Cancer stem cell hypothesis: a brief summary and two proposals

The investigation and development of the cancer stem cell (CSC) model has received much focus during these years. CSC is characterized as a small fraction of cancer cells that have an indefinite ability for self-renewal and pluripotency and are responsible for initiating and sustaining of the bulk of cancer. So, whether current treatment strategies, most of which target the rapid division of cancer cells, could interfere with the slow-cycling CSCs is broadly questioned. Meanwhile, however, the new understanding of tumorigenesis has led to the development of new drug screening strategies. Both stem cells and mesenchymal stem cells have been vigorously used in pre-clinical studies of their anti-tumor potential, mainly due to their inherent tumoritropic migratory properties and their ability to carry anti-tumor transgenes. Here, based on the tumorigenic and tumoritropic characteristics of CSCs, we proposed two hypotheses exploring possible usage of CSCs as novel anti-tumor agents and potential sources for tissue regeneration. Further experimental validation of these hypotheses may unravel some new research topics.     

Convergence of normal stem cell and cancer stem cell developmental stage: Implication for differential therapies

Increased evidence shows that normal stem cells may contribute to cancer development and progression by acting as cancer-initiating cells through their interactions with abnormal environmental elements.We postulate that normal stem cells and cancer stem cells (CSC) possess similar mechanisms of self-renewal and differentiation.CSC can be the key to the elaboration of anti-cancer-based therapy.In this article,we focus on a controversial new theme relating to CSC.Tumorigenesis may have a critical stage characterized as a "therapeutic window",which can be identified by asso-ciation of molecular,biochemical and biological events.Identifying such a stage can allow the production of more effective therapies (e.g.manipulated stem cells) to treat several cancers.More importantly,confirming the existence of a similar therapeutic window during the conversion of normal stem cells to malignant CSC may lead to targeted therapy specifically against CSC.This conversion information may be derived from investigating the biological behaviour of both normal stem cells and cancerous stem cells.Currently,there is little knowledge about the cellular and molecular mechanisms that govern the initiation and maintenance of CSC.Studies on co-evolution and interdependence of cancer with normal tissues may lead to a useful treatment paradigm of cancer.The crosstalk between normal stem cells and cancer formation may converge developmental stages of different types of stem cells (e.g.normal stem cells,CSC and embryonic stem cells).The differential studies of the convergence may result in novel therapies for treating cancers.     

肿瘤干细胞与肿瘤转移

近年来,肿瘤干细胞(cancer stem cell,CSC)学说研究认为CSC与肿瘤发生、发展、转移和复发关系极为密切。研究还发现CSC具有明显的异质性,即CSC可分为增生、耐药、侵袭和转移等行为不同的亚群细胞,其中具有转移生物学特性的CSC亚群细胞称为肿瘤转移干细胞(migrating cancer stem cell,MCSC)。目前认为,上皮-间质转变、趋化因子和靶器官微环境可能在肿瘤转移过程中起着重要作用。针对MCSC及其相关机制的靶向治疗有望能更有效地遏制肿瘤的转移。     

Growth of cancer cell lines under stem cell-like conditions has the potential to unveil therapeutic targets

Malignant tumors comprise a small proportion of cancer-initiating cells (CIC), capable of sustaining tumor formation and growth. CIC are the main potential target for anticancer therapy. However, the identification of molecular therapeutic targets in CIC isolated from primary tumors is an extremely difficult task. Here, we show that after years of passaging under differentiating conditions, glioblastoma, mammary carcinoma, and melanoma cell lines contained a fraction of cells capable of forming spheroids upon in vitro growth under stem cell-like conditions. We found an increased expression of surface markers associated with the stem cell phenotype and of oncogenes in cell lines and clones cultured as spheroids vs. adherent cultures. Also, spheroid-forming cells displayed increased tumorigenicity and an altered pattern of chemosensitivity. Interestingly, also from single retrovirally marked clones, it was possible to isolate cells able to grow as spheroids and associated with increased tumorigenicity. Our findings indicate that short-term selection and propagation of CIC as spheroid cultures from established cancer cell lines, coupled with gene expression profiling, represents a suitable tool to study and therapeutically target CIC: the notion of which genes have been down-regulated during growth under differentiating conditions will help find CIC-associated therapeutic targets.     

Limitations of the cancer stem cell theory

Cancer stem cells (CSCs) can be operationally defined as a subset of neoplastic cells which are responsible for the growth and re-growth of primary and metastatic tumors. Although the existence of perpetually dividing cells is a logical necessity to explain the malignant properties of human tumors, experimental data supporting their existence have only recently been obtained. New knowledge in basic stem cell biology and the availability of several cell surface markers for the definition and isolation of small subsets of immature cells coupled to the use of the classical model of xenotransplantation in immune deficient mice has identified putative CSCs in several solid tumors such as mammary, colon, brain, pancreas, prostate, melanoma and others. However, the theory must be considered as still in its infancy, since tumors grown in mice only partially recapitulate the biology of human cells. In addition, whether the “transformed” cell is the neoplastic counterpart of a normal stem cell or whether complete malignant behaviour can occur in a more differentiated cell has still to be demonstrated. In spite of these difficulties, the CSC hypothesis could be of clinical relevance, especially in the definition of new ways to assay drug sensitivity of primary human tumors.     

Implications of cancer stem cell theory for cancer chemoprevention by natural dietary compounds

The emergence of cancer stem cell theory has profound implications for cancer chemoprevention and therapy. Cancer stem cells give rise to the tumor bulk through continuous self-renewal and differentiation. Understanding the mechanisms that regulate self-renewal is of greatest importance for discovery of anticancer drugs targeting cancer stem cells. Naturally occurring dietary compounds have received increasing attention in cancer chemoprevention. The anticancer effects of many dietary components have been reported for both in vitro and in vivo studies. Recently, a number of studies have found that several dietary compounds can directly or indirectly affect cancer stem cell self-renewal pathways. Herein we review the current knowledge of most common natural dietary compounds for their impact on self-renewal pathways and potential effect against cancer stem cells. Three pathways (Wnt/β-catenin, Hedgehog and Notch) are summarized for their functions in self-renewal of cancer stem cells. The dietary compounds, including curcumin, sulforaphane, soy isoflavone, epigallocatechin-3-gallate, resveratrol, lycopene, piperine and vitamin D₃, are discussed for their direct or indirect effect on these self-renewal pathways. Curcumin and piperine have been demonstrated to target breast cancer stem cells. Sulforaphane has been reported to inhibit pancreatic tumor-initiating cells and breast cancer stem cells. These studies provide a basis for preclinical and clinical evaluation of dietary compounds for chemoprevention of cancer stem cells. This may enable us to discover more preventive strategies for cancer management by reducing cancer resistance and recurrence and improving patient survival.     

Beyond tumorigenesis： cancer stem cells in metastasis

The importance of cancer stem cells （CSCs） in tumor-initiation has been firmly established in leukemia and recently reported for a variety of solid tumors. However, the role of CSCs in multistage cancer progression, particularly with respect to metastasis, has not been well-defined. Cancer metastasis requires the seeding and successful colonization of specialized CSCs at distant organs. The biology of normal stem cells and CSCs share remarkable similarities and may have important implications when applied to the study of cancer metastasis. Furthermore, overlapping sets of molecules and pathways have recently been identified to regulate both stem cell migration and cancer metastasis. These molecules constitute a complex network of cellular interactions that facilitate both the initiation of the pre-metastasis niche by the primary tumor and the formation of a nurturing organ microenvironment for migrating CSCs. In this review, we surveyed the recent advances in this dynamic field and propose a unified model of cancer progression in which CSCs assume a central role in both tumorigenesis and metastasis. Better understanding of CSCs as a fundamental component of the metastatic cascade will lead to novel therapeutic strategies against metastatic cancer.     

Characterization of a stem cell population in lung cancer A549 cells

We isolated a stem cell subpopulation from human lung cancer A549 cells using FACS/Hoechst 33342. This side population (SP), which comprised 24% of the total cell population, totally disappeared after treatment with the selective ABCG 2 inhibitor fumitremorgin C. In a repopulation study, isolated SP and non-SP cells were each able to generate a heterogeneous population of SP and non-SP cells, but this repopulation occurred more rapidly in SP cells than non-SP. An MTT assay and cell cycle distribution analysis reveal a similar profile between SP and non-SP groups. However, in the presence of doxorubicin (DOX) and methotrexate (MTX), SP cells showed significantly lower Annexin V staining when compared to non-SP cells. Taken together, these results demonstrate that SP cells have an active regeneration capacity and high anti-apoptotic activity compared with non-SP cells. Furthermore, our GeneChip^® data revealed a heightened mRNA expression of ABCG2 and ABCC2 in SP cells. Overall these data explain why the SP of A549 has a unique ability to resist DOX and MTX treatments. Therefore, we suggest that the expression of the ABCG2 transporter plays an important role in the multidrug resistance phenotype of A549 SP cells.     

DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers

DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint.     

p53: The barrier to cancer stem cell formation

The role of p53 as the “guardian of the genome” in differentiated somatic cells, triggering various biological processes, is well established. Recent studies in the stem cell field have highlighted a profound role of p53 in stem cell biology as well. These studies, combined with basic data obtained 20 years ago, provide insight into how p53 governs the quantity and quality of various stem cells, ensuring a sufficient repertoire of normal stem cells to enable proper development, tissue regeneration and a cancer free life. In this review we address the role of p53 in genomically stable embryonic stem cells, a unique predisposed cancer stem cell model and adult stem cells, its role in the generation of induced pluripotent stem cells, as well as its role as the barrier to cancer stem cell formation.     

Mesenchymal stem cell secretome and regenerative therapy after cancer

Cancer treatment generally relies on tumor ablative techniques that can lead to major functional or disfiguring defects. These post-therapy impairments require the development of safe regenerative therapy strategies during cancer remission. Many current tissue repair approaches exploit paracrine (immunomodulatory, pro-angiogenic, anti-apoptotic and pro-survival effects) or restoring (functional or structural tissue repair) properties of mesenchymal stem/stromal cells (MSC). Yet, a major concern in the application of regenerative therapies during cancer remission remains the possible triggering of cancer recurrence. Tumor relapse implies the persistence of rare subsets of tumor-initiating cancer cells which can escape anti-cancer therapies and lie dormant in specific niches awaiting reactivation via unknown stimuli. Many of the components required for successful regenerative therapy (revascularization, immunosuppression, cellular homing, tissue growth promotion) are also critical for tumor progression and metastasis. While bi-directional crosstalk between tumorigenic cells (especially aggressive cancer cell lines) and MSC (including tumor stroma-resident populations) has been demonstrated in a variety of cancers, the effects of local or systemic MSC delivery for regenerative purposes on persisting cancer cells during remission remain controversial. Both pro- and anti-tumorigenic effects of MSC have been reported in the literature. Our own data using breast cancer clinical isolates have suggested that dormant-like tumor-initiating cells do not respond to MSC signals, unlike actively dividing cancer cells which benefited from the presence of supportive MSC. The secretome of MSC isolated from various tissues may partially diverge, but it includes a core of cytokines (i.e. CCL2, CCL5, IL-6, TGFβ, VEGF), which have been implicated in tumor growth and/or metastasis. This article reviews published models for studying interactions between MSC and cancer cells with a focus on the impact of MSC secretome on cancer cell activity, and discusses the implications for regenerative therapy after cancer.     

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133⁺ cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.     

A rat mammary gland cancer cell with stem cell properties of self-renewal and multi-lineage differentiation

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing cancer-initiating cells with stem cell properties at the single cell level has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell, derived from rat mammary adenocarcinoma has: the ability to serially re-generate mammospheres in long-term non-adherent cultures, the differentiation potential to generate all the cell lineages of the mammary gland and branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multi-lineage differentiation and the tubular-like structure formation potential suggest that LA7 cells is a cancer stem model system to study the dynamics of tumor formation at the single cell level. Cinzia Cocola, Sveva Sanzone and Simonetta Astigiano have contributed equally to this work.     

CD9 correlates with cancer stem cell potentials in human B-acute lymphoblastic leukemia cells

Cancer stem cell (CSC) theory suggests that only a small subpopulation of cells having stem cell-like potentials can initiate tumor development. While recent data on acute lymphoblastic leukemia (ALL) are conflicting, some studies have demonstrated the existence of such cells following CD34-targeted isolation of primary samples. Although CD34 is a useful marker for the isolation of CSCs in leukemias, the identification of other specific markers besides CD34 has been relatively unsuccessful. To identify new markers, we first performed extensive analysis of surface markers on several B-ALL cell lines. Our data demonstrated that every B-ALL cell line tested did not express CD34 but certain lines contained cell populations with marked heterogeneity in marker expression. Moreover, the CD9⁺ cell population possessed stem cell characteristics within the clone, as demonstrated by in vitro and transplantation experiments. These results suggest that CD9 is a useful positive-selection marker for the identification of CSCs in B-ALL.     

Synthesis and evaluation of 2,5-diamino and 2,5-dianilinomethyl pyridine analogues as potential CXCR4 antagonists

CXCR4 and its cognate ligand CXCL12 has been linked to various pathways such as cancer metastasis, inflammation, HIV-1 proliferation, and auto-immune diseases. Small molecules have shown potential as CXCR4 inhibitors and modulators, and therefore can mitigate diseases related to the CXCR4-CXCL12 pathway. We have designed and synthesized a series of 2,5-diamino and 2,5-dianilinomethyl pyridine derivatives as potential CXCR4 antagonists. Thirteen compounds have an effective concentration (EC) of 100?nM or less in a binding affinity assay and nine of these have at least 75% inhibition of invasion in Matrigel binding assay. Compounds 3l, 7f, 7j, and 7p show a minimal reduction in inflammation when carrageenan paw edema test is conducted. Overall, these compounds show potential as CXCR4 antagonist.     

Nicotine increases cancer stem cell population in MCF-7 cells

Epidemiological studies have suggested that cigarette smoking is related to increased breast cancer risk. Nicotine is most likely related to the risk in cigarette smoking. However, the mechanisms by which nicotine promotes cancer development are not fully understood. It has recently been suggested that development of breast cancer are originated from cancer stem cells, which are a minor population of breast cancer. In the present study, we investigated the effects of nicotine on the population of cancer stem cells in MCF-7 human breast cancer cells, using flow cytometry with a cancer stem cell marker aldehyde dehydrogenase (ALDH). We found that nicotine increased ALDH-positive cell population in a dose-dependent manner. We further demonstrated that a PKC-Notch pathway is involved in the effect of nicotine. In addition, the effect of nicotine was blocked by treatment with the α7 subunit-selective antagonist of nicotinic acetylcholine receptors (nAChR) α-Bungarotoxin. These data suggest that nicotine increases the stem cell population via α7-nAChR and the PKC-Notch dependent pathway in MCF-7 cells. These findings reveal a relationship between nicotine and the cancer stem cells in human breast cancer.     

Oral epithelial stem cells—Implications in normal development and cancer metastasis

Oral mucosa is continuously exposed to environmental forces and has to be constantly renewed. Accordingly, the oral mucosa epithelium contains a large reservoir of epithelial stem cells necessary for tissue homeostasis. Despite considerable scientific advances in stem cell behavior in a number of tissues, fewer studies have been devoted to the stem cells in the oral epithelium. Most of oral mucosa stem cells studies are focused on identifying cancer stem cells (CSC) in oral squamous cell carcinomas (OSCCs) among other head and neck cancers. OSCCs are the most prevalent epithelial tumors of the head and neck region, marked by their aggressiveness and invasiveness. Due to their highly tumorigenic properties, it has been suggested that CSC may be the critical population of cancer cells in the development of OSCC metastasis. This review presents a brief overview of epithelium stem cells with implications in oral health, and the clinical implications of the CSC concept in OSCC metastatic dissemination.     

Isolation and characterization of cancer stem cells from cervical cancer HeLa cells

Cervical cancer is one of the most common gynecologic malignancies and poses a serious health problem worldwide. Identification and characterization of cervical cancer stem cells may facilitate the development of novel strategies for the treatment of advanced and metastatic cervical cancer. Breast cancer-resistance protein (Bcrp1)-positive cells were selected from a population of parent HeLa cells using flow cytometry. The invasion capacity of Bcrp1-positive and -negative cells was analyzed with a Boyden chamber invasion test. The tumorigenicity of these cells was determined by in vivo transplantation in non-obesity diabetes/severe combined immunodeficiency (NOD/SCID) mice. The Bcrp1-positive subpopulation accounted for about 7% of the parent HeLa cell population. The proliferative capacity of the Bcrp1-positive cells was greater than that of the Bcrp1-negative cells (P < 0.05). In the invasion assay, the Bcrp1-positive cells demonstrated a greater invasive capacity through the artificial basement membrane than their Bcrp1-negative counterparts. Following transplantation of 10⁴ cells, only the Bcrp1-positive cells formed tumors in NOD/SCID mice. When 10⁵ or 10⁶ cells were transplanted, the tumor incidence and the tumor mass were greater in the Bcrp1-positive groups than those in the Bcrp1-negative groups (P < 0.05). The Bcrp1-positive subpopulation cervical cancer stem cells.     

肿瘤干细胞与表观遗传学调控

关于恶性肿瘤发生、复发与转移机制的研究由来已久,但目前的临床治疗方法依然不能克服肿瘤复发与转移的难题,肿瘤患者的生存率并未得到显著改善。近年来的研究提示肿瘤的起源、复发与转移的真正原因可能是存在于肿瘤内的极少数具有干细胞特性的细胞,即肿瘤干细胞（cancer stem cells,CSC）。与此同时,越来越多的研究表明,对于肿瘤干细胞的发生与功能维持,表观遗传学的调控机制可能发挥着极其重要的作用。该文简要综述目前肿瘤干细胞和表观遗传学相关领域的研究进展,并对肿瘤干细胞形成及发展过程中表观遗传学的调控作用及机制进行重点介绍。     

Mitochondria as therapeutic targets for cancer stem cells

Cancer stem cells(CSCs) are maintained by theirsomatic stem cells and are responsible for tumor initiation, chemoresistance, and metastasis. Evidence for the CSCs existence has been reported for a number of human cancers. The CSC mitochondria have been shown recently to be an important target for cancer treatment, but clinical significance of CSCs and their mitochondria properties remain unclear. Mitochondriatargeted agents are considerably more effective compared to other agents in triggering apoptosis of CSCs, as well as general cancer cells, via mitochondrial dysfunction. Mitochondrial metabolism is altered in cancer cells because of their reliance on glycolytic intermediates, which are normally destined for oxidative phosphorylation. Therefore, inhibiting cancer-specific modifications in mitochondrial metabolism, increasing reactive oxygen species production, or stimulating mitochondrial permeabilization transition could be promising new therapeutic strategies to activate cell death in CSCs as well, as in general cancer cells. This review analyzed mitochondrial function and its potential as a therapeutic target to induce cell death in CSCs. Furthermore, combined treatment with mitochondriatargeted drugs will be a promising strategy for the treatment of relapsed and refractory cancer.