Expression of OB-Rb in diverse classes of stem cells and role of leptin in sustaining pluripotency. (A) Immunoblot of lysates prepared from mouse CD133⁺ liver TISCs (lanes 1 and 8), E14 mESCs (lane 2), and human liver hepatoblasts and hepatocytes (lanes 3 and 4). OB-Rb expression is also shown for the H1 and H9 human ESCs (lanes 5 and 6), rat iPS2 cells (lane 7), and human HepG2 hepatocellular carcinoma cells (lane 9). (B) Leptin sustains pluripotency in cultured E14 mouse embryonic stem cells. E14 Oct4-GFP cells were cultured for 11 d in media with or without LIF (1,000 U/mL) or in the presence of leptin (200 ng/mL), harvested, and analyzed by FACS for quantification of GFP-expressing cells. The graphs shown are representative of results from three independent biological replicates.

Functional analysis of leptin and downstream effectors OCT4 and SOX2 in proliferation and TISC-mediated oncogenesis. (A) In vitro cell proliferation assay. Equal numbers (1.5 × 10⁵) of TISCs expressing either nontargeting, scrambled shRNA (sh-Scr) or shRNAs targeting either SOX2 or OCT4 were plated and cultured in low-serum media or in the same media supplemented with recombinant mouse leptin (200 ng/mL). At 48-h intervals, cells were harvested and viable cells were counted. Error bars represent SD from at least three independent biological replicates (**P < 0.01 and *P < 0.05 relative to identically treated sh-Scr controls harvested at the same time). (B) Ki67 and Annexin V immunostaining shows decreased proliferation in TISCs stably expressing shRNAs targeting SOX2 or OCT4. Cells were cultured for 96 h in media supplemented with leptin (200 ng/mL) before harvest, fixation, and immunostaining. Asterisks indicate statistical significance (*P < 0.05) relative to identically cultured sh-Scr controls. (C) Leptin stimulates tumor development by implanted TISCs. CD133⁺ TISCs (4 × 10⁴) were implanted s.c. into the dorsal hind flanks of NOG mice. Mice were randomly assigned to cohorts (n = 8 mice) receiving i.p. injections of either PBS (vehicle) or recombinant mouse leptin (2 μg/g body weight) every other day for 2 wk. (D) TISCs (4 × 10⁴) were implanted s.c. into the dorsal hind flanks of ob/ob or Lepr^db/db mice or into lean littermate controls. (Lower) Mouse body weight. (E) Immunoblot analysis of phospho-STAT3 (p-Y705) and STAT3 levels in lysates prepared from TISC tumors recovered from ob/ob, Lepr^db/db, or lean control hosts. Tumors from all hosts were allowed to attain similar volumes (400–500 mm³) before surgical recovery and preparation of lysates.

