Accepted Manuscript Title: Cancer stem cells: a challenging paradigm for designing targeted drug therapies Author: Ishaq N. Khan Saleh Al-Karim Roop S. Bora Adeel G. Chaudhary Kulvinder S. Saini PII: DOI: Reference:

S1359-6446(15)00260-3 http://dx.doi.org/doi:10.1016/j.drudis.2015.06.013 DRUDIS 1641

To appear in: Received date: Revised date: Accepted date:

18-2-2015 19-6-2015 24-6-2015

Please cite this article as: Khan, I.N., Al-Karim, S., Bora, R.S., Chaudhary, A.G., Saini, K.S.,Cancer stem cells: a challenging paradigm for designing targeted drug therapies, Drug Discovery Today (2015), http://dx.doi.org/10.1016/j.drudis.2015.06.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research highlights  New drug discovery and molecules in clinical development against cancer stem cells  Surface marker, transcription factors and signal transduction pathways in CSCs  RNAi, miRNA, biologics, vaccines, etc for targeting therapeutics against CSCs

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 Plasticity model remains the most attractive hypothesis for designing targeted therapies

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Cancer stem cells: a challenging paradigm for designing targeted drug therapies Ishaq N. Khan1,2,3, Saleh Al-Karim1,4, Roop S. Bora1,5,6, Adeel G. Chaudhary2,3, and Kulvinder S. Saini1,5,6 1

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Embryonic & Cancer Stem Cell Research Group, Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah-21589, Saudi Arabia 2 Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah-21589, Saudi Arabia 3 Centre of Innovation for Personalized Medicine, King Abdulaziz University, Jeddah-21589, Saudi Arabia 4 Embryonic Stem Cell Unit, King Fahd Medical Research Centre, King Abdulaziz University, Jeddah-21589, Saudi Arabia 5 Biotechnology Research Group, Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah-21589, Saudi Arabia 6 School of Biotechnology, Eternal University, Baru Sahib-173 101, Himachal Pradesh, India

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Corresponding author: Saini, K.S. ([email protected]; [email protected])

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Keywords: CSCs; ESCs; new drug discovery; drug resistance; miRNA; RNAi. Teaser: Recently acquired knowledge about the survival of CSCs during chemo- and radiation therapy have resulted in renewed interest from the pharmaceutical industry to target new drug discovery efforts against key gene targets.

Introduction

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Despite earlier controversies about their role and existence within tumors, cancer stem cells (CSCs) are now emerging as a plausible target for new drug discovery. Research and development (R&D) efforts are being directed against key gene(s) driving initiation, growth, and metastatic pathways in CSCs and the tumor microenvironment (TME). However, the niche signals that enable these pluripotent CSCs to evade radio- and chemotherapy, and to travel to secondary tissues remain enigmatic. Small-molecule drugs, biologics, miRNA, RNA interference (RNAi), and vaccines, among others, are under active investigation. Here, we examine the feasibility of leveraging current knowhow of the molecular biology of CSCs and their cellular milieu to design futuristic, targeted drugs with potentially lower toxicity that can override the multiple drug-resistance issues currently observed with existing therapeutics.

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CSCs or cancer-initiating cells (CICs) are cells localized within solid tumors, or in hematological cancers, that have traits associated with normal stem cells. CSCs are tumorigenic and have the potential to give rise to all cell types found in a particular cancer. They have the inherent ability to generate tumors through the stem cell processes of self-renewal and differentiation. These cells can survive and usually persist in tumors for a substantial length of time as a distinct population, and can eventually cause relapse and metastasis by generating new tumors [1–9]. Several studies have suggested that CSCs exist only as small subpopulations, sometimes as high as 25%, [LM1]within tumors and are usually found in slow-growing cells [10,11]. Other studies have indicated that, within a tumor, as little as 0.01–1% of human cancer cells are able to develop into tumors in immunodeficient mice [12]. Given their ability to develop new tumors, and evade chemo- and/or radiotherapy, CSCs were implicated in the exacerbation of tumor metastatic potential and subsequent cancer recurrence [13–16]. CSCs further have the ability to migrate and undergo rapid clonal proliferation [3,5,17,18] and differ from normal cancer cells in the expression of key cancer-specific markers, which are primarily located in hypoxic regions and subject to modulation by niche signal(s) [2,5,6,8,19,20]. These cells exhibit phenotypic and functional heterogeneity, which arises through their clonal evolution [1,4–6,9,18,21]. The origin of CSCs is still not clear and various theories have been proposed to explain their origin (Figure 1). According to one school of thought, CSCs are generated by a mutation in a stem cell niche population during development. It has been postulated that developing stem cells are mutated and undergo further expansion resulting in daughter stem cells harboring the same mutation(s). These daughter stem cells have the potential to become a tumor. Another hypothesis associates adult stem cells with the generation of CSCs and appears to overlook the contribution of differentiated cells towards the development of malignancy. In tissues such as skin and gut, which have a high rate of cell turnover, it has been speculated that adult stem cells are responsible for tumor formation. The long lifespan of adult stem cells along with frequent cell division provides the ideal environment for the accumulation of mutations that leads to cancer development. A third theory postulates that the dedifferentiation of mutated cells might help them acquire stem cell-like characteristics after the availability of ‘desirable niche’ and favorable environment[LM2], possibly as a consequence of aberrant activation of the epithelial-mesenchymal transition (EMT) [11,22,23].

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There are two forms of CSCs: stationary CSCs (SCS) and migrating CSCs (MCS). SCS cells are embedded in carcinomas and persist in the differentiated central areas of tumors. MCS are cells within the tumor that have acquired the ability to spread and migrate to distant secondary sites and promote metastasis. During tumor progression, distinct CSCs (CSC*) can originate as a result of additional mutations or epigenetic modifications. Some of these new CSCs can undergo the EMT, retaining stem cell characteristics and giving rise to mCSCs, which provide the ‘seeds of metastasis’. There is further evidence to support the hypothesis that mCSCs are derived from SCS, because induction of EMT in the SCSs converts these cells into mobile CSCs or mCSCs (Figure 2) [3,5,6,24,25]. In addition, the notion that EMT induces the CSC phenotype might provide a potential mechanistic basis for therapeutics resistance, metastasis, tumor dormancy, and delayed relapse [23]. From the new drug discovery point of view, the plasticity model remains the most attractive hypothesis to leverage current understanding of the molecular basis of tumorigenesis and CSC biology for designing targeted therapies against various malignancies. Molecular biomarkers of CSCs

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CSCs have now been reported in most human tumors, including those in breast, brain, colon, ovary, pancreas, and prostate, and melanoma and multiple myeloma [14,26–29]. To isolate and identify CSCs from solid and hematological tumors, biomarkers that are specific for normal stem cells of the same organ(s) can be deployed. Different cell surface markers that are commonly used for isolating and characterizing CSCs, such as CD133, CD44, CD24, epithelial cell adhesion molecule (EpCAM), THY1, and ATP-binding cassette subfamily B member 5 (ABCB5), are outlined in Table 1.

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CSCs were initially isolated and characterized from breast cancer. Work from different laboratories identified key biomarkers for breast CSCs, including CD44, Serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5), Topoisomerase (DNA) II Alpha (TOP2A), Cytokeratin 5 (CK5), Tumor protein p63 (TP63), SRY (sex determining region Y)-box 4 (SOX4), CD24, Adhesion regulating molecule 1 (ADRM1), Delta and Notch-like epidermal growth factor-related receptor (DNER), Delta-like 1 (DLL1), Jagged 1 (JAG1), and aldehyde dehydrogenase (ALDH) [30–33]. Additional evidence suggests that breast CSCs are phenotypically diverse and that the expression of CSC markers in breast cancer is heterogeneous, with evidence for the presence of many subsets of breast CSCs [34]. In brain tumors, stem-like tumor cells have been identified using cell surface markers, such as CD133, CD44, stage-specific embryonic antigen-1 (SSEA-1) and epidermal growth factor receptor (EGFR) [35–38]. For the identification of CSCs in human colon cancer, cell surface markers such as CD133, CD44, and ABCB5 [39– 41] have been exploited. In addition, the AC133 epitope is specifically expressed in colon CSCs and seems to have lower expression after differentiation [42]. Biomarkers for lung CSCs have been identified that include CD133, ALDH, CD90, CD44, CD13, EpCAM, and oncofetal protein 5T4 [13,43–46].

