Current treatment regimens for rhabdomyosarcoma (RMS), the most common pediatric soft tissue cancer, rely on conventional chemotherapy, and although they show clinical benefit, there is a significant risk of adverse side effects and secondary tumors later in life. Therefore, identifying and targeting sub-populations with higher tumorigenic potential and self-renewing capacity would offer improved patient management strategies. Hedgehog signaling has been linked to the development of embryonal RMS (ERMS) through mouse genetics and rare human syndromes. However, activating mutations in this pathway in sporadic RMS are rare and therefore the contribution of hedgehog signaling to oncogenesis remains unclear. Here, we show by genetic loss- and gain-of-function experiments and the use of clinically relevant small molecule modulators that hedgehog signaling is important for controlling self-renewal of a subpopulation of RMS cells in vitro and tumor initiation in vivo. In addition, hedgehog activity altered chemoresistance, motility and differentiation status. The core stem cell gene NANOG was determined to be important for ERMS self-renewal, possibly acting downstream of hedgehog signaling. Crucially, evaluating the presence of a subpopulation of tumor-propagating cells in patient biopsies identified by GLI1 and NANOG expression had prognostic significance. Hence, this work identifies novel functional aspects of hedgehog signaling in ERMS, redefines the rationale for its targeting as means to control ERMS self-renewal and underscores the importance of studying functional tumor heterogeneity in pediatric cancers.

BackgroundThe transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells.ResultsTransgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B) was shown to be restricted to the inner cell mass (ICM) of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF.ConclusionOverexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent manner as STAT3 or Nanog.

Naive pluripotency is established in preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of naive pluripotency. 2i culture has optimized this state, leading to a gene signature and DNA hypomethylation closely comparable to preimplantation epiblast, the developmental ground state. Here we show that Pramel7 (PRAME-like 7), a protein highly expressed in the inner cell mass (ICM) but expressed at low levels in ESCs, targets for proteasomal degradation UHRF1, a key factor for DNA methylation maintenance. Increasing Pramel7 expression in serum-cultured ESCs promotes a preimplantation epiblast-like gene signature, reduces UHRF1 levels and causes global DNA hypomethylation. Pramel7 is required for blastocyst formation and its forced expression locks ESCs in pluripotency. Pramel7/UHRF1 expression is mutually exclusive in ICMs whereas Pramel7-knockout embryos express high levels of UHRF1. Our data reveal an as-yet-unappreciated dynamic nature of DNA methylation through proteasome pathways and offer insights that might help to improve ESC culture to reproduce in vitro the in vivo ground-state pluripotency.

Developmental biology, regenerative medicine and cancer biology are more and more interested in understanding the molecular mechanisms controlling pluripotency and self-renewal in stem cells. Pluripotency is maintained by a synergistic interplay between extrinsic stimuli and intrinsic circuitries, which allow sustainment of the undifferentiated and self-renewing state. Nevertheless, even though a lot of efforts have been made in the past years, the precise mechanisms regulating these processes remain unclear. One of the key extrinsic factors is leukemia inhibitory factor (LIF) that is largely used for the cultivation and derivation of mouse embryonic and induced pluripotent stem cells. LIF acts through the LIFR/gp130 receptor and activates STAT3, an important regulator of mouse embryonic stem cell self-renewal. STAT3 is known to inhibit differentiation into both mesoderm and endoderm lineages by preventing the activation of lineage-specific differentiation programs. However, LIF activates also parallel circuitries like the PI3K-pathway and the MEK/ERK-pathway, but its mechanisms of action remain to be better elucidated. This review article aims at summarizing the actual knowledge on the importance of LIF in the maintenance of pluripotency and self-renewal in embryonic and induced pluripotent stem cells.