Sohee Jun

REV3 and REV1 Play Major Roles in Recombination-independent Repair of DNA Interstrand Cross-links Mediated by Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA)

DNA interstrand cross-links (ICLs) are the most cytotoxic lesions to eukaryotic genome and are repaired by both homologous recombination-dependent and -independent mechanisms. To better understand the role of lesion bypass polymerases in ICL repair, we investigated recombination-independent repair of ICLs in REV3 and REV1 deletion mutants constructed in avian DT40 cells and mouse embryonic fibroblast cells. Our results showed that Rev3 plays a major role in recombination-independent ICL repair, which may account for the extreme sensitivity of REV3 mutants to cross-linking agents. This result raised the possibility that the NER gap synthesis, when encountering an adducted base present in the ICL repair intermediate, can lead to recruitment of Rev3, analogous to the recruitment of polymerase η during replicative synthesis. Indeed, the monoubiquitination-defective Proliferating Cell Nuclear Antigen (PCNA) mutant exhibits impaired recombination-independent ICL repair as well as drastically reduced mutation rate, indicating that the PCNA switch is utilized to enable lesion bypass during DNA repair synthesis. Analyses of a REV1 deletion mutant also revealed a significant reduction in recombination-independent ICL repair, suggesting that Rev1 cooperates with Rev3 in recombination-independent ICL repair. Moreover, deletion of REV3 or REV1 significantly altered the spectrum of mutations resulting from ICL repair, further confirming their involvement in mutagenic repair of ICLs.

PAF and EZH2 Induce Wnt/β-Catenin Signaling Hyperactivation

Fine control of Wnt signaling is essential for various cellular and developmental decision-making processes. However, deregulation of Wnt signaling leads to pathological consequences, one of which is cancer. Here, we identify a function of PAF, a component of translesion DNA synthesis, in modulating Wnt signaling. PAF is specifically overexpressed in colon cancer cells and intestinal stem cells and is required for colon cancer cell proliferation. In Xenopus laevis, ventrovegetal expression of PAF hyperactivates Wnt signaling, developing a secondary axis with β-catenin target gene upregulation. Upon Wnt signaling activation, PAF dissociates from PCNA and binds directly to β-catenin. Then, PAF recruits EZH2 to the β-catenin transcriptional complex and specifically enhances Wnt target gene transactivation, independently of EZH2s methyltransferase activity. In mice, conditional expression of PAF induces intestinal neoplasia via Wnt signaling hyperactivation. Our studies reveal an unexpected role of PAF in regulating Wnt signaling and propose a regulatory mechanism of Wnt signaling during tumorigenesis.

FOXKs Promote Wnt/β-Catenin Signaling by Translocating DVL into the Nucleus

Dishevelled (DVL) proteins serve as crucial regulators that transduce canonical Wnt signals to the GSK3β-destruction complex, resulting in the stabilization of β-catenin. Emerging evidence underscores the nuclear functions of DVLs, which are critical for Wnt/β-catenin signaling. However, the mechanism underlying DVL nuclear localization remains poorly understood. Here we discovered two Forkhead box (FOX) transcription factors, FOXK1 and FOXK2, as bona fide DVL-interacting proteins. FOXK1 and FOXK2 positively regulate Wnt/β-catenin signaling by translocating DVL into the nucleus. Moreover, FOXK1 and FOXK2 protein levels are elevated in human colorectal cancers and correlate with DVL nuclear localization. Conditional expression of Foxk2 in mice induced intestinal hyper-proliferation that featured enhanced DVL nuclear localization and upregulated Wnt/β-catenin signaling. Together, our results not only reveal a mechanism by which DVL is translocated into the nucleus but also suggest unexpected roles of FOXK1 and FOXK2 in regulating Wnt/β-catenin signaling.

Dyrk2-associated EDD-DDB1-VprBP E3 Ligase Inhibits Telomerase by TERT Degradation

Background: Telomerase is an essential enzyme for the immortalization of stem and cancer cells. Results: Dyrk2-associated E3 ligase targets telomerase reverse transcriptase, a catalytic subunit of telomerase. Conclusion: Dyrk2-E3 ligase is necessary to negatively regulate telomerase activity. Significance: Learning how telomerase is regulated is crucial for understanding telomerase regulatory mechanism in cancer and stem cells. Telomerase maintains the telomere, a specialized chromosomal end structure that is essential for genomic stability and cell immortalization. Telomerase is not active in most somatic cells, but its reactivation is one of the hallmarks of cancer. In this study, we found that dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 2 (Dyrk2) negatively regulates telomerase activity. Dyrk2 phosphorylates TERT protein, a catalytic subunit of telomerase. Phosphorylated TERT is then associated with the EDD-DDB1-VprBP E3 ligase complex for subsequent ubiquitin-mediated TERT protein degradation. During the cell cycle, Dyrk2 interacts with TERT at the G2/M phase and induces degradation. In contrast, depletion of endogenous Dyrk2 disrupts the cell cycle-dependent regulation of TERT and elicits the constitutive activation of telomerase. Similarly, a Dyrk2 nonsense mutation identified in breast cancer compromises ubiquitination-mediated TERT protein degradation. Our findings suggest the novel molecular mechanism of kinase-associated telomerase regulation.

HIV-1 Vpr protein inhibits telomerase activity via the EDD-DDB1-VPRBP E3 ligase complex.

Background: Telomerase is an essential enzyme for chromosome stability. Results: The HIV-1 accessory protein Vpr targets TERT, a catalytic subunit of telomerase, via ubiquitin-mediated degradation. Conclusion: Vpr inhibits telomerase activity by TERT down-regulation. Significance: Learning how telomerase is deregulated by HIV-1 Vpr is crucial for understanding HIV-1-associated pathogenesis. Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis.

