CELL CYCLE 2016, VOL. 15, NO. 18, 2393–2397 http://dx.doi.org/10.1080/15384101.2016.1196305
PERSPECTIVE
Reprogramming strategies for the establishment of novel human cancer models Ignacio Sancho-Martineza,b and Juan Carlos Izpisua Belmontec a Institute of Hepatology, Foundation for Liver Research, London, UK; bCentre for Stem Cells and Regenerative Medicine, King’s College London, Guy's Hospital, London, UK; cGene Expression Laboratory Belmonte, Salk Institute for Biological Studies, La Jolla, CA, USA
ABSTRACT
ARTICLE HISTORY
Cancer comprises heterogeneous cells, ranging from highly proliferative immature precursors to more differentiated cell lineages. The emergence of the “cancer stem cell” (CSC) hypothesis that they are the cells responsible for resistance, metastasis and secondary tumor appearance identifies these populations as novel obligatory targets for the treatment of cancer. CSCs, like their normal tissue-specific stem cell counterparts, are multipotent, partially differentiated, self-sustaining, yet transformed cells. To date, most studies on CSC biology have relied on the use of murine models and primary human material. In spite of much progress, the use of primary material presents several limitations that limit our understanding of the mechanisms underlying CSC formation, the similarities between normal stem cells and CSCs and ultimately, the possibility for developing targeted therapies. Recently, different strategies for controlling cell fate have been applied to the modeling of human cancer initiation and for the generation of human CSC models. Here we will summarize recent developments in the establishment and application of reprogramming strategies for the modeling of human cancer initiation and CSC formation.
Received 29 April 2016 Revised 20 May 2016 Accepted 25 May 2016
Stem cells and cancer The CSC hypothesis was originally proposed for hematological malignancies when John Dick first identified cancer cells bearing stem cell properties in leukemias.1,2 In an analogous manner to the traditional landscape of development postulated by Conrad Hal Waddington, the CSC theory stipulates that tumor growth and heterogeneity is maintained in a hierarchical manner by a subpopulation of cells with stem cell properties.3-7 CSCs at the top of the hierarchy are thought to differentiate into less tumorigenic cancer cell populations unable to selfrenew and initiate tumor formation as well as presenting increased susceptibility to conventional therapies.8,9 Unfortunately, whereas current anticancer therapies can in most cases kill differentiated cancer cell populations, they marginally affect the undifferentiated CSC compartment. Thus, developing therapeutic strategies targeting undifferentiated CSCs remains an urgent medical need. Traditionally, studies on CSCs have been limited by the availability of tumor samples from which CSCs could be isolated and cultures established based on the presence of selected surface markers. In addition, it is now well-accepted that so-called CSCs markers are not universal and should be exclusively considered as a mean to enrich, yet not define, cancer cell populations with stem-like properties.10 Instead, functional testing of stem cell features has been proposed as the most reliable approach to characterize putative CSC populations. Three major functional tests define functional CSCs3,4,11: a) the
KEYWORDS
cancer stem cells; driver mutations; iPSCs; reprogramming; transformation
ability to initiate tumor formation upon transplantation of limited cell numbers; b) the ability of a given cell population to differentiate into all different lineages comprising the tumor mass; and c) the ability to self-renew as undifferentiated stem cells and thus, to form secondary tumors upon serial transplantation. Another limitation compromising our understanding on CSCs biology relates to potential inter-patient variability as a result of the different genetic and epigenetic backgrounds present in the human population. In addition, cancer arises as a consequence of transforming “driver” mutations able to initiate tumor formation. Upon tumor initiation, positive selection and clonal progression further leads to the accumulation of “passenger” mutations conferring additional growth advantages. Identification of driver mutations might therefore allow not only for the establishment of targeted therapies but also, for the elucidation and targeting of the early events underlying cancer formation. Unfortunately, current bioinformatics are thought to underestimate the role that low frequency mutations might play during carcinogenesis. Therefore, development of novel strategies allowing for the functional evaluation of putative driver mutations and studying the molecular dynamics underlying human cancer initiation are in urgent need.10,12-22 Lastly, it remains unclear whether stem cell properties arise by dedifferentiation of cancer cells during tumor progression and as a result of the accumulation of passengers mutations, whether they are a direct consequence of mutations in multipotent adult stem cell
CONTACT Ignacio Sancho-Martinez [email protected] Centre for Stem Cells and Regenerative Medicine, King’s College London, 28th Floor, Tower Wing, Guy’s Hospital, Great Maze Pond, SE1 9RT, London, UK; Juan Carlos Izpisua Belmonte [email protected] Gene Expression Laboratory Belmonte, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 93037, USA. Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/kccy © 2016 Taylor & Francis
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Figure 1. CSCs can originate by different mechanisms. CSCs can theoretically emerge by mutations on embryonic stem cell populations and therefore lead to the formation of embryonic tumors (a). In addition, the embryonal rest theory speculated that undifferentiated remnants of developmental processes could contribute to tumor initiation. The prevailing theories for adult tumor formation speculate that accumulation of mutations might lead to the dynamic reversion of a differentiated phenotype into a malignant stem cell-like state (b). Alternatively, the similarities between CSCs and adult stem cell populations bring about the possibility that mutations directly affecting tissues-specific progenitor cells underlie CSC generation (c). Nowadays, it is hardly believed that a single hypotheses bears true for all different tumor types. Instead, different mechanisms might underlie the appearance of CSCs in a tumor, or even patient, specific manner.
