Tumor heterogeneity of gastric cancer: From the perspective of tumor-initiating cell

Gastric cancer(GC) remains one of the most common and malignant types of cancer due to its rapid progression, distant metastasis, and resistance to conventional chemotherapy, although efforts have been made to understand the underlying mechanism of this resistance and to improve clinical outcome. It is well recognized that tumor heterogeneity, a fundamental feature of malignancy, plays an essential role in the cancer development and chemoresistance. The model of tumor-initiating cell(TIC) has been proposed to explain the genetic, histological, and phenotypical heterogeneity of GC. TIC accounts for a minor subpopulation of tumor cells with key characteristics including high tumorigenicity, maintenance of self-renewal potential, giving rise to both tumorigenic and non-tumorigenic cancer cells, and resistance to chemotherapy. Regarding tumor-initiating cell of GC(GATIC), substantial studies have been performed to(1) identify the putative specific cell markers for purification and functional validation of GATICs;(2) trace the origin of GATICs; and(3) decode the regulatory mechanism of GATICs. Furthermore, recent studies demonstrate the plasticity of GATIC and the interaction between GATIC and its surrounding factors(TIC niche or tumor microenvironment). All these investigations pave the way for the development of GATIC-targeted therapy, which is in the phase of preclinical studies and clinical trials. Here, we interpret the heterogeneity of GC from the perspectives of TIC by reviewing the above-mentioned fundamental and clinical studies of GATICs. Problems encountered during the GATIC investigations and the potential solutions are also discussed.

All-trans retinoic acid(ATRA)inhibits insufficient radiofrequency ablation(IRFA)-induced enrichment of tumor-initiating cells in hepatocellular carcinoma

Objective:Local recurrence of hepatocellular carcinoma(HCC)after radiofrequency ablation(RFA)treatment remains a serious problem.Tumor-initiating cells(TICs)are thought to be responsible for tumor relapse.Here,we investigated the effect of the TIC differentiation inducer,all-trans retinoic acid(ATRA),on RFA and explored the potential molecular mechanisms.Methods:The proportions of CD133+and epithelial cell adhesion molecule(Ep CAM);TICs in recurrent HCC after RFA and primary HCC were first determined in clinic.Then,the effect of heat intervention or insufficient RFA(IRFA)on the malignant potential of HCC cells,including cell migration,sphere formation ability,tumor growth,the proportion of CD133+and Ep CAM+TICs and expression of stem cell-related genes,was evaluated in vitro and in vivo.Finally,the effect of ATRA on the tumor growth and the proportion of TICs was evaluated.Results:In clinical data,a higher proportion of CD133+and Ep CAM+TICs was found in recurrent tumors than in primary tumors.In vitro heat intervention promoted the cell migration and sphere formation ability.Additionally,it increased the proportion of CD133+and Ep CAM+TICs and the expression of stem cell-related genes.In addition,after IRFA the residual tumors in xenografts grew faster and had more TICs than untreated tumors.ATRA remarkably inhibited residual tumor growth after IRFA by elimination of TICs though the PI3 K/AKT pathway.Combination treatment with ATRA resulted in longer survival outcomes in mouse xenografts than RFA alone.Conclusions:ATRA,as a TIC differentiation inducer,could help to improve the effect of RFA treatment,which was partially attributed to its effect against TICs.The data indicated its potential as an alternative drug in the development of better therapeutic strategies for use in combination with RFA.

Could gastrointestinal tumor-initiating cells originate from cell-cell fusion in vivo?