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Relation between embryonic stem cells and CSCs

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The concept of CSCs in solid tumors was initially described in 1952 by a Japanese pathologist Yoshida[LM3], as outlined by Tanaka [47]. In 1970, an experiment for tracing the lineage of ESCs from early-stage embryos supported the concept of CSCs [48]. Then, four decades later, the most significant and comprehensive lineagetracing studies [49,50] showed that CSCs have the ability to develop and sustain tumor growth just like embryonic stem cells (ESCs). In addition, CSCs [LM4]have the ability to develop into blastocysts and provide sustenance for fetal growth. CSCs exhibit similarities with, and are closely related to, ESCs with respect to various genetic and biochemical parameters, such as their mechanism for self-renewal, the generation of tumor cells from normal somatic cells and different types of stem cell, the impact of the niche for differentiation, and their ability of proliferation and migration. The relation between ESCs and CSCs can be interpreted from different perspectives; for example, they have common transcription factors (e.g., Sox2, Octamer 4 (Oct4), and Nanog), similar putative surface markers (Nestin, CD133, etc.), and ESC-associated pathways are activated in CSCs [51]. Table 1 lists key transcription factors [2,6,8,25,52–66], surface markers [6–8,11,18,19,25,52,53,59,60,67–72], and signaling pathways [1,4,6,8,11,17,18,52,58,60,71,73] that are common in ESCs and CSCs. However, CSCs in glioblastoma multiforme (GBM) and melanomas express Nestin, whereas Bmi-1 is expressed in CSCs of breast and prostate cancers, and neuroblastomas, and leukemias [19]. In addition, there are other surface markers that are unique to the ESCs, including CD31 (PECAM-1), CD49f (Integrin α6/CD29) and CD326 [52]. Another recent study reported that Doublecortin-like kinase 1 (DCLK1) is a specific marker for the identification of a functionally rare subpopulation of CSCs in intestinal and pancreatic tumors [74,75]. Taken together, these studies highlight the novelty of biochemical markers and pathways operating within CSCs and that are often unique to individual cancers.

TME and CSC metastasis

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The TME has a crucial rule in tumor progression and metastasis. There are various types of stromal cells, such as tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and endothelial cells, which are usually in direct contact with tumor cells and help these cells to maintain their stemness, thereby facilitating tumor progression [73]. The cellular and molecular factors that have important roles in mediating communication between tumor cells and TME include cytokines [e.g., interleukin (IL)-6, IL-8, transforming growth factor (TGF)-β, and oncostatin M (OSM)] [76–78], chemokines [e.g., CXC chemokine receptor 1 and 4 (CXCR1 and CXCR4)] [79,80], and growth factors, such as EGF and b-FGF (basic fibroblast growth factor) [81], which are crucial for tumor growth and survival. It has also been shown that IL-6, IL-8, and OSM, which are secreted by TAMs and CAFs, are responsible for the stemness ability of tumor cells. In various human carcinomas, a correlation has been demonstrated between the induction of EMT with enhanced secretion of multiple cytokines, chemokines, and growth factors [76–78]. Other factors involved in the TME include angiogenic factors, vasculogenic factors, and hypoxia inducible factors (HIFs), which have synergistic roles in tumor progression and metastasis [73].

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Extracellular matrix (ECM) remodeling and hardening have a vital role in tumor progression by modulating the tissue architecture and TME. Pioneer studies in breast cancer have shown that the induction of collagen crosslinking leads to ECM stiffening, enhanced focal adhesions, and metastasis [82]. In addition, enhanced tissue stiffness observed during increased collagen deposition resulted in tremendous endothelial cell proliferation and the formation of new blood vessels [82,83]. A metastatic role of CSCs has been reported in studies of diverse types of cancers, including pancreatic cancer, breast cancer, and colorectal cancer [84–86]. The metastasis of CSCs from the primary site to a distant and distinct part of the body involves a series of mechanistic alterations, called the ‘invasion-metastasis cascade’, which includes: invasion, angiogenesis, intravasation, dissemination in blood, extravasation, and colonization. It has been proposed recently that, in most cases, these metastatic colonies remain dormant and might re-emerge after a lag phase, resulting in tumor recurrence [87]. Molecular signaling in CSCs

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To maintain stemness, CSCs are likely to require multiple signaling pathways that include: EpCAM, Sonic Hedgehog (SHH), Wnt/β-catenin, spalt-like transcription factor 4 (SALL4), TGF-β, Neurogenic locus Notch homolog protein (Notch), IL-6, and receptor tyrosine kinases (RTK). These signal transduction pathways often have crucial roles in the self-renewal and pluripotency of stem cell phenotypes, and, thus, regulate genes that are instrumental in the formation of CSCs [13,73,88]. CSCs exhibit many characteristics of ESCs or adult tissue stem cells because they tend to retain activation of few key and highly conserved signaling pathways involved in growth and tissues homeostasis, such as Wnt, Notch, HH, and phosphatidylinositol 3'-kinase (PI3K)/Akt signaling pathways [11,47]. Table 2 outlines current new drug discovery efforts against these key signal transduction pathways [11,47,73,89–94]. Wnt signal transduction has been found to be responsible for the maintenance of the CSC population in several types of cancer [11], including gastric cancer [95], colon cancer [96,97] and lung cancer [98]. The Notch signaling pathway has been implicated in CSC progression, particularly during the initiation of the EMT phenotype. In brain and breast tumors, the Notch pathway is currently an attractive target for new drug discovery R&D [23,91]. SHH signal transduction has an important role in the regulation of self-renewal aspects of stem cells and in embryonic growth, and is implicated in EMT. Similar to the Wnt and Notch pathways, the SHH pathway has also been found to be significant in CSC biology [11,47], and is an essential component in the maintenance of CSCs in gastric [99], colon [100], and pancreatic [101] cancers. In addition to these highly conserved pathways, the PI3K/Akt pathway has been shown to have a significant role in the self-renewal potential of normal stem cells. The preferential activation of the PI3K/Akt signaling pathway was recently reported in the CSCs of brain and colon [47,73]. Therapies targeting CSCs

Current treatments for cancer have severe limitations primarily because of the development of resistance over time to chemo- and radiotherapy. Several studies over the past decade have conclusively demonstrated that CSCs are more resistant to current cancer therapies compared with their nonCSCs counterparts. Recent therapeutic evidence points to the fact that the elimination of CSCs should be the ultimate goal for improving the safety and efficacy of any current cancer drug, thereby improving the chances of overall recovery and quality of life of the patient [49,102,103]. Several strategies are being devised to not only specifically eliminate CSCs, but also manipulate the microenvironment, or the microniches, that support CSC proliferation. Desirable therapeutic targets for eliminating CSC have been identified and characterized. Key gene targets can be found in the Wnt/β-catenin, HH, EGFR, and Notch pathways, the ABC superfamily, anti-apoptotic factors, DNA repair enzymes, detoxifying enzymes, and other cell surface markers [49]. Some of the possible therapeutic interventions for CSCs are highlighted in Box 1.

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Cell surface proteins, such as CD44, IL-3R,and T-cell immunoglobulin domain and mucin domain 3 (TIM-3), which are differentially expressed CSCs, have been exploited to target leukemia stem cells in human acute myeloid leukemia (AML). These studies have shown that monoclonal antibodies targeting these cell surface proteins were effective in eliminating CSCs in mice [104–106]. Gemtuzumab ozogamicin, a humanized anti-CD33 mouse monoclonal antibody conjugated to the cytotoxic drug calicheamicin, was generated to target CD33, which is overexpressed in AML, and is currently being used to treat patients with AML [107]. Expression of a cell surface molecule, CD133, is upregulated in several types of CSC, such as glioma, breast cancer, lung cancer, colon cancer, and prostate cancer, and this enhanced CD133 expression correlates with a high risk of mortality in patients with cancer [108,109]. Wang and colleagues utilized an anti-CD-133 monoclonal antibody conjugated with carbon nanotubes to selectively target CD133+ CSCs in glioblastoma, and subsequent irradiation with near-infrared laser light resulted in specific targeting and eradication of CD133+ cells without affecting CD133– cells [110]. In addition, the tumorigenic and self-renewal potential of the treated CD133+ CSCs were significantly inhibited in vitro, and these cells failed to induce tumor growth in nude mice [110]. This approach of targeting CD133+ CSC could be a promising strategy for treating human cancers, but requires further clinical validation.