populations already presenting a dedifferentiated phenotype or whether different mechanisms will be present in different tumor types or even different patients. Thus, 2 major theories attempting to explain the appearance of cancer cells with stem cell properties prevail nowadays3,4,7: a) that dynamic dedifferentiation, or reversion, to a stem cell phenotype occurs as a consequence of mutations and transformation; b) that mutations directly transform tissue-specific stem cell populations into malignant CSCs (Fig. 1). Collectively, the generation of reliable and tractable models functionally recapitulating CSC properties will represent a valuable model for studying the molecular mechanisms underlying tumorigenesis and for the future development of therapeutic strategies.23-27 Notwithstanding, the recent application of cell reprogramming methodologies to cancer biology has opened unexpected new avenues for studying human tumorigenesis in a personalized manner.
methodologies, factors and even the use of virtually any somatic cell lineage as starting cell material for iPSC generation. Yet, despite the technical advancements that rapidly followed the initial reports on iPSCs, translating the promises of reprogramming technologies into the clinic (i.e.: cellular products for transplantation and regenerative medicine) has faced important roadblocks. Notwithstanding, the observations that differentiation of iPSCs in vitro could be used as a surrogate for studying early development and disease in a rapid and controllable manner while avoiding ethical implications, further highlighted that reprogramming technologies are not limited to future cell therapy applications but that can also be applied to advance basic developmental and disease knowledge and even for the establishment and testing of novel therapeutics.30 Disease modeling thus leverages the ability of iPSCs to recapitulate key disease phenotypes that are linked to specific genetic mutations (i.e.,: Fanconi Anemia31,32) or even aberrant epigenetic patterns (i.e.: Angelman Syndrome33).
Reprogramming and disease modeling
Leveraging reprogramming for the modeling of cancer initiation
In 2006, Takahashi and Yamanaka revealed to the world the ability to experimentally generate, mouse28 (Takahashi and Yamanaka, 2006) and later human29 (Takahashi et al., 2007), induced Pluripotent Stem cells (iPSCs) by reprogramming differentiated somatic cell lineages. In their initial study, Takahashi and Yamanaka focused on 24 genes previously identified as playing pivotal roles in the maintenance of pluripotency in Embryonic Stem Cells (ESCs). Four transcription factors (Oct3/4, Sox2, c-Myc and Klf4) were ultimately found to suffice for the dedifferentiation of fibroblasts back to an ESC-like phenotype when maintained in pluripotent culture conditions.28,29 This major breakthrough generated incredible excitement in the scientific community due to the possibilities that iPSCs opened for generating unlimited patient-specific cell lineages for regenerative medicine. Since the initial reports describing iPSC generation by viral-based/integrative approaches, laboratories worldwide rapidly developed alternative reprogramming strategies based on the use of different transgene delivery
Cancer formation can originate by accumulation of mutations occurring de novo in otherwise wild-type cells (Fig. 1). In addition, certain mutations predisposing for cancer initiation can be inherited with additional mutations acquired during the lifetime of the individual ultimately contributing to cancer initiation. It is thus clear that cancer can be considered a genetic disorder in where genetic mutations lead to cell transformation and the acquisition of aberrant epigenetic patterns resulting in malignancy. Consequently, strategies based on the use of pluripotent stem cells (PSCs) could be applied to the modeling of human cancer in an analogous manner to “conventional” disease modeling. Up to date several reports have described the modeling of human cancer by applying reprogramming methodologies, including: the generation of iPSCs by reprogramming of cancer cells,34-43 the generation of iPSCs from somatic lineages bearing mutations associated with inherited cancer,44 and the
