TBX3 functions as a tumor suppressor downstream of activated CTNNB1 mutants during hepatocarcinogenesis

TBX3 expression is negatively correlated with TP53 mutations in human HCCs.

“TBX3 expression was found to be negatively correlated with TP53 mutations (Fig. 1B and Fig. S1A, C).”

• cites [15] Ahn, 2014, Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification, Hepatology sourceCould not verify

  The Clinical Features section reports TP53 mutation rates and associations with tumor grade and other genetic alterations, but not with TBX3 expression. Searches for “TBX3 expression TP53 mutations” and “TBX3” found no passage addressing the claimed negative correlation; page numbers were not available in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

GOF CTNNB1 mutant human and mouse HCCs are known to be chromosomally stable.

“It is also worth noting that activation of YAP has been shown to induce chromosomal instability in HCC,[27] whereas GOF CTNNB1 mutant human and mouse HCCs are known to be chromosomally stable.[28]”

• cites [28] Calvisi, 2004, Disruption of beta-catenin pathway or genomic instability define two distinct categories of liver cancer in transgenic mice, Gastroenterology sourcePartly supported

  The ‘Frequency of β-Catenin Mutations’ section (page unavailable) reports that β-catenin mutations were found more often in human HCCs with stable genomes, and the Abstract describes the β-catenin-activated c-myc/E2F-1 mouse tumors as having relatively stable genomes. But the paper also finds β-catenin mutations in c-myc/TGF-α tumors, the model characterized by chromosomal instability. It supports an association, not the categorical claim that GOF CTNNB1-mutant human and mouse HCCs are chromosomally stable. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

YAP/TAZ downstream targets Ctgf and Cyr61 were significantly increased in Tbx3 KO tumors.

“Nuclear YAP/TAZ levels were upregulated (Fig. 2F) and their downstream targets, including Ctgf and Cyr61, were significantly increased in Tbx3 KO tumors (Fig. 2H).”

Nuclear expression of SOX9 was prominent in Tbx3 KO tumor cells.

“Intriguingly, the nuclear expression of SOX9 was prominent in Tbx3 KO tumor cells (Fig. 2D, E).”

Overexpression of TBX3 significantly inhibited HCC cell growth in vitro.

“Overexpression of TBX3 significantly inhibited HCC cell growth in vitro (Fig. S4C).”

NOTCH2 protein levels were augmented in Tbx3 KO tumors.

“Accordingly, we found augmented NOTCH2 protein levels in Tbx3 KO tumors (Fig. 2D).”

A total of 1,596 genes positively correlated with TBX3 expression and 3,632 genes negatively correlated with TBX3 expression were identified in human HCCs.

“A total of 1,596 genes positively correlated with TBX3 expression, and 3,632 genes negatively correlated with TBX3 expression were identified (Table S5, 6).”

Tbx3 KO tumors displayed significantly higher Ki67 immunoreactivity.

“Tbx3 KO tumors displayed significantly higher Ki67 immunoreactivity (Fig. 2E, G), suggesting that increased tumor cell proliferation might be the major cellular mechanism leading to accelerated HCC growth in the Tbx3 KO genetic background.”

Nuclear YAP/TAZ levels were upregulated in Tbx3 KO tumors.

“Nuclear YAP/TAZ levels were upregulated (Fig. 2F) and their downstream targets, including Ctgf and Cyr61, were significantly increased in Tbx3 KO tumors (Fig. 2H).”

No significant correlation between TBX3 expression levels and other genetic events in human HCCs was observed.

“No significant correlation between TBX3 expression levels and other genetic events in human HCCs was observed (Fig. S1A).”

TBX3 negatively co-expressed genes were implicated in oncogenic pathways and cellular mechanisms, such as PI3K-Akt signaling pathway and cell cycle.

“TBX3 negatively co-expressed genes were implicated in oncogenic pathways and cellular mechanisms, such as “PI3K-Akt signaling pathway” and “cell cycle” (Fig. 1E and Fig. S2B).”

