Cancer stem cells and post-therapy tumour recurrence: a systematic review of mechanistic pathways and translational gaps

Cancer Stem Cell–Directed Therapies: Recent Data From the Laboratory and Clinic

Comparison of hematopoietic cancer stem cells with normal stem cells leads to discovery of novel differentially expressed SSRs

Moving Forward in Human Mammary Stem Cell Biology and Breast Cancer Prognostication Using ALDH1

1942P IL6 pre-treatment promotes chemosensitivity by eliminating quiescent cancer stem cells

Methylation of Cancer-Stem-Cell-Associated Wnt Target Genes Predicts Poor Prognosis in Colorectal Cancer Patients

We have used hTERT (the catalytic component of telomerase) to immortalize a variety of human cell types (sometimes in combination with Cdk4 to bypass cell culture stress). Cell types immortalized include skin keratinocytes and fibroblasts, muscle satellite cells, breast epithelial and stromal cells, corneal epithelial cells and fibroblasts (keratocytes), and human colonic epithelial cells. In addition, we have immortalized human bronchial epithelial cells (HBEC) and have determined that these cells can terminally differentiate into both central and peripheral lung cell types. These immortalized HBECs have been used to study the molecular pathogenesis of lung cancer by stable “knock down” of TP53 and also by over-expression of C-myc and mutant K-rasV12. When these cells containing multiple genetic alterations are introduced into immunosuppressed mice, the experimentally transformed cells make tumors that represent several distinct histological types. This suggests that these immortalized and transformed HBECs have bronchiolar-alveolar stem-like characteristics that can differentiate into multiple lineages. Stem cells are defined by both their ability to make more stem cells (self renewal) and their ability to produce cells that can differentiate. Experimentally immortalized human bronchial epithelial cells fulfill this definition of normal stem cells by continuous self renewal and by retaining the capability of differentiating into several cell types. Since experimentally transformed cells make lung tumors representing several major lineages, this is also an indication that the HBECs are derived from a multi-potent lung stem cell. Similar to normal stem cells, cancer (initiating) stem cells also have the ability to self-renew as well as undergo differentiation to give rise to phenotypically diverse types of cancer cells. There is mounting evidence that these rare cancer stem cells may be multidrug resistant and responsible for tumor relapse and metastasis. Targeted cancer therapeutic approaches seek to identify pathways that are more tumor specific, resulting in fewer side-effects and that may produce long-term durable responses. Telomerase is a novel cancer therapeutic target since it is activated in the vast majority of human cancers and telomeres of almost all human tumor cells are maintained at short but stable lengths. In addition, telomerase is not expressed or is expressed at levels that do not fully maintain telomeres in normal tissues, and telomeres are generally longer in normal stem cells compared to cancer cells. This potentially provides a therapeutic advantage for targeting telomerase over approaches that affect both normal and cancer cells equally. We have previously reported that telomerase positive cancer cells that are experimentally induced to undergo quiescence, down regulate telomerase. As part of our anti-telomerase therapeutics program, we have addressed the following questions: Are putative cancer stem cell populations quiescent and do they have short or long telomeres? While the molecular characteristics of cancer stem cells are not completely defined and subject to some controversies, we have isolated and examined cells expressing these cancer stem cell putative markers reported for breast, brain, prostate, pancreas, and lung cancer. In each case we have observed that purified cancer stem cell populations are positive for telomerase activity, indicating they are not quiescent. In addition, a telomerase inhibitor currently being tested in clinical trials robustly inhibits the activity of telomerase in these sorted sub-populations of putative cancer stem cells as well as the mass population of cancer cells. Finally, we have observed that cancer stem cells have short telomeres in comparison to normal stem cells. These findings support the idea that there may be a therapeutic window of opportunity to target cancer stem cells by inhibiting telomerase, thus driving telomeres progressively shorter leading to cancer stem cell death, potentially without irreversible damage to normal stem cells. Cancer remains a major cause of death in spite of substantial progress towards understanding the molecular basis of many types of cancers. The discovery of new drugs is a high priority, and telomerase inhibitors have the potential to act by a novel mechanism that will provide new options for cancer therapy. A review of ongoing anti-telomerase clinical trials will be presented. In summary, telomerase inhibitors might not only directly limit or stop the growth of human tumors including cancer stem cells, but might also act in an additive or synergistic fashion with existing therapies to amplify their effectiveness. Citation Format: Jerry W. Shay. Role of telomerase in normal and neoplastic stem cells [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY33-03