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The therapeutic antibody trastuzumab (i.e., Herceptin™) has effectively increased the survival of patients with human epidermal growth factor receptor 2 (HER2)+ breast cancer; however, disease relapse has been observed in more than 70% of treated patients. This has now been attributed to the presence of CD44highCD24low breast CSCs, which are resistant to radiotherapy and chemotherapy, and cannot be targeted by trastuzumab efficiently because of the low expression of HER2 in breast CSCs. Diessner et al. tested an innovative approach to specifically target HER2+ breast cancers using an antibody-–drug conjugate (T-DM1), which comprised the potent chemotherapeutic drug DM1 coupled to trastuzumab. Their study revealed that CD44highCD24low breast CSCs were effectively depleted after treatment with T-DM1, and their colony formation ability was suppressed. It was also observed that co-culturing of treated tumor cells with natural killer cells dramatically enhanced the antibody-dependent cellmediated cytotoxicity and inhibited the EMT-induced stem cell-like properties of differentiated tumor cells [111]. Targeted alteration of individual cytokine signaling molecules and surface markers in solid tumors and CSCs might be a better therapeutic strategy, because it aims to block the transition switch of tumor cells from an epithelial to mesenchymal phenotype. MiRNA targeting CSCs

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MiRNA are 19–24 nucleotide (nt)-long noncoding RNAs (ncRNAs) that inhibit translation of mRNA by binding to the 3 untranslated region (3UTR) of its target mRNA. These ncRNAs are initially transcribed in the nucleus as large molecules called pri-miRNAs, which are processed by Drosha to yield an approximately 70 nt-long transcript called pre-miRNA, which has the capacity to form a stem-loop structure. The pre-miRNA is then transported by Exportin-5 to the cytoplasm and further processed into a 19–24-nt long transcript by the enzymatic action of Dicer. These duplex miRNAs bind to a protein complex known as the RNA induced silencing complex (RISC), where one of the miRNA strands is selected that normally guides this complex to the 3UTR of the target mRNA. If the miRNA and target mRNA show perfect complementarity, it leads to the degradation of target mRNA; however, imperfect base pairing inhibits translation of the mRNA into protein [112,113]. Several studies have confirmed that miRNA has a crucial role in gene regulation, because each miRNA appears to regulate the translation of multiple genes and many genes appear to be regulated by multiple miRNAs [113]. MiRNAs have a significant role in a variety of biological processes, particularly the regulation of cell proliferation, apoptosis, developmental timing, stem cell selfrenewal and differentiation, aging, DNA methylation, and chromatin remodeling [113]. MiRNA deregulation has been observed in several diseases, including cancer. Genome-wide analysis using microarrays, RNA-seq, and nextgeneration sequencing has revealed the aberrant expression of miRNA in all types of human cancer [114,115].

Key miRNAs are now known to have vital roles in cancer development and progression because they can function as oncomirs, or tumor suppressor miRNAs, in human malignancies. The role of miRNAs in cancer was first discovered in chronic lymphocytic leukaemia (CLL), wherein the miRNA cluster, miR-15a-miR-16-1 was deleted or downregulated in 69% of patients [116]. Further studies confirmed that miR-15a-miR-16-1 acts as a tumor suppressor and its deletion in patients with CLL leads to cancer development. Several other tumor suppressor miRNAs, such as the miR-34 and let-7 family, were identified and characterized [117]. These tumor suppressor miRNAs perform their activity by targeting and inhibiting the expression of oncogenic proteins [118]. The miR-15a-miR-16-1 cluster further inhibits the expression of the gene encoding the anti-apoptotic protein B-cell CLL/lymphoma 2 (BCL2), and induces expression of myeloid leukaemia cell differentiation protein (MCL1). Similarly, let-7 was found to downregulate the expression of the oncogenes Kras and myc, and miR-34 regulates the p53 pathway by targeting cyclin-dependent kinase 4, myc, and c-Met [119]. In addition, several miRNAs, such as the miR-21, miR-155, and miR-17-miR-92 clusters, were found to display oncogenic properties and are upregulated in various cancer types [120]. Several studies have demonstrated that miR-21 overexpression causes B cell lymphoma in mice, induces lung carcinogenesis by activating the RAS–Mitogen-activated protein kinase kinase

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(MEK)–Extracellular signal-regulated kinase (ERK) pathway, and promotes metastasis of breast cancer by inhibiting programmed cell death protein 4 [121–123]. Several studies have now clearly demonstrated that CSCs have a distinct expression profile of key miRNAs. Yu et al. analyzed the miRNA expression profile in self-renewing breast CSCs, differentiated cells from breast cancer cell lines, and primary breast tumors. They observed that miRNA let-7 was downregulated in self-renewing, tumorinitiating cells compared with nontumorigenic cancer cells [124]. However, the expression of miRNA let-7 was upregulated once the cells had differentiated into nontumorigenic cancer cells. The let-7 family of miRNAs acts as tumor-suppressive miRNAs and regulates the expression of the ras oncogene and the gene encoding high mobility group AT-hook 2 (HMGA2) involved in mesenchymal cell differentiation and tumorigenesis. Lentiviral-mediated overexpression of let-7 was shown to inhibit cell proliferation and also drastically reduced the number of undifferentiated, self-renewing stem cells in vitro. Moreover, let-7 overexpression resulted in reduced tumorigenic and metastatic potential in NOD/SCID mice in vivo [124]. Given that miRNA let-7 is a common tumor suppressor and its downregulation has been reported in several cancers, it is likely that let-7 is one of the key regulators of multiple stem cell-like features of breast CSCs and, hence, has tremendous potential as a therapeutic target for cancer treatment [124–127].

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The expression of the miR-30 family was also found to be downregulated in tumor-initiating breast CSCs [124]. Overexpression of miR-30e in breast CSCs impaired their self-renewal potential by inhibiting expression of ubiquitin-conjugating enzyme 9 (Ubc9) and integrin 3 (ITGB3) and resulted in the induction of apoptosis. Overexpression of miR-30e also prevented breast cancer progression and lung metastases in NOD/SCID mice. By contrast, inhibition of miR-30e expression resulted in enhancement of the self-renewal capacity of CSCs, tumor formation, and metastasis. These studies suggest that, by manipulating miR-30 expression, the stem-like properties of breast CSCs can be controlled by targeted therapeutic intervention [128]. Zhu and colleagues demonstrated that expression of miR-128 was significantly downregulated in CSCs isolated from patients with primary breast cancer, subsequently resulting in increased expression levels of target proteins of miR-128, such as polycomb ring finger, Bmi-1 and ATP-binding cassette subfamily C member 5 (ABCC5). Furthermore, reduced expression of miR-128 in breast tumors was found to be associated with chemoresistance and poor survival of patients with cancer [129]. Overexpression of miR-128 in breast CSCs combined with doxorubicin treatment led to the downregulation of Bmi-1 and ABCC5 levels, which resulted in enhanced apoptosis [129]. Taken together, these studies highlight the therapeutic potential of miR-128 for systematic and efficient targeting of CSCs.

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The miR-34c is another important tumor suppressor that has been shown to inhibit cell proliferation and metastasis [130]. Expression of miR-34c was significantly reduced in human breast cancer cell lines, enriched for Breast cancer suppressor candidate (BCSC)[LM5], which led to the increased self-renewal potential and EMT of these cells. Overexpression of miR-34c resulted in downregulation of its target gene Notch4 and inhibited EMT, and the self-renewal capacity and metastatic potential of the cells [130]. Bu and colleagues demonstrated that high levels of miR-34a expression inhibited Notch signaling and stimulated daughter cells to become non-colon CSCs, whereas low expression levels of miR-34a induced Notch signaling, which directed daughter cells to remain as colon CSCs [131]. This study suggests that miR-34a maintains a crucial homeostasis between self-renewing stem cells versus differentiated cells. Manipulation of such functional aspects of miRNA turnover and metabolism in CSCs could represent a novel therapeutic strategy for cancer treatment in the near future. Exploiting RNAi for targeting CSCs

Gene silencing using RNAi has tremendous potential to knock down specifically and efficiently the expression of unwanted oncogenes in various cell types without causing significant adverse effects. Several studies have indicated that small interfering (si)-RNA can be used for inhibiting the expression of target genes in human hematopoietic stem cells (HSCs). Scherr et al. showed that the lentiviral vector-mediated transfer of short hairpin (sh)-RNA into HSC can lead to long-term gene silencing of hematopoietic-specific genes. They demonstrated that shRNA targeting the human receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF; beta-GMR), which have a crucial role in the regulation of cell proliferation, survival, and differentiation of hematopoietic cells, resulted in marked decrease in expression levels of beta-GMR and inhibited the functional activity of the receptor. In addition, lentiviral-mediated transfer of beta-GMR-specific shRNA into primary normal CD34+ cells selectively inhibited the colony formation of transduced progenitors cells when stimulated with GM-CSF and/or IL-3 [132]. This study indicated that lentiviral-mediated transfer of shRNA can be exploited to induce long-term gene silencing in human hematopoietic stem and progenitor cells for potential therapeutic intervention. Treatment of CML with imatinib, a tyrosine kinase inhibitor, does not eradicate leukemic stem cells completely, thus increasing the possibility of early relapses. It has been revealed that Abelson helper integration site-1 (Ahi-1) is highly upregulated in leukemic stem cells that also have elevated levels of bcr-abl. It was observed that overexpression of Ahi-1 in hematopoietic cells promoted cell proliferation in vitro and caused leukaemia in vivo in a murine model. In addition, expression of Ahi-1 was able to reverse the effect of bcr-abl downregulation, and was also found to be involved in persistent phosphorylation of bcr-abl and activation of Janus kinase 2 (JAK2)-Signal