Ablation of Tbx3 significantly accelerated tumor growth in c-Met/β-Catenin mice.

“Importantly, we found that ablation of Tbx3 significantly accelerated tumor growth in c-Met/β-Catenin mice.”

• Also stated in: § Results: · plain

  “Using Tbx3flox/flox mice, we found that ablation of Tbx3 significantly accelerates c-Met/β-Catenin driven HCC formation.”

No remarkable differences such as lineage shift, including no cholangiocarcinoma or mixed tumors, were detected between c-Met/β-Catenin/Cre and control tumors.

“However, no remarkable differences such as lineage shift (no cholangiocarcinoma/mixed tumors) were detected (Fig. S6).”

TBX3 positively co-expressed genes were enriched in metabolic pathways.

“In addition, KEGG pathway analysis revealed that TBX3 positively co-expressed genes were enriched in metabolic pathways (Fig. 1E and Fig. S2B).”

HCCs with high TBX3 expression were mainly characterized by CTNNB1 mutations.

“Consistent with TBX3 as a Wnt/β-Catenin target, [12] HCCs with high TBX3 expression were mainly characterized by CTNNB1 mutations (Fig. 1B and Fig. S1A, B).”

• cites [12] Renard, 2007, Tbx3 is a downstream target of the Wnt/beta-catenin pathway and a critical mediator of beta-catenin survival functions in liver cancer, Canc Res sourceNot checked — no full text of the source

TBX3 protein was expressed at high levels in c-Met/β-Catenin and Yap/β-Catenin driven mouse hepatoblastoma tumors.

“Consistent with human HCC data,[12] TBX3 protein was expressed at high levels in c-Met/β-Catenin and Yap/β-Catenin driven mouse HB tumors.”

• cites [12] Renard, 2007, Tbx3 is a downstream target of the Wnt/beta-catenin pathway and a critical mediator of beta-catenin survival functions in liver cancer, Canc Res sourceNot checked — no full text of the source

The top genes positively correlated with TBX3 expression were canonical Wnt/β-Catenin targets, including GLUL, CYP2E1, LGR5, and SP5.

“Noticeably, the top positive correlated genes were all canonical Wnt/β-Catenin targets, including GLUL, CYP2E1, LGR5, and SP5, providing further evidence that TBX3 is a main Wnt/β-Catenin target (Fig. S2A).”

Notch2 and other canonical Notch target genes were upregulated at mRNA level in Tbx3 KO tumors.

“Notch2 and other canonical Notch target genes were also upregulated at mRNA level (Fig. 2H and Fig. S9C).”

No difference between control and Tbx3 KO tumors was found in the ERK, AKT, and STAT3 pathways.

“We analyzed the major pathways regulating tumor proliferation, including ERK, AKT, and STAT3 pathways, but found no difference between control and Tbx3 KO tumors (Fig. S7).”

TBX3 expression was positively correlated with expression of cytochrome P450 enzymes, such as CYP1A1, CYP1A2, CYP2E1, and CYP3A4.

“For instance, TBX3 expression was positively correlated with the expression of cytochrome P450 enzymes, such as CYP1A1, CYP1A2, CYP2E1, and CYP3A4 (Fig. S3).”

Ablation of Tbx3 has a limited impact on liver tumor development driven by constitutively activated YAP.

“In summary, the present data indicate that ablation of Tbx3 has a limited impact on liver tumor development driven by constitutively activated YAP.”

ChIP-PCR assay confirmed binding of TBX3 to the PLD1 promoter sequence in human HCC cells with HA-tagged TBX3.

“HA-tagged TBX3 was transfected into the cells, and ChIP-PCR assay confirmed the binding of TBX3 to the PLD1 promoter sequence (Fig. 7E).”

Forced overexpression of TBX3 in human HCC cell lines significantly reduced PLD1 mRNA expression.

“In addition, forced overexpression of TBX3 in human HCC cell lines significantly reduced PLD1 mRNA expression (Fig. 7B), whereas Pld1 mRNA levels were significantly elevated in Tbx3(−) c-Met/β-Catenin tumors when compared to Tbx3(+) liver tumors (Fig. 7C).”