See related article in EMBO Molecular Medicine http://dx.doi.org/10.1002/emmm.201201516

The hypothesis that tumorigenic stem cells arises from the mutated stem cells suggests that cancer can be viewed as an aberrant organ initiated by tumorigenic cancer cells with stem-cell qualities. The tumor initiator cell or tumor stem cell/ cancer stem cell (CSC) is possible origins ofnormal stem cell or progenitor cells. The existence of such a CSC population may represent only a small fraction of a tumor possessing the ability to regenerate tumors through aberrant proliferation and self-renewal, but most cancer cells lack this ability. A central question in stem cell and CSC biology is, which cells can be transformed to generate tumors? With the growing evidence that CSC exist in malignancies of haematopoietic origin and in most solid tumors, it is becoming increasingly important to understand the molecular mechanisms that regulate self-renewal and differentiation because mutation of genes involved in these pathways likely participates in tumor growth. Studies of normal stem cell and CSC from the same tissue have suggested that the ontogeny of tumors. That signaling pathways such as Wnt, BMP and Notch have similar effects on normal stem cell and CSC self-renewal implys that common molecular pathways regulate both populations. Stem cell biology has provided a platform to address many questions in developmental biology. Further understanding the biology of stem cell, cancer cell, and CSC will contribute to the identification of molecular targets important for future therapies. To know the biology of CSC, we first should identify the unique characteristics of stem cells. A normal adult stem cell is defined as a somatic cell undergoing extensive cell division and has the potential to give rise to both stem cells and cells that differentiate into specialized cells. A normal stem cell must possess two qualities to play its natural function: self-renewal and differentiation. Self-renewal is an important ability of stem cells, indicating a special cell division that enables a stem cell to develop another stem cell with essentially the same development and replication potential. The capability to self-renewal causes the stem cell expansion in response to local or systemic signals, which start massive proliferation and maintenance of a tissue specific undifferentiated pool of cells in the organ or tissue. Differentiation is the second function of a stem cell and involves the development of daughter cells that become tissue specific specialized cells. Studies have demonstrated that the CSCs are likely to share many of the properties of normal stem cells, and that the CSCs are source of tumor recurrences and/or metastasis after apparently complete remissions. Although there is an argument about CSC model that may overlook the complexity of the interactions which occur within a tumor and between a tumor and its environment, evidence that many cancers are driven by CSC has important clinical implications. For example, conventional therapies may shrink tumors by killing mainly cells with limited proliferative potential, however, CSCs are less sensitive to these therapies, then they will remain viable after therapy and re-establish the tumor. Therefore, CSC model has favored the conceptual development of targeted therapies towards CSC’s “stemness”. However, how to achieve CSC eradication is emergent to overcome the major barrier. Here we describe latest therapeutic approaches based on the “stemness” of CSC that may allow us to discover the effective methods for eradicating seeds of malignant tumor cells. This includes targeted therapy directed toward CSC by surface specific markers, survival niche, blocking CSC critical signal pathways, inhibiting CSC efficient DNA repair, manipulation of ncRNAs, screening drug-resistance of CSC in three dimensional culture, CSC vaccines as well as CSC nanotheranostics.

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.

Poised with purpose: Cell plasticity enhances tumorigenicity

Replicator dynamics of cancer stem cell: Selection in the presence of differentiation and plasticity