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transformation of fibroblasts by oncogenes and tumor suppressor genes to a CSC phenotype.45 Reprogramming of cancer cells into pluripotent cells, or “induced pluripotent cancer cells (iPCCs),” provides a potential platform for the screening of novel therapeutic compounds.46 However, the pluripotent nature of undifferentiated iPCCs would rather resemble an embryonic origin for a given tumor. The first idea that cancer could arise from embryonic cells appeared in the early 19th century and was formally presented by Durante and Conheim as the “embryonal rest theory of cancer.”24 This theory stated that remnants of embryonic tissue remain in adult organs. A change in the environment, or “disequilibrium” in the surrounding tissue (field theory), would allow the embryonic tissue to resume cell proliferation and to produce masses of cells that resembled fetal tissues. By the turn of the 20th century, the embryonic rest theory was generally discredited. Regardless of whether some tumors arise from a pluripotent embryonic cell population as opposed to multipotent progenitor cells, the reprogramming of cancer cells to iPCCs can in certain instances lead to an erasure of malignant phenotypes47 that might limit the applicability of such strategies.48,49 Another report has demonstrated the use of undifferentiated iPSCs for the modeling of brain tumors and screening purposes.50 Modeling cancer by using non-transformed pluripotent cells can provide a useful platform for the identification of novel compounds targeting cell populations with stem cell properties.51 However, and similar to the use of iPCCs, generation of neoplastic pluripotent stem cell models imply a pluripotent origin for a somatic tumor, a hypothesis long time debunked, and prevents the establishment of reliable in vivo models recapitulating non-embryonic tumor-specific features. Indeed, injection of undifferentiated iPSCs into the murine brain did not result in the formation of glioma-like tumors but rather lead to the formation of embryonic teratomas into the brain.50,52 As an alternative approach, reprogramming strategies have been used for the reprogramming of differentiated glioma cells to a CSCs-like phenotype.53 The use of primary or reprogrammed CSCs therefore represents a reliable platform for screening studies focused on the development of novel therapeutics specifically targeting CSC populations. Furthermore, the conversion of differentiated cancer cells to CSCs by reprogramming allows for studying the epigenetic reprogramming events underlying CSC generation from more differentiated cancer cells and potentially contribute to the development of strategies altogether preventing the appearance of CSC populations. As a trade-off, leveraging cellular material that has already undergone transformation and clonal selection for studies on cancer initiation prevents the identification and mechanistic study on the role that different mutations might play as drivers of tumorigenesis.3-5, 7,10, 53-56 Lastly, a complementary strategy could make use of our current understanding of the genetics of different tumors. For example, a combination of reprogramming strategies, whether iPSC generation or lineage conversion, alongside the generation of “cancer-specific” backgrounds by introducing in specific mutations and/or upon manipulation of pro-oncogenic signaling pathways pathways, could result in the generation of novel human cancer models for the study of cellular transformation.
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In such a case, the generation of specific lineages by differentiation could provide a tractable platform for studying tumor initiation processes, the functional testing of putative driver mutations. In addition, applying reprogramming strategies for the study of human cancer could ease studies on the role that different patient-specific genetics play during cancer initiation and therapy susceptibility as discussed hereafter.52 Reprogramming as a platform for the functional identification of driver mutations Identification of driver mutations generally relies on the use of complex algorithms for the analysis of genomic data obtained from tumor samples. Whereas these approaches have generally been successful, they tend to rely on sequencing data and the relative frequencies by which a given mutation is found in patient-tumors while underestimating the role that low frequency mutations might play during carcinogenesis.15-17,57-59 Thus, generation of reliable in vitro and in vivo models might additionally be used for functionally evaluating whether a given mutation act as a driver or passenger as well as for the