Background: Ovarian carcinoma is a highly lethal gynecological malignancy due to its frequent relapses and adoption of chemoresistance. To develop new biomarkers for disease progression in ovarian carcinoma, CSCs, which are considered to contribute to disease relapse and metastasis, were isolated from human ovarian carcinoma tissues, and differentially expressed microRNAs (miRNAs) in CSCs were identified and assessed the clinical implication of expression of these miRNAs.Methods: Primary cancer cells derived from human ovarian carcinomas were cultured and spheroid-forming cells (SFCs) were isolated. Profiles of miRNA expression in CSC-like SFCs were identified by miRNA microarray and the results were validated by quantitative real-time RT-PCR (qRT-PCR). We also assessed the correlations between miRNA expression levels and clinicopathological parameters in ovarian carcinomas.Results: Five miRNAs (miR-5703, miR-630, miR-1246, miR-424-5p, and miR-320b) were significantly dysregulated in CSC-like SFCs compared with primary cancer cells. The qRT-PCR showed that miR-5703 and miR-1246 expression was significantly higher in ovarian cancer cells than in normal control cells, whereas the miR-424-5p level was significantly lower. Decreased expression of miR-424-5p was significantly associated with distant metastasis in high stage (stage IIII & IV) carcinomas (35.5% vs. 72.2%, respectively, p=0.013)Conclusion: Taken together, miR-5703, miR-630, miR-1246, miR-424-5p, and miR-320b are useful markers for enriching ovarian CSCs. Decreased expression of miR-424-5p in ovarian carcinoma might be a putative biomarker for distant metastasis in ovarian carcinoma.

The mechanisms through which cancer-upregulated gene 2 (CUG2), a novel oncogene, affects Wnt/β-catenin signaling, essential for tumorigenesis, are unclear. In this study, we aimed to elucidate some of these mechanisms in A549 lung cancer cells. Under the overexpression of CUG2, the protein levels and activity of β-catenin were evaluated by western blot analysis and luciferase assay. To examine a biological consequence of β-catenin under CUG2 overexpression, cell migration, invasion and sphere formation assay were performed. The upregulation of β-catenin induced by CUG2 overexpression was also accessed by xenotransplantation in mice. We first found that CUG2 overexpression increased β-catenin expression and activity. The suppression of β-catenin decreased cancer stem cell (CSC)-like phenotypes, indicating that β-catenin is involved in CUG2-mediated CSC-like phenotypes. Notably, CUG2 overexpression increased the phosphorylation of β-catenin at Ser33/Ser37, which is known to recruit E3 ligase for β-catenin degradation. Moreover, CUG2 interacted with and enhanced the expression and kinase activity of never in mitosis gene A-related kinase 2 (NEK2). Recombinant NEK2 phosphorylated β-catenin at Ser33/Ser37, while NEK2 knockdown decreased the phosphorylation of β-catenin, suggesting that NEK2 is involved in the phosphorylation of β-catenin at Ser33/Ser37. Treatment with CGK062, a small chemical molecule, which promotes the phosphorylation of β-catenin at Ser33/Ser37 through protein kinase C (PKC)α to induce its degradation, reduced β-catenin levels and inhibited the CUG2-induced features of malignant tumors, including increased cell migration, invasion and sphere formation. Furthermore, CGK062 treatment suppressed CUG2-mediated tumor formation in nude mice. Taken together, the findings of this study suggest that CUG2 enhances the phosphorylation of β-catenin at Ser33/Ser37 by activating NEK2, thus stabilizing β-catenin. CGK062 may thus have potential for use as a therapeutic drug against CUG2-overexpressing lung cancer cells.