Ovarian cancer is most lethal among gynecological cancers with often fatal consequences due to lack of effective biomarkers and relapse, which propels ovarian cancer research into unique directions to establish solid targeted therapeutics. "Ovarian stem cells" expressing germline pluripotent markers serve as novel paradigm with potential to address infertility, menopause, and probably influence tumor initiation. Cancer stem cells (CSCs) pose vital role in tumor recurrence and hence it is extremely important to study them with respect to ovarian stem cells across various cancer stages and normal ovaries. Pluripotent (OCT4, NANOG, SOX2, SSEA1, and SSEA4), germline (IFITM3, VASA/DDX4), and cancer stem (CD44, LGR5) cell specific markers were characterized for protein and mRNA expression in tumor tissues to understand their distribution in the surface epithelium and ovarian cortex in benign, borderline, and high-grade malignant stages. To elucidate whether pluripotent ovarian germline stem cells and CSCs are common subset of stem cells in tumor tissues, VASA was colocalized with known pluripotent stem (OCT4, SSEA1, SSEA4) and CSC (CD44, LGR5) specific markers by confocal microscopy. Single, smaller spherical (≤5 μm), and larger elliptical fibroblast like (≥10 μm) cells (also in clusters or multiples) were detected implying probable functional behavioral significance of cells in tumor initiation and metastasis across various cancer stages. Cells revealed characteristic staining pattern in ovarian surface epithelium (OSE) and cortex regions exclusive for each marker. Co-expression studies revealed specific subpopulations existing simultaneously in OSE and cortex and that a dynamic hierarchy of (cancer) stem cells with germline properties prevails in normal ovaries and cancer stages. Novel insights into CSC biology with respect to ovarian and germline stem cell perspective were obtained. Understanding molecular signatures and distribution within ovarian tissue may enable identification of precise tumor-initiating CSC populations and signaling pathways thus improving their efficient targeting and strategies to prevent their dissemination causing fatal relapse.

Cancer stem cells are proposed to initiate and maintain tumor growth. Dysregulation of normal stem cell signaling may lead to the generation of cancer stem cells (CSCs), however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional co-activator YAP1 through direct regulation of SOX9 promotes the generation of CSCs and that the inhibition of YAP1 and SOX9 attenuates CSC formation. SOX9 transcripts and expression are upregulated upon YAP1 activation and several lines of evidence indicate that SOX9 is a direct target of YAP1. The Chromatin Immunoprecipitation analysis and luciferase assays demonstrate direct binding of YAP1 to the SOX9 promoter through a conserved TEAD binding site. Mutation of this site abrogates transcriptional regulation of SOX9 by YAP1 and Tead2. Functional studies demonstrate that YAP1 regulation of SOX9 is necessary and sufficient to confer CSC properties and tumorigenesis in vitro and in vivo. The small molecule inhibitor of YAP1, Verteporfin (VP) significantly blocks CSC self-renewal properties in cells with high YAP1 and a high proportion of the CSC marker aldehyde dehydrogenase 1 (ALDH1) indicating that VP targets the CSC population. These data identify YAP1 as a driver of esophageal cancer (EC) stem cells, in part, by regulation of SOX9 and suggest that pharmacological inhibition of YAP1 may be an effective means of specifically targeting EC stem cells. Citation Format: Shumei Song, Jaffer A. Ajani, Soichiro Honjo, Dipen M. Maru, Qiongrong Chen, Jiankang Jin, Ailing W. Scott, Todd R. Heallen, Lianchun Xiao, Wayne L. Hofstetter, Brian Weston, Jeffrey H. Lee, Roopma Wadhwa, Kazuki Sudo, James F. Martin, John R. Stroehlein, Mien-Chie Hung, Randy L. Johnson. The Hippo coactivator YAP1 upregulates SOX9 and endows cancer stem cell properties in non-transformed cells and esophageal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3896. doi:10.1158/1538-7445.AM2014-3896