establishment of therapies altogether preventing malignant cell transformation and cancer formation. The Tabar laboratory first demonstrated the use of pluripotent stem cells studying driver mutations and the modeling of pediatric brain tumors.60 Funato et al differentiated human Embryonic Stem Cells (ESCs) into neural progenitor cells (NPCs) and induced cellular transformation by over expressing a constitutively active form of the oncogene PDGFRA, knocking down the tumor suppressor gene p53 or by expressing the K27M mutant form of histone H3.3, to model the genetics found in pediatric gliomas.60 These manipulated neural stem cells demonstrated tumor-initiating capacity with the resulting tumors recapitulating key pathological features of human pediatric gliomas as well as allowed for the screening of chemicals targeting human GSC populations. However, whereas the use of ESCs allows for the functional study on the role that different mutations or epigenetic alterations play in CSC formation and tumor initiation, it is worth noting that it limits the possibility of studying the influence that different patient-specific genetic backgrounds might play in tumorigenesis and malignancy. In addition, the use of human embryonic material still faces ethical controversy. To tackle these caveats, we have recently reported on the establishment of tractable human iPSC models for the study of driver mutations and GSC formation. More recently, we reported on the use of human iPSCs for studying adult glioma initiation. iPSC-derived NPC (iNPCs) transformation was induced by combining different mutations typically observed in human gliomas that collectively affect the PI3K, MAPK and p53 signaling pathways.61 Genetic manipulation of human iNPCs led to the generation of human cellular models presenting functional GSC features.52 Indeed, our results indicated that the use of different genetic alterations allowed for the generation of different human glioma subtypes. In vivo transplantation resulted in the generation of tumors displaying histopathological features hardly recapitulated with conventional xenograft models.34,62,63 Most interestingly, leveraging the established models alongside the use chemical inhibitors we were able to demonstrate the causative role of
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PI3K and MAPK signaling during the metabolic reprogramming underlying the transformation of NPCs to GSCs. Lastly, a pilot screening of 101 FDA-approved compounds further identified 3 different compounds able to compromise GSC viability and migration while isogenic wild-type NPCs remained unaffected.52 In summary, modeling cancer by reprogramming approaches might ultimately provide platforms for studying the mechanisms underlying human tumor initiation and cancer cell plasticity as well as facilitate the identification and testing of novel therapeutic compounds and the development of differentiation therapies.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding Work in the laboratory of I.S.-M. was supported by The Foundation for Liver Research and King’s College London. Work in the laboratory of J.C. I.B. was supported by the G. Harold and Leila Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust, The Moxie Foundation and UCAM.
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pluripotency-based reprogramming. Oncogene 2013; 32:2249-60, 60 e1-21; PMID:22777357; http://dx.doi.org/10.1038/onc.2012.237 Stricker S, Pollard S. Reprogramming cancer cells to pluripotency: an experimental tool for exploring cancer epigenetics. Epigenetics 2014; 9:798-802; PMID:24686321; http://dx.doi.org/10.4161/epi.28600 Stricker SH, Feber A, Engstrom PG, Caren H, Kurian KM, Takashima Y, Watts C, Way M, Dirks P, Bertone P, et al. Widespread resetting of DNA methylation in glioblastoma-initiating cells suppresses malignant cellular behavior in a lineage-dependent manner. Genes Dev 2013; 27:654-69; PMID:23512659; http://dx.doi.org/ 10.1101/gad.212662.112 Sachlos E, Risueno RM, Laronde S, Shapovalova Z, Lee JH, Russell J, Malig M, McNicol JD, Fiebig-Comyn A, Graham M, et al. Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells. Cell 2012; 149:1284-97; PMID:22632761; http://dx.doi.org/10.1016/j.cell.2012.03.049 Duan S, Yuan G, Liu X, Ren R, Li J, Zhang W, Wu J, Xu X, Fu L, Li Y, et al. PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype. Nat Commun 2015; 6:10068; PMID:26632666; http://dx.doi.org/10.1038/ncomms10068 Sancho-Martinez I, Nivet E, Xia Y, Hishida T, Aguirre A, Ocampo A, Ma L, Morey R, Krause MN, Zembrzycki A, et al. Establishment of human iPSC-based models for the study and targeting of glioma initiating cells. Nat Commun 2016; 7:10743; PMID:26899176; http://dx. doi.org/10.1038/ncomms10743 Suva ML, Rheinbay E, Gillespie SM, Patel AP, Wakimoto H, Rabkin SD, Riggi N, Chi AS, Cahill DP, Nahed BV, et al. Reconstructing and reprogramming the tumor-propagating potential of glioblastoma stem-like cells. Cell 2014; 157:580-94; PMID:24726434; http://dx.doi. org/10.1016/j.cell.2014.02.030 Flavahan WA, Wu Q, Hitomi M, Rahim N, Kim Y, Sloan AE, Weil RJ, Nakano I, Sarkaria JN, Stringer BW, et al. Brain tumor initiating cells adapt to restricted nutrition through preferential glucose uptake. Nature neuroscience 2013; 16:1373-82; PMID:23995067; http://dx. doi.org/10.1038/nn.3510 Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, Bayani J, Head R, Lee M, Bernstein M, et al. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell stem cell 2009; 4:568-80; PMID:19497285; http://dx.doi.org/10.1016/j.stem.2009.03.014 Koso H, Takeda H, Yew CC, Ward JM, Nariai N, Ueno K, Nagasaki M, Watanabe S, Rust AG, Adams DJ, et al. Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells. Proc Natl Acad Sci U S A 2012; 109:E2998-3007; PMID:23045694; http://dx.doi.org/10.1073/pnas.1215899109 Krogan NJ, Lippman S, Agard DA, Ashworth A, Ideker T. The Cancer Cell Map Initiative: Defining the Hallmark Networks of Cancer. Mol Cell 2015; 58:690-8; PMID:26000852; http://dx.doi.org/10.1016/ j.molcel.2015.05.008 Hartmaier RJ, Priedigkeit N, Lee AV. Who’s driving anyway? Herculean efforts to identify the drivers of breast cancer. Breast Cancer Res 2012; 14:323; PMID:23113888; http://dx.doi.org/10.1186/bcr3325 Ding L, Wendl MC, McMichael JF, Raphael BJ. Expanding the computational toolbox for mining cancer genomes. Nat Rev Genet 2014; 15:556-70; PMID:25001846; http://dx.doi.org/10.1038/nrg3767 Funato K, Major T, Lewis PW, Allis CD, Tabar V. Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation. Science 2014; 346:1529-33; PMID:25525250; http://dx.doi.org/10.1126/science.1253799 Brennan CW, Verhaak RG, McKenna A, Campos B, Noushmehr H, Salama SR, Zheng S, Chakravarty D, Sanborn JZ, Berman SH, et al. The somatic genomic landscape of glioblastoma. Cell 2013; 155:46277; PMID:24120142; http://dx.doi.org/10.1016/j.cell.2013.09.034 Guerrero-Cazares H, Chen L, Quinones-Hinojosa A. Glioblastoma heterogeneity and more accurate representation in research models. World Neurosurg 2012; 78:594-6; PMID:22120218; http://dx.doi. org/10.1016/j.wneu.2011.10.022 Sancho-Martinez I, Martin-Villalba A. Tyrosine phosphorylation and CD95: a FAScinating switch. Cell Cycle 2009; 8:838-42; PMID:19221505; http://dx.doi.org/10.4161/cc.8.6.7906
PERSPECTIVE
Reprogramming strategies for the establishment of novel human cancer models Ignacio Sancho-Martineza,b and Juan Carlos Izpisua Belmontec a Institute of Hepatology, Foundation for Liver Research, London, UK; bCentre for Stem Cells and Regenerative Medicine, King’s College London, Guy's Hospital, London, UK; cGene Expression Laboratory Belmonte, Salk Institute for Biological Studies, La Jolla, CA, USA
ABSTRACT
ARTICLE HISTORY
Cancer comprises heterogeneous cells, ranging from highly proliferative immature precursors to more differentiated cell lineages. The emergence of the “cancer stem cell” (CSC) hypothesis that they are the cells responsible for resistance, metastasis and secondary tumor appearance identifies these populations as novel obligatory targets for the treatment of cancer. CSCs, like their normal tissue-specific stem cell counterparts, are multipotent, partially differentiated, self-sustaining, yet transformed cells. To date, most studies on CSC biology have relied on the use of murine models and primary human material. In spite of much progress, the use of primary material presents several limitations that limit our understanding of the mechanisms underlying CSC formation, the similarities between normal stem cells and CSCs and ultimately, the possibility for developing targeted therapies. Recently, different strategies for controlling cell fate have been applied to the modeling of human cancer initiation and for the generation of human CSC models. Here we will summarize recent developments in the establishment and application of reprogramming strategies for the modeling of human cancer initiation and CSC formation.
