SIX1 Downregulation Suppresses Self-renewal Capacity and THY1 Expression in Hepatocellular Carcinoma and SIX1 Dominate the Survival in Liver Cancer.

BACKGROUND/AIMS: Sine oculis homeoprotein 1 exerts an essential role in embryonic development, and it was also identified to be reactivated in various types of mammalian cancer. The sine oculis homeoprotein 1 transcription factor was demonstrated to induce epithelial-mesenchymal transition, regulate crucial genes associated with cancer progression, and increase the oncogenic potential of cells. Therefore, the present study aimed to identify the role of sine oculis homeoprotein 1 in cancer. MATERIALS AND METHODS: Sine oculis homeoprotein 1 gene expression was tested with real-time quantitative polymerase chain reaction (PCR) in different cancer types. Sine oculis homeoprotein 1 expression was suppressed by short hairpin RNA transduction in the SNU398 hepatocellular carcinoma cell line. The effects of sine oculis homeoprotein 1 on cell proliferation, drug resistance, and sphere formation were assessed in shSIX1 cells. Immunohistochemical and in silico analyses were performed to determine the prognostic role of sine oculis homeoprotein 1 expression. RESULTS: The upregulated expression levels of sine oculis homeoprotein 1 were revealed to be correlated with the stage of the disease in breast, colon, and liver cancer, with liver cancer exhibiting the highest expression profile. Sine oculis homeoprotein 1 downregulation significantly affected cell proliferation and suppressed sorafenib resistance and sphere-forming ability. Furthermore, sine oculis homeoprotein 1 knockdown cells were identified to have decreased CD90 levels, essential for cancer stem cell properties. Finally, sine oculis homeoprotein 1 expression was a CD90-independent biomarker for the clinical prognosis of liver cancer. CONCLUSION: The results of this study showed that the knockdown of sine oculis homeoprotein 1 expression might help to prevent hepatocarcinogenesis by increasing drug sensitivity and controlling tumor sphere formation. Overall, these results indicated that sine oculis homeoprotein 1 expression might be useful as a diagnostic marker for patients with hepatocellular carcinoma.

Arginine mediated photodynamic therapy with silicon(IV) phthalocyanine photosensitizers.

In the current study, we synthesized a new SiPc derivative conjugated with arginine at the axial positions, for a novel phthalocyanine-based photosensitizer for photodynamic therapy (PDT) applications in cancer cells. Axially-di-arginine substituted new silicon(IV) phthalocyanine photosensitizer (PS-5a) has been thoroughly researched for its anti-cancer properties. Various spectroscopic techniques were used to characterize this conjugate, including 1H NMR, 13C NMR, FT-IR, UV-vis, and MS spectral data. The in vitro PDT activities of the conjugate on cancer cells were tested through its cytotoxic, clonogenic, apoptotic effects on, and its capacity to induce DNA damage, and the disruption of mitochondrial membrane potential in cancer cell lines (liver; HuH-7, cervix; HeLa and breast; MCF7). Cancer cells exposed to the light illumination following uptake of the PS-5a as a photosensitizer revealed DNA breakage and collapsed mitochondrial membrane potential. The results of the present investigation demonstrate that PS-5a has a significant photo-cytotoxic effect on cancer cells. So, axially-di-arginine substituted silicon(IV) phthalocyanine could be an effective PDT agent for PDT treatment.

An investigation of bacteriocin nisin anti-cancer effects and FZD7 protein interactions in liver cancer cells.

The bacteriocin, nisin, produced by Lactococcus and Streptococcus species during fermentation, is widely used for bio preservatives in a wide variety of foods. Liver cancer has a high mortality rate and is the fourth leading cause of cancer-related deaths worldwide. Recently, researchers have shown the anti-cancer effects of nisin through in vitro and in vivo studies. This study aimed to investigate the effect of nisin on liver cancer cell lines, which represented two subgroups of the disease model. Nisin exhibited significant growth inhibition and apoptosis in both cell lines, HuH-7, and SNU182. Drug resistance is the main problem in liver cancer and the epithelial-to-mesenchymal transition has a role in the development of drug resistance in hepatocellular carcinoma. The expression of EMT transcription factors ZEB1, SNAI1, and TWIST1 were analyzed depending on nisin treatment, TWIST1 expression was down-regulated after nisin treatment compared to the untreated SNU182 and HuH-7 cell lines. Besides, due to the reported correlation between the overexpression of Frizzled (FZD) proteins, specifically FZD7, in primary hepatocellular carcinomas (HCCs), molecular docking was assessed for Nisin A in the binding site of FZD7. Results confirmed that Nisin A was able to form important hydrogen bonding with key residues. This research not only determined the role of nisin in different liver cancer cell models but it also provided the first result of FZD7 and nisin interaction.

Epithelial-to-Mesenchymal Plasticity in Circulating Tumor Cell Lines Sequentially Derived from a Patient with Colorectal Cancer.

