JARID1B has recently been identified as a H3K4 demethylase (Christensen et al., 2007
; Yamane et al., 2007
). H3K4 methylation has been proposed as a critical component in regulating gene expression and cellular identities and as an epigenetic mark in embryonic stem cells (Guenther et al., 2007
). Demethylation of H3K4 by JARID1B was shown to play a role in cell fate decisions of embryonic stem cells by blockage of terminal differentiation (Dey et al., 2008
). In cancer, it is known that demethylation of H3K4 supports transformation from hematopoietic precursors to leukemia stem cells via regulation of the developmental Hox gene family (Krivtsov and Armstrong, 2007
). As previously suggested for other chromatin remodeling factors such as HDACs or the histone methylase SUV39H1 (Macaluso et al., 2006
), also JARID1B maybe part of a multi-molecular complex consisting of DNA, histones, transcription factors such as PAX9, FOXG1B, or LMO2, and pRB (Roesch et al., 2008
; Tan et al., 2003
). Former studies by us indicated that JARID1B can be actively involved in the maintenance of the slow-cycling state in melanoma via stabilization of pRB (Ser795)-mediated cell cycle control (Roesch et al., 2006
). Interestingly, stabilization of hypophosphorylated pRB is usually understood as a tumor-suppressive mechanism because of its anti-proliferative effect, but in the long run, slow proliferation can be also associated with tumor maintenance as we suggest here. Thus, JARID1B may have a dual role over time, immediately anti-proliferative but long-term tumor maintaining.
JARID1B is overexpressed in breast cancer (Lu et al., 1999
) raising the question of whether it determines also in epithelial malignancies a population with increased stemness capacity. Our unpublished observations from head and neck squamous cell carcinoma (HNSCC) revealed a broad JARID1B immunoreactivity throughout the entire epithelial portion and, thus, in a pattern very different from that of melanoma. A possible explanation for this discrepancy is provided by publications on another family member, JARID2. JARID2 can be expressed in an undulating fashion. It is strongly expressed in embryonic stem cells, decreases when cells proliferate and tissues expand, but increases again after terminal differentiation (Takeuchi et al., 2006
). Thus, the observed immunostaining pattern of HNSCCs could reflect a high expression of JARID1B also in differentiated carcinoma cells. Since epithelial carcinomas generally harbor more differentiated cells than melanomas, it was also not surprising that sorted label-retaining esophageal carcinoma cells showed decreased stemness (differentiated cells were less clonogenic, not shown). Consequently, to unravel the role of JARID1B as a marker for epithelial CSCs, further biological markers are needed to exclude the differentiated progeny.
According to the traditional CSC concept, tumor initiation is regarded as an exclusive characteristic of CSCs (Clarke et al., 2006
). Thus, our finding that tumor initiation in immunodeficient animals is independent of JARID1B expression challenges the role of JARID1B as a classic CSC marker. In highly susceptible xenotransplantation assays like the one we used, the ratio of tumor initiating cells can increase up to 25% of unsorted cells (Quintana et al., 2008
). This suggests that either virtually every melanoma cell is a CSC because it can induce de novo
tumors in xenograft assays irrespective of any known stem cell marker, or that melanoma is not hierarchically organized into subpopulations of tumorigenic and non-tumorigenic cells and the CSC model does not apply. However, tumor initiation in xenograft models could be more dependent on secondary factors, such as the adaptation to host-derived growth factors (Adams and Strasser, 2008
). For example, congenic transplantation of murine lymphoma cells revealed that tumor initiation is not necessarily restricted to a minority population (Kelly et al., 2007
). Thus, it is generally questionable whether the ability of melanoma cells to initiate de novo
tumors really reflects the same potential as to maintain growth within an already established tumor microenvironment. Particularly the normalized growth data in support that this assay measures more the gradual loss of tumor growth than the loss of tumor initiation.
The Weinberg group recently observed a dependency of self-renewal on exogenous factors. Treatment of immortal human mammary epithelial cells with recombinant TGFβ could induce the stem like CD44high
phenotype known from breast CSCs (Mani et al., 2008
). Ours and Weinberg’s data suggest that at least some stem-like cells from solid tumors may actually not be static entities, but rather tumor cells that transiently acquire stemness properties depending on the tumor context. This supports a model of dynamic stemness in which at any given point of time a slow-cycling, self-renewing but dynamic subpopulation of tumor cells exists among the bulk of tumor cells. This subpopulation might continually arise or disappear (‘moving target’). Certainly, improved assays, e.g. humanized mouse models, are critically needed to address these questions in the future.
Since the study by Mani et al. suggested epithelial-mesenchymal transition (EMT) as an alternative stemness-associated mechanism, we asked if also JARID1B-positive melanoma cells show EMT or, at least, an EMT-like phenotype (because of their neuroectodermal origin, melanoma cells may not undergo classic EMT). However, none of our genome-wide profiling experiments (1. JARID1B knockdown vs. scrambled, 2. transient JARID1B overexpression vs. mock, and 3. J/EGFP-positive vs. J/EGFP-negative) detected a consistent classic EMT signature in correlation with high JARID1B expression. We also did not find a consistent overlap with previously suggested stemness markers, like CD133 (Monzani et al., 2007
) and p75/NGFR (Wong et al., 2006
). A trend towards higher expression of CD20 (Fang et al., 2005
) in J/EGFP-positive cells was assumed but due to the low overall expression frequency (0.7’2%), there was a lack of experimental consistency in replicate analyses (not shown).
The existence of a slow-cycling subpopulation is of high clinical importance because almost all current therapeutic regimens predominantly target the rapidly proliferating tumor bulk (Blagosklonny, 2005
). Indeed, we observed that anti-cancer therapies in vitro
(cytotoxic or BRAFV600E-targeted) uniformly result in an enrichment for JARID1B-positive cells (data not shown). Thus, we postulate that targeting of the slow-cycling subpopulation, e.g. by inhibition of its H3K4 demethylase activity, in combination with a conventional debulking strategy could help eradicate all melanoma cells and increase the low therapy response rate in malignant melanoma.