- Email: [email protected]
iPSCs from cancer cells: challenges and opportunities
Spotlight
iPSCs from cancer cells: challenges and opportunities Vero´nica Ramos-Mejia1, Mario F. Fraga2,3 and Pablo Menendez1 1
Pfizer-University of Granada-Andalusian Government Centre for Genomics and Oncological Research (GENyO), Avda de la Ilustracio´n 114, 18007, Granada, Spain 2 Cancer Epigenetics Laboratory, Instituto Universitario de Oncologı´a del Principado de Asturias, HUCA, Universidad de Oviedo, 33006, Oviedo, Spain 3 Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a/CNB-CSIC, Cantoblanco, 28049, Madrid, Spain
Reprogramming and oncogenic transformation are stepwise processes that share many similarities, and induced pluripotent stem cells (iPSCs) generated from cancer cells could illuminate molecular mechanisms underlying the pathogenesis of human cancer. Deciphering the barriers underlying the reprogramming process of primary cancer cells could reveal information on the links between pluripotency and oncogenic transformation that would be instrumental for therapy development.
Reprogramming human cancer cells: state-of-the-art The discovery of methods for reprogramming somatic cells into induced pluripotent stem cells (iPSCs) through ectopic expression of a few pluripotency factors holds the promise of generating custom-tailored cells for disease modeling, drug screening studies, and treatment of numerous diseases [1]. Indeed, iPSCs are routinely generated from tissues obtained from healthy donors and patients as well as cell types at different ontogeny and developmental stages. Intriguingly, reprogramming human primary cancer cells remains a challenge. Despite significant interest in generating iPSCs from cancer cells to help determine the mechanisms that underlie oncogenic transformation, there are only five reports demonstrating successful reprogramming of malignant human cells and, unfortunately, only one of these reports reprogrammed human primary cancer cells (the remaining studies used cell lines). Similar to non-cancerous tissues, a variety of techniques have been used to reprogram cancer cells. Carrete et al. [2] generated iPSCs from the chronic myeloid leukemia (CML) cell line KBM7 carrying the BCR-ABL fusion oncogene by expressing four ectopic reprogramming factors (OCT4, KLF4, SOX2, and c-MYC [OKSM]). In another study, Choi et al. [3] reprogrammed EBV-immortalized B lymphocytes to pluripotency using non-integrative episomal vectors. Lin et al. [4] reprogrammed human skin cancer cell lines to pluripotency using the microRNA miR-302. Miyoshi et al. [5] reprogrammed gastrointestinal transformed cell lines using retroviral vectors expressing c-MYC and BCL2. Finally, Hu et al. [6] reported what is probably the most interesting piece of work describing cancer cell reprogramming. These authors successfully reprogrammed primary human lymphoblasts from a BCR-ABL+ CML patient using
Corresponding author: Menendez, P. ([email protected]). Keywords: induced pluripotent stem cells; cancer cells; reprogramming; transformationoncogenesis.
transgene-free iPSC technology to ectopically express OKSM and LIN28. Reprogramming human primary cancer cells: biological or technical reprogramming barriers? This small number of successes represents only a minor fraction of attempts to reprogram human primary cancer cells to pluripotency. These experiments remain a challenge, and the hurdles for reprogramming primary cancer cells are yet to be determined. Although technical constraints cannot be excluded as the cause of failure, elegant nuclear transplantation studies by the Jaenisch group [7] in 2004 support the idea that uncharacterized, fundamental biological barriers exist (Figure 1) that prevent the reestablishment of developmental pluripotency in malignant cancer cells. When nuclei from melanoma, leukemia, lymphoma, and breast cancer cells were transplanted into oocytes, the reprogramming activity of the cytoplasm was only effective for the melanoma genome, arguing that not all cancer genomes can be epigenetically reprogrammed to pluripotency. Although cancer-specific genetic lesions may be an obstacle to the reprogramming process, several lines of evidence indicate that genetic lesions on their own may not impede reprogramming. First, leukemic cell lines carrying fusion oncogenes have been reprogrammed, presenting a proof-of-principle that cells carrying chromosomal translocations can be reprogrammed. Second, limiting tumor suppressor activity can be important for modulating the reprogramming process. Indeed, reduced