Based on knowledge of the reprogramming steps, we hypothesized that overcoming MEF-specific networks is the first step in the process. We observed that specific siRNA-mediated knockdown of MEF genes encoding catalytic or regulatory proteins, including WISP1, PRRX1, HMGA2, NFIX, PRKG2, COX2, 6720477E09RIK, and TGFβ3, significantly enhanced reprogramming (). To accelerate screening of small molecules, we employed a computational screening method using the NextBio data-mining framework [15
] and identified six molecules (), including Nabumetone, OHTM, Corynanthine, Moclobemide, NiSO4, and lectin, which function together to reprogram MEFs without Sox2 (). One of those factors alone, OHTM, could partially replace the Sox2 transgene during reprogramming by inducing endogenous Sox2 expression (). We further showed that Nabumetone enhances reprogramming by inhibiting COX2 activity (). Finally, we showed that Nabumetone also promotes reprogramming in the absence of c-Myc or Sox2 function without compromising self-renewal and pluripotency of small molecule-derived iPS cells ().
Nabumetone is a non-steroidal anti-inflammatory drug (NSAID) clinically used primarily to treat pain and inflammation associated with osteoarthritis (OA) or rheumatoid arthritis (RA) [19
]. Nabumetone exerts anti-inflammatory activity by inhibiting COX2 function through its metabolite 6-methoxy-2-naphthylacetic acid (6-MNA). Moreover, it is reported that NSAIDs compromise tumor growth in clinical cases and experimental models of cancer, and also that two isoforms cyclooxygenase-1 and -2 function in a variety of pathophysiological processes, such as modulating apoptosis, angiogenesis, invasion, and carcinogenesis [21
]. Preliminary in vitro
and in vivo
studies show that following COX inhibition, signals regulating cell proliferation and apoptosis networks, including EGFR, KRas, PI3K, JAK1, STAT3, c-jun, PCNA, TGFβ3, BAX, TUNEL, Bcl-2, c-jun, p21, p27, p53, and NM23, are widely altered in tumor cells [27
]. However, the roles of COX inhibitors in tumorigenesis remain controversial, since COX2 expression differs widely in different types of cancer cells [23
]. In this study we showed that COX2 is highly expressed in MEFs and serves as a barrier to reprogramming. Therefore, further analysis is required to understand the biological function and molecular regulation of COX2 in both cancer and reprogramming biology.
Tamoxifen is a standard chemotherapy used to treat primary and advanced breast cancer by blocking the estrogen receptor (ER) via its metabolites OHTM and endoxifen. OHTM activity has been addressed primarily through its effect on the ER [28
]. However, we did not observe detectable levels of ER expression in MEFs (data not shown). OHTM-induced programmed cell death can reportedly be induced through ER-independent pathways in HeLa cells [29
], suggesting that other factors respond to OHTM. Moreover, 3, 4-dihydroxytamoxifen, a more hydroxylated form of OHTM, can interact with both proteins and DNA [28
], suggesting the possibility of numerous targets in vivo
Reprogramming of somatic cells to iPS cells by small molecules could facilitate pharmaceutical and medical applications of pluripotent stem cells [30
]. A number of studies have identified small molecules that enhance reprogramming by targeting various pathways including TGFβ and GSK3 [13
]. Although iPS cells can be generated in the absence of Sox2 [13
], only RepSox has been shown to partially induce Nanog expression in partial iPS cells [13
]. Here, we are the first to report that Sox2 can be induced by OHTM treatment during reprogramming. Further investigation is required to identify pathways modulated by OHTM in MEFs during reprogramming.
Increasing evidence shows that overcoming the security of somatic cell identity is a critical step initiating the transition from mesenchymal to epithelial status [38
]. This step requires large-scale regulation of opposing genes within only few days during the first 8 days of reprogramming, including Cdh1, Epcam, Crb3, Ocln, Snail, Slug, Zeb1, Zeb2, BMP, and TGFβ pathways [40
]. Since TGFβ3 is also on our list and TGFβ3 knock-down greatly enhances reprogramming efficiency, these data support our idea that down-regulating MEF regulatory factors is an effective approach to enhance reprogramming. Furthermore, our study confirms that downregulation of MEF genes encoding catalytic factors constitutes some of the earliest steps of reprogramming and that attenuating key somatic genes is critical to enhance reprogramming efficiency. Further study is needed to reveal how the individual network of these MEF-enriched enzymes functions in the process.
In summary, mouse ES cells express regulatory genes that differ from those expressed in MEFs. Reasoning that the latter encode factors that maintain MEF function and establish fibroblast identity, we manipulated them using either siRNAs or small molecules in an effort to enhance reprogramming efficiency. This hypothesis-driven approach provides an alternative to shot-gun screening, which should accelerate understanding of the molecular mechanism underlying generation of iPS cells and suggest novel therapeutic methodologies.