of a DA depend critically not only on the dosage and timing of the drug application, but also on the tumor nature and its environment.

Etomoxir, a carnitine palmitoyltransferase 1 inhibitor, combined with temozolomide reduces stemness and invasiveness in patient-derived glioblastoma tumorspheres.

INTRODUCTION: The importance of fatty acid oxidation (FAO) in the bioenergetics of glioblastoma (GBM) is being realized. Etomoxir (ETO), a carnitine palmitoyltransferase 1 (CPT1) inhibitor exerts cytotoxic effects in GBM, which involve interrupting the FAO pathway. We hypothesized that FAO inhibition could affect the outcomes of current standard temozolomide (TMZ) chemotherapy against GBM. METHODS: The FAO-related gene expression was compared between GBM and the tumor-free cortex. Using four different GBM tumorspheres (TSs), the effects of ETO and/or TMZ was analyzed on cell viability, tricarboxylate (TCA) cycle intermediates and adenosine triphosphate (ATP) production to assess metabolic changes. Alterations in tumor stemness, invasiveness, and associated transcriptional changes were also measured. Mouse orthotopic xenograft model was used to elucidate the combinatory effect of TMZ and ETO. RESULTS: GBM tissues exhibited overexpression of FAO-related genes, especially CPT1A, compared to the tumor-free cortex. The combined use of ETO and TMZ further inhibited TCA cycle and ATP production than single uses. This combination treatment showed superior suppression effects compared to treatment with individual agents on the viability, stemness, and invasiveness of GBM TSs, as well as better downregulation of FAO-related gene expression. The results of in vivo study showed prolonged survival outcomes in the combination treatment group. CONCLUSION: ETO, an FAO inhibitor, causes a lethal energy reduction in the GBM TSs. When used in combination with TMZ, ETO effectively reduces GBM cell stemness and invasiveness and further improves survival. These results suggest a potential novel treatment option for GBM.

A novel biguanide (IM1761065) inhibits bioenergetics of glioblastoma tumorspheres.

PURPOSE: Glioblastoma (GBM) is a rapidly growing tumor in the central nervous system with altered metabolism. Depleting the bioenergetics of tumors with biguanides have been suggested as an effective therapeutic approach for treating GBMs. The purpose of this study was to determine the effects of IM1761065, a novel biguanide with improved pharmacokinetics, on GBM-tumorspheres (TSs). METHODS: The biological activities of IM1761065 on GBM-TSs, including their effects on viability, ATP levels, cell cycle, stemness, invasive properties, and transcriptomes were examined. The in vivo efficacy of IM1761065 was tested in a mouse orthotopic xenograft model. RESULTS: IM1761065 decreased the viability and ATP levels of GBM-TSs in a dose-dependent manner, and reduced basal and spare respiratory capacity in patient-derived GBM-TS, as measured by the oxygen consumption rate. Sphere formation, expression of stemness-related proteins, and invasive capacity of GBM-TSs were also significantly suppressed by IM1761065. A gene-ontology comparison of IM1761065-treated groups showed that the expression levels of stemness-related, epithelial mesenchymal transition-related, and mitochondrial complex I genes were also significantly downregulated by IM1761065. An orthotopic xenograft mouse model showed decreased bioluminescence in IM1761065-treated cell-injected mice at 5 weeks. IM1761065-treated group showed longer survival than the control group (P = 0.0289, log-rank test). CONCLUSION: IM1761065 is a potent inhibitor of oxidative phosphorylation. The inhibitory effect of IM1761065 on the bioenergetics of GBM-TS suggests that this novel compound could be used as a new drug for the treatment of GBM.

Understanding the influence of substrate when growing tumorspheres.

BACKGROUND: Cancer stem cells are important for the development of many solid tumors. These cells receive promoting and inhibitory signals that depend on the nature of their environment (their niche) and determine cell dynamics. Mechanical stresses are crucial to the initiation and interpretation of these signals. METHODS: A two-population mathematical model of tumorsphere growth is used to interpret the results of a series of experiments recently carried out in Tianjin, China, and extract information about the intraspecific and interspecific interactions between cancer stem cell and differentiated cancer cell populations. RESULTS: The model allows us to reconstruct the time evolution of the cancer stem cell fraction, which was not directly measured. We find that, in the presence of stem cell growth factors, the interspecific cooperation between cancer stem cells and differentiated cancer cells induces a positive feedback loop that determines growth, independently of substrate hardness. In a frustrated attempt to reconstitute the stem cell niche, the number of cancer stem cells increases continuously with a reproduction rate that is enhanced by a hard substrate. For growth on soft agar, intraspecific interactions are always inhibitory, but on hard agar the interactions between stem cells are collaborative while those between differentiated cells are strongly inhibitory. Evidence also suggests that a hard substrate brings about a large fraction of asymmetric stem cell divisions. In the absence of stem cell growth factors, the barrier to differentiation is broken and overall growth is faster, even if the stem cell number is conserved. CONCLUSIONS: Our interpretation of the experimental results validates the centrality of the concept of stem cell niche when tumor growth is fueled by cancer stem cells. Niche memory is found to be responsible for the characteristic population dynamics observed in tumorspheres. The model also shows why substratum stiffness has a deep influence on the behavior of cancer stem cells, stiffer substrates leading to a larger proportion of asymmetric doublings. A specific condition for the growth of the cancer stem cell number is also obtained.

PC3-Secreted Microprotein Is Expressed in Glioblastoma Stem-Like Cells and Human Glioma Tissues.

Glioblastoma multiforme (GBM) is the most prevalent malignant primary brain tumor with a high recurrence rate. Despite multimodal therapy including surgical resection, chemotherapy, and radiotherapy, the median survival time after the initial diagnosis of GBM is approximately 14 months. Since cancer stem cells (CSCs) are considered the leading cause of cancer recurrence, glioblastoma stem cell-targeted therapy is a promising strategy for the treatment of GBM. However, because CSC heterogeneity has been implicated in the difficulties of CSC-target therapy, more in-depth knowledge of CSC biology is still required to develop novel therapies. In this study, we established single cell-derived tumorspheres from human glioblastoma U87MG cells. One of these tumorspheres, P4E8 clone, showed CSC-like phenotypes, such as self-renewal capacity, expression of CSC markers, resistance to anti-cancer agents, and in vivo tumorigenicity. Therefore, we used P4E8 cells as a cell-based model of glioblastoma stem cells (GSCs). Gene expression analysis using microarray indicated that the most highly expressed genes in P4E8 cells compared to the parental U87MG were PC3-secreted microprotein (MSMP). Furthermore, MSMP was expressed in patient-derived GSCs and human glioma tissues at the protein level, implying that MSMP might contribute to glioma development and progression.

Potential Antitumor Effects of 6-Gingerol in p53-Dependent Mitochondrial Apoptosis and Inhibition of Tumor Sphere Formation in Breast Cancer Cells.

Hormone-specific anticancer drugs for breast cancer treatment can cause serious side effects. Thus, treatment with natural compounds has been considered a better approach as this minimizes side effects and has multiple targets. 6-Gingerol is an active polyphenol in ginger with various modalities, including anticancer activity, although its mechanism of action remains unknown. Increases in the level of reactive oxygen species (ROS) can lead to DNA damage and the induction of DNA damage response (DDR) mechanism, leading to cell cycle arrest apoptosis and tumorsphere suppression. Epidermal growth factor receptor (EGFR) promotes tumor growth by stimulating signaling of downstream targets that in turn activates tumor protein 53 (p53) to promote apoptosis. Here we assessed the effect of 6-gingerol treatment on MDA-MB-231 and MCF-7 breast cancer cell lines. 6-Gingerol induced cellular and mitochondrial ROS that elevated DDR through ataxia-telangiectasia mutated and p53 activation. 6-Gingerol also induced G0/G1 cell cycle arrest and mitochondrial apoptosis by mediating the BAX/BCL-2 ratio and release of cytochrome c. It also exhibited a suppression ability of tumorsphere formation in breast cancer cells. EGFR/Src/STAT3 signaling was also determined to be responsible for p53 activation and that 6-gingerol induced p53-dependent intrinsic apoptosis in breast cancer cells. Therefore, 6-gingerol may be used as a candidate drug against hormone-dependent breast cancer cells.