expression of p53, p16INK4a/Rb, p19ARF, or p21CIP1 leads to more efficient and faster cellular reprogramming [8]. Because most tumors have dismantled the p53 and p16INK4a/Rb tumor suppressor pathways, one would expect enhanced or accelerated reprogramming of human primary cancer cells with p53 and/or p16INK4a/Rb inactivation. However, iPSCs generated from primary cancer cells with p53 and/or p16INK4a/ Rb inactivation has not yet been reported. Third, iPSCs have been generated from patients with a variety of genetic diseases [9], further indicating that inherited genetic lesions themselves do not impose an insurmountable barrier for reprogramming. DNA damage response (DDR) programs preserve genome integrity and suppress malignant transformation. One can hypothesize that cells with inefficient DDR may be refractory to reprogramming; however, increasing evidence suggests that many forms of DNA damage are still compatible with reprogramming. First, ataxia 245
Spotlight
Trends in Molecular Medicine May 2012, Vol. 18, No. 5
(a)
Primary non-malignant cell OCT4, KLF4, SOX2, C-MYC
(b)
Primary cancer cell e.g. OCT4, KLF4, SOX2, C-MYC, LIN28, miR302
Bona fide iPSCs Efficient reprogramming to pluripotency
Directed differentiation
Inability to reprogram human primary cancer cells
Bona fide iPSCs
Valuable information on reprogramming and oncogenic transformation
Reprogramming barriers ? Genetic Lesions ?
Epigenetic marks ?
Genomic instability and poor DDR? Reprogramming-induced senescence? Technical constrains?
Cell lineage A
Cell lineage B
Cell lineage C TRENDS in Molecular Medicine
Figure 1. Potential biological barriers impeding the reprogramming of human primary cancer cells. (a) iPSCs can be routinely generated from a variety of non-cancerous tissues or cell types at different ontogeny and developmental stages. Directed differentiation of iPSCs holds the promise of generating custom-tailored cells for many downstream applications such as cell therapy, disease modeling, drug screening, and developmental biology. (b) Reprogramming of human primary cancer cells to pluripotency remains a challenge. The difficulties in reprogramming cancer cells do not seem exclusively due to either technical barriers or the need for improved reprogramming technologies. Rather, it seems that biological barriers such as cancer-specific genetic mutations, epigenetic remodeling, accumulation of DNA damage, or reprogramming-induced cellular senescence may influence the reprogramming efficiency of human primary cancer cells.
telangiectasia mutated (ATM), which is critical for sensing and responding to DNA double-strand breaks, may play an important role during reprogramming. ATM / iPSCs are, however, identical to wild type iPSCs, indicating that ATM and possible other key DDR proteins do not have an essential role during the reprogramming process. Second, Hutchinson–Gilford progeria syndrome (HGPS), a fatal premature aging syndrome characterized by genomic instability and profound defects in the DDR machinery has recently been recapitulated with iPSCs. The successful generation of iPSCs from HGPS patients represents a proof-of-principle that reprogramming to pluripotency may be successfully attained in cells accumulating unrepaired DNA damage. These data suggest that accumulation of DNA damage does not seem to represent an insurmountable ‘reprogramming barrier’. Epigenetic modifications are central to many developmental processes and are a hallmark of cancer. Genome sequencing studies reveal that some tumors lack somatic mutations or copy number variations (i.e. MLL-AF4+ proB acute lymphoblastic leukemia), indicating that epigenetic alterations rather than somatic genetic mutations may drive the oncogenic process. Cancers with very few irreversible genetic lesions represent an ideal system in which to address the extent potential epigenetic alterations influence the reprogramming process of a malignant cell. Both cancer cells and iPSCs display global hypomethylation, and it is vital to address whether these epigenetic changes are necessary for nuclear reprogramming or a consequence of the process to determine to what extent epigenetic alterations influence the ‘reprogramming process’ in tumors. Although demethylation of pluripotency genes is necessary, the functional importance of de novo 246
