Patient and tumor characteristics in Japanese patients.

<p>Abbreviations: B, hepatitis B virus; C, hepatitis C virus; CH, chronic hepatitis; LC, liver cirrhosis; Max. dia., maximum diameter; MD, moderately differentiated; PD, poorly differentiated; Peri-T, peri-tumor; Vas. Inv., vascular invasion; WD, well differentiated.</p

Loss of the SxxSS motif in TCF-4 isoforms promotes tumorigenesis.

<p>(A) Representative experiment demonstrating xenograft tumor development and growth rate. EV2, control; J1, J cell; K2 and K5, K cell clones. (B) Protein expression of the indicated molecules in J1 tumors (1–5) and K2 tumors (13–17) by immunoblot analysis. Nuclear (N) and cytoplasmic (C) proteins expressed in the 150 µM CoCl₂-treated J cells (Hypo-J) were used as positive controls. (Bottom panel) TCF-4J and K mRNA expression was verified by RT-PCR in J1 and K2 tumors; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Immunohistochemistry for HIF-2α and EGFR expression in representative J- and K-cell derived tumors. Original magnification, 200x. HE, hematoxylin and eosin; and (-), negative staining. (D) Magnified view (400x) for the corresponding squared areas in (C) for HIF-2α and EGFR expression.</p

Protein expression of TCF-4 isoforms J and K.

<p>(A) Organization of the “short” (A, B) and the “long” (J, K) isoforms; TCF-4B and J lack the SxxSS motif. (B) Confocal laser-scanning microscopy demonstrates nuclear localization of TCF-4J and TCF-4K (green). Nuclei are counterstained by DAPI. (C) Nuclear (Nuc)/cytoplasmic (Cyto) fractionation followed by immunoblot analysis confirms the nuclear localization of TCF-4J and K isoforms. Lamin (lamin A/C) and γ-tubulin were used as nuclear and cytoplasmic markers, respectively.</p

Loss of the SxxSS motif in TCF-4 isoforms increases expression of HIF-αs under hypoxia.

<p>(A and B) Immunoblot analysis of empty vector-transfected (EV), TCF-4J-overexpressing (J), and TCF-4K-overexpressing cells (K). The cells were treated with 0 and 150 µM CoCl₂ (A) or cultured under 1% oxygen tension for 48 hr (B). Cell lysates were subjected to detect HIF-αs and Myc-tag TCF-4 isoform protein expression. Expression levels were plotted as a ratio to actin (right panel). (C) Confocal microscopy for nuclear localization of HIF-2α protein. Cells were treated with 0 µM (Normoxia) or 150 µM (Hypoxia) CoCl₂ and stained with an anti-HIF-2α antibody (green). DAPI was employed for nuclear staining. (D) Immunoblot analysis of total cell lysates from cells treated with 0 or 150 µM CoCl₂ for 48 hr. A and B represent HAK-1A cells overexpressing TCF-4A (“short form” of TCF-4K) and TCF-4B (“short form” of TCF-4J), respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039981#pone-0039981-g002" target="_blank">Figure 2A</a>). Note the robust increase in expressions of HIF-1α and HIF-2α in B cells.</p

Lack of SxxSS motif contributes to protein stability of HIF-αs in J cells.

<p>(A) Cells were treated with 150 µM CoCl₂ for 36 hr followed by incubation with or without MG-132 (10 µM) for 2 hr. Total cell lysates were employed for immunoblot analysis. Relative expression of HIF-αs was normalized to actin. (B) Protein stability of HIF-αs was evaluated by immunoblot analysis, where cells were exposed to 5 µg/mL cycloheximide (CHX) to inhibit protein synthesis for 0 or 60 min at the last phase of the 48 hr CoCl₂ treatment period. (C) Expression of VHL in TCF-4J and K cells. Cells were treated 0 or 150 µM CoCl₂ and nuclear and cytoplamic fractions was prepared for immunoblot analysis. Expression level of HIF-2α and VHL was normalized by lamin (bottom panel); VHL-C, cytoplasmic VHL. (D) Interaction between VHL and HIF-2α in nucleus of TCF-4J and K cells. (E) Polyubiquitination of HIF-2α in cytoplasmic and nuclear fractions of TCF-4J and K cells. Immunoprecipitation (IP)/immunoblot (IB) analysis was performed by using antibodies for HIF-2α and ubiquitin (Ub); *, IgG heavy chain. Note the decreased ubiquitination of HIF-2α and weak VHL-HIF-2α interaction in J cells, which was in contrast to the observations in K cells.</p

Relation between clinicopathological factors and TCF-4J and K expression in patients.