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transducer and activator of transcription 5 (STAT5) [133]. RNAi-mediated silencing of Ahi-1 in bcr-abl-transduced line (–) CD34 (+) cord blood cells and primary CML stem cells resulted in the inhibition of cell proliferation. This study clearly highlights that Ahi-1 is a potential target in CML and silencing of Ahi-1 gene expression using RNAi can be exploited as a therapeutic for CML. GBM is the most common human brain tumor and is generally nonresponsive to drugs. It has been reported that a relatively small subset of glioma stem cells (GSCs) residing within the tumor might be responsible for tumor growth and subsequent drug resistance. It was observed that currently used cancer drugs and radiation therapy primarily target CD133– cells, but can not eliminate the CD133+ population [134], with these latter cells having stem cell properties [135]. These studies indicate that current therapies might successfully eliminate only differentiated cells, but are unable to target effectively a minor population of GSCs, which can lead to tumor recurrence. Hence, it is necessary to devise and examine new therapeutic strategies that can effectively target and eliminate GSCs. The Notch signaling pathway has an important role in maintaining a balance between proliferation and apoptosis. The pathway is deregulated in CSCs, which results in generation of tumors because of the expansion of CSCs (Table 2). Wang et al. demonstrated that introduction of Notch-1-specific siRNA in GSCs resulted in a drastic decrease in Notch-1 expression levels and inhibited the proliferation of GSC in vitro [136]. Moreover, GSCs with silenced Notch-1 exhibited a dramatic reduction in oncogenicity when xenografted into Balb/c nude mice. These results suggest that siRNA-mediated silencing of Notch-1 can be further exploited as a new therapeutic modality to treat glioma. Elimination of GSCs is vital to achieve long-lasting treatment for glioma and prevent tumor recurrence.

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Aldehyde dehydrogenase-1A1 (ALDH1A1) is an important marker of CSCs in various malignant and nonmalignant tumors. ALDH1A1 expression has been detected in several cancers, including breast, colon, pancreatic, lung, and liver [137–141]. ALDH1A1 is involved in the oxidation of aldehydes, conversion of retinol to retinoic acid, and the regulation of differentiation pathways. ALDH1A1 is considered to be an important marker of stemness and has a crucial role in the initiation of tumor formation [142], because ALDH1A1+ cells exhibited enhanced invasive properties compared with ALDH1A1– cells. In addition, expression of ALDH1A1 is associated with chemoresistance in patients with cancer and reduced chances of survival [137,143,144]. From these studies, it has become clear that ALDH1A1 mediates platinum resistance in ovarian cancer by modulating the regulation of cell cycle checkpoints and DNA repair pathways [144]. Landen et al. evaluated the potential of targeting ALDH1A1 expression to sensitize resistant tumor cells to chemotherapy and, hence, develop a new strategy to effectively eradicate CSCs. The authors demonstrated that inhibition of ALDH1A1 expression using ALDH1A1-specific siRNA encapsulated in nanoliposomal particles, effectively sensitized taxane- and platinum-resistant cell lines to chemotherapy and resulted in reduced tumor growth in a mouse model of ovarian cancer. Reduction in tumor growth was found to be significant compared with chemotherapy alone [145]. Taken together, these preclinical data sets suggest that targeting CSCs using RNAi-mediated gene silencing could be an effective approach to target CSCs and might reduce the probability of recurrence and, thus, improve the survival rate of patients with cancer.

Establishing proof-of-concept in human studies

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The discovery and development of targeted therapeutic drugs, particularly new chemical entities (NCEs) and new biological entities (NBEs) against CSCs remains a challenge, although few molecules have completed phase II human clinical studies and are moving into phase III, thereby establishing the proof-of-concept (PoC) and safety and efficacy of these new and novel approaches. As shown in Table 3 [146,147], and also outlined in a recent commentary [148], it is heartening to see the progress of small-molecule drugs against specific gene targets of CSCs over the past 5 years. Verastem Inc. is testing VS-6063, a small molecule that inhibits focal adhesion kinase (FAK), in phase II trials in patients with lung cancer and/or mesothelioma. Inhibition of FAK, an enzyme involved in cellular adhesion and cell mobility, is believed to kill CSCs directly, thereby preventing metastasis. OncoMed Pharmaceuticals Inc. is developing tarextumab, a fully humanized monoclonal antibody that targets Notch 2 and Notch receptors. Tarextumab exhibits anti-CSC effects by inhibiting the Notch pathway signaling and also affects the stromal microenvironment and TME. In a Phase Ib study that combined tarextumab with conventional drugs for pancreatic cancer, 83% of the tumors of 29 patients were stable or shrank significantly during trial periods of 8 weeks to 1 year. Biomarker analyses further revealed that those patients whose tumor samples had elevated levels of Notch3 gene expression, showed better response and longer survival compared with patients with low Notch3 expression. Oncomed has recently initiated phase II clinical trials for tarextumab in patients with pancreatic and/or lung cancer. Genus Oncology is targeting Mucin 1 (MUC-1), an oncoprotein that has a pivotal role in tumor progression in AML, multiple myeloma, colon cancer, non-small cell lung carcinoma and breast cancer [149–152]. The company has recently moved its drug candidate, GO-203-2c, into phase Ib/IIa clinical development. Earlier in preclinical studies, GO-203-2c was shown to be two to three times more sensitive towards leukaemia blast and stem cells, compared with tumor cells [147]. These encouraging clinical data sets seem to represent a new surge of interest from academic labs and pharmaceutical and/or biotech companies in targeting tumor-specific CSCs and also examining the feasibility of

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finding appropriate diagnostic and prognostic markers. Another key point is the success of combo therapy, where a small-molecule drug is mixed with a biologic molecule for targeting CSCs. Clearly, recent data from the cancer genome initiatives involving next-generation sequencing technologies, better bio- and chemoinformatic approaches coupled with the gene expression signatures of CSCs are helping researchers design better, safer, and more efficacious drugs against a variety of cancers. Concluding remarks and prospects

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Over the past 25 years, we have learned that the complex interplay of genetic diversity, epigenetic mechanisms, and the TME contribute to tumor heterogeneity and drug resistance, thereby rendering current therapies unable to put a dent in disease progression and overall patient survival. The six ‘hallmarks of cancer’ plus two enabling characteristics constitute well-programmed and systematically orchestrated phenomena for delineating the complexity of neoplastic disease progression [153]. Over the past decade, these guiding principles of genetic regulation of tumor progression and metastasis have coalesced with recently acquired understanding of CSC biology, thus making the dynamics of tumor homeostasis amenable to better-targeted therapies. These latest developments are now helping unravel the mystery of how CSCs might evade radiation and chemotherapy. Clearly, the role of TME in cell–cell communication among tumor cells and in providing shelter to CSCs during chemo- and radiotherapy requires further investigation. How certain growth factors, such as FGF-2, provide selective growth advantage to CSCs or neighboring tumor cells and, thus, may be themselves a prime drug target, also requires additional research Cell–cell interactions among CSCs, tumor cells, and other cell types within these tumors and the TME necessitate transcriptomic and metabolomics approaches to delineate the molecular drivers of tumor growth, progression, and metastasis. Various high-throughput assays for discovering NCEs/NBEs could be deployed against ‘key ring leader’ genes, which are amenable to targeted drug therapy. Efforts are underway to discover and develop a single NCE that will target two different genes from parallel signal transduction pathways of tumorigenesis. As observed in Table 3, a combination approach, in which a small-molecule drug may be combined with a biologics, or an RNAi, seem to be clinically beneficial against targeting CSCs.