DNA methylation of developmental genes is not obvious and recent studies suggest that de novo DNA methylation is dispensable for the nuclear reprogramming of somatic cells. Thus, if there were epigenetic marks that would make cancer cells difficult to reprogram, such marks would probably be associated with pluripotency factors. Nuclear reprogramming induces the demethylation and expression of pluripotency genes that otherwise become naturally repressed by promoter hypermethylation during differentiation. If other, unknown pluripotency factors exist that are not repressed by DNA methylation during differentiation and become aberrantly hypermethylated in cancer, they may be targets for therapeutic demethylation. A future task should be to identify these potential pluripotency factors, determining their susceptibility to epigenetic reprogramming in cancer cells. It has been recently established that aberrant oncogene expression triggers senescence (OIS; oncogene-induced senescence) in primary cells, thus limiting oncogenic transformation [8]. Importantly, reprogramming and senescence are related processes as shown by studies demonstrating that reprogramming cells is more challenging the closer cells are to the onset of senescence [8]. The expression of reprogramming factors triggers senescence (RIS; reprogramming-Induced senescence) by activating several tugene mor-suppressive mechanisms. In addition, expression profiling studies have revealed that signature genes activated during reprogramming are common to these anti-proliferative responses [8]. As a consequence, one might expect that malignant tumors which have bypassed OIS reprogram to pluripotency easier than those benign/pre-malignant tumors that are highly senescent. But this is not the case, suggesting that RIS might confer a
Spotlight reprogramming barrier, a topic that has been excellently reviewed elsewhere [8]. The small number of reports on reprogramming human primary cancer cells is a limitation to deciphering the biological or technical barriers preventing the reprogramming of cancer cells. Scientists working in stem cells, reprogramming, and cancer biology should be encouraged to report scientific failures and negative data as this would provide relevant clues to define specific hurdles that need to be circumvented and whether these obstacles are cancer type- or cancer stage-dependent. Cancer cell-specific iPSCs: a unique system for modeling cancer pathogenesis and drug screening The mechanisms of oncogenic transformation are not amenable to analysis with patient samples because the cancers are studied once the transformation events have already occurred. Compelling evidence indicates that reprogramming, pluripotency, lineage-specification, and oncogenic transformation are deeply intertwined processes [8]. For instance, cancerous mutations in oncogenes and tumor suppressor genes or chromosomal rearrangements leading to constitutive expression of fusion oncogenes are usually affiliated with a specific tissue or cell type, indicating that the effects of cancer-relevant mutations are highly influenced by the environment and differentiation state of a given cell. It is also well accepted that cancer arises from ‘less differentiated’ cells and that cancer propagation is sustained by so-called cancer stem cells (CSCs) that are multipotent and have self-renewal capabilities. Also, aggressive poorly differentiated human cancers express high levels of pluripotency-associated factors, suggesting that reprogramming to a more dedifferentiated state occurs during tumor progression. It is also worth noting that iPSCs (and ESCs) cause teratomas (a benign germ cell tumor) upon injection into immunodeficient mice Therefore, an unprecedented approach to study the interaction of specific oncogenic mutations with different tissue types and their association with specific developmental states is to reprogram human primary cancer cells to pluripotency. Re-differentiation of these cancer cell-specific iPSCs towards disease-affected and unaffected lineages represents a unique cellular system for modeling cancer pathogenesis by understanding how specific oncogenic mutations impose the tumor phenotype on a particular lineage and developmental state. The availability of widely banked patient-specific cancer cells and cord blood units from newborns who later developed childhood cancer will also offer a unique opportunity to unravel the developmental and molecular mechanisms underlying the stepwise transformation process from a preleukemic to a leukemic clone. A corollary to the malignant phenotype being reversed to a pluripotent state is that if a cancer cell can be fully reprogrammed it would suggest that the so-called ‘cancer state’ is not irreversible, a concept that would have significant implications for cancer biology.