Combinatorial Therapeutic Effect of Inhibitors of Aldehyde Dehydrogenase and Mitochondrial Complex I, and the Chemotherapeutic Drug, Temozolomide against Glioblastoma Tumorspheres.

Resident cancer cells with stem cell-like features induce drug tolerance, facilitating survival of glioblastoma (GBM). We previously showed that strategies targeting tumor bioenergetics present a novel emerging avenue for treatment of GBM. The objective of this study was to enhance the therapeutic effects of dual inhibition of tumor bioenergetics by combination of gossypol, an aldehyde dehydrogenase inhibitor, and phenformin, a biguanide compound that depletes oxidative phosphorylation, with the chemotherapeutic drug, temozolomide (TMZ), to block proliferation, stemness, and invasiveness of GBM tumorspheres (TSs). Combination therapy with gossypol, phenformin, and TMZ induced a significant reduction in ATP levels, cell viability, stemness, and invasiveness compared to TMZ monotherapy and dual therapy with gossypol and phenformin. Analysis of differentially expressed genes revealed up-regulation of genes involved in programmed cell death, autophagy, and protein metabolism and down-regulation of those associated with cell metabolism, cycle, and adhesion. Combination of TMZ with dual inhibitors of tumor bioenergetics may, therefore, present an effective strategy against GBM by enhancing therapeutic effects through multiple mechanisms of action.

LncRNA ADAMTS9-AS2 suppresses the proliferation of gastric cancer cells and the tumorigenicity of cancer stem cells through regulating SPOP.

Nowadays, research on CSCs is still in an initial stage, and there are few studies reporting the successful isolation and identification of CSCs. In the present study, we attempted to isolate CSCs through cultivating the cell line MKN45 in defined serum-free medium and study the expression of stem cell markers or related proteins (Oct3/4, Sox2, Nanog and CD44) in CSCs. Moreover, immunofluorescence staining was performed to validate the stem cell markers of spheroid body-forming cells. Further experiments were used to evaluate the SPOP expression in tumorsphere cells. In addition, ADAMTS9-AS2 is a lncRNA that contributes to the genesis and development of many cancers, including gastric cancer (GC). We found ADAMTS9-AS2 functioned as an anti-oncogene and positively correlated with the expression of SPOP in GC tissues by combining bioinformatics analyses. Furthermore, we reported that ADAMTS9-AS2 regulated the expression of SPOP in GC cells and tumorsphere cells to inhibit GC progression. Together, our results demonstrated that SPOP and ADAMTS9-AS2 can be potential targets for GC treatment.

THG-1 suppresses SALL4 degradation to induce stemness genes and tumorsphere formation through antagonizing NRBP1 in squamous cell carcinoma cells.

Knockdown of THG-1 in TE13 esophageal squamous cell carcinoma (ESCC) cells is known to suppress tumorsphere growth. THG-1 was identified as an NRBP1 binding protein, and NRBP1 was reported to downregulate an stemness-related transcriptional factor SALL4, so we decided to examine the possibility that tumorigenic function of THG-1 is achieved by the competition to the tumor-suppressive function of NRBP1. SALL4 was decreased in THG-1 deficient TE13 cells with reduced tumorsphere formation, while exogenous SALL4 expression in THG-1 deficient TE13 cells recovered expression of stemness genes (NANOG and OCT4) and partially, but significantly, recovered tumorsphere formation ability. Additionally, we found that NRBP1 induced ubiquitination of SALL4, and THG-1 interrupted the ubiquitination of SALL4 by antagonizing NRBP1 binding to SALL4. These results suggest that THG-1 promotes tumorsphere growth of ESCC cells by the stabilization of SALL4 protein and induction of the target stemness genes through competitive binding to NRBP1.