Received 29 April 2016 Revised 20 May 2016 Accepted 25 May 2016
Stem cells and cancer The CSC hypothesis was originally proposed for hematological malignancies when John Dick first identified cancer cells bearing stem cell properties in leukemias.1,2 In an analogous manner to the traditional landscape of development postulated by Conrad Hal Waddington, the CSC theory stipulates that tumor growth and heterogeneity is maintained in a hierarchical manner by a subpopulation of cells with stem cell properties.3-7 CSCs at the top of the hierarchy are thought to differentiate into less tumorigenic cancer cell populations unable to selfrenew and initiate tumor formation as well as presenting increased susceptibility to conventional therapies.8,9 Unfortunately, whereas current anticancer therapies can in most cases kill differentiated cancer cell populations, they marginally affect the undifferentiated CSC compartment. Thus, developing therapeutic strategies targeting undifferentiated CSCs remains an urgent medical need. Traditionally, studies on CSCs have been limited by the availability of tumor samples from which CSCs could be isolated and cultures established based on the presence of selected surface markers. In addition, it is now well-accepted that so-called CSCs markers are not universal and should be exclusively considered as a mean to enrich, yet not define, cancer cell populations with stem-like properties.10 Instead, functional testing of stem cell features has been proposed as the most reliable approach to characterize putative CSC populations. Three major functional tests define functional CSCs3,4,11: a) the
KEYWORDS
cancer stem cells; driver mutations; iPSCs; reprogramming; transformation
ability to initiate tumor formation upon transplantation of limited cell numbers; b) the ability of a given cell population to differentiate into all different lineages comprising the tumor mass; and c) the ability to self-renew as undifferentiated stem cells and thus, to form secondary tumors upon serial transplantation. Another limitation compromising our understanding on CSCs biology relates to potential inter-patient variability as a result of the different genetic and epigenetic backgrounds present in the human population. In addition, cancer arises as a consequence of transforming “driver” mutations able to initiate tumor formation. Upon tumor initiation, positive selection and clonal progression further leads to the accumulation of “passenger” mutations conferring additional growth advantages. Identification of driver mutations might therefore allow not only for the establishment of targeted therapies but also, for the elucidation and targeting of the early events underlying cancer formation. Unfortunately, current bioinformatics are thought to underestimate the role that low frequency mutations might play during carcinogenesis. Therefore, development of novel strategies allowing for the functional evaluation of putative driver mutations and studying the molecular dynamics underlying human cancer initiation are in urgent need.10,12-22 Lastly, it remains unclear whether stem cell properties arise by dedifferentiation of cancer cells during tumor progression and as a result of the accumulation of passengers mutations, whether they are a direct consequence of mutations in multipotent adult stem cell
CONTACT Ignacio Sancho-Martinez [email protected] Centre for Stem Cells and Regenerative Medicine, King’s College London, 28th Floor, Tower Wing, Guy’s Hospital, Great Maze Pond, SE1 9RT, London, UK; Juan Carlos Izpisua Belmonte [email protected] Gene Expression Laboratory Belmonte, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 93037, USA. Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/kccy © 2016 Taylor & Francis
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Figure 1. CSCs can originate by different mechanisms. CSCs can theoretically emerge by mutations on embryonic stem cell populations and therefore lead to the formation of embryonic tumors (a). In addition, the embryonal rest theory speculated that undifferentiated remnants of developmental processes could contribute to tumor initiation. The prevailing theories for adult tumor formation speculate that accumulation of mutations might lead to the dynamic reversion of a differentiated phenotype into a malignant stem cell-like state (b). Alternatively, the similarities between CSCs and adult stem cell populations bring about the possibility that mutations directly affecting tissues-specific progenitor cells underlie CSC generation (c). Nowadays, it is hardly believed that a single hypotheses bears true for all different tumor types. Instead, different mechanisms might underlie the appearance of CSCs in a tumor, or even patient, specific manner.
populations already presenting a dedifferentiated phenotype or whether different mechanisms will be present in different tumor types or even different patients. Thus, 2 major theories attempting to explain the appearance of cancer cells with stem cell properties prevail nowadays3,4,7: a) that dynamic dedifferentiation, or reversion, to a stem cell phenotype occurs as a consequence of mutations and transformation; b) that mutations directly transform tissue-specific stem cell populations into malignant CSCs (Fig. 1). Collectively, the generation of reliable and tractable models functionally recapitulating CSC properties will represent a valuable model for studying the molecular mechanisms underlying tumorigenesis and for the future development of therapeutic strategies.23-27 Notwithstanding, the recent application of cell reprogramming methodologies to cancer biology has opened unexpected new avenues for studying human tumorigenesis in a personalized manner.