Trends in Molecular Medicine May 2012, Vol. 18, No. 5
In addition, drug screening/development assays leading to useful therapeutic approaches may be undertaken after directed differentiation of cancer cell-specific iPSCs into the cells of interest. The rationale for such an application is at least two-fold: (i) if the oncogenic mutations present in cancer cell-iPSCs prevent cellular differentiation, differentiation-inducing compounds that can cause regression of some tumors, such as all-trans retinoid acid in the case of acute promyelocytic leukemia, can be assayed [10]; and (ii) drugs selectively eliminating cells carrying a specific oncogenic mutation may be tested in a range of cell types, including iPSCs and their differentiated derivatives, generated from a variety of cancers. Concluding remarks Reprogramming, pluripotency, and oncogenic transformation are connected processes that share many similarities, as outlined above [8]. Collectively, the fact that the same alterations that drive tumorigenesis robustly influence the reprogramming of non-cancer somatic cells is a doubleedged sword: it poses safety concerns for future cell therapy applications with iPSCs while at the same time it encourages prospective future studies aimed at analyzing the mechanisms and barriers underlying the direct reprogramming of cancer cells (Figure 1) in an attempt to reveal valuable new information on the links between reprogramming and cell transformation. Acknowledgments This work was supported by The Junta de Andalucia/FEDER (P08-CTS3678), The FIS/FEDER (PI10/00449), The MICINN (Fondo Especial del Estado para Dinamizacio´n de la Economı´a y Empleo-PLE-2009-0111) and The Spanish Association Against Cancer/Junta Provincial de Albacete (CI110023).
References 1 Takahashi, K. and Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 2 Carette, J.E. et al. (2010) Generation of iPSCs from cultured human malignant cells. Blood 115, 4039–4042 3 Choi, S.M. et al. (2011) Reprogramming of EBV-immortalized Blymphocyte cell lines into induced pluripotent stem cells. Blood 118, 1801–1805 4 Lin, S.L. et al. (2008) Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA 14, 2115–2124 5 Miyoshi, N. et al. (2010) Defined factors induce reprogramming of gastrointestinal cancer cells. Proc. Natl. Acad. Sci. U.S.A. 107, 40–45 6 Hu, K. et al. (2011) Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood 117, e109–e119 7 Hochedlinger, K. et al. (2004) Reprogramming of a melanoma genome by nuclear transplantation. Genes Dev. 18, 1875–1885 8 Banito, A. and Gil, J. (2010) Induced pluripotent stem cells and senescence: learning the biology to improve the technology. EMBO Rep. 11, 353–359 9 Park, I.H. et al. (2008) Disease-specific induced pluripotent stem cells. Cell 134, 877–886 10 Menendez, P. et al. (2006) Human embryonic stem cells: a journey beyond cell replacement therapies. Cytotherapy 8, 530–541 1471-4914/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2012.04.001 Trends in Molecular Medicine, May 2012, Vol. 18, No. 5
247
iPSCs from cancer cells: challenges and opportunities Vero´nica Ramos-Mejia1, Mario F. Fraga2,3 and Pablo Menendez1 1
Pfizer-University of Granada-Andalusian Government Centre for Genomics and Oncological Research (GENyO), Avda de la Ilustracio´n 114, 18007, Granada, Spain 2 Cancer Epigenetics Laboratory, Instituto Universitario de Oncologı´a del Principado de Asturias, HUCA, Universidad de Oviedo, 33006, Oviedo, Spain 3 Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a/CNB-CSIC, Cantoblanco, 28049, Madrid, Spain
Reprogramming and oncogenic transformation are stepwise processes that share many similarities, and induced pluripotent stem cells (iPSCs) generated from cancer cells could illuminate molecular mechanisms underlying the pathogenesis of human cancer. Deciphering the barriers underlying the reprogramming process of primary cancer cells could reveal information on the links between pluripotency and oncogenic transformation that would be instrumental for therapy development.
Reprogramming human cancer cells: state-of-the-art The discovery of methods for reprogramming somatic cells into induced pluripotent stem cells (iPSCs) through ectopic expression of a few pluripotency factors holds the promise of generating custom-tailored cells for disease modeling, drug screening studies, and treatment of numerous diseases [1]. Indeed, iPSCs are routinely generated from tissues obtained from healthy donors and patients as well as cell types at different ontogeny and developmental stages. Intriguingly, reprogramming human primary cancer cells remains a challenge. Despite significant interest in generating iPSCs from cancer cells to help determine the mechanisms that underlie oncogenic transformation, there are only five reports demonstrating successful reprogramming of malignant human cells and, unfortunately, only one of these reports reprogrammed human primary cancer cells (the remaining studies used cell lines). Similar to non-cancerous tissues, a variety of techniques have been used to reprogram cancer cells. Carrete et al. [2] generated iPSCs from the chronic myeloid leukemia (CML) cell line KBM7 carrying the BCR-ABL fusion oncogene by expressing four ectopic reprogramming factors (OCT4, KLF4, SOX2, and c-MYC [OKSM]). In another study, Choi et al. [3] reprogrammed EBV-immortalized B lymphocytes to pluripotency using non-integrative episomal vectors. Lin et al. [4] reprogrammed human skin cancer cell lines to pluripotency using the microRNA miR-302. Miyoshi et al. [5] reprogrammed gastrointestinal transformed cell lines using retroviral vectors expressing c-MYC and BCL2. Finally, Hu et al. [6] reported what is probably the most interesting piece of work describing cancer cell reprogramming. These authors successfully reprogrammed primary human lymphoblasts from a BCR-ABL+ CML patient using
Corresponding author: Menendez, P. ([email protected]). Keywords: induced pluripotent stem cells; cancer cells; reprogramming; transformationoncogenesis.