Co-expression of cancer driver genes: IDH-wildtype glioblastoma-derived tumorspheres.

BACKGROUND: Driver genes of GBM may be crucial for the onset of isocitrate dehydrogenase (IDH)-wildtype (WT) glioblastoma (GBM). However, it is still unknown whether the genes are expressed in the identical cluster of cells. Here, we have examined the gene expression patterns of GBM tissues and patient-derived tumorspheres (TSs) and aimed to find a progression-related gene. METHODS: We retrospectively collected primary IDH-WT GBM tissue samples (n = 58) and tumor-free cortical tissue samples (control, n = 20). TSs are isolated from the IDH-WT GBM tissue with B27 neurobasal medium. Associations among the driver genes were explored in the bulk tissue, bulk cell, and a single cell RNAsequencing techniques (scRNAseq) considering the alteration status of TP53, PTEN, EGFR, and TERT promoter as well as MGMT promoter methylation. Transcriptomic perturbation by temozolomide (TMZ) was examined in the two TSs. RESULTS: We comprehensively compared the gene expression of the known driver genes as well as MGMT, PTPRZ1, or IDH1. Bulk RNAseq databases of the primary GBM tissue revealed a significant association between TERT and TP53 (p < 0.001, R = 0.28) and its association increased in the recurrent tumor (p < 0.001, R = 0.86). TSs reflected the tissue-level patterns of association between the two genes (p < 0.01, R = 0.59, n = 20). A scRNAseq data of a TS revealed the TERT and TP53 expressing cells are in a same single cell cluster. The driver-enriched cluster dominantly expressed the glioma-associated long noncoding RNAs. Most of the driver-associated genes were downregulated after TMZ except IGFBP5. CONCLUSIONS: GBM tissue level expression patterns of EGFR, TERT, PTEN, IDH1, PTPRZ1, and MGMT are observed in the GBM TSs. The driver gene-associated cluster of the GBM single cells were enriched with the glioma-associated long noncoding RNAs.

A tumorsphere model of glioblastoma multiforme with intratumoral heterogeneity for quantitative analysis of cellular migration and drug response.

Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumor and is characterized by its sudden onset and invasive growth into the brain parenchyma. The invasive tumor cells evade conventional treatments and are thought to be responsible for the ubiquitous tumor regrowth. Understanding the behavior of these invasive tumor cells and their response to therapeutic agents could help improve patient outcome. In this study, we present a GBM tumorsphere migration model with high biological complexity to study migrating GBM cells in a quantitative and qualitative manner. We demonstrated that the in vitro migration model could be used to investigate both inhibition and stimulation of cell migration with oxaliplatin and GBM-derived extracellular vesicles, respectively. The intercellular heterogeneity within the GBM tumorspheres was examined by immunofluorescent staining of nestin/vimentin and GFAP, which showed nestin and vimentin being highly expressed in the periphery of tumorspheres and GFAP mostly in cells in the tumorsphere core. We further showed that this phenotypic gradient was present in vivo after implanting dissociated GBM tumorspheres, with the cells migrating away from the tumor being nestin-positive and GFAP-negative. These results indicate that GBM tumorsphere migration models, such as the one presented here, could provide a more detailed insight into GBM cell biology and prove highly relevant as a pre-clinical platform for drug screening and assessing drug response in the treatment of GBM.

Berberine and palmatine inhibit the growth of human rhabdomyosarcoma cells.