Tumor cell proliferation was significantly inhibited by LATS2 overexpression.

“Tumor cell proliferation was significantly inhibited (Fig. 3D, H).”

Overexpression of TBX3 in three human HCC cell lines significantly reduced protein levels of total YAP/TAZ and nuclear YAP/TAZ.

“We overexpressed TBX3 in three human HCC cell lines. TBX3 significantly reduced the protein levels of total YAP/TAZ as well as nuclear YAP/TAZ (Fig. 6A, B).”

No difference in hepatocyte differentiation markers (Cyp1a1, Cyp1a2, Cyp2e1) and markers of HCC and hepatoblasts (Afp, Epcam) was found between Tbx3(+) and Tbx3(−) tumor samples.

“No difference in hepatocyte differentiation markers (Cyp1a1, Cyp1a2, and Cyp2e1), as well as markers of HCC and hepatoblasts (Afp and Epcam), was found between Tbx3(+) and Tbx3(−) tumor samples (Fig. S15B, C).”

Gene sequencing revealed no mutations in exon 3 of the mouse Ctnnb1 allele in c-Myc mouse HCCs.

“Gene sequencing revealed no mutations in exon 3 of the mouse Ctnnb1 allele (Fig. S19B).”

A strong negative correlation between PLD1 and TBX3 mRNA expression was noted in the TCGA-LIHC database.

“In the TCGA-LIHC database, a strong negative correlation between PLD1 and TBX3 mRNA expression was noted (Fig. 7A).”

LATS2 overexpression led to decreased SOX9 expression.

“As expected, LATS2 overexpression led to decreased total YAP/TAZ as well as nuclear YAP/TAZ protein levels (Fig. 3E, F), as well as decline of their downstream targets Ctgf and Cyr61 mRNA (Fig. 3G), as well as decreased SOX9 expression (Fig. 3D, E).”

Similar expression of Yap/Notch pathway genes including Ctgf, Cyr61, and Hes1 was observed in tumor tissues in the two mouse cohorts.

“qRT-PCR analysis suggested a similar expression of Yap/Notch pathway genes, including Ctgf, Cyr61, and Hes1, in tumor tissues in the two mouse cohorts (Fig. 4G and Fig. S15A).”

PLD1 levels positively correlate with YAP/TAZ target genes including CCN2, JAG1, NOTCH2, and AXL in human HCCs.

“Consistent with previous studies, we found that PLD1 levels positively correlate with those of YAP/TAZ target genes, including CCN2, JAG1, NOTCH2, and AXL in human HCCs (Fig. S22).”

Additional genetic events such as TP53 or ARID1A mutations are present in the 13 CTNNB1 mutant HCC samples with low TBX3 expression.

“Further analysis revealed the presence of additional genetic events in the 13 CTNNB1 mutant HCC samples with low TBX3 expression, such as TP53 or ARID1A mutations (Fig. S20).”

Overexpression of PLD1 resulted in significantly higher tumor burden in c-Met/β-Catenin/PLD1 mice than in c-Met/β-Catenin/pT3 mice.

“We found a significantly higher tumor burden in c-Met/β-Catenin/PLD1 than that of c-Met/β-Catenin/pT3 mice (Fig. 7J).”

mRNA levels of LATS2, YAP, and TAZ were not regulated by TBX3.

“However, the mRNA levels of LATS2, YAP, and TAZ were not regulated by TBX3 (Fig. S16), suggesting that TBX3 likely modulates YAP/TAZ activity at the post-transcriptional level.”

LATS2 overexpression led to decreased total YAP/TAZ and nuclear YAP/TAZ protein levels.

“As expected, LATS2 overexpression led to decreased total YAP/TAZ as well as nuclear YAP/TAZ protein levels (Fig. 3E, F), as well as decline of their downstream targets Ctgf and Cyr61 mRNA (Fig. 3G), as well as decreased SOX9 expression (Fig. 3D, E).”