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RNAi technology has the added advantage of silencing multiple genes in various pathways regulating tumor growth and proliferation. However, the major bottleneck remains the targeted delivery of these therapeutic agents into tumors [154]. One of the promising technological advances here is the targeted delivery of a chemical drug or miRNA packaged into a minicell, which can be targeted to tumors via minicell surface-attached bispecific antibodies, which has made tremendous progress in the clinic [155–157]. To avoid toxicity, similar technological innovations and tailored treatments for patients based on their existing specific gene expression signatures will be required to target CSCs and at lower doses currently used by clinical oncologists. We need to leverage tissue- and cell-specific biomarkers and genetic signatures of individual tumors to assess toxicity related issues in preclinical studies. These unique data sets needs to be coupled with targeted delivery tools to achieve nanomolar doses that effectively kill CSCs and tumor cells without affecting normal stem cells, TME, and normal tissue homeostasis. The proper identification, characterization, and gene expression profiling of circulating tumor cells and CSCs, which are responsible for metastatic relapse, will provide better prognostic biomarkers and suitable therapeutic intervention strategies [158]. Toxicogenomics coupled with traditional pathology as well as in silico tools needs to be leveraged early during the drug discovery process to develop and take forward safe and efficacious compounds into the clinic [159]. Finally, as pointed out in a recent commentary in Nature, individual patient-centric data sets collected during the process of clinical development of new drugs and also in during IV trials, will go a long way in selecting ‘responders versus nonresponders’ and also predicting possible toxicity issues, ultimately taking us away from ‘onesize fit-all’ model and a step closer towards personalized medicine [160]. Acknowledgments The authors acknowledge financial assistance from the Science and Technology Unit, Deanship of Scientific Research, and Deanship of Graduate Studies, King Abdulaziz University, Jeddah, Saudi Arabia. We would like to thank Peter Thomas, Hassan Mukhtar, and Surender Kharbanda for critical comments and suggestions. We would also like to extend special appreciation to Deema Hussein for her help and valuable insights during the preparation of this manuscript.

References 1 Reya, T. et al. (2001) Stem cells, cancer, and cancer stem cells. Nature 414, 105–111 2 Li, L. and Neaves, W.B. (2006) Normal stem cells and cancer stem cells: the niche matters. Cancer Res. 66, 4553–4557 3 Li, F. et al. (2007) Beyond tumorigenesis: cancer stem cells in metastasis. Cell Res. 17, 3–14 4 Gil, J. et al. (2008) Cancer stem cells: the theory and perspectives in cancer therapy. J. Appl. Genet. 49, 193–199 5 Liu, H.G. et al. (2011) Cancer stem cell subsets and their relationships. J. Transl. Med. 9, 1479–5876 6 Unai, S.A. et al. (2011) Embryonic and cancer stem cells: two views of the same landscape. In Embryonic Stem Cells: Recent Advances in Pluripotent Stem Cell-based Regenerative Medicine (Atwood, C., ed.), pp. 371–398, Intech 7 Magee, J.A. et al. (2012) Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 21, 283–296 8 Brianna, H.U., S. (2013) Cancer stem cells: a review of the literature and the implications in head and neck cancer. J. Cancer Res. Updates 2, 186–193 9 Friedmann-Morvinski, D. and Verma, I.M. (2014) Dedifferentiation and reprogramming: origins of cancer stem cells. EMBO Rep. 15, 244– 253 10 Gupta, P.B. et al. (2009) Cancer stem cells: mirage or reality? Nat. Med. 15, 1010–1012 11 Takebe, N. et al. (2015) Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update. Nat. Rev. Clin. Oncol. Published online April 7, 2015. http://dx.doi.org/10.1038/nrclinonc.2015.61 12 Meacham, C.E. and Morrison, S.J. (2013) Tumor heterogeneity and cancer cell plasticity. Nature 501, 328–337 13 Oishi, N. et al. (2014) Molecular biology of liver cancer stem cells. Liver Cancer 3, 71–84

Page 8 of 21

Ac ce

pt

ed

M

an

us

cr

ip t

14 Hussein, D. et al. (2011) Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion. Neurol. Oncol. 13, 70–83 15 Scatena, R. et al. (2012) Advances in Cancer Stem Cell Biology, Springer 16 Khan, I. et al. (2014) Initial characterization of drug resistant cancer stem cells isolated from primary brain tumors (astrocytoma) cell lines generated from Saudi patients. BMC Genomics 15 (Suppl. 2), P53 17 Dreesen, O. and Brivanlou, A.H. (2007) Signaling pathways in cancer and embryonic stem cells. Stem Cell Rev. 3, 7–17 18 Roy, S. and Majumdar, A. (2012) Signaling in colon cancer stem cells. J. Mol. Signal. 7, 11 19 Karsten, U. and Goletz, S. (2013) What makes cancer stem cell markers different? Springerplus 2, 1–8 20 Sell, S. (1993) Cellular origin of cancer: dedifferentiation or stem cell maturation arrest? Environ. Health Perspect. 101 (Suppl. 5), 15 21 Brabletz, T. et al. (2005) Migrating cancer stem cells: an integrated concept of malignant tumor progression. Nat. Rev. Cancer 5, 744–749 22 NIH (2013) Stem Cell Information, National Institutes of Health 23 Espinoza, I. and Miele, L. (2013) Deadly crosstalk: Notch signaling at the intersection of EMT and cancer stem cells. Cancer Lett. 341, 41– 45 24 Donovan, P.J. (1998) The germ cell-the mother of all stem cells. Int. J. Dev. Biol. 42, 1043–1050 25 Ratajczak, M.Z. et al. (2014) The embryonic rest hypothesis of cancer development: an old XIX century theory revisited. J. Cancer Stem Cell Res. 2, e1001 26 Liu, C. et al. (2011) The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44. Nat. Med. 17, 211–215 27 Silva, I.A. et al. (2011) Aldehyde dehydrogenase in combination with CD133 defines angiogenic ovarian cancer stem cells that portend poor patient survival. Cancer Res. 71, 3991–4001 28 Naujokat, C. and Steinhart, R. (2012) Salinomycin as a drug for targeting human cancer stem cells. J. Biomed. Biotechnol. 2012, 950658 29 Iacopino, F. et al. (2014) Isolation of cancer stem cells from three human glioblastoma cell lines: characterization of two selected clones. PLoS ONE 9, e105166 30 Al-Hajj, M. et al. (2003) Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U. S. A. 100, 3983–3988 31 Ginestier, C. et al. (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567 32 Pece, S. et al. (2010) Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content. Cell 140, 62–73 33 Miyoshi, Y. et al. (2008) Basal-like subtype and BRCA1 dysfunction in breast cancers. Int. J. Clin. Oncol. 13, 395–400 34 Deng, S. et al. (2010) Distinct expression levels and patterns of stem cell marker, aldehyde dehydrogenase isoform 1 (ALDH1), in human epithelial cancers. PLoS ONE 5, 0010277 35 Singh, S.K. et al. (2004) Identification of human brain tumor initiating cells. Nature 432, 396–401 36 Son, M.J. et al. (2009) SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma. Cell Stem Cell 4, 440–452 37 Mazzoleni, S. et al. (2010) Epidermal growth factor receptor expression identifies functionally and molecularly distinct tumor-initiating cells in human glioblastoma multiforme and is required for gliomagenesis. Cancer Res. 70, 7500–7513 38 Anido, J. et al. (2010) TGF-beta receptor inhibitors target the CD44(high)/Id1(high) glioma-initiating cell population in human glioblastoma. Cancer Cell 18, 655–668 39 O’Brien, C.A. et al. (2007) A human colon cancer cell capable of initiating tumor growth in immunodeficient mice. Nature 445, 106–110 40 Dalerba, P. et al. (2007) Phenotypic characterization of human colorectal cancer stem cells. Proc. Natl. Acad. Sci. U. S. A. 104, 10158–10163 41 Wilson, B.J. et al. (2011) ABCB5 identifies a therapy-refractory tumor cell population in colorectal cancer patients. Cancer Res. 71, 5307– 5316 42 Kemper, K. et al. (2010) The AC133 epitope, but not the CD133 protein, is lost upon cancer stem cell differentiation. Cancer Res. 70, 719– 729 43 Eramo, A. et al. (2008) Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 15, 504–514 44 Sullivan, J.P. et al. (2010) Evidence for self-renewing lung cancer stem cells and their implications in tumor initiation, progression, and targeted therapy. Cancer Metastasis Rev. 29, 61–72 45 Leung, E.L. et al. (2010) Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS ONE 5, 0014062 46 Damelin, M. et al. (2011) Delineation of a cellular hierarchy in lung cancer reveals an oncofetal antigen expressed on tumor-initiating cells. Cancer Res. 71, 4236–4246 47 Tanaka, S. (2015) Cancer stem cells as therapeutic targets of hepato-biliary-pancreatic cancers. J. Hepatobiliary Pancreat. Sci. 14, 248 48 Stevens, L.C. (1970) The development of transplantable teratocarcinomas from intratesticular grafts of pre- and postimplantation mouse embryos. Dev. Biol. 21, 364–382 49 Chen, J. et al. (2012) A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488, 522–526 50 Driessens, G. et al. (2012) Defining the mode of tumor growth by clonal analysis. Nature 488, 527–530 51 Guo, Y. et al. (2011) Expression profile of embryonic stem cell-associated genes Oct4, Sox2 and Nanog in human gliomas. Histopathology 59, 763–775 52 Zhao, W. et al. (2012) Embryonic stem cell markers. Molecules 17, 6196–6236 53 Holland, J.D. et al. (2013) Wnt signaling in stem and cancer stem cells. Curr. Opin. Cell Biol. 25, 254–264 54 Fujikura, J. et al. (2002) Differentiation of embryonic stem cells is induced by GATA factors. Genes Dev. 16, 784–789 55 Rippon, H. and Bishop, A. (2004) Embryonic stem cells. Cell Prolif. 37, 23–34 56 Genander, M. and Frisén, J. (2010) Ephrins and Eph receptors in stem cells and cancer. Curr. Opin. Cell Biol. 22, 611–616 57 Kim, J. et al. (2010) A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs. Cell 143, 313– 324 58 Smart, N. and Riley, P.R. (2008) The stem cell movement. Circulat. Res. 102, 1155–1168 59 Islam, F. et al. (2015) Cancer stem cells in oesophageal squamous cell carcinoma: identification, prognostic and treatment perspectives. Crit. Rev. Oncol. Hematol. Published online April 16, 2015. http://dx.doi.org/10.1016/j.critrevonc.2015.04.007 60 Singh, A. and Settleman, J. (2010) EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29, 4741–4751 61 Pavlov, K. et al. (2015) GATA6 expression in Barrett’s oesophagus and oesophageal adenocarcinoma. Dig. Liver Dis. 47, 73–80 62 Chhabra, R. (2015) Cervical cancer stem cells: opportunities and challenges. J. Cancer Res. Clin. Oncol. Published online January 7, 2015. http://dx.doi.org/10.1007/s00432-014-1905-y 63 Zhang, Y. et al. (2012) NAC1 modulates sensitivity of ovarian cancer cells to cisplatin by altering the HMGB1-mediated autophagic response. Oncogene 31, 1055–1064 64 Emani, M.R. et al. (2015) The L1TD1 protein interactome reveals the importance of post-transcriptional regulation in human pluripotency. Stem Cell Rep. 4, 519–528