A natural isoquinoline alkaloid, berberine, has been known to exhibit anti-tumor activity in various cancer cells via inducing cell cycle arrest. However, it has not been investigated whether berberine and its analogs inhibit the growth of rhabdomyosarcoma (RMS), which is the most frequent soft tissue tumor in children. The present study examined the anti-tumor effects of berberine and palmatine on expansions of three human embryonal RMS cell lines; ERMS1, KYM1, and RD. Intracellular incorporation of berberine was relatively higher than that of palmatine in every RMS cell line. Berberine significantly inhibited the cell cycle of all RMS cells at G1 phase. On the other hand, palmatine only suppressed the growth of RD cells. Both of berberine and palmatine strongly inhibited the growth of tumorsphere of RD cells in three-dimensional culture. These results indicate that berberine derivatives have the potential of anti-tumor drugs for RMS therapy.Abbreviations: ARMS: alveolar rhabdomyosarcoma; ERMS: embryonal rhabdomyosarcoma; RMS: rhabdomyosarcoma.

Fighting breast cancer stem cells through the immune-targeting of the xCT cystine-glutamate antiporter.

Tumor relapse and metastatic spreading act as major hindrances to achieve complete cure of breast cancer. Evidence suggests that cancer stem cells (CSC) would function as a reservoir for the local and distant recurrence of the disease, due to their resistance to radio- and chemotherapy and their ability to regenerate the tumor. Therefore, the identification of appropriate molecular targets expressed by CSC may be critical in the development of more effective therapies. Our studies focused on the identification of mammary CSC antigens and on the development of CSC-targeting vaccines. We compared the transcriptional profile of CSC-enriched tumorspheres from an Her2+ breast cancer cell line with that of the more differentiated parental cells. Among the molecules strongly upregulated in tumorspheres we selected the transmembrane amino-acid antiporter xCT. In this review, we summarize the results we obtained with different xCT-targeting vaccines. We show that, despite xCT being a self-antigen, vaccination was able to induce a humoral immune response that delayed primary tumor growth and strongly impaired pulmonary metastasis formation in mice challenged with tumorsphere-derived cells. Moreover, immunotargeting of xCT was able to increase CSC chemosensitivity to doxorubicin, suggesting that it may act as an adjuvant to chemotherapy. In conclusion, our approach based on the comparison of the transcriptome of tumorspheres and parental cells allowed us to identify a novel CSC-related target and to develop preclinical therapeutic approaches able to impact on CSC biology, and therefore, hampering tumor growth and dissemination.

Stimulative role of ST6GALNAC1 in proliferation, migration and invasion of ovarian cancer stem cells via the Akt signaling pathway.

BACKGROUND: Ovarian cancer is known as one of the most common cancers in the world among women. ST6GALNAC1 is highly expressed in cancer stem cells (CSCs), which correlates to high tumor-initiating, self-renewal and differentiation abilities. This present study aims to investigate how ST6GALNAC1 affects ovarian cancer stem cells (OCSCs). METHODS: In order to identify the differentially expressed genes related to ovarian cancer, microarray-based gene expression profiling of ovarian cancer was used, and ST6GALANC1 was one of the identified targets. After that, levels of ST6GALNAC1 in OCSCs and ovarian cancer cells were examined. Subsequently, an Akt signaling pathway inhibitor LY294002 was introduced into the cluster of differentiation 90+ (CD90+) stem cells, and cell proliferation, migration and invasion, levels of CXCL16, EGFR, CD44, Nanog and Oct4, as well as tumorigenicity of OCSCs were examined. RESULTS: By using a comprehensive microarray analysis, it was determined that ST6GALNAC1 was highly expressed in ovarian cancer and it regulated the Akt signaling pathway. High levels of ST6GALNAC1 were observed in OCSCs and ovarian cancer cells. Silencing ST6GALNAC1 was shown to be able to reduce cell proliferation, migration, invasion, self-renewal ability, tumorigenicity of OCSCs. In accordance with these results, the effects of ST6GALNAC1 in OCSCs were dependent on the Akt signaling pathway. CONCLUSIONS: When taken together, our findings defined the potential stimulative roles of ST6GALNAC1 in ovarian cancer and OCSCs, which relied on the Akt signaling pathway.

Combined treatment with 2'-hydroxycinnamaldehyde and temozolomide suppresses glioblastoma tumorspheres by decreasing stemness and invasiveness.