methodologies, factors and even the use of virtually any somatic cell lineage as starting cell material for iPSC generation. Yet, despite the technical advancements that rapidly followed the initial reports on iPSCs, translating the promises of reprogramming technologies into the clinic (i.e.: cellular products for transplantation and regenerative medicine) has faced important roadblocks. Notwithstanding, the observations that differentiation of iPSCs in vitro could be used as a surrogate for studying early development and disease in a rapid and controllable manner while avoiding ethical implications, further highlighted that reprogramming technologies are not limited to future cell therapy applications but that can also be applied to advance basic developmental and disease knowledge and even for the establishment and testing of novel therapeutics.30 Disease modeling thus leverages the ability of iPSCs to recapitulate key disease phenotypes that are linked to specific genetic mutations (i.e.,: Fanconi Anemia31,32) or even aberrant epigenetic patterns (i.e.: Angelman Syndrome33).
Reprogramming and disease modeling
Leveraging reprogramming for the modeling of cancer initiation
In 2006, Takahashi and Yamanaka revealed to the world the ability to experimentally generate, mouse28 (Takahashi and Yamanaka, 2006) and later human29 (Takahashi et al., 2007), induced Pluripotent Stem cells (iPSCs) by reprogramming differentiated somatic cell lineages. In their initial study, Takahashi and Yamanaka focused on 24 genes previously identified as playing pivotal roles in the maintenance of pluripotency in Embryonic Stem Cells (ESCs). Four transcription factors (Oct3/4, Sox2, c-Myc and Klf4) were ultimately found to suffice for the dedifferentiation of fibroblasts back to an ESC-like phenotype when maintained in pluripotent culture conditions.28,29 This major breakthrough generated incredible excitement in the scientific community due to the possibilities that iPSCs opened for generating unlimited patient-specific cell lineages for regenerative medicine. Since the initial reports describing iPSC generation by viral-based/integrative approaches, laboratories worldwide rapidly developed alternative reprogramming strategies based on the use of different transgene delivery
Cancer formation can originate by accumulation of mutations occurring de novo in otherwise wild-type cells (Fig. 1). In addition, certain mutations predisposing for cancer initiation can be inherited with additional mutations acquired during the lifetime of the individual ultimately contributing to cancer initiation. It is thus clear that cancer can be considered a genetic disorder in where genetic mutations lead to cell transformation and the acquisition of aberrant epigenetic patterns resulting in malignancy. Consequently, strategies based on the use of pluripotent stem cells (PSCs) could be applied to the modeling of human cancer in an analogous manner to “conventional” disease modeling. Up to date several reports have described the modeling of human cancer by applying reprogramming methodologies, including: the generation of iPSCs by reprogramming of cancer cells,34-43 the generation of iPSCs from somatic lineages bearing mutations associated with inherited cancer,44 and the
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transformation of fibroblasts by oncogenes and tumor suppressor genes to a CSC phenotype.45 Reprogramming of cancer cells into pluripotent cells, or “induced pluripotent cancer cells (iPCCs),” provides a potential platform for the screening of novel therapeutic compounds.46 However, the pluripotent nature of undifferentiated iPCCs would rather resemble an embryonic origin for a given tumor. The first idea that cancer could arise from embryonic cells appeared in the early 19th century and was formally presented by Durante and Conheim as the “embryonal rest theory of cancer.”24 This theory stated that remnants of embryonic tissue remain in adult organs. A change in the environment, or “disequilibrium” in the surrounding tissue (field theory), would allow the embryonic tissue to resume cell proliferation and to produce masses of cells that resembled fetal tissues. By the turn of the 20th century, the embryonic rest theory was generally discredited. Regardless of whether some tumors arise from a pluripotent embryonic cell population as opposed to multipotent progenitor cells, the reprogramming of cancer cells to iPCCs can in certain instances lead to an erasure of malignant phenotypes47 that might limit the applicability of such strategies.48,49 Another report has demonstrated the use of undifferentiated iPSCs for the modeling of brain tumors and screening purposes.50 Modeling cancer by using non-transformed pluripotent cells can provide a useful platform for the identification of novel compounds targeting cell populations with stem cell properties.51 However, and similar to the use of iPCCs, generation of neoplastic pluripotent stem cell models imply a pluripotent origin for a somatic tumor, a hypothesis long time debunked, and prevents the establishment of reliable in vivo models recapitulating non-embryonic tumor-specific features. Indeed, injection of undifferentiated iPSCs into the murine brain did not result in the formation of glioma-like tumors but rather lead to the formation of embryonic teratomas into the brain.50,52 As an alternative approach, reprogramming strategies have been used for the reprogramming of differentiated glioma cells to a CSCs-like phenotype.53 The use of primary or reprogrammed CSCs therefore represents a reliable platform for screening studies focused on the development of novel therapeutics specifically targeting CSC populations. Furthermore, the conversion of differentiated cancer cells to CSCs by reprogramming allows for studying the epigenetic reprogramming events underlying CSC generation from more differentiated cancer cells and potentially contribute to the development of strategies altogether preventing the appearance of CSC populations. As a trade-off, leveraging cellular material that has already undergone transformation and clonal selection for studies on cancer initiation prevents the identification and mechanistic study on the role that different mutations might play as drivers of tumorigenesis.3-5, 7,10, 53-56 Lastly, a complementary strategy could make use of our current understanding of the genetics of different tumors. For example, a combination of reprogramming strategies, whether iPSC generation or lineage conversion, alongside the generation of “cancer-specific” backgrounds by introducing in specific mutations and/or upon manipulation of pro-oncogenic signaling pathways pathways, could result in the generation of novel human cancer models for the study of cellular transformation.
