Phosphorylation-Mediated Activation of β-Catenin-TCF4-CEGRs/ALCDs Pathway Is an Essential Event in Development of Aggressive Hepatoblastoma

Abstract

Background and Aims: Hepatoblastoma (HBL), a deadly malignancy in children, is the most common type of pediatric liver cancer. We recently demonstrated that β-catenin, phosphorylated at S675 (ph-S675-β-catenin), causes pathological alterations in fibrolamellar hepatocellular carcinoma (FLC), by activating oncogenes and fibrotic genes via human genomic regions, known as cancer-enhancing genomic regions or aggressive liver cancer domains (CEGRs/ALCDs). The aim of this study was to determine the role of the ph-S675-β-catenin-TCF4-CEGRs/ALCDs pathway in HBL. Methods: The ph-S675-β-catenin-TCF4-CEGRs/ALCDs pathway was examined in a large cohort of HBL specimens, in HBL cell lines HepG2 and Huh6, and in patient-derived xenografts (PDXs). Results: β-catenin is phosphorylated at S675 in a large portion of tested HBL patients. In these patients, ph-S675-β-catenin forms complexes with TCF4 and opens CEGRs/ALCDs-dependent oncogenes for transcription, leading to a massive overexpression of the oncogenes. The inhibition of the β-catenin-TCF4-CEGRs/ALCDs axis inhibits the proliferation of cancer cells and tumor growth in HBL cell lines and HBL-PDXs. The ph-S675-β-catenin is abundant in mitotic cells. We found that markers of HBL Glypican 3 (GPC3) and Alpha Fetoprotein (AFP) are increased in HBL patients by β-catenin-TCF4-p300 complexes. Conclusions: The phosphorylation-mediated activation of the β-catenin-TCF4-p300-CEGRs/ALCDs pathway increases oncogene expression in patients with aggressive liver cancer and promotes the development of hepatoblastoma.

Cellular origin and molecular mechanisms of lung metastases in patients with aggressive hepatoblastoma

“…Preceding studies have identified genomic regions of human DNA that promote oncogene expression in aggressive HBL. 7 8 9 10 11 12 Since the RELI algorithm identified ZBTB3 as a protein that frequently binds to CEGRs/ALCDs, 10 we searched the CEGRs/ALCDs and found a perfect binding site for ZBTB3 (Figure 1 A). ZBTB3 has previously been identified as an oncogene**** exhibiting oncogenic activity when it forms complexes with the chromatin remodeling protein SRCAP.…”

“…Further studies with a bigger number of patients are needed to understand if the higher increase of markers of neurons in lung metastases is a common feature of repetitive lung metastases. Because our group previously found that β-catenin phosphorylation at Ser675 was elevated in patients with HBL, 11 we examined the expression of ph-S675-β-catenin in HBL lung metastases. Western blot showed that ph-S675-β-catenin and total β-catenin were elevated in lung metastases (Figure 2 F, right).…”

“…Our previous work found that the ph-S675-β-catenin-CEGRs/ALCDs pathway is active in aggressive HBL, causing overexpression of a marker of HBL, GPC3. 11 Therefore, we examined if the ph-S675-β-catenin-GPC3 pathway is preserved in hbl cells. We found that β-catenin, ph-S675-β-catenin, and GPC3 are highly expressed in hbl23 and hbl11t1/2 cells (Figure 6 A and Supplemental Figure S8A, http://links.lww.com/HC9/A764 ).…”

“…The previous study showed that GPC3 is detected in the blood of patients with HBL who had active ph-S675-β-catenin in primary tumors 11 ; therefore, we examined if the generated ph-S675-β-catenin–positive hbl cells might secrete GPC3 into the media. Proteins in the hbl-conditioned media were concentrated by loading on ion exchange column UnoQ and by subsequent elution by a sharp gradient of NaCl (Figure 6 F and Supplemental Figure S10D, http://links.lww.com/HC9/A764 ).…”

“…Particularly, our recent reports showed that ph-S675-β-catenin is abundant in primary tumors of patients with fibrolamellar HCC and HBL and that it increases the expression of the oncogenes through the CEGRs/ALCDs axis. 10 11 …”