Tumor-initiating cells(TICs)or cancer stem cells are believed to be responsible for gastrointestinal tumor initiation,progression,metastasis,and drug resistance.It is hypothesized that gastrointestinal TICs(giTICs)might originate from cell-cell fusion.Here,we systemically evaluate the evidence that supports or opposes the hypothesis of giTIC generation from cell-cell fusion both in vitro and in vivo.We review giTICs that are capable of initiating tumors in vivo with 5000 or fewer in vivo fused cells.Under this restriction,there is currently little evidence demonstrating that giTICs originate from cell-cell fusion in vivo.However,there are many reports showing that tumor generation in vitro occurs with more than 5000 fused cells.In addition,the mechanisms of giTIC generation via cell-cell fusion are poorly understood,and thus,we propose its potential mechanisms of action.We suggest that future research should focus on giTIC origination from cell-cell fusion in vivo,isolation or enrichment of giTICs that have tumor-initiating capabilities with 5000 or less in vivo fused cells,and further clarification of the underlying mechanisms.Our review of the current advances in our understanding of giTIC origination from cell-cell fusion may have significant implications for the understanding of carcinogenesis and future cancer therapeutic strategies targeting giTICs.

ATP-citrate lyase regulates stemness and metastasis in hepatocellular carcinoma via the Wnt/β-catenin signaling pathway

Background: Hepatocellular carcinoma(HCC) is one of the most highly malignant tumors. Liver tumor-initiating cells(LTICs) have been considered to contribute to HCC progression and metastasis. ATP-citrate lyase(ACLY), as a key enzyme for de novo lipogenesis, has been reported to be upregulated in various tumors. However, its expression and role in HCC and LTICs remain unknown. Methods: The expressions of ACLY in HCC tissues were detected by quantitative real-time PCR(q RT-PCR), Western blotting and immunohistochemistry. Kaplan-Meier curves and Chi-square test were used to determine the clinical significance of ACLY expression in HCC patients. A series of assays were performed to determine the function of ACLY on stemness, migration and invasion of HCC cells. Luciferase reporter assay, Western blotting and immunoprecipitation were used to study the regulation of the Wnt/β-catenin signaling by ACLY. Rescue experiments were performed to investigate whether β-catenin was the mediator of ACLY-regulated stemness and migration in HCC cells. Results: ACLY was highly expressed in HCC tissues and LTICs. Overexpression of ACLY was significantly correlated with poor prognosis, progression and metastasis of HCC patients. Knockdown of ACLY remarkably suppressed stemness properties, migration and invasion in HCC cells. Mechanistically, ACLY could regulate the canonical Wnt pathway by affecting the stability of β-catenin, and Lys49 acetylation of β-catenin might mediate ACLY-regulated β-catenin level in HCC cells. Conclusions: ACLY is a potent regulator of Wnt/β-catenin signaling in modulating LTICs stemness and metastasis in HCC. ACLY may serve as a new target for the diagnosis and treatment of HCC.

Hallmarks in colorectal cancer:Angiogenesis and cancer stem-like cells

Carcinogenesis is a multistep process that requires the accumulation of various genetic and epigenetic aberrations to drive the progressive malignant transformation of normal human cells.Two major hallmarks of carcinogenesis that have been described are angiogenesis and the stem cell characteristic of limitless replicative potential.These properties have been targeted over the past decade in the development of therapeutic treatments for colorectal cancer(CRC),one of the most commonly diagnosed and lethal cancers worldwide.The treatment of solid tumor cancers such as CRC has been challenging due to the heterogeneity of the tumor itself and the chemoresistance of the malignant cells.Furthermore,the same microenvironment that maintains the pool of intestinal stem cells that contribute to the continuous renewal of the intestinal epithelia also provides the necessary conditions for proliferative growth of cancer stem-like cells.These cancer stem-like cells are responsible for the resistance to therapy and cancer recurrence,though they represent less than 2.5%of the tumor mass.The stromal environment surrounding the tumor cells,referred to as the tumor niche,also supports angiogenesis,which supplies the oxygen and nutrients needed for tumor development.Anti-angiogenic therapy,such as with bevacizumab,a monoclonal antibody against vascular-endothelial growth factor,significantly prolongs the survival of metastatic CRC patients.However,such treatments are not completely curative,and a large proportion of patient tumors retain chemoresistance or show recurrence.This article reviews the current knowledge regarding the molecular phenotype of CRC cancer cells,as well as discusses the mechanisms contributing to their maintenance.Future personalized therapeutic approaches that are based on the interaction of the carcinogenic hallmarks,namely angiogenic and proliferative attributes,could improve survival and decrease adverse effects induced by unnecessary chemotherapy.

Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction

AIM: To investigate adenoviral transduction in mesenchymal stem cells(MSCs) and effects on stemness in vitro and function as a cell therapy in vivo.METHODS: Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine(PEI)-mediated transfection of pc DNA3-e GFP or adenoviral transduction of green fluorescent protein(GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness(i.e., cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2(BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and histology.RESULTS: PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs(81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs(78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs(7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression(0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs(1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteogenic differentiation in vitrowere not affected by Ad transduction in both fetal and adult MSCs, but fetal MSCs had reduced chondrogenic differentiation in vitro when compared to adult(P < 0.01). Chondrogenic differentiation was also significantly reduced in Ad-GFP transduced cells(P < 0.05). AdBMP2 transduced adult MSCs induced new bone formation in more thighs than Ad-BMP2 transduced fetal MSCs(83% vs 17% of the six treated thighs per group, P < 0.05) and resulted in increased femur midshaft diameter due to greater extent of periosteal new bone(1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P < 0.05).CONCLUSION: Fetal MSCs may be genetically manipulated ex vivo with adenoviral vectors. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications in vivo.

Identification and analysis of a minority fraction in a U87 cell line Do side-population cells represent bona fide stem cell-like cancer cells?

BACKGROUND： Overwhelming evidence suggests that tumor bulks are comprised of differentiated tumor cells and cancer stem cells （CSCs）. The stem cell-like side-population （SP） cells account for a minor fraction of the total tumor cells, yet are apparently the cells capable of tumor initiation, growth, maintenance, and recurrence. OBJECTIVE： To identify potential stem cell-like cancer cells in a U87 human brain glioma cell line on the basis of dye efflux, clone formation, and multi-drug resistance capacity. DESIGN, TIME AND SETTING： The cellular and molecular biology experiment was performed at the Laboratory of Shanghai Institute of Hematology and Laboratory of Shanghai Institute of Endocrinology in Ruijin Hospital; in vivo contrast observational animal trial was performed at Experimental Animal Center, School of Medicine, Shanghai Jiao Tong University from June 2007 to May 2008. MATERIALS： The U87 cell line was provided by the Shanghai Institute of Cancer Research, Chinese Academy of Science; DMEM/F12 （1 ： 1） and fetal bovine serum were purchased from Gibco Invitrogen, USA; human recombinant basic fibroblast growth factors were purchased from BD Bioscience, USA; Hoechst 33342, Verapamil, and methyl thiazolyl tetrazolium were purchased from Sigma, USA; phycoerythrin-labeled anti-human-CD133 was purchased from Milteny Biotec, Germany; SYBR PrimeScriptTM RT-PCR kit was purchased from TaKaRa Biotechnology, Dalian, China. METHODS： Monolayer cultured cells were harvested by 0.25% Trypsin-EDTA and suspended at a 1 ×10^6/mL dilution in PBS containing 2% FBS, and were stained with Hoechst 33342 dye, either alone or in combination with Verapamil. Following fluorescence-activated cell sorting, SP and non-SP subsets were cultivated with serum-containing （DMEM plus 10% fetal bovine serum） or serum-free culture medium [DMEM/F12 （1： 1） ＋ 1× B27 supplement ＋ 10 ng/mL basic fibroblast growth factors ＋ 1× L-glutamine] to determine growth characteristics in vitro. Finally, single free U87 cells and subsets （SP or non-SP cells） were subcutaneously injected into the backs of 5-week-old nude mice for in vivo tumorigenicity. MAIN OUTCOME MEASURES： Cell morphology and clonogenicity were observed under inverted microscope; SP phenotype and fluorescent antibody labeling were analyzed by MoFIoTM flow cytometry; ABC transporter mRNA expression was evaluated by semi-quantitative real-time RT-PCR; efflux capacity for anti-neoplastic drugs from the U87 cell line and subsets was measured with the MTT assay, then detected by enzyme-linked immunosorbent assay at a wavelength of 490 nm; in vivo tumorigenicity in immunodeficient nude mice was evaluated by diameter size. RESULTS： During in vitro passages, human U87 cells maintained a stable SP fraction profile and exhibited the ability to form neurosphere-like clones. SP cell proliferation decreased compared with non-treated U87 cells. CD133 expression was reduced in the SP and non-SP cells. Freshly sorted SP fractions expressed higher levels of ABC drug transporter genes, and exhibited increased potential for cytotoxic drug resistance. The in vivo malignancy of U87 cells was largely dependent on non-SP cells in nude mice, and tumors that formed from the non-SP fraction developed faster and larger compared with tumors from the SP fraction. CONCLUSION： The SP cell component was a key factor that influenced mRNA expression and cytotoxic drug resistance. In particular, cancer stem cells or tumor-initiating cells were not exclusively enriched in the SP subset of the U87 cell line, and non-SP cells were even more tumorigenic.