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In such a case, the generation of specific lineages by differentiation could provide a tractable platform for studying tumor initiation processes, the functional testing of putative driver mutations. In addition, applying reprogramming strategies for the study of human cancer could ease studies on the role that different patient-specific genetics play during cancer initiation and therapy susceptibility as discussed hereafter.52 Reprogramming as a platform for the functional identification of driver mutations Identification of driver mutations generally relies on the use of complex algorithms for the analysis of genomic data obtained from tumor samples. Whereas these approaches have generally been successful, they tend to rely on sequencing data and the relative frequencies by which a given mutation is found in patient-tumors while underestimating the role that low frequency mutations might play during carcinogenesis.15-17,57-59 Thus, generation of reliable in vitro and in vivo models might additionally be used for functionally evaluating whether a given mutation act as a driver or passenger as well as for the establishment of therapies altogether preventing malignant cell transformation and cancer formation. The Tabar laboratory first demonstrated the use of pluripotent stem cells studying driver mutations and the modeling of pediatric brain tumors.60 Funato et al differentiated human Embryonic Stem Cells (ESCs) into neural progenitor cells (NPCs) and induced cellular transformation by over expressing a constitutively active form of the oncogene PDGFRA, knocking down the tumor suppressor gene p53 or by expressing the K27M mutant form of histone H3.3, to model the genetics found in pediatric gliomas.60 These manipulated neural stem cells demonstrated tumor-initiating capacity with the resulting tumors recapitulating key pathological features of human pediatric gliomas as well as allowed for the screening of chemicals targeting human GSC populations. However, whereas the use of ESCs allows for the functional study on the role that different mutations or epigenetic alterations play in CSC formation and tumor initiation, it is worth noting that it limits the possibility of studying the influence that different patient-specific genetic backgrounds might play in tumorigenesis and malignancy. In addition, the use of human embryonic material still faces ethical controversy. To tackle these caveats, we have recently reported on the establishment of tractable human iPSC models for the study of driver mutations and GSC formation. More recently, we reported on the use of human iPSCs for studying adult glioma initiation. iPSC-derived NPC (iNPCs) transformation was induced by combining different mutations typically observed in human gliomas that collectively affect the PI3K, MAPK and p53 signaling pathways.61 Genetic manipulation of human iNPCs led to the generation of human cellular models presenting functional GSC features.52 Indeed, our results indicated that the use of different genetic alterations allowed for the generation of different human glioma subtypes. In vivo transplantation resulted in the generation of tumors displaying histopathological features hardly recapitulated with conventional xenograft models.34,62,63 Most interestingly, leveraging the established models alongside the use chemical inhibitors we were able to demonstrate the causative role of
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PI3K and MAPK signaling during the metabolic reprogramming underlying the transformation of NPCs to GSCs. Lastly, a pilot screening of 101 FDA-approved compounds further identified 3 different compounds able to compromise GSC viability and migration while isogenic wild-type NPCs remained unaffected.52 In summary, modeling cancer by reprogramming approaches might ultimately provide platforms for studying the mechanisms underlying human tumor initiation and cancer cell plasticity as well as facilitate the identification and testing of novel therapeutic compounds and the development of differentiation therapies.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding Work in the laboratory of I.S.-M. was supported by The Foundation for Liver Research and King’s College London. Work in the laboratory of J.C. I.B. was supported by the G. Harold and Leila Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust, The Moxie Foundation and UCAM.
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