PAF-Mediated MAPK Signaling Hyperactivation via LAMTOR3 Induces Pancreatic Tumorigenesis

Deregulation of mitogen-activated protein kinase (MAPK) signaling leads to development of pancreatic cancer. Although Ras-mutation-driven pancreatic tumorigenesis is well understood, the underlying mechanism of Ras-independent MAPK hyperactivation remains elusive. Here, we have identified a distinct function of PCNA-associated factor (PAF) in modulating MAPK signaling. PAF is overexpressed in pancreatic cancer and required for pancreatic cancer cell proliferation. In mouse models, PAF expression induced pancreatic intraepithelial neoplasia with expression of pancreatic cancer stem cell markers. PAF-induced ductal epithelial cell hyperproliferation was accompanied by extracellular signal-regulated kinase (ERK) phosphorylation independently of Ras or Raf mutations. Intriguingly, PAF transcriptionally activated the expression of late endosomal/lysosomal adaptor, MAPK and mTOR activator 3 (LAMTOR3), which hyperphosphorylates MEK and ERK and is necessary for pancreatic cancer cell proliferation. Our results reveal an unsuspected mechanism of mitogenic signaling activation via LAMTOR3 and suggest that PAF-induced MAPK hyperactivation contributes to pancreatic tumorigenesis.

PAF-Wnt signaling-induced cell plasticity is required for maintenance of breast cancer cell stemness

Cancer stem cells (CSCs) contribute to tumour heterogeneity, therapy resistance and metastasis. However, the regulatory mechanisms of cancer cell stemness remain elusive. Here we identify PCNA-associated factor (PAF) as a key molecule that controls cancer cell stemness. PAF is highly expressed in breast cancer cells but not in mammary epithelial cells (MECs). In MECs, ectopic expression of PAF induces anchorage-independent cell growth and breast CSC marker expression. In mouse models, conditional PAF expression induces mammary ductal hyperplasia. Moreover, PAF expression endows MECs with a self-renewing capacity and cell heterogeneity generation via Wnt signalling. Conversely, ablation of endogenous PAF induces the loss of breast cancer cell stemness. Further cancer drug repurposing approaches reveal that NVP-AUY922 downregulates PAF and decreases breast cancer cell stemness. Our results unveil an unsuspected role of the PAF-Wnt signalling axis in modulating cell plasticity, which is required for the maintenance of breast cancer cell stemness.

LIG4 mediates Wnt signalling-induced radioresistance

Despite the implication of Wnt signalling in radioresistance, the underlying mechanisms are unknown. Here we find that high Wnt signalling is associated with radioresistance in colorectal cancer (CRC) cells and intestinal stem cells (ISCs). We find that LIG4, a DNA ligase in DNA double-strand break repair, is a direct target of β-catenin. Wnt signalling enhances non-homologous end-joining repair in CRC, which is mediated by LIG4 transactivated by β-catenin. During radiation-induced intestinal regeneration, LIG4 mainly expressed in the crypts is conditionally upregulated in ISCs, accompanied by Wnt/β-catenin signalling activation. Importantly, among the DNA repair genes, LIG4 is highly upregulated in human CRC cells, in correlation with β-catenin hyperactivation. Furthermore, blocking LIG4 sensitizes CRC cells to radiation. Our results reveal the molecular mechanism of Wnt signalling-induced radioresistance in CRC and ISCs, and further unveils the unexpected convergence between Wnt signalling and DNA repair pathways in tumorigenesis and tissue regeneration.

Wnt2 complements Wnt/β-catenin signaling in colorectal cancer.

Wnt2 is implicated in various human cancers. However, it remains unknown how Wnt2 is upregulated in human cancer and contributes to tumorigenesis. Here we found that Wnt2 is highly expressed in colorectal cancer (CRC) cells. In addition to co-expression of Wnt2 with Wnt/β-catenin target genes in CRC, knockdown or knockout of Wnt2 significantly downregulates Wnt/β-catenin target gene expression in CRC cells. Importantly, depletion or ablation of endogenous Wnt2 inhibits CRC cell proliferation. Similarly, neutralizing secreted Wnt2 reduces Wnt target gene expression and suppresses CRC cell proliferation. Conversely, Wnt2 increases cell proliferation of intestinal epithelial cells. Intriguingly, WNT2 expression is transcriptionally silenced by EZH2-mediated H3K27me3 histone modification in non-CRC cells, However, WNT2 expression is de-repressed by the loss of PRC2s promoter occupancy in CRC cells. Our results reveal the unexpected roles of Wnt2 in complementing Wnt/β-catenin signaling for CRC cell proliferation.

Identification of KIAA1199 as a Biomarker for Pancreatic Intraepithelial Neoplasia

Pancreatic cancer is one of the most aggressive cancers and has an extremely poor prognosis. Despite recent progress in both basic and clinical research, most pancreatic cancers are detected at an incurable stage owing to the absence of disease-specific symptoms. Thus, developing novel approaches for detecting pancreatic cancer at an early stage is imperative. Our in silico and immunohistochemical analyses showed that KIAA1199 is specifically expressed in human pancreatic cancer cells and pancreatic intraepithelial neoplasia, the early lesion of pancreatic cancer, in a genetically engineered mouse model and in human patient samples. We also detected secreted KIAA1199 protein in blood samples obtained from pancreatic cancer mouse models, but not in normal mice. Furthermore, we found that assessing KIAA1199 autoantibody increased the sensitivity of detecting pancreatic cancer. These results indicate the potential benefits of using KIAA1199 as a biomarker for early-stage pancreatic cancer.