transgene-free iPSC technology to ectopically express OKSM and LIN28. Reprogramming human primary cancer cells: biological or technical reprogramming barriers? This small number of successes represents only a minor fraction of attempts to reprogram human primary cancer cells to pluripotency. These experiments remain a challenge, and the hurdles for reprogramming primary cancer cells are yet to be determined. Although technical constraints cannot be excluded as the cause of failure, elegant nuclear transplantation studies by the Jaenisch group [7] in 2004 support the idea that uncharacterized, fundamental biological barriers exist (Figure 1) that prevent the reestablishment of developmental pluripotency in malignant cancer cells. When nuclei from melanoma, leukemia, lymphoma, and breast cancer cells were transplanted into oocytes, the reprogramming activity of the cytoplasm was only effective for the melanoma genome, arguing that not all cancer genomes can be epigenetically reprogrammed to pluripotency. Although cancer-specific genetic lesions may be an obstacle to the reprogramming process, several lines of evidence indicate that genetic lesions on their own may not impede reprogramming. First, leukemic cell lines carrying fusion oncogenes have been reprogrammed, presenting a proof-of-principle that cells carrying chromosomal translocations can be reprogrammed. Second, limiting tumor suppressor activity can be important for modulating the reprogramming process. Indeed, reduced expression of p53, p16INK4a/Rb, p19ARF, or p21CIP1 leads to more efficient and faster cellular reprogramming [8]. Because most tumors have dismantled the p53 and p16INK4a/Rb tumor suppressor pathways, one would expect enhanced or accelerated reprogramming of human primary cancer cells with p53 and/or p16INK4a/Rb inactivation. However, iPSCs generated from primary cancer cells with p53 and/or p16INK4a/ Rb inactivation has not yet been reported. Third, iPSCs have been generated from patients with a variety of genetic diseases [9], further indicating that inherited genetic lesions themselves do not impose an insurmountable barrier for reprogramming. DNA damage response (DDR) programs preserve genome integrity and suppress malignant transformation. One can hypothesize that cells with inefficient DDR may be refractory to reprogramming; however, increasing evidence suggests that many forms of DNA damage are still compatible with reprogramming. First, ataxia 245
Spotlight
Trends in Molecular Medicine May 2012, Vol. 18, No. 5
(a)
Primary non-malignant cell OCT4, KLF4, SOX2, C-MYC
(b)
Primary cancer cell e.g. OCT4, KLF4, SOX2, C-MYC, LIN28, miR302
Bona fide iPSCs Efficient reprogramming to pluripotency
Directed differentiation
Inability to reprogram human primary cancer cells
Bona fide iPSCs
Valuable information on reprogramming and oncogenic transformation
Reprogramming barriers ? Genetic Lesions ?
Epigenetic marks ?
Genomic instability and poor DDR? Reprogramming-induced senescence? Technical constrains?
Cell lineage A
Cell lineage B
Cell lineage C TRENDS in Molecular Medicine
Figure 1. Potential biological barriers impeding the reprogramming of human primary cancer cells. (a) iPSCs can be routinely generated from a variety of non-cancerous tissues or cell types at different ontogeny and developmental stages. Directed differentiation of iPSCs holds the promise of generating custom-tailored cells for many downstream applications such as cell therapy, disease modeling, drug screening, and developmental biology. (b) Reprogramming of human primary cancer cells to pluripotency remains a challenge. The difficulties in reprogramming cancer cells do not seem exclusively due to either technical barriers or the need for improved reprogramming technologies. Rather, it seems that biological barriers such as cancer-specific genetic mutations, epigenetic remodeling, accumulation of DNA damage, or reprogramming-induced cellular senescence may influence the reprogramming efficiency of human primary cancer cells.