PAUF, a tumor-promoting protein secreted by cancer cells, exerts paracrine effects on immune cells through TLR4 receptors expressed on immune cell surfaces. This study aimed to investigate if PAUF elicits autocrine effects on pancreatic cancer (PC) cells through TLR4, a receptor that is overexpressed on PC cells. In this study, TLR4 expression was detected in PC cells only, but not normal pancreatic cells. The migration of TLR4 high-expressing PC cells (i.e., BxPC-3) was reduced by a selective TLR4 inhibitor, in a dose-dependent manner. Using TLR4 overexpressed and knockout PC cell lines, we observed direct PAUF-TLR4 binding on the PC cell surfaces, and that PAUF-induced cancer migration may be mediated exclusively through the TLR4 receptor. Further experiments showed that PAUF signaling was passed down through the TLR4/MyD88 pathway without the involvement of the TLR4/TRIF pathway. TLR4 knockout also downregulated PC membrane PD-L1 expression, which was not influenced by PAUF. To the best of our knowledge, TLR4 is the first receptor identified on cancer cells that mediates PAUF’s migration-promoting effect. The results of this study enhanced our understanding of the mechanism of PAUF-induced tumor-promoting effects and suggests that TLR4 expression on cancer cells may be an important biomarker for anti-PAUF treatment.

Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of β-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator. [BMB Reports 2022; 55(2): 98-103]

Background: Pancreatic adenocarcinoma up-regulated factor (PAUF) is a cancer-secreted protein which is overexpressed on pancreatic cancer (PC) cells and promotes tumor growth in an autocrine way. However, the receptor of PAUF on PC cells has yet to be identified. Meanwhile, it is known that PAUF, as a tumor microenvironment (TME) modulator, plays a role in immune evasion and suppression through toll-like receptors notably TLR4 which are mainly expressed on immune cells. Previous studies show that TLR4 is also highly expressed in PC tumor tissues, compared to normal tissues, and activation of TLR4 signaling pathway induces the migration and invasion of PC cells. Objective: This study aimed to investigate if TLR4 expressed on cancer cells can act as a receptor for PAUF to mediate its tumor promoting effects. Methods and Results: We tested the TLR4 expression in six PC cell lines and one normal pancreatic cell line. And confirmed that TLR4 is expressed in all PC cell lines at variable levels, but not in the normal pancreatic cell line.Next, we found that a chemical TLR4-specific inhibitor TAK-242 significantly reduced the migration of PC cells with high TLR4 expression (BxPC-3), but it did not impact the migration of PC cells with low TLR4 expression (Panc-1). This experiment suggests that some endogenous ligands of TLR4 may have mediated PC cell migration.Using TLR4 overexpressed and knockout PC cell lines, we showed that treatment with recombinant PAUF increased the migration and invasion of TLR4 overexpressed PC cells, but not in the TLR4 knockout cells. Likewise, treatment of anti-PAUF antibody reduced the migration and invasion of TLR4 overexpressed PC cells, but not in the TLR4 knockout cells. These results shows that PAUF’s migration and invasion promoting effects of PC cells is TLR4 dependent.To further understand the molecular mechanism of PAUF-induced metastasis (migration/invasion)-promoting effects via TLR4, we conducted a series of in vitro studies including immunoprecipitation (IP), western blot analysis, flow cytometry, and luciferase reporter assay. We demonstrated that PAUF activates TLR4/MyD88 signaling pathway, but not TLR4/TRIF pathway. Conclusions: In conclusion, for the first time this study demonstrates that TLR4 expressed on PC cell surfaces functions as a receptor of PAUF to mediate its metastasis-promoting effects, which are exclusively through the MyD88/NF-κB signaling pathway. This study also suggests TLR4 as a potential biomarker for identification of optimal patients, and a new therapeutic target to treat PC. Anti-PAUF antibody used in this study is currently evaluated by clinical trials in France, Spain, and US FDA. Citation Format: Fen Jiang, So Eun Youn, Da Eun Hong, Tae Heung Kang, Hye Yun Won, Yun Yong Park, Sang Seok Koh. PAUF induces migration of human pancreatic cancer cells exclusively via the TLR4/MyD88/NF-κB signaling pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3611.