LATS2 overexpression led to decline of Ctgf and Cyr61 mRNA.

“As expected, LATS2 overexpression led to decreased total YAP/TAZ as well as nuclear YAP/TAZ protein levels (Fig. 3E, F), as well as decline of their downstream targets Ctgf and Cyr61 mRNA (Fig. 3G), as well as decreased SOX9 expression (Fig. 3D, E).”

TBX3 mRNA expression was negatively correlated with mRNA levels of YAP/TAZ downstream targets including CCN2, AXL, and JAG1, as well as canonical Notch signaling targets (NOTCH1, NOTCH2, JAG2, HES1, HEYL, NRARP) in human HCCs.

“We found that TBX3 mRNA expression was negatively correlated with the mRNA levels of YAP/TAZ downstream targets, including CCN2, AXL, and JAG1, as well as the canonical Notch signaling targets (NOTCH1, NOTCH2, JAG2, HES1, HEYL, and NRARP) in human HCCs (Fig. 5).”

c-Met/β-Catenin/Cre/Lats2 Tbx3flox/flox mice developed liver tumors at similar latency as c-Met/β-Catenin/pCMV injected Tbx3flox/flox mice.

“Indeed, c-Met/β-Catenin/Cre/Lats2 Tbx3flox/flox mice developed liver tumors at the similar latency as c-Met/β-Catenin/pCMV injected Tbx3flox/flox mice (Fig. 3B, C).”

No significant changes in expressions of E-Cadherin, HNF4α, GS, and Vimentin were observed in the LATS2 experiment.

“No significant changes in the expressions of E-Cadherin, HNF4α, GS, and Vimentin were observed (Fig. S11).”

LATS2 and p-YAPSer397 levels were consistently lower in Tbx3(−) tumors in the Yap/β-Catenin experiment.

“However, LATS2 and p-YAPSer397 levels were consistently lower in Tbx3(−) tumors (Fig. 4C).”

Almost 50% of HCCs with elevated TBX3 do not show CTNNB1 mutations in the TCGA-LIHC dataset.

“Indeed, analysis of the TCGA-LIHC dataset revealed that almost 50% of HCCs with elevated TBX3 do not show CTNNB1 mutations (Fig. S1A, B).”

In HepG2 cells, ChIP with anti-TBX3 antibody demonstrated binding of endogenous TBX3 to the PLD1 promoter.

“Furthermore, in HepG2 cells, characterized by high TBX3 expression, ChIP with anti-TBX3 antibody demonstrated the binding of endogenous TBX3 to the PLD1 promoter (Fig. 7F).”

No difference in levels of ERK, AKT, and STAT3 cascades was noted in the Yap/β-Catenin experiment.

“No difference in the levels of ERK, AKT, and STAT3 cascades was noted (Fig. S14).”

No significant changes in expressions of E-Cadherin, HNF4α, GS, and Vimentin were observed in the Yap/β-Catenin experiment.

“No significant changes in the expressions of E-Cadherin, HNF4α, GS, and Vimentin were observed (Fig. S13).”

YAP/TAZ targets CTGF and CYR61 mRNA expression were significantly downregulated by TBX3 overexpression.

“YAP/TAZ targets CTGF and CYR61 mRNA expression were significantly downregulated (Fig. 6C).”

Tumor cells in the Yap/β-Catenin experiment were highly proliferative, showing similar proliferation rates as indicated by Ki67 index.

“Tumor cells were highly proliferative, showing similar proliferation rates as indicated by Ki67 index (Fig. 4D, F).”

Overexpression of PLD1 together with TBX3 prevented TBX3-induced downregulation of CTGF and CYR61 in HCC cell lines.

“Furthermore, overexpression of PLD1 together TBX3 prevented TBX3 induced downregulation of CTGF and CYR61 in HCC cell lines (Fig. 7H).”

Overexpression of TBX3 led to decreased cellular PA levels in Li-7 human HCC cells.

“Importantly, overexpression of TBX3 led to decreased cellular PA levels in Li-7 human HCC cells (Fig. 7D).”