Matrigel modulates a stem cell phenotype and promotes tumor formation in a mantle cell lymphoma cell line

Tumors may be maintained by subpopulations of cells possessing stem cell-like properties. We evaluated the stem cell-like and tumor-forming properties of side population (SP) and CD133+/ CD44+ cells in Granta 519, a human mantle cell lymphoma cell line. The in-vitro Cobblestone Area Forming Cell (CAFC) assay, designed to detect stem and progenitor cells, revealed that SP cells contained the greatest proportion of stem cell-like cells. The addition of Matrigel to CAFC assays of SP and non-SP cells both increased their respective stem cell frequencies in comparison to those cultures without Matrigel, and additionally resulted in observed stem cell frequencies which were the same between SP and non-SP cells. Contrary, Matrigel decreased the stem cell frequencies of CD133+/CD44+ or CD133-/CD44- cells. In-vivo assays revealed tumor formation from Matrigel-mixed SP and non-SP cells, and in one instance, occurred with as few as one Matrigel-mixed SP cell. Vehicle-mixed injections of SP and non-SP tumor cells resulted in tumor formation from SP cells only. Tumor formation did not occur from Matrigel nor hyaluronan (cellular substrate for CD44-expressing cells)-mixed populations of CD133+/CD44+ or CD133-/CD44- cells. These data demonstrate that Matrigel modulates a stem cell phenotype and promotes tumor formation from SP and non-SP cells. The tumor micro-environmental niche and tumor cell to micro-environmental interactions may be important future targets for novel chemotherapeutic agents.

DNA damage and breast cancer

Cancer is intimately related to the accumulation of DNA damage,and repair failures(including mutation prone repair and hyperactive repair systems).This article relates current clinical categories for breast cancer and their common DNA damage repair defects.Information is included on the potential for accumulation of DNA damage in the breast tissue of a woman during her lifetime and the role of DNA damage in breast cancer development.We then cover endogenous and exogenous sources of DNA damage,types of DNA damage repair and basic signal transduction pathways for three gene products involved in the DNA damage response system;namely BRCA1,BRIT1 and PARP-1.These genes are often considered tumor suppressors because of their roles in DNA damage response and some are under clinical investigation as likely sources for effective new drugs to treat breast cancers.Finally we discuss some of the problems of DNA damage repair systems in cancer and the conundrum of hyper-active repair systems which can introduce mutations and confer a survival advantage to certain types of cancer cells.

rtcisE2F promotes the self-renewal and metastasis of liver tumorinitiating cells via N^(6)-methyladenosine-dependent E2F3/E2F6 mRNA stability