Page 9 of 21

Ac ce

pt

ed

M

an

us

cr

ip t

65 Peterson, H. et al. (2013) From structural to molecular systems biology: experimental and computational approaches to unravel mechanisms of kinase activity regulation in cancer and neurodegeneration: qualitative modeling identifies IL-11 as a novel regulator in maintaining self–renewal in human pluripotent stem cells. Front. Physiol. 4, 303 66 Dong, C.Y. et al. (2014) Twist-1, a novel regulator of hematopoietic stem cell self-renewal and myeloid lineage development. Stem Cells 32, 3173–3182 67 Malanchi, I. et al. (2012) Interactions between cancer stem cells and their niche govern metastatic colonization. Nature 481, 85–89 68 Keller, G. (2005) Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 19, 1129–1155 69 Korin, N. and Levenberg, S. (2007) Engineering human embryonic stem cell differentiation. Biotechnol. Genet. Eng. Rev. 24, 243–262 70 Darini, C. et al. (2013) Targeting cancer stem cells expressing an embryonic signature with anti-proteases to decrease their tumor potential. Cell Death Dis. 4, e706 71 Rodríguez-Gómez, J.A. et al. (2012) T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal. Am. J. Physiol. Cell Physiol. 302, C494–C504 72 Wiese, C. et al. (2004) Nestin expression: a property of multi-lineage progenitor cells? Cell Mol. Life Sci. 61, 2510–2522 73 Hamaı, A. et al. (2014) Cancer stem cells and autophagy: facts and perspectives. J. Cancer Stem Cell Res. 2, e1005 74 Nakanishi, Y. et al. (2013) Dclk1 distinguishes between tumor and normal stem cells in the intestine. Nat. Genet. 45, 98–103 75 Bailey, J.M. et al. (2014) DCLK1 marks a morphologically distinct subpopulation of cells with stem cell properties in preinvasive pancreatic cancer. Gastroenterology 146, 245–256 76 Fernando, R.I. et al. (2011) IL-8 signaling plays a critical role in the epithelial-mesenchymal transition of human carcinoma cells. Cancer Res. 71, 5296–5306 77 Kim, S.Y. et al. (2013) Role of the IL-6-JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells. Cell Signal 25, 961–969 78 West, N.R. et al. (2014) Oncostatin-M promotes phenotypic changes associated with mesenchymal and stem cell-like differentiation in breast cancer. Oncogene 33, 1485–1494 79 Gassenmaier, M. et al. (2013) CXC chemokine receptor 4 is essential for maintenance of renal cell carcinoma-initiating cells and predicts metastasis. Stem Cells 31, 1467–1476 80 Fulmer, T. (2010) Targeting chemokines in breast cancer. SciBX 3, 4 81 Feng, Y. et al. (2012) EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis. Cell Prolif. 45, 413–419 82 Levental, K.R. et al. (2009) Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139, 891–906 83 Northcott, J.M. et al. (2015) Fighting the force: potential of homeobox genes for tumor microenvironment regulation. Biochim. Biophys. Acta Rev. Cancer 1855, 248–253 84 Hermann, P.C. et al. (2007) Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1, 313–323 85 Charafe-Jauffret, E. et al. (2009) Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res. 69, 1302–1313 86 Pang, R. et al. (2010) A subpopulation of CD26+ cancer stem cells with metastatic capacity in human colorectal cancer. Cell Stem Cell 6, 603–615 87 Giancotti, F.G. (2013) Mechanisms governing metastatic dormancy and reactivation. Cell 155, 750–764 88 Takebe, N. et al. (2011) Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways. Nat. Rev. Clin. Oncol. 8, 97–106 89 Takahashi-Yanaga, F. and Kahn, M. (2010) Targeting Wnt signaling: can we safely eradicate cancer stem cells? Clin. Cancer Res. 16, 3153– 3162 90 Jiang, X. et al. (2013) Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal adenocarcinoma. Proc. Natl. Acad. Sci. U. S. A. 110, 12649–12654 91 Pannuti, A. et al. (2010) Targeting Notch to target cancer stem cells. Clin. Cancer Res. 16, 3141–3152 92 O’Reilly, E.M. et al. (2015) Final results of phase Ib of anticancer stem cell antibody tarextumab (OMP-59R5, TRXT, anti-Notch 2/3) in combination with nab-paclitaxel and gemcitabine (Nab-P+ Gem) in patients (pts) with untreated metastatic pancreatic cancer (mPC). ASCO Annu. Meet. Proc. 33, 278 93 Hoffman, L.M. et al. (2015) Phase I trial of weekly MK-0752 in children with refractory central nervous system malignancies: a pediatric brain tumor consortium study. Childs Nerv. Syst. Published online May 1, 2015. http://dx.doi.org/10.1007/s00381-015-2725-3 94 Basset-Seguin, N. et al. (2015) Efficacy of Hedgehog pathway inhibitors in basal cell carcinoma. Mol. Cancer Ther. 14, 633–641 95 Cai, C. and Zhu, X. (2012) The Wnt/β-catenin pathway regulates self-renewal of cancer stem-like cells in human gastric cancer. Mol. Med. Rep. 5, 1191–1196 96 Vermeulen, L. et al. (2010) Wnt activity defines colon cancer stem cells and is regulated by the microenvironment. Nat. Cell Biol. 12, 468– 476 97 de Sousa, E.M. et al. (2011) Targeting Wnt signaling in colon cancer stem cells. Clin. Cancer Res. 17, 647–653 98 Teng, Y. et al. (2010) Wnt/β-catenin signaling regulates cancer stem cells in lung cancer A549 cells. Biochem. Biophys. Res. Commun. 392, 373–379 99 Song, Z. et al. (2011) Sonic hedgehog pathway is essential for maintenance of cancer stem-like cells in human gastric cancer. PLoS ONE 6, e17687 100 Medema, J.P. and Vermeulen, L. (2011) Microenvironmental regulation of stem cells in intestinal homeostasis and cancer. Nature 474, 318– 326 101 Rodova, M. et al. (2012) Sonic hedgehog signaling inhibition provides opportunities for targeted therapy by sulforaphane in regulating pancreatic cancer stem cell self-renewal. PLoS ONE 7, e46083 102 LaBarge, M.A. (2010) The difficulty of targeting cancer stem cell niches. Clin. Cancer Res. 16, 3121–3129 103 Lacerda, L. et al. (2010) The role of tumor initiating cells in drug resistance of breast cancer: Implications for future therapeutic approaches. Drug Resist. Updat. 13, 99–108 104 Jin, L. et al. (2006) Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat. Med. 12, 1167–1174 105 Jin, L. et al. (2009) Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 5, 31–42 106 Kikushige, Y. et al. (2010) TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells. Cell Stem Cell 7, 708–717 107 Curiel, T.J. (2012) Immunotherapy: a useful strategy to help combat multidrug resistance. Drug Resist. Updat. 15, 106–113 108 Mehra, N. et al. (2006) Progenitor marker CD133 mRNA is elevated in peripheral blood of cancer patients with bone metastases. Clin. Cancer Res. 12, 4859–4866 109 Lin, E.H. et al. (2007) Elevated circulating endothelial progenitor marker CD133 messenger RNA levels predict colon cancer recurrence. Cancer 110, 534–542 110 Wang, C.H. et al. (2011) Photothermolysis of glioblastoma stem-like cells targeted by carbon nanotubes conjugated with CD133 monoclonal antibody. Nanomedicine 7, 69–79

