Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells

<p>Abstract</p> <p>Background</p> <p>β-catenin mutations that constitutively activate the canonical Wnt signaling have been observed in a subset of hepatocellular carcinomas (HCCs). These mutations are associated with chromosomal stability, low histological grade, low tumor invasion and better patient survival. We hypothesized that canonical Wnt signaling is selectively activated in well-differentiated, but repressed in poorly differentiated HCCs. To this aim, we characterized differentiation status of HCC cell lines and compared their expression status of Wnt pathway genes, and explored their activity of canonical Wnt signaling.</p> <p>Results</p> <p>We classified human HCC cell lines into "well-differentiated" and "poorly differentiated" subtypes, based on the expression of hepatocyte lineage, epithelial and mesenchymal markers. Poorly differentiated cell lines lost epithelial and hepatocyte lineage markers, and overexpressed mesenchymal markers. Also, they were highly motile and invasive. We compared the expression of 45 Wnt pathway genes between two subtypes. TCF1 and TCF4 factors, and LRP5 and LRP6 co-receptors were ubiquitously expressed. Likewise, six Frizzled receptors, and canonical Wnt3 ligand were expressed in both subtypes. In contrast, canonical ligand Wnt8b and noncanonical ligands Wnt4, Wnt5a, Wnt5b and Wnt7b were expressed selectively in well- and poorly differentiated cell lines, respectively. Canonical Wnt signaling activity, as tested by a TCF reporter assay was detected in 80% of well-differentiated, contrary to 14% of poorly differentiated cell lines. TCF activity generated by ectopic mutant β-catenin was weak in poorly differentiated SNU449 cell line, suggesting a repressive mechanism. We tested Wnt5a as a candidate antagonist. It strongly inhibited canonical Wnt signaling that is activated by mutant β-catenin in HCC cell lines.</p> <p>Conclusion</p> <p>Differential expression of Wnt ligands in HCC cells is associated with selective activation of canonical Wnt signaling in well-differentiated, and its repression in poorly differentiated cell lines. One potential mechanism of repression involved Wnt5a, acting as an antagonist of canonical Wnt signaling. Our observations support the hypothesis that Wnt pathway is selectively activated or repressed depending on differentiation status of HCC cells. We propose that canonical and noncanonical Wnt pathways have complementary roles in HCC, where the canonical signaling contributes to tumor initiation, and noncanonical signaling to tumor progression.</p

WNT/(formula)-Catenin signaling pathway activation in epithelial cancers

Cataloged from PDF version of article.Wnt signaling is involved in a large set of cellular and developmental processes, and when mis-regulated can lead to both degenerative diseases and many types of cancer. The involvement of Wnt signaling was already well demonstrated in several types of human cancers such as colorectal cancer. However, in some others such as hepatocellular carcinoma (HCC) and breast cancer, the role of Wnt signaling is not fully understood. To study the role of Wnt pathway in liver cancer, we first classified human hepatoma cell lines into well-differentiated and poorly differentiated groups using hepatocyte-specific biomarkers. Wnt/β-catenin signaling activity was measured using TCF/LEF-dependent reporter assay. Canonical Wnt/β-catenin signaling was constitutively active in 80% of well differentiated and 14% of poorly differentiated cell lines, respectively. Furthermore, ectopic expression mutant of S33Y β-catenin resulted in strong canonical Wnt/β-catenin activity in well differentiated, but not in poorly differentiated HCC cells. Comprehensive analysis of major Wnt signaling components by a rapid RT-PCR assay showed redundant expression of many Wnt ligands, Frizzled receptors, co-receptors and TCF/LEF factors in HCC. In contrast, canonical signaling-inhibitory Wnt5A and Wnt5B ligands were selectively expressed in poorly differentiated HCC cell lines. Our observations indicate that canonical Wnt/β-catenin signaling is active in well differentiated, but repressed in poorly differentiated HCC cells. Thus, canonical Wnt/β-catenin signaling plays a dual role in HCC. To study the role of Wnt pathway in breast cancer, we performed a comprehensive expression analysis, by RT-PCR, of Wnt signaling molecules, including 19 Wnt ligands, ten Frizzled receptors, two LRP co-receptors and four Lef/TCF transcription factors in immortalized normal human mammary epithelial cells (HMECs), six breast cancer cell lines (BCCL) and 14 primary breast tumors (PBT). BCCL expressed/over-expressed all Frizzleds except FZD10, LRP5/6 and Lef/TCFs. They also overexpressed WNT4, WNT7B, WNT8B, WNT9A and WNT10B, but the expression of WNT1, WNT2B, WNT3, WNT5A, WNT5B and WNT16 was lost or decreased in most BCCL. Wnt expression correlated with nuclear β-catenin accumulation and cyclin D1 induction in BCCL, compared to HMECs, indicating a reactivation of the canonical Wnt signaling in malignant cells. Furthermore, the expression of FZD1, WNT-4, WNT7B, WNT8B, WNT9A and WNT10B, all implicated in canonical Wnt signaling, was upregulated in PBT, whereas the non-canonical WNT5A expression was down-regulated. Our study gave strong evidences for the differential involvement of Wnt pathway in liver and breast cancers. In liver cancer, Wnt pathway activity seems to be linked to the differentiation status of HCC cell lines. Furthermore, our data showed that the canonical Wnt pathway was active in well-differentiated HCC cell lines and repressed in poorly differentiated ones. In contrast, the study of Wnt pathway in breast cancer cell lines showed similarities rather than differences. Indeed, our study revealed a significant correlation between Wnt ligands mRNA expression profile and the induction of Cyclin D and nuclear β-catenin protein accumulation in all breast cancer cell lines studied. We concluded that, although involved in both types of cancers, Wnt signaling is acting differently in liver and breast cancers. More interestingly, in the same type of cancer such as HCC, Wnt signaling displayed differential activity depending on the cell differentiation status.Benhaj, KhemaisPh.D