Metastasis is a complicated and only partially understood multi-step process of cancer progression. A subset of cancer cells that can leave the primary tumor, intravasate, and circulate to reach distant organs are called circulating tumor cells (CTCs). Multiple lines of evidence suggest that in metastatic cancer cells, epithelial and mesenchymal markers are co-expressed to facilitate the cells' ability to go back and forth between cellular states. This feature is called epithelial-to-mesenchymal plasticity (EMP). CTCs represent a unique source to understand the EMP features in metastatic cascade biology. Our group previously established and characterized nine serial CTC lines from a patient with metastatic colon cancer. Here, we assessed the expression of markers involved in epithelial-mesenchymal (EMT) and mesenchymal-epithelial (MET) transition in these unique CTC lines, to define their EMP profile. We found that the oncogenes MYC and ezrin were expressed by all CTC lines, but not SIX1, one of their common regulators (also an EMT inducer). Moreover, the MET activator GRHL2 and its putative targets were strongly expressed in all CTC lines, revealing their plasticity in favor of an increased MET state that promotes metastasis formation.

ROR1 Expression and Its Functional Significance in Hepatocellular Carcinoma Cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common and deadly cancer; however, very little improvement has been made towards its diagnosis and prognosis. The expression and functional contribution of the receptor tyrosine kinase ROR1 have not been investigated in HCC before. Hence, we investigated the expression of ROR1 in HCC cells and assessed its involvement in hepatocarcinogenesis. METHODS: Recombinant bacterial ROR1 protein was used as an immunogen to generate ROR1 monoclonal antibodies. ROR1 transcript levels were detected by RT-qPCR and the protein expression of ROR1 in HCC was assessed by Western blotting by using homemade anti-ROR1 monoclonal antibodies. Apoptosis, cell cycle, trans-well migration, and drug efflux assays were performed in shRNA-ROR1 HCC cell clones to uncover the functional contribution of ROR1 to hepatocarcinogenesis. RESULTS: New ROR1 antibodies specifically detected endogenous ROR1 protein in human and mouse HCC cell lines. ROR1-knockdown resulted in decreased proliferation and migration but enhanced resistance to apoptosis and anoikis. The observed chemotherapy-resistant phenotype of ROR1-knockdown cells was due to enhanced drug efflux and increased expression of multi-drug resistance genes. CONCLUSIONS: ROR1 is expressed in HCC and contributes to disease development by interfering with multiple pathways. Acquired ROR1 expression may have diagnostic and prognostic value in HCC.

ETS1 is coexpressed with ZEB2 and mediates ZEB2-induced epithelial-mesenchymal transition in human tumors.

Epithelial-mesenchymal transition (EMT) is an embryonic program that is reactivated in cancer and regulates the invasion and metastasis of tumor cells. Zinc finger E-box binding homeobox 2 (ZEB2) induces EMT by upregulating matrix metalloproteinases (MMP), yet MMP genes lack ZEB2 binding motif in their promoters. Recently, expression of MMPs was associated to the activation of ETS1 transcription factor; however, a link between ZEB2 and ETS proto-oncogene 1, transcription factor (ETS1) remains to be elucidated. Hence, we investigated the transcriptional regulation of ETS1 by ZEB2 after our initial observation that ZEB2 and ETS1 are coexpressed in hepatocellular carcinoma cells (HCCs). Chromatin immunoprecipitation and luciferase reporter assays clearly showed that ZEB2 binds to E-box sequences on the promoter of ETS1. Elevated expression of ETS1 was found in DLD-ZEB2 and A431-ZEB2 inducible systems, and knockdown of ZEB2 caused an explicit downregulation of ETS1 in shZEB2-SNU398 and shZEB2-SK-HEP-1 cells. Repression of ETS1 expression in ZEB2-induced conditions substantially impaired the migration and invasive capacities of DLD1 cells. Mechanistically, knockdown of ETS1 in ZEB2-expressing cells resulted in the downregulation of established ZEB2 targets TWIST and MMP9. Correlation analyses in HCC lines, cancer complementary DNA arrays, and The Cancer Genome Atlas RNA-sequencing data set revealed that ZEB2 and ETS1 are coexpressed, and their expressions in human tumors show a highly significant positive correlation. Our results demonstrated that ZEB2 acts as an upstream regulator of ETS1 and, in turn, ETS1 maintains ZEB2-induced EMT. These findings add another level of complexity to the understanding of ZEB2 in the invasion and metastasis of cancer cells, and put ZEB2/ETS1 axis as a novel therapeutic target in human malignancies.

Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors.

BACKGROUND: ZEB2 is a transcriptional repressor that regulates epithelial-to-mesenchymal transition (EMT) through binding to bipartite E-box motifs in gene regulatory regions. Despite the abundant presence of E-boxes within the human genome and the multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets has been identified so far. Hence, we explored genome-wide ZEB2 binding by chromatin immunoprecipitation-sequencing (ChIP-seq) under endogenous ZEB2 expression conditions. METHODS: For ChