Ovarian Cancer Stem Cells

Author's accepted version. The final publication is available at Springer via http://dx.doi.org/10.1007/9783-642-27841-9_7203-1 Ovarian Cancer Stem C...
Author: Jessie Elliott
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Author's accepted version. The final publication is available at Springer via http://dx.doi.org/10.1007/9783-642-27841-9_7203-1

Ovarian Cancer Stem Cells Synonyms Ovarian cancer initiating cells; ovarian cancer stem-like cells Definition Consistent with the cancer stem cell (CSC) hypothesis, ovarian cancer stem cells represent a subset of cancer cells lying at the top of hierarchy of all malignant cells that form the bulk of ovarian cancers. Cancer stem cells display ability to continually sustain tumor growth through their indefinite self-renewal and ability to generate all cancer cell lineages (transit-amplifying cells, differentiated cells) found in particular tumors through their (aberrant) differentiation. Links Ovarian Cancer Stem Cells Adult Stem Cells Transit amplifying cells Cancer Stem Cells Targeted Drug Development Hypoxia Apoptosis Characteristics Classification of ovarian cancers Ovarian cancers represent highly heterogeneous group of diseases with distinct cellular origin, epidemiology, pathogenesis, morphological and molecular characteristics and clinical course. Considering cell types from which ovarian cancers presumably originate, most ovarian cancers can be classified into 3 major categories: epithelial (~90%), sex cord-stromal (~8%) and germ cell (~2%) malignancies, each of which includes a number of subtypes. Serous ovarian carcinomas, which represent approximately 70% of epithelial ovarian cancers, can be sub-classified based on their cytological features (degree of nuclear atypia and mitotic rate) into high-grade serous ovarian carcinoma (HGSOC) and low-grade serous ovarian carcinoma (LGSOC). HGSOC and LGSOC do not represent two grades of the same disease but rather two distinct tumor types with different molecular pathogenesis, clinical behavior and prognosis. HGSOC accounts for 60-80% of epithelial ovarian cancers and majority of ovarian cancer stem cell research employed cells and tissues related to this specific tumor type. As a result, the body of knowledge on ovarian cancer stem cell available at the present time and discussed in this text largely 1

reflects characteristics of cancer stem cells relevant to the high-grade serous ovarian carcinoma. Isolation, identification and characterization of ovarian CSCs Originally, cancer stem cells were identified as a small subset (< 0.2%) of the total population of leukemic cells isolated from clinical specimens of acute myeloid leukemia (AML). The proportion of CSCs in solid tumors appears to be highly variable between tumors of the same type but seems to be higher than that in leukemias. For example, colorectal carcinomas display highly variable and in some cases relatively high proportion of CSCs (2-25%). In HGSOC, the reported frequency of stem cells (defined as tumor-initiating cells) is low (≤0.046%) ) and varies substantially among patients (up to 100-fold). On the other hand, the frequency of CSCs appears to be similar in primary tumors and matched omental metastasis in the same patients, suggesting that the frequency of CSC (tumor-initiating cells) might be an intrinsic property of ovarian tumors. Ovarian cancer stem cells were isolated from ovarian solid tumor masses, cells floating in malignant ascites and from established ovarian cancer cell lines, and they were identified based on their ability to self-renew and differentiate. Self-renewal and differentiation of ovarian CSCs were demonstrated by xenograft assays, in which ovarian cancer stem cells upon serial transplantations into immunodeficient mice generated tumors that histologically resembled parental tumors from which they were derived. Alternatively, self-renewal and ability to differentiate were demonstrated using sphere-forming assays in vitro. Isolation of ovarian cancer stem cells or their experimental enrichment in tumor cell populations has been accomplished by various methods. Method that exploits resistance of cancer stem cells to anoikis (apoptosis induced by lack of cell-matrix interactions) employs in vitro culture of cancer cells in suspension under conditions that prevent cell attachment and support proliferation of cancer stem cells. Other method exploits higher resistance of CSCs against cytotoxic effects of certain anticancer drugs and selects CSC upon drug treatment of mixed populations ovarian cancer cells under conditions that promote cell death in more differentiated cells. Methods based of Fluorescence-Activated Cell Sorting (FACS) isolate putative ovarian cancer stem cells based on their expression of aldehyde dehydrogenase 1 (ALDH1) or ability to exclude the Hoechst 33342 dye. Cells that exclude the fluorescent Hoechst dye display side population (SP) phenotype, which is associated with expression of multidrug transporter ABCG2 or ABCB1. SP cell compartment often includes ovarian cancer stem cells in a heterogeneous mixture of multiple phenotypically distinct subpopulations. Putative ovarian cancer stem cells have been characterized by the expression of one or more of the following cell surface proteins: CD44 (receptor for hyaluronic acid), CD133 (prominin-1), CD117 (c-Kit), and CD24 (HSA); however, no single marker clearly identifies ovarian cancer stem cells and distinct populations of ovarian cancer stem cells appear to display distinct marker profiles. Different combinations of these surface 2

proteins with or without functional assays results for cellular ALDH1 activity and SP phenotype were employed to define various isolates of ovarian cancer stem cells, such as CD44+/CD117+, CD133+, CD24+, CD133+/ALDH1+, CD44+/MyD88+, CD44+/CD24+, CD44+/CD24+/EpCAM+/SP and others. Biology of ovarian cancer stem cells Cell-of-origin: Cancer stem cell hypothesis does not address the origin of cancer stem cells and it does not postulate that cancer stem cells originate from the non-malignant adult stem cells. In fact, more differentiated progeny of adult stem cells, including transit amplifying cells and terminally differentiated cells, may also undergo oncogenic mutations and acquire ability to sustain tumor growth. As a result, the cells-of-origin of ovarian cancer stem cells have not yet been identified. Adult stem cells have been suggested as possible cells-of-origin for cancer stem cells, because adult stem cells are present long enough in various tissues from which malignancies commonly arise (e.g. intestinal epithelium), and therefore, they can accumulate multiple mutations necessary for malignant transformations. In addition, adult stem cells already possess activated selfrenewal program, which is indispensable for CSCs. Taken together, adult stem cells might be possible cells-of-origin of ovarian cancer stem cells. These adult stem cells may be associated with different tissues of origin (ovarian, endometrial or distal fallopian tube) giving rise to ovarian cancer stem cells associated with different types of epithelial ovarian cancers (low-grade serous, endometroid, clear cell and high-grade serous ovarian cancers). Cell division and differentiation: Cancer stem cells are defined by their ability to produce stem cells (self-renewal) and ability to produce more differentiated progeny. These two tasks can be accomplished by asymmetric and symmetric cell division. Symmetric stemcell division generates two identical daughter cancer stem cells and results in the expansion of cancer stem cell pool. On the other hand, asymmetric cell division produces non-identical daughter cells with one cell retaining parental stem cell properties and another cell displaying more differentiated phenotype. Asymmetric stem cell division generates phenotypically diverse cancer cells that comprise the bulk of cells in tumors and but have little capacity to contribute to the tumor progression (nontumorigenic cancer cells). The balance between these two modes of stem cell division is controlled by cell signaling to produce appropriate numbers of stem cells and their more differentiated progeny. Considering ability for multilineage differentiation, adult stem cells are considered to be multipotent, since they are capable to differentiate into multiple, albeit limited range of cell types. On the other hand, CSCs do not have to display capacity for differentiation into distinctly different cell lineages, and working definition of CSCs requires that they are able to generate upon transplantation to a susceptible host tumors resembling parental tumors from which they were isolated. Tumors display variable heterogeneity of differentiated cancer cells from those that exhibit one differentiated state to those that display variety of distinctly differentiated cancer cells that express markers of various tissues. In this context, ovarian cancer stem cells demonstrate multipotency and showed ability to differentiate into cells displaying endothelial cell phenotype, which may contribute to tumor neovascularization. 3

Heterogeneity of ovarian cancer stem cells: ovarian cancer stem cells differ in their molecular characteristics among different cases of ovarian cancers, but individual ovarian tumors may also contain more than one type of cancer stem cells. Solid tumor masses, as well as malignant cells from ascites in some high-grade serous ovarian cancer cases contain heterogeneous population of cancer stem cells, some of which are CD133positive while other are CD133-negative, but both can recapitulate morphology of original tumors in transplantation assays. Similarly, some cancer stem cell-associated properties such as anticancer drug resistance, or ability to exclude Hoechst 3342 dye can be mediated by distinct mechanisms found in various cancer stem cell populations isolated from a single cell line. In addition, ovarian cancer stem cell phenotype appears to be unstable, since xenografted tumors originated from CD133-positive cancer stem cells may contain CD133-negative cancer stem cells (plasticity phenomenon). This heterogeneity of ovarian cancer stem cell phenotypes may complicate clinical applications of cancer stem cell theory in ovarian cancers. Cell signaling: Self-renewal and cell-fate determination in ovarian cancer stem cells is mediated by several developmental pathways, including Notch, Wnt/β-catenin, Hedgehog, and TGFβ cellular signaling pathways. These pathways, which are also found in normal stem cells, are aberrantly activated in cancer stem cells. For example, alteration of Notch pathway via over-expression of Notch3 gene is prevalent and associated with poor clinical outcome in high-grade serous ovarian cancers. Consequently, these pathways represent attractive targets for designing new anticancer agents effective against CSC. Stem cell niche: Normal adult stem cells are maintained in a quiescent state in stem cell niches - specialized tissue locations that provide microenvironment with stemnesssupporting cell-cell interactions, cell-extracellular matrix interactions, as well as interactions with various other factors such as growth factors, cytokines, metabolites and reduced oxygen tension. Certain events such as tissue injury trigger signals from stem cell niches that promote renewal and/or differentiation of adult stem cells to form new tissues. Although CSCs may be more autonomous than normal adult stem cells, and their self-renewal may be less dependent on stem cell niche, a growing body of evidence suggests that microenvironment can contribute to the maintenance of cancer stem cells. In ovarian cancers, hypoxia maintains ovarian cancer stem cell properties through the activation of hypoxia inducible factors (HIFs) and over-expression of embryonic stem cell markers Oct3/4 (POU5F1) and Sox2 known to play critical roles in self-renewal of embryonic stem cells. The role of microRNAs: miRNAs are small non-coding RNAs with ~22 nt size that regulate gene expression predominantly through decreased mRNA stability and/or inhibition of translation. Human genome encodes more than 2000 different miRNAs that modulate broad range of cellular processes including cell proliferation, differentiation, metabolism, stem cell phenotype and apoptosis. Aberrant expression of miRNAs correlates with various diseases and disease-associated miRNAs represent prospective diagnostic biomarkers and therapeutic targets. Several miRNAs are important regulators 4

of ovarian cancer stem cells. For example, miR-199a decreases expression of CD44 in CD44+/CD117+ ovarian cancer stem cells, reduces their tumorigenicity in animal model in vivo and enhances their sensitivity to anticancer drugs cisplatin, paclitaxel and doxorubicin. Similarly, miR-200a and let-7 suppress ovarian cancer stem cell phenotype via reduced migration and invasion of CD133-positive cancer stem cells and negative regulation of ALDH-positive cancer stem cells, respectively. In contrast, miRNA-27a and miR-503 are overexpressed in ALDH-positive ovarian cancer stem cells and correlate with tumor resistance to anticancer drugs, advanced clinical stage (miR-503) and distant metastasis (miR-27a). Drug resistance: Cancer stem cells share some properties of the normal stem cells, including relative quiescence and resistance to cytotoxic agents and ionizing radiation. Ovarian cancer stem cells isolated from cell lines or clinical specimens displayed resistance to traditional (cytotoxic) anticancer agents used in the first- or second-line treatment of ovarian cancers such as cisplatin, paclitaxel, docetaxel, topotecan, etoposide and epirubicin, but also to some experimental targeted therapeutics (e.g. inhibitors of NFκB and COX-2). The molecular mechanisms reported to play role in the drug resistance of ovarian CSCs include: (i) drug efflux via copper transporters ATP7A and ATP7B and ATP-binding cassette multidrug transporters ABCG2, ABCB1 and ABCA3, (ii) DNA repair (ERCC1), (iii) detoxification by cellular glutathione, and (iv) increased activity of antiapoptotic (BCL2, CLU, MET, BIRC5) and decreased activity of proapoptotic (BID, HRK) factors. Although the link between ovarian cancer stem cells and quiescence has been demonstrated, the role of quiescence in anticancer drug resistance of ovarian CSCs is not self-evident. Clinical significance of ovarian cancer stem cells Ovarian cancer is the most lethal of all gynecological cancers and the fifth most common malignancy in women in developed countries. It is estimated that in 2014 there will be nearly 22,000 new ovarian cancer cases and over 14,000 ovarian cancer-related deaths in the United States alone. Current treatment of advanced ovarian cancer, which includes debulking surgery and chemotherapy, is initially effective in the majority of patients; however, 70-90% of them eventually develops disease recurrence with limited treatment options. As a result, the 5-year relative survival for advanced stages III and IV (FIGO system) is only 39% and 17%, respectively. Since cancer stem cells are considered to be therapy-resistant cells (TRCs), they may remain as a minimal residual disease after the bulk of ovarian tumor mass has been removed by surgery and/or chemotherapy. These cells can repopulate the tumor and contribute to the disease recurrence, as well as cancer invasion and metastasis through epithelial-mesenchymal transition program. This concept is supported by the finding that primary tumor cells collected from patients immediately after chemotherapy for ovarian cancer displayed increased expression of CD133, CD44 and ALDH1A1 markers associated with ovarian cancer stem cell phenotype. Likewise, human ovarian cancer tissue transplanted into immunocompromised mice displayed increased expression of ovarian cancer stem cell markers CD117 and OCT4 upon treatment of animals with paclitaxel. Thus, conventional chemotherapy may not be able

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to eradicate this special sub-population of cancer cells and development of new therapeutic agents specifically targeting ovarian cancer stem cells are needed. Ovarian cancer stem cells, or their markers have been also associated with clinical outcome. Among patients with early stage epithelial ovarian cancers, those with > 20% CD44-positive cells tend to display poorer response to carboplatin-paclitaxel treatment and significantly shorter progression-free survival compared to patients with < 20% of CD44-positive ovarian cancer cells. Likewise, expression of CD133, ALDH1 or their combination in tumor cells was associated with significantly poorer disease outcome. Therapeutic targeting of ovarian cancer stem cells Several approaches have been implemented in order to find drugs that target ovarian cancer stem cells: (i) high-throughput screening (HTS) of chemical libraries to identify compounds inhibiting viability of cancer stem cell-enriched cell cultures, (ii) search for agents modulating signaling pathways involved in the regulation of CSC self-renewal and maintenance of CSC phenotype. e.g. Notch, Hedgehog and Wnt pathway. HTS identified several prospective ovarian CSC-targeting drugs that are currently used for different therapeutic purposes, including dactinomycin and mithramycin A (antineoplastic agents), mepacrine (antimalarial drug) and niclosamide (antihelmintic). Prospective agents modulating CSC-relevant signaling pathways include γ-secretase inhibitor I (prevents activation of Notch 3), cyclopamine and LDE225 (inhibit Smo component of Hedgehog pathway). Future ovarian cancer therapy will likely combine both: CSC-targeting agents to eliminate intrinsically resistant cancer stem cells, but also conventional anticancer drugs to eliminate more differentiated cancer cells, since they may be able to dedifferentiate and generate new cancer stem cells. Another promising approach targeting ovarian cancer stem cells is immunotherapy, e.g. therapy that employs dendritic cells loaded with antigens originating from patients' autologous ovarian cancer stem cells (Phase II clinical trial in 2014). Controversies Cancer stem cell hypothesis and the existence of cancer stem cells has been subjected to considerable criticism. Proponents of the CSC hypothesis suggest that this concept should guide the development of new anticancer therapies while dissenting scientists believe that cancer stem cells in solid tumors are just an artifact resulting from methods used to identify cancer initiating (tumorigenic) cells. The first and still most convincing study supporting the existence of CSCs relates to acute myeloid leukemia where only CD34+/CD38- displayed tumorigenicity in transplantation assays, ability to differentiate into leukemic blasts and resistance to anticancer drug daunorubicin. The criticism towards cancer stem cell hypothesis also stems from the disputes over terminology, inconsistencies in reported frequency among cancer cells, and inability to explain the cell-of-origin of cancer stem cells. Cancer stem cell hypothesis is not indispensable to explain observed heterogeneity of tumor cell populations. In addition, transplantation (xenograft) assays used for the identification of CSCs produce inconsistent results reflecting tumor host-related factors. For example, using less immunodeficient NOD.SCID mice, melanoma CSCs have been identified as rare tumor-initiating cells that 6

expressed ABCB5 gene, while using NSG mice with more severely impaired immune system identified CSC as a sizable (~28%) subpopulation of melanoma cells with no expression of ABCB5. Moreover, normal stem cells display potential to differentiate into multiple distinct cell types, while most reported CSCs did not demonstrate ability to differentiate into more than single cell type. Nevertheless, multilineage differentiation capacity has been shown in leukemia, colorectal carcinoma and ovarian cancer stem cells. The existence of cancer stem cells and their cell-of-origin is now strongly supported in cells associated with myelodysplastic syndrome (MDS). References 1. Visvader JE, Lindeman GJ (2012) Cancer stem cells: Current status and evolving complexities. Cancer Stem Cell 10:717-728 2. Ahmed N, Abubaker K, Findlay JK (2014) Ovarian cancer stem cells: Molecular concepts and relevance as therapeutic targets. Molecular Aspects of Medicine 39:110-125 3. Burgos-Ojeda D, Rueda BR, Buckanovich RJ (2012) Ovarian cancer stem cell markers: Prognostic and therapeutic implications. Cancer Letters 322:1-7 4. Pattabiraman DR, Weinberg RA (2014) Tackling the cancer stem cells - what challenges do they pose? Nature Reviews Drug Discovery 13:497-512 5. Shah MJ, Landen CN (2014) Ovarian cancer stem cells: Are they real and why are they important? Gynecologic Oncology 132:483-489

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Figure 1

Ovarian cancer stem cells isolated from OVCAR-3 cell line forming tumor spheres with up-regulated expression of CD44, CD133, ALDH1A1, CD117, SCA1, and SOX2 relative to parental OVCAR-3 cells.

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