telangiectasia mutated (ATM), which is critical for sensing and responding to DNA double-strand breaks, may play an important role during reprogramming. ATM / iPSCs are, however, identical to wild type iPSCs, indicating that ATM and possible other key DDR proteins do not have an essential role during the reprogramming process. Second, Hutchinson–Gilford progeria syndrome (HGPS), a fatal premature aging syndrome characterized by genomic instability and profound defects in the DDR machinery has recently been recapitulated with iPSCs. The successful generation of iPSCs from HGPS patients represents a proof-of-principle that reprogramming to pluripotency may be successfully attained in cells accumulating unrepaired DNA damage. These data suggest that accumulation of DNA damage does not seem to represent an insurmountable ‘reprogramming barrier’. Epigenetic modifications are central to many developmental processes and are a hallmark of cancer. Genome sequencing studies reveal that some tumors lack somatic mutations or copy number variations (i.e. MLL-AF4+ proB acute lymphoblastic leukemia), indicating that epigenetic alterations rather than somatic genetic mutations may drive the oncogenic process. Cancers with very few irreversible genetic lesions represent an ideal system in which to address the extent potential epigenetic alterations influence the reprogramming process of a malignant cell. Both cancer cells and iPSCs display global hypomethylation, and it is vital to address whether these epigenetic changes are necessary for nuclear reprogramming or a consequence of the process to determine to what extent epigenetic alterations influence the ‘reprogramming process’ in tumors. Although demethylation of pluripotency genes is necessary, the functional importance of de novo 246
DNA methylation of developmental genes is not obvious and recent studies suggest that de novo DNA methylation is dispensable for the nuclear reprogramming of somatic cells. Thus, if there were epigenetic marks that would make cancer cells difficult to reprogram, such marks would probably be associated with pluripotency factors. Nuclear reprogramming induces the demethylation and expression of pluripotency genes that otherwise become naturally repressed by promoter hypermethylation during differentiation. If other, unknown pluripotency factors exist that are not repressed by DNA methylation during differentiation and become aberrantly hypermethylated in cancer, they may be targets for therapeutic demethylation. A future task should be to identify these potential pluripotency factors, determining their susceptibility to epigenetic reprogramming in cancer cells. It has been recently established that aberrant oncogene expression triggers senescence (OIS; oncogene-induced senescence) in primary cells, thus limiting oncogenic transformation [8]. Importantly, reprogramming and senescence are related processes as shown by studies demonstrating that reprogramming cells is more challenging the closer cells are to the onset of senescence [8]. The expression of reprogramming factors triggers senescence (RIS; reprogramming-Induced senescence) by activating several tugene mor-suppressive mechanisms. In addition, expression profiling studies have revealed that signature genes activated during reprogramming are common to these anti-proliferative responses [8]. As a consequence, one might expect that malignant tumors which have bypassed OIS reprogram to pluripotency easier than those benign/pre-malignant tumors that are highly senescent. But this is not the case, suggesting that RIS might confer a
Spotlight reprogramming barrier, a topic that has been excellently reviewed elsewhere [8]. The small number of reports on reprogramming human primary cancer cells is a limitation to deciphering the biological or technical barriers preventing the reprogramming of cancer cells. Scientists working in stem cells, reprogramming, and cancer biology should be encouraged to report scientific failures and negative data as this would provide relevant clues to define specific hurdles that need to be circumvented and whether these obstacles are cancer type- or cancer stage-dependent. Cancer cell-specific iPSCs: a unique system for modeling cancer pathogenesis and drug screening The mechanisms of oncogenic transformation are not amenable to analysis with patient samples because the cancers are studied once the transformation events have already occurred. Compelling evidence indicates that reprogramming, pluripotency, lineage-specification, and oncogenic transformation are deeply intertwined processes [8]. For instance, cancerous mutations in oncogenes and tumor suppressor genes or chromosomal rearrangements leading to constitutive expression of fusion oncogenes are usually affiliated with a specific tissue or cell type, indicating that the effects of