Introduction: Cancer stem cells (CSC) are characterized by stem cell properties such as self-renewal potential and multilineage differentiation capacity. CSC's are thought to be the main drivers of tumor formation and progression and the main cause of therapy resistance and metastatic spread. In contrast to other cancer types such as glioblastoma and colon cancer, attempts thus far have not resulted in the identification of a proven CSC population in esophageal cancer. Therefore in this study we aim to identify and characterize CSC's in esophageal adenocarcinoma (EAC). Methods and Results: To address this goal, we attempted to culture CSC's from primary untreated EAC biopsy material using serum-free medium formulations that, however, have not been successful thus far. As an alternative model we employed two EAC cell lines, OE19 and OE33, which we were able to maintain as spheroids in serum-free Neural Basal Medium thought to enrich for stem cell growth. Dissociation of spheres and limiting dilution assays showed enhanced spheroid forming potential in OE19 but not in OE33 when compared to cells obtained from normal 10% FCS monolayer cultured counterparts. Next, the expression of several known CSC markers was determined by FACS analysis to determine self-renewal potential of sorted fractions in subsequent limited dilution assays. Side population, ALDH1, CD44/CD24 fractions were tested of which only CD44+ /CD24+ cells showed enhanced capacity to form spheroids. Tumor growths of these populations are currently being evaluated on immune-compromised mice. Other stem cell markers such as OCT4 and SOX2 were enhanced in OE19 spheroids compared to 10% FCS cultured cells. However, in OE33 spheroids expression of these markers were unchanged or downregulated. Since EAC derives from dysplastic epithelia with intestinal characteristics in Barrett's esophagus, we examined the status of the Wnt pathway known to be involved in maintenance of intestinal/ colon (cancer) stem cells. In the EAC cells β-catenin expression was present that could be enhanced by LiCl. Reporter assays to determine Wnt- activity are in progress. Finally, the sensitivity of spheroid versus monolayer cultured OE33 cells to 5-FU, cisplatin and paclitaxel was evaluated. Sensitivity did not differ much, although monolayer cultured OE33 cells were more resistant for 5-FU. Conclusion: Given the differences in the two spheroid models regarding spheroid forming potential, CSC-marker expression and chemosensitivity, caution is warranted when using esophageal adenocarcinoma cell line derived spheroids as model for CSC's. However, CD44 and CD24 might have a potential as CSC-markers in EAC, which is currently further corroborated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5360. doi:1538-7445.AM2012-5360

Gastric cancer (GC) stem cells play an important role in GC progression. Circular RNAs (circRNAs) act as microRNA (miRNA) sponges and inhibit the biological function of miRNAs in GC cytoplasm. MiRNAs also participate in GC progress. circ_0051246 was shown to be associated with miR-375 after analyzing GC microarray data GSE78091 and GSE83521. The oncoprotein Yes-associated protein 1 (YAP1) is targeted by miR-375 and can be inactivated via the Hippo tumor suppressor pathway. Due to insufficient research on circ_0051246, this study aimed to investigate its relationship with miR-375 and YAP1 in cancer stem cells (CSCs). SGC-7901 CSCs were used to establish knockdown/overexpression models of circ_0051246, miR-375, and YAP1. Malignant phenotypes of CSCs were assessed using Cell Counting Kit 8, colony/sphere formation, 5-Ethynyl-2'-deoxyuridine assay, flow cytometry, Transwell, and wound healing assays. To detect the interactions between circ_0051246, miR-375, and YAP1 in CSCs, a dual-luciferase reporter assay and fluorescence in situ hybridization were performed. In addition, 24 BALB/c nude mice were used to establish orthotopic xenograft tumor models. Four groups of mice were injected with CSCs (1 × 106 cells/100 µL) with circ_0051246 knockdown, miR-375 overexpression, or their respective control cells, and tumor progression and gene expression were observed by hematoxylin-eosin staining, immunohistochemistry. Western blot and quantitative real-time PCR were utilized to examine protein and gene expression, respectively. Circ_0051246 silencing reduced viability, promoted apoptosis, and inhibited proliferation, migration and invasion of CSCs. The functional effects of miR-375 mimics were comparable to those of circ_0051246 knockdown; however, the opposite was observed after miR-375 inhibitors treatment of CSCs. Furthermore, circ_0051246-overexpression antagonized the miR-375 mimics' effects on CSCs. Additionally, YAP1 overexpression promoted CSC features, such as self-renewal, migration, and invasion, inhibited apoptosis and E-cadherin levels, and upregulated the expression of N-cadherin, vimentin, YAP1, neurogenic locus notch homolog protein 1, and jagged canonical notch ligand 1. Conversely, YAP1-silenced produced the opposite effect. Moreover, miR-375 treatment antagonized the malignant effects of YAP1 overexpression in CSCs. Importantly, circ_0051246 knockdown and miR-375 activation suppressed CSC tumorigenicity in vivo. This study highlights the promotion of circ_0051246-miR-375-YAP1 axis activation in GC progression and provides a scientific basis for research on the molecular mechanism of CSCs.