INTRODUCTION: Glioblastoma (GBM) is the most common and aggressive human primary brain malignancy. The key properties of GBM, stemness and invasiveness, are known to be associated with a highly unfavorable prognosis. Notably, the process of epithelial-mesenchymal transition (EMT) is closely related to the progression of GBM. On the basis of reports that 2'-hydroxycinnamaldehyde (HCA) and its derivative, 2'-benzoyloxycinnamaldehyde (BCA), suppresses EMT in several human cancer cells, we sought to evaluate the therapeutic efficacy of HCA and BCA, alone and in combination with temozolomide (TMZ), on GBM tumorspheres (TSs). METHODS: Two human GBM TSs were treated with HCA, BCA, or TMZ. Therapeutic effects were evaluated by measuring ATP levels, neurosphere formation, 3D-invasion in collagen matrix, and viability. Protein expression profiles after drug treatment were evaluated by western blotting. In vivo anticancer efficacy of drugs was examined in a mouse orthotopic xenograft model. RESULTS: Combined treatment of GBM TSs with HCA or BCA and TMZ significantly reduced cell viability, stemness, and invasiveness. Expression levels of stemness-, invasiveness-, and mesenchymal transition-associated markers, Zeb1, N-cadherin, and ß-catenin, were also substantially decreased by the combined treatment. The combined treatment also reduced tumor growth in a mouse orthotopic xenograft model. CONCLUSION: Our findings suggest that HCA and BCA, combined with TMZ, are potential therapeutic agents in the treatment of GBM.

Metastatic Breast Cancer Cells Enter Into Dormant State and Express Cancer Stem Cells Phenotype Under Chronic Hypoxia.

Tumor dormancy is a poorly understood stage in cancer progression characterized by mitotic cycle arrest in G0/G1 phase and low metabolism. The cells survive in a quiescent state and wait for appropriate environmental conditions to begin proliferation again giving rise to metastasis. Despite their key role in cancer development and metastasis, the knowledge about their biology and origin is still very limited due to the poorness of established in vitro models that faithfully recapitulated tumor dormancy. Using at least three cycles of 1% O2 hypoxia and reoxygenation, we establish and characterize the hypoxia-resistant human breast cancer cell line chMDA-MB-231 that can stably survive under 1% O2 condition by entering into dormant state characterized by arrest in G0/G1 phase and low metabolism. This dormant state is reversible since once replaced in normoxia the cells recover the proliferation rate in 2 weeks. We show that chronic hypoxia induces autophagy that may be the survival mechanism of chMDA-MB-231 cells. Furthermore, the data in this work demonstrate that cycling hypoxic/reoxygenation stress selects MDA-MB-231 population that presents the cancer stem-like phenotype characterized by CD24- /CD44+ /ESA+ expression and spheroid forming capacity. We believe that our study presents a promising approach to select dormant breast cancer cells with stem-like phenotype using the hypoxia/reoxygenation regimen that may represent an area with profound implications for therapeutic developments in oncology. J. Cell. Biochem. 118: 3237-3248, 2017. © 2017 Wiley Periodicals, Inc.

The role of cancer stem cells in tumor heterogeneity and resistance to therapy.

Cancer is a heterogenous disease displaying marked inter- and intra-tumoral diversity. The existence of cancer stem cells (CSCs) has been experimentally demonstrated in a number of cancer types as a subpopulation of tumor cells that drives the tumorigenic and metastatic properties of the entire cancer. Thus, eradication of the CSC population is critical for the complete ablation of a tumor. This is, however, confounded by the inherent resistance of CSCs to standard anticancer therapies, eventually leading to the outgrowth of resistant tumor cells and relapse in patients. The cellular mechanisms of therapy resistance in CSCs are ascribed to several factors including a state of quiescence, an enhanced DNA damage response and active repair mechanisms, up-regulated expression of drug efflux transporters, as well as the activation of pro-survival signaling pathways and inactivation of apoptotic signaling. Understanding the mechanisms underlying the acquisition of resistance to therapy may hold the key to targeting the CSC population.