Page 10 of 21

Ac ce

pt

ed

M

an

us

cr

ip t

111 Diessner, J. et al. (2014) Targeting of preexisting and induced breast cancer stem cells with trastuzumab and trastuzumab emtansine (TDM1). Cell Death Dis. 27, 115 112 Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 113 Bartel, D.P. (2009) MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 114 Lu, J. et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435, 834–838 115 Volinia, S. et al. (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc. Natl. Acad. Sci. U. S. A. 103, 2257–2261 116 Calin, G.A. et al. (2004) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. U. S. A. 101, 2999–3004 117 Esquela-Kerscher, A. and Slack, F.J. (2006) Oncomirs: microRNAs with a role in cancer. Nat. Rev. Cancer 6, 259–269 118 Kasinski, A.L. and Slack, F.J. (2011) Epigenetics and genetics. MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy. Nat. Rev. Cancer 11, 849–864 119 Spizzo, R. et al. (2009) SnapShot: microRNAs in Cancer. Cell 137, 586–586 120 Volinia, S. et al. (2010) Reprogramming of miRNA networks in cancer and leukemia. Genome Res. 20, 589–599 121 Medina, P.P. et al. (2010) OncomiR addiction in an in vivo model of microRNA-21-induced pre-B-cell lymphoma. Nature 467, 86–90 122 Hatley, M.E. et al. (2010) Modulation of K-Ras-dependent lung tumorigenesis by MicroRNA-21. Cancer Cell 18, 282–293 123 Frankel, L.B. et al. (2008) Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J. Biol. Chem. 283, 1026–1033 124 Yu, F. et al. (2007) let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131, 1109–1123 125 Johnson, C.D. et al. (2007) The let-7 microRNA represses cell proliferation pathways in human cells. Cancer Res. 67, 7713–7722 126 Kumar, M.S. et al. (2008) Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc. Natl. Acad. Sci. U. S. A. 105, 3903–3908 127 Barh, D. et al. (2010) MicroRNA let-7: an emerging next-generation cancer therapeutic. Curr. Oncol. 17, 70–80 128 Yu, F. et al. (2010) Mir-30 reduction maintains self-renewal and inhibits apoptosis in breast tumor-initiating cells. Oncogene 29, 4194–4204 129 Zhu, Y. et al. (2011) Reduced miR-128 in breast tumor-initiating cells induces chemotherapeutic resistance via Bmi-1 and ABCC5. Clin. Cancer Res. 17, 7105–7115 130 Yu, F. et al. (2012) MicroRNA 34c gene down-regulation via DNA methylation promotes self-renewal and epithelial-mesenchymal transition in breast tumor-initiating cells. J. Biol. Chem. 287, 465–473 131 Bu, P. et al. (2013) A microRNA miR-34a-regulated bimodal switch targets Notch in colon cancer stem cells. Cell Stem Cell 12, 602–615 132 Scherr, M. et al. (2003) Inhibition of GM-CSF receptor function by stable RNA interference in a NOD/SCID mouse hematopoietic stem cell transplantation model. Oligonucleotides 13, 353–363 133 Zhou, L.L. et al. (2008) AHI-1 interacts with BCR-ABL and modulates BCR-ABL transforming activity and imatinib response of CML stem/progenitor cells. J. Exp. Med. 205, 2657–2671 134 Bao, S. et al. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444, 756–760 135 Patrawala, L. et al. (2005) Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic. Cancer Res. 65, 6207–6219 136 Wang, J. et al. (2012) siRNA targeting Notch-1 decreases glioma stem cell proliferation and tumor growth. Mol. Biol. Rep. 39, 2497–2503 137 Ma, S. et al. (2007) Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 132, 2542–2556 138 Huang, E.H. et al. (2009) Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res. 69, 3382–3389 139 Dembinski, J.L. and Krauss, S. (2009) Characterization and functional analysis of a slow cycling stem cell-like subpopulation in pancreas adenocarcinoma. Clin. Exp. Metastasis 26, 611–623 140 Ucar, D. et al. (2009) Aldehyde dehydrogenase activity as a functional marker for lung cancer. Chem. Biol. Interact. 178, 48–55 141 Charafe-Jauffret, E. et al. (2010) Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin. Cancer Res. 16, 45–55 142 Yokota, A. et al. (2009) GM-CSF and IL-4 synergistically trigger dendritic cells to acquire retinoic acid-producing capacity. Int. Immunol. 21, 361–377 143 Wang, Y.C. et al. (2012) ALDH1-bright epithelial ovarian cancer cells are associated with CD44 expression, drug resistance, and poor clinical outcome. Am. J. Pathol. 180, 1159–1169 144 Meng, E. et al. (2014) ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling. PLoS ONE 9, e107142 145 Landen, C.N., Jr. et al. (2010) Targeting aldehyde dehydrogenase cancer stem cells in ovarian cancer. Mol. Cancer Ther. 9, 3186–3199 146 ClinicalTrials.gov. (2015) Clinical Studies of Human Participants Conducted Around the World, US National Institutes of Health 147 Genus Oncology (2015) MUC1-C and Cancer Stem Cells, Genus Oncology 148 Kaiser, J. (2015) The cancer stem cell gamble. Science 347, 226–229 149 Stroopinsky, D. et al. (2013) MUC1 is a potential target for the treatment of acute myeloid leukemia stem cells. Cancer Res. 73, 5569–5579 150 Alam, M. et al. (2013) MUC1-C oncoprotein activates ERK→ C/EBPβ signaling and induction of aldehyde dehydrogenase 1A1 in breast cancer cells. J. Biol. Chem. 288, 30892–30903 151 Alam, M. et al. (2014) MUC1-C induces the LIN28B→ LET-7→ HMGA2 axis to regulate self-renewal in NSCLC. Molecular Cancer Res. 13, 449–460 152 Alam, M. et al. (2014) Targeting the MUC1-C oncoprotein inhibits self-renewal capacity of breast cancer cells. Oncotarget 5, 2622–2634 153 Hanahan, D. and Weinberg, R.A. (2011) Hallmarks of cancer: the next generation. Cell 144, 646–674 154 Bora, R.S. et al. (2012) RNA interference therapeutics for cancer: challenges and opportunities (review). Mol. Med. Rep. 6, 9–15 155 MacDiarmid, J.A. et al. (2009) Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug. Nat. Biotechnol. 27, 643–651 156 MacDiarmid, J.A. and Brahmbhatt, H. (2011) Minicells: versatile vectors for targeted drug or si/shRNA cancer therapy. Curr. Opin. Biotechnol. 22, 909–916 157 EnGeneIC (2009) XXXXX, EnGeneIC Cancer Research Foundation[LM6] 158 Grover, P. et al. (2014) Circulating tumor cells: the evolving concept and the inadequacy of their enrichment by EpCAM-based methodology for basic and clinical cancer research. Ann. Oncol. 25, 1506–1516 159 SM Sabir, J. et al. (2013) Role of toxicogenomics in the development of safe, efficacious and novel anti-microbial therapies. Infect. Disord. Drug Targets 13, 206–214 160 Schork, N.J. (2015) Personalized medicine: time for one-person trials. Nature 520, 609–611 Figure 1. [LM7]Current hypotheses about how cancer stem cells (CSCs) might arise. (A) A stem cell itself undergoes genetic and/or epigenetic changes while maintaining the ability of stemness; (B) a progenitor cell experiences a few genetic and/or epigenetic changes, resulting in it recovering its stem cell-like characteristics; or (C) a fully differentiated cell undergoes a couple of mutations, genetically and/or epigenetically, until it rediscovers its self-renewal capacity. Moreover, differentiated

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cells can also undergo an epithelial to mesenchymal transition (EMT) to acquire stemness characteristics. All three of these genetically diverse models can result in the generation of CSCs. Figure 2. [LM8]Stochastic (A), hierarchical (B), and plasticity (C) models of cancer stem cells (CSCs) in tumorigenesis. (A) Every cell in the tumor has tumorigenic potential; (B) the cells with stem cell-like characteristics (i.e., CSC) have important roles in tumor progression; (C) both CSCs and nonCSCs have important roles in tumorigenesis. Data from the literature suggest that induction of an epithelial to mesenchymal transition (EMT) delivers a potential mechanistic basis for therapeutics resistance, metastasis, tumor dormancy, and delayed relapse. We and others believe that the plasticity model (C) is the most attractive for targeted drug discovery and development against CSCs. Abbreviation: mCSCs, migrating cancer stem cells.