cancer-relevant mutations are highly influenced by the environment and differentiation state of a given cell. It is also well accepted that cancer arises from ‘less differentiated’ cells and that cancer propagation is sustained by so-called cancer stem cells (CSCs) that are multipotent and have self-renewal capabilities. Also, aggressive poorly differentiated human cancers express high levels of pluripotency-associated factors, suggesting that reprogramming to a more dedifferentiated state occurs during tumor progression. It is also worth noting that iPSCs (and ESCs) cause teratomas (a benign germ cell tumor) upon injection into immunodeficient mice Therefore, an unprecedented approach to study the interaction of specific oncogenic mutations with different tissue types and their association with specific developmental states is to reprogram human primary cancer cells to pluripotency. Re-differentiation of these cancer cell-specific iPSCs towards disease-affected and unaffected lineages represents a unique cellular system for modeling cancer pathogenesis by understanding how specific oncogenic mutations impose the tumor phenotype on a particular lineage and developmental state. The availability of widely banked patient-specific cancer cells and cord blood units from newborns who later developed childhood cancer will also offer a unique opportunity to unravel the developmental and molecular mechanisms underlying the stepwise transformation process from a preleukemic to a leukemic clone. A corollary to the malignant phenotype being reversed to a pluripotent state is that if a cancer cell can be fully reprogrammed it would suggest that the so-called ‘cancer state’ is not irreversible, a concept that would have significant implications for cancer biology.
Trends in Molecular Medicine May 2012, Vol. 18, No. 5
In addition, drug screening/development assays leading to useful therapeutic approaches may be undertaken after directed differentiation of cancer cell-specific iPSCs into the cells of interest. The rationale for such an application is at least two-fold: (i) if the oncogenic mutations present in cancer cell-iPSCs prevent cellular differentiation, differentiation-inducing compounds that can cause regression of some tumors, such as all-trans retinoid acid in the case of acute promyelocytic leukemia, can be assayed [10]; and (ii) drugs selectively eliminating cells carrying a specific oncogenic mutation may be tested in a range of cell types, including iPSCs and their differentiated derivatives, generated from a variety of cancers. Concluding remarks Reprogramming, pluripotency, and oncogenic transformation are connected processes that share many similarities, as outlined above [8]. Collectively, the fact that the same alterations that drive tumorigenesis robustly influence the reprogramming of non-cancer somatic cells is a doubleedged sword: it poses safety concerns for future cell therapy applications with iPSCs while at the same time it encourages prospective future studies aimed at analyzing the mechanisms and barriers underlying the direct reprogramming of cancer cells (Figure 1) in an attempt to reveal valuable new information on the links between reprogramming and cell transformation. Acknowledgments This work was supported by The Junta de Andalucia/FEDER (P08-CTS3678), The FIS/FEDER (PI10/00449), The MICINN (Fondo Especial del Estado para Dinamizacio´n de la Economı´a y Empleo-PLE-2009-0111) and The Spanish Association Against Cancer/Junta Provincial de Albacete (CI110023).
References 1 Takahashi, K. and Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 2 Carette, J.E. et al. (2010) Generation of iPSCs from cultured human malignant cells. Blood 115, 4039–4042 3 Choi, S.M. et al. (2011) Reprogramming of EBV-immortalized Blymphocyte cell lines into induced pluripotent stem cells. Blood 118, 1801–1805 4 Lin, S.L. et al. (2008) Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA 14, 2115–2124 5 Miyoshi, N. et al. (2010) Defined factors induce reprogramming of gastrointestinal cancer cells. Proc. Natl. Acad. Sci. U.S.A. 107, 40–45 6 Hu, K. et al. (2011) Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood 117, e109–e119 7 Hochedlinger, K. et al. (2004) Reprogramming of a melanoma genome by nuclear transplantation. Genes Dev. 18, 1875–1885 8 Banito, A. and Gil, J. (2010) Induced pluripotent stem cells and senescence: learning the biology to improve the technology. EMBO Rep. 11, 353–359 9 Park, I.H. et al. (2008) Disease-specific induced pluripotent stem cells. Cell 134, 877–886 10 Menendez, P. et al. (2006) Human embryonic stem cells: a journey beyond cell replacement therapies. Cytotherapy 8, 530–541 1471-4914/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2012.04.001 Trends in Molecular Medicine, May 2012, Vol. 18, No. 5
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