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Pluripotency factor-mediated expression of the leptin receptor (OB-R) links obesity to oncogenesis through tumor-initiating stem cells

By Stuart P. Atkinson

Tumour initiating stem cells (TISCs) are rare, highly malignant cells identified within diverse tumor types that share important similarities with embryonic stem cells (ESCs) (Clark and FullerVisvader and Lindeman and Clevers) including the mis-regulated expression of OCT4, SOX2, and NANOG (Chen et al and Kim ­et al). Studies have begun to give insight into the events behind TISC function, such as the loss of function of the type II TGF-β receptor and excessive activation of the IL-6 cytokine-signaling pathway, including the downstream effector STAT3 human hepatocellular carcinoma (HCC) (Baek et al and Tang et al). In a mouse model of HCC, isolation of highly tumourigenic CD133+/Nanog+ liver TISCs (Machida et al) on the basis of cell-surface receptors has suggested that identification of TISC-associated cell surface receptor expression and associated signal transduction pathways may be important for TISC function. Therefore researchers from the laboratories of Douglas Edmund Feldman and Keigo Machida at the University of Southern California sought to study this hypothesis and report that the leptin receptor (OB-R or Lepr), a transmembrane receptor for the adipocyte-derived peptide hormone leptin, is important for the tumourigenic nature of TISCs and also for the pluripotent nature of ESCs and induced pluripotent stem cells (iPSCs) (Feldman et al).

Q-PCR analysis of liver TISCs (CD133+/Nanog+) isolated from the transgenic mouse model of HCC demonstrated comparable expression levels of cytokine and adipokine receptors to those observed in non-progenitor liver counterparts (CD133-). However, OB-R was over-expressed in TISCs both at the mRNA and protein level and immunohistochemical and FACS analysis found a significant overlap between CD133 and OB-R in mouse tumour specimens. Detailed analysis of the OB-R gene promoter found a sequence encompassing the proximal 0.5 kb downstream of the transcription start site in exon 1 as a critical mediator of TISC-specific expression of OB-R which contained consensus target motifs for Oct4, Sox2, Nanog, Stat3 and Klf4. Further, an Oct4/Sox2 heterodimer consensus binding site was found near the TSS, and mutation of this site diminished promoter strength, suggesting that this is a major control element. In agreement with this hypothesis, it was found by chromatin immunoprecipitation (ChIP) and electrophoretic mobility-shift assays (EMSA) that Oct4 and Sox2 directly bound this site and further, that lentiviral shRNA-mediated knockdown of Oct4 or Sox2 resulted in a reduction in the level of OB-R expression in TISCs

The proposed link between OB-R and pluripotency was further investigated in other stem cell lines, and a direct correspondence between OB-R expression and pluripotency was found in mouse ESCs, human ESCs and rat iPSCs, indicating that OB-R expression is a general feature of pluripotent cells. Indeed, addition of Leptin to culture medium could keep mouse ESCs in a pluripotent state in the absence of Lif. When TISCs were exposed to Leptin, Stat3, a downstream effector of OB-R, became phosphorylated and enriched at the promoter regions of Oct4 and Sox2. Analysis of Oct4 and Sox2 promoter activity showed increased activity upon Leptin exposure with a corresponding increase at the protein level, although no affect was observed for Nanog. Stable expression of a non-phosphorylatable, dominant-negative Stat3 mutant in TISCs abolished the induction of endogenous Oct4 and Sox2 by leptin suggesting that activation of Stat3 is the basis for induction by Leptin of downstream components of the pluripotency-associated transcription factor network.   TISCs that stably expressed lentiviral shRNAs for Oct4 or Sox2 showed a decreased rate of cell growth, with lower levels of Ki67 than control cells, and were refractory to the addition of Leptin. Oct4/Sox2 knockdown also negatively affected tumour growth when TISCs were implanted into immune-compromised NOG (NOD/Shi-scid/IL-2Rγnull) mice.

Next, TISCS where implanted into obese ob/ob mice, which lack Leptin, and Leprdb/db mice, which lack the Leptin receptor, and while the obese hosts formed larger tumours than lean controls, TISCs implanted into the Leprdb/db grew more rapidly than TISCs in the obese mice. This was correlative to a reduced level of phosphorylated Stat3 in tumours from ob/ob mice compared to the Leprdb/db mice suggesting that Leptin may act as a central activator in TISCs. Added significance was given to these findings upon the finding that OB-R was highly co-expressed in CD133+ cells from human HCC specimens compared to CD133- cells, and further, a link was made between OCT4 and OB-R expression in a diverse array of human cancers extending the clinical significance of the causal relationship between OCT4 and OB-R first identified in mouse TISCs.

Overall these studies suggest that OB-R expression is a vital feature of the pluripotent nature of ESCs and iPSCs and reveal a signaling route from white adipose tissue to tumor stem cell subpopulations. Moreover, this study has implications to understanding how systemic hormonal cues can influence tumor growth and malignant progression. This study provides a mechanism behind the published correlation between excess body fat and cancer mortality (Calle et al and Renehan et al) and may provide a model system to investigate mechanisms by which we can intervene therapeutically for those patients at high risk. Recent evidence implicating Leptin in supporting both the proliferation and the pluripotency of cultured human embryonic stem cells (Anisimov et al) suggests that the data provided herein may also hold true for investigations in human.




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