Table 1. Transcription factors, surface markers, and signaling pathways involved in ESCs and CSCsa Refs

Surface marker(s)

Refs

Signaling pathway(s)

Refs

Oct-3 (Pou5f1) Oct-4 (Pou5f1) Nanog c-Myc

[52,53] [6,25,52–57] [2,6,8,52,53,55,57,58]

CD324 (E-Cadherin) CD90 (Thy-1) CD117 (c-KIT, SCFR) CD29 (β1 integrin)

[52,53,59,67]

Wnt/β-catenin Wnt/Ca2+ Wnt pathway TGF-β SHH

[1,4,8,11,18,58] [6, 8] [1,4,11,18,58,60]

[54,6l]

M

[6,52,62] [6,52]

ed

[52,60] [6,52,63]

pt

[6,52,64]

[6, 52, 65]

[59, 66]

cr

[6,8,52,53,59,70]

[6,7,18,25,52,59,70]

an

[6,52,53,57,59,60]

[52,68,69]

Factor Bmi-1 Hox gene products JAK/STAT NOTCH MAP-Kinase/ERK PI3K/AKT NFkB SMAD1/5/8 SMAD2/3 SMAD4 β-catenin T-type Ca2+ (Cav3.2)

us

[6,52]

CD24 (HAS), CD24- /low CD59 (Protectin) CD133 TRA-1-60 TRA-1-81alkaline phosphatase ALDH 1 CD147 CD34+ CD38CD44+ SSEA-1(CD15/ Lewis x) SSEA-3 SSEA-4 TIM3 CD326 CD96 CD45 CD176/TF CD174 Lewis ESA+ ABCB5+ 21integrin ABCG2 (CDw338) Nestin Pygo2 MAML1 Musashi1 CD271 Podoplanin EpCAM

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ABCB1 MDR-1 HMGA2 Stat3 LEF1 TCF3 Sall4 KLF4 Sox2 Rex1 (Zfp42) Gata-4 Gata-6 UTF1 ZFX TBN FoxD3 HMGA2 NAC1 GCNF (NR6A1) Ecat1 ECAT11/L1td1 Fbxo15 ECAT9(Gdf3) ECAT9 Dppa2 Dppa3 Dppa 4 Dppa5 FGF-4 Twist1

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Transcription factor

[19,52,53,71]

[1,11,17,18,58,73]

[52,60]

[71]

[52,67]

[19] [6,8]

[19,59] [53,72] [11,59]

[60]

a A detailed description of some of these transcription factors, surface markers, and signaling pathways can be found in the main text under the subheadings of ‘Molecular biomarkers of CSCs’ and ‘Relation between ESCs and CSCs’.

Table 2. New drug discovery and development against specific pathways operating in tumors and CSCs Signal Drug molecule transduction pathway

Development stage

Refs

Wnt

Phase Ia, Ib/II Phase I Phase I/ Ib Preclinical trials

[11,47,89, 90]

Phase I/II

[11,91–93]

Notch

CBP/β-catenin antagonist Porcupine inhibitor Anti-Frizzled antibodies SiRNA-based therapeutics GSIs (γ-secretase inhibitors) Anti-DLL4 antibodies Anti-Notch antibodies MAML1-stapled peptide

Phase I Phase I, Ib/II Preclinical trials

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Preclinical trials Preclinical trials Preclinical trials [11,47,73, 94]

Phase I/II Phase I/II Phase I/II/III Phase I/II Phase I Phase I

Table 3. Current status of drugs in clinical development targeting CSCsa

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SHH

Notch soluble receptor decoy SiRNA-based therapeutics MiRNA-based therapeutics Vismodegib BMS
833923 Sonidegib Glasdegib LEQ506 TAK
441

Drug agent

Therapeutic target(s)

Cancer type(s)

Dompe Genus Oncology OncoMed

Reparixin GO-203-2c Tarextumab Demcizumab Vantictumab Ipafricept SL-401 BBI608

Chemokine receptors 1,2 MUC1-C Notch-2,3 receptors DLL4 (Notch ligand) Wnt pathway Wnt pathway IL-3 receptor STAT3, -catenin[LM9] Nanog Nanog Multiple kinases Focal adhesion kinase Focal adhesion kinase Focal adhesion kinase PI3K and mTORC1/2

Breast Leukemias and solid tumors Pancreatic, lung Ovarian Solid tumor Pancreatic Leukemia Colon Gastric, esophageal Colon, other cancers Solid tumors Mesothelioma, lung Ovarian cancer Solid tumors Solid tumor and lymphomas

BBI503 VS-6063 VS-6063 and paclitaxel VS-4718 VS-5584

a

us

Development stage

Phase II Phase Ib/II a Phase II Phase II Phase Ia Phase Ia Phase I/II Phase III Phase III Phase II Phase II Phase II Phase I Phase I/Ib Phase I/Ib

ed

Data from corporate websites and [146–148].

an

Verastem

M

Stemline Therapeutics Sumitomo Dainippon

cr

Sponsor

Box 1. Possible therapeutic interventions for targeting CSCs From a drug discovery point of view, the various small-molecule drugs and biotech approaches targeting the CSCs include: (i) the blockade of key signaling pathways regulated by, for example, EpCAM, SHH, Wnt/β-catenin, SALL4, TGF-β, Notch, SERPINB5, TOP2A, TP63, and SOX4,

pt

and also specific kinases and phosphatases regulating signal transduction; (ii) interfering with the tumor microenvironment around CSCs, particularly developing new chemical entities and/or new biological entities (NCEs/NBEs) that would target key cell and biochemical processes, such as hypoxia, vasculogenesis, angiogenesis, cell adhesion, invasion, and metastasis; and (iii) blocking cell surface markers that control the initiation, development, and metastatic phenotype of CSCs, such as CD44, CK5, CD24, ADRM1, DNER, DLL1, JAGI, ALDH, CD133, CD15

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(SSEA-1), EGFR, ABCB5, CD90, and oncofetal protein 5T4.

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Ishaq N. Khan is currently pursuing his PhD at King AbdulAziz University which awarded him a doctoral scholarship in cancer biology. His major area of research is on the characterization of drug resistant cancer stem cells in primary brain tumours from Saudi patients. Part of his doctorate work was presented at the 2nd International Genomic Medicine Conference, Jeddah, Saudi Arabia in November 2013 and at the Advances in Cancer Drug Discovery Conference, Cambridge, UK (March 2014). He obtained his MSc in Drug Discovery and Development from University of Sunderland, UK, and Bachelor in Biotechnology from University of Peshawar, Pakistan.

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Prof. Kulvinder S. Saini is currently Professor of Biotechnology and Genomics at King Abdulaziz University (www.kau.edu.sa), Jeddah, Saudi Arabia with major focus on New Drug Discovery Research targeting cancer and inflammation. Earlier, he was Director-R & D at Eternal University (www.eternaluniversity.edu.in) Baru Sahib, India. His most recent industrial assignment (2002-2010) was as a Director of Biotechnology and Bioinformatics at Ranbaxy (www.ranbaxy.com), India. International appointments includes 3.5 years at Harvard Medical School, Boston-USA as a Research Fellow, and at the Princess Alexandra Hospital, Brisbane-Australia as a Senior Research Fellow. He has a Ph.D. in Animal Biochemistry from Sydney University, Australia.

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Saleh A. Alkarim is a Professor in the Department of Biological Sciences, at King Abdulaziz University (KAU), Saudi Arabia. In addition to the faculty appointment, he is also the Head of Embryonic Stem Cell unit, and Embryonic & Cancer Stem Cell research group at King Fahd Medical Research Centre. He also served as a Dean of the Faculty of Science and Dean of Student Affairs. Currently he is the Editor in Chief of Aljamia Journal (KAU) and AL-Eiejas Alelmuy Journal. He has a Ph.D. in Embryology and Molecular Biology from Nottingham University (1987), United Kingdom.

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Figure

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Figure

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Khan photo

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Saini photo

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al Karim photo

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