Selective expression of leptin receptor OB-Rb in CD133⁺ TISCs is mediated by the pluripotency factors OCT4 and SOX2. (A) Quantitative RT-PCR analysis of mRNA transcript levels for the IL-6 receptor (IL-6R), adiponectin receptor (AdipoR1), and leptin receptor (OB-R) in CD133⁺/Nanog⁺ TISCs and CD133⁻ cells. (B) Selective expression of the long-form leptin receptor OB-Rb in CD133⁺/Nanog⁺ TISCs. Two independent sets of CD133⁺ and matched CD133⁻ cells were isolated in parallel by dissociation of Ns5a-Tg mouse liver tumors. Tumor lysates were probed using antisera specific for OB-R (Top) or NANOG (Middle). Asterisk (*) indicates a nonspecific cross-reacting band and is included as a loading control. (C) Immunostaining of mouse liver tumors showing distribution of CD133 and OB-R. CD133 localizes prominently to plasma membranes. Colocalization of OB-R and CD133 occurs in a subpopulation of tumor cells. (Scale bar, 50 μm.) (D) FACS analysis and quantification of OB-R expression in CD133⁻ controls and CD133⁺/Nanog⁺ TISCs isolated from dissociated tumors. (E) Lepr promoter-reporter assay. The indicated Lepr-luciferase fusions or a promoter-less vector were examined in CD133⁺/Nanog⁺ TISCs and PIL-4 hepatoblasts. A candidate OCT4/SOX2-binding site identified by sequence homology search is indicated by the blue triangle. Promoter activity is expressed as relative light units (RLU) normalized to the activity of cotransfected Renilla luciferase. Error bars represent the SD from at least three independent biological replicates (**P < 0.01). (F) ChIP-qPCR assay showing direct association of OCT4 and SOX2 on Lepr. Error bars represent SD from at least three independent biological replicates. (**P < 0.01; *P < 0.05). (G) Lentiviral-mediated shRNA knockdown of OCT4 and SOX2. Lysates prepared from CD133⁺ TISCs infected with lentivirus encoding nontargeting, scrambled control shRNA (sh-Scr), sh-SOX2 (lane 2), or sh-OCT4 (lanes 3 and 4) were analyzed by immunoblotting to determine the levels of SOX2 and OCT4. An actin immunoblot of the same lysates is included as a loading control. (H) Immunoblot analysis of OB-Rb in lysates prepared from CD133⁺ TISCs stably expressing a nontargeting shRNA control (sh-Scr, lane 1) or shRNAs targeting either SOX2 (lane 2) or OCT4 (lanes 3 and 4).

Leptin stimulates the activity of a pluripotency-associated TF network in TISCs. (A) CD133⁻ nonprogenitor tumor cells or CD133⁺ TISCs were exposed to leptin (150 or 300 ng/mL) for the indicated times, followed by lysis and immunoblotting using antisera specific for STAT3 or phospho-STAT3 (p-Y705). (B and C) STAT3 ChIP-qPCR analysis. Fragmented, cross-linked chromatin isolated from leptin-treated (150 ng/mL, 24 h) TISCs was immunoprecipitated using anti-STAT3 antibody, followed by qPCR using primers designed to amplify the indicated sites at the Oct4 (B) or Sox2 (C) genomic loci. (D) Promoter-reporter assays. Cultured TISCs transfected with the indicated reporter constructs were either untreated or exposed to recombinant mouse leptin (100 or 200 ng/mL, 24 h) before harvest and determination of luciferase activity. (E) Immunoblot analysis of lysates from TISCs exposed to leptin (150 ng/mL), LPS (5 ng/mL), or both ligands for 24 h. (F) qRT-PCR analysis of OCT4 and SOX2 in TISCs stably transfected with either empty vector or dominant-negative STAT3 (STAT3 Y705F). Fold induction is calculated as the ratio of transcript levels present in cells exposed to leptin (200 ng/mL) for 24 h relative to untreated controls.

Elevated OB-R expression in human HCC and correspondence of OB-R and OCT4 in multiple tumor types. (A) Immunohistochemical analysis of HCC tumors. Surgically resected HCC patient tumor specimens from age-matched specimens were fixed and stained with antisera to CD133 and OB-R and counterstained with DAPI to indicate nuclei. White arrows indicate examples of cells strongly coexpressing CD133 and OB-R. (Scale bar, 100 μm.) (B) Quantification of CD133 and OB-R reactivity. CD133⁺ and CD133⁻ cells were scored for the degree of OB-R immunopositivity. Brackets indicate statistically significant (**P < 0.01) comparisons between groups. (C) Immunoblot analysis of lysates prepared from surgically resected HCC patient specimens (T) and matched normal tissue (N). Immunoblot of actin serves as a control for protein loading. (D) Scatter plots showing results from meta-analysis comparing the expression levels of OB-R and OCT4 across multiple tumor samples. Expression data were collected from pancreatic and breast cancers, multiple myeloma, and CaCo-2 colon adenocarcinoma cells. (E) Model of leptin action on TISCs in promoting tumor growth. Leptin signaling triggers phosphorylation and activation of STAT3 to induce OCT4 and SOX2, thereby completing a self-reinforcing oncogenic signaling circuit in TISCs. Cytokines such as IL-6 and TNF may also contribute to this signaling network.