<p>Abbreviations: CH, chronic hepatitis; HBV, hepatitis B virus; HCV, hepatitis C virus; LC, liver cirrhosis; MD, moderately differentiated; PD, poorly differentiated; WD, well differentiated.</p

TCF-4J-overexpressing cells proliferate under hypoxic conditions.

<p>(A) Phase-contrast microscopy of parental HAK-1A (1A) and 1A-derived stable clones, including the empty vector-transfected clone (EV2), the TCF-4J-transfected clones (J1 and J15), and the TCF-4K-transfected clones (K2 and K5). The morphologic appearance of the highly malignant HAK-1B (1B) cell line, a clonally dedifferentiated cell type from 1A, is also presented for comparison. Bar  = 50 µm. (B) Immunoblot analysis of stable cell clones. Positive signals for Myc-tag are shown in J1, J15, K2, and K5. HepG2 cells (G2) known to express both full-length (92 kDa) and truncated β-catenin (75 kDa), exhibited a lower band for β-catenin (β-Cat) HepG2 was also used as a positive control for EpCAM and K-19 expression. HEK293 was used as a negative control for K-19. (C) Cell growth rates of stable clones under the hypoxic conditions generated by CoCl₂ (150 µM) for 7 days. Growth rate is represented as the fold-increase compared to day 0. (D) Cell growth of stable clones in normoxia (20% O₂) or hypoxic conditions (1% O₂) Cells were exposed to either 20% or 1% oxygen for 5 days. Note that reduction of cell growth in hypoxic contidions was less in J cells (13%) compared to EV (22%) and K (27%) cells. (E) Anchorage-independent growth assay (sphere assay). Phase-contrast microscopic views of representative cell aggregates are displayed at 0, 75, and 150 µM of CoCl₂. Bar  = 300 µm. Note the striking difference in colony growth rate and appearance at 150 µM of CoCl₂. *<i>p</i><0.05; **<i>p</i><0.01.</p

Patient and tumor characteristics in South Korean patients.

<p>Abbreviations: B, hepatitis B virus; CH, chronic hepatitis; HCC/CCC, combined hepatocellular carcinoma and cholangiocarcinoma; LC, liver cirrhosis; MD, moderately differentiated; NBNC, non-B, non-C; PD, poorly differentiated; Peri-T, peri-tumor; Vas. Inv., vascular invasion.</p

Proposed role(s) of TCF-4J and K isoforms during hepatic oncogenesis.

<p>Loss of SxxSS motif in TCF-4J (ΔSxxSS) supports high level nuclear expression of HIF-2α with increasing degrees of hypoxia. This phenomenon upregulates and activates the EGFR signaling cascade including downstream genes that inhibit apoptosis to promote tumor development. In contrast, the TCF-4K isoform containing the SxxS motif degrades HIF-2α under increasing hypoxic conditions via the ubiquitination pathway and, therefore, downregulates HIF-2α mediated gene expression. The net effect is to inhibit the characteristics of a more aggressive malignant phenotype. Thus, TCF-4 isoform expression may play an important role in the molecular pathogenesis of poorly differentiated HCC where severe hypoxia is likely to occur and particularly when TCF-4 isoforms that lack a regulatory SxxSS motif are expressed.</p

Time-to-progression, time-to-local progression, progression-free-survival and overall survival after TACE among groups.

<p>Time-to-progression, time-to-local progression, progression-free-survival and overall survival after TACE among groups.</p