Similar expression levels for most Hippo/YAP/TAZ proteins, including nuclear localized YAP and TAZ, were detected in Tbx3(+) and Tbx3(−) Yap/β-Catenin tumors.

“As concerns the Hippo/YAP/TAZ pathway, similar expression levels for most of the proteins (Fig. 4C), including nuclear localized YAP and TAZ (Fig. 4E) were detected in Tbx3(+) and Tbx3(−) Yap/β-Catenin tumors.”

Pld1 mRNA levels were significantly elevated in Tbx3(−) c-Met/β-Catenin tumors compared to Tbx3(+) liver tumors.

“In addition, forced overexpression of TBX3 in human HCC cell lines significantly reduced PLD1 mRNA expression (Fig. 7B), whereas Pld1 mRNA levels were significantly elevated in Tbx3(−) c-Met/β-Catenin tumors when compared to Tbx3(+) liver tumors (Fig. 7C).”

Among 96 human HCCs with CTNNB1 mutations in the TCGA-LIHC dataset, 83 samples showed high TBX3 expression, whereas 13 samples exhibited low TBX3 levels.

“It should be also noted that in the TCGA-LIHC dataset, among 96 human HCCs with CTNNB1 mutations, 83 samples showed high TBX3 expression, whereas 13 samples exhibited low TBX3 levels.”

Similar tumor lesions were observed in c-Met/β-Catenin/Cre/Lats2 and c-Met/β-Catenin/pCMV injected Tbx3flox/flox mice.

“Histologically, similar tumor lesions were observed in c-Met/β-Catenin/Cre/Lats2 Tbx3flox/flox and c-Met/β-Catenin/pCMV injected Tbx3flox/flox mice (Fig. S10).”

Increased tumor cell proliferation might be the major cellular mechanism leading to accelerated HCC growth in the Tbx3 KO genetic background.

“Tbx3 KO tumors displayed significantly higher Ki67 immunoreactivity (Fig. 2E, G), suggesting that increased tumor cell proliferation might be the major cellular mechanism leading to accelerated HCC growth in the Tbx3 KO genetic background.”

TBX3 is a main Wnt/β-Catenin target in human HCC.

“Noticeably, the top positive correlated genes were all canonical Wnt/β-Catenin targets, including GLUL, CYP2E1, LGR5, and SP5, providing further evidence that TBX3 is a main Wnt/β-Catenin target (Fig. S2A).”

• cites [12] Renard, 2007, Tbx3 is a downstream target of the Wnt/beta-catenin pathway and a critical mediator of beta-catenin survival functions in liver cancer, Canc Res sourceNot checked — no full text of the source

TBX3 likely modulates YAP/TAZ activity at the post-transcriptional level.

“However, the mRNA levels of LATS2, YAP, and TAZ were not regulated by TBX3 (Fig. S16), suggesting that TBX3 likely modulates YAP/TAZ activity at the post-transcriptional level.”

TBX3 may function downstream of activated Wnt/β-Catenin signaling in maintaining the distinctive phenotypes of GOF CTNNB1 mutant HCCs.

“Our data suggest TBX3 may function downstream of activated Wnt/β-Catenin signaling in maintaining these unique phenotypes.”

c-Myc may induce TBX3 expression independent of the Wnt/β-Catenin pathway.

“The data suggest that c-Myc may induce TBX3 expression independent of the Wnt/β-Catenin pathway.”

The β-Catenin/TBX3/Hippo cascade might contribute to maintaining a relatively less aggressive status of HCC.

“This might contribute to maintaining a relatively less aggressive status of HCC.”

Activation of YAP has been shown to induce chromosomal instability in HCC.

“It is also worth noting that activation of YAP has been shown to induce chromosomal instability in HCC,[27] whereas GOF CTNNB1 mutant human and mouse HCCs are known to be chromosomally stable.[28]”

• cites [27] Weiler, 2017, Induction of chromosome instability by activation of yes-associated protein and forkhead box M1 in liver cancer, Gastroenterology sourceNot checked — no full text of the source