Liver cancer is highly heterogeneous,and the tumor tissue harbors a variety of cell types.Liver tumor initiating cells(TICs)well contribute to tumor heterogeneity and account for tumor initiation and metastasis,but the molecular mechanisms of liver TIC self-renewal are elusive.Here,we identified a functional read-through rt-circRNA,termed rtcisE2F,that is highly expressed in liver cancer and liver TICs.rtcisE2F plays essential roles in the self-renewal and activities of liver TICs.rtcisE2F targets E2F6 and E2F3 mRNAs,attenuates mRNA turnover,and increases E2F6/E2F3 expression.Mechanistically,rtcisE2F functions as a scaffold of N^(6)-methyladenosine(m^(6)A)reader IGF2BP2 and E2F6/E2F3 mRNA.rtcisE2F promotes the association of E2F6/E2F3 mRNAs with IGF2BP2,and inhibits their association with another m^(6)A reader,YTHDF2.IGF2BP2 inhibits E2F6/E2F3 mRNA decay,whereas YTHDF2 promotes E2F6/E2F3 mRNA decay.By switching m^(6)A readers,rtcisE2F enhances E2F6/E2F3 mRNA stability.E2F6 and E2F3 are both required for liver TIC self-renewal and Wnt/β-catenin activation,and inhibition of these pathways is a potential strategy for preventing liver tumorigenesis and metastasis.In conclusion,the rtcisE2F-IGF2BP2/YTHDF2-E2F6/E2F3-Wnt/β-catenin axis drives liver TIC self-renewal and initiates liver tumorigenesis and metastasis,and may provide a strategy to eliminate liver TICs.

When fats commit crimes: fatty acid metabolism, cancer stemness and therapeutic resistance

The role of fatty acid metabolism,including both anabolic and catabolic reactions in cancer has gained increas-ing attention in recent years.Many studies have shown that aberrant expression of the genes involved in fatty acid synthesis or fatty acid oxidation correlate with malignant phenotypes including metastasis,therapeutic resistance and relapse.Such phenotypes are also strongly associated with the presence of a small percentage of unique cells among the total tumor cell population.This distinct group of cells may have the ability to self-renew and propagate or may be able to develop resistance to cancer therapies independent of genetic alterations.Therefore,these cells are referred to as cancer stem cells/tumor-initiating cells/drug-tolerant persisters,which are often refractory to cancer treatment and difficult to target.Moreover,interconversion between cancer cells and cancer stem cells/tumor-initiating cells/drug-tolerant persisters may occur and makes treatment even more challenging.This review highlights recent findings on the relationship between fatty acid metabolism,cancer stemness and therapeutic resistance and prompts discussion about the potential mechanisms by which fatty acid metabolism regulates the fate of cancer cells and therapeutic resistance.

Neuro-oncogenesis and the adult human sub-ventricular zone in high grade glioma

The last fifteen years have seen the application of the cancer stem cell hypothesis to tumors of the central nervous system,in particular to high grade glioma(HGG),the most aggressive and common brain cancer in adults.Seminal studies have shown that cancer stem cells(alternatively named tumor-initiating cells)are capable of self-renew and multipotency,similar to their normal counterpart.More importantly they give rise to tumors that closely mimic the phenotype and genotype of human HGG.The identification of neurogenic niches in adult rodent and human brain has further reinforced the hypothesis that HGG might derive from the malignant transformation occurring in these areas,especially in the sub-ventricular zone(SVZ),the largest and most well characterised stem cell niche.Following from evidence of animal model studies supporting this hypothesis,recently we investigated the role of the SVZ in neuro-oncogenesis using tissue material derived from HGG patients.We also described response to conventional chemo-therapies of cancer stem cells isolated from the SVZ and the tumor mass(T)of the same patients and reconstructed tumor evolution.In this review,such findings will be discussed in the context of the current literature on the biology of the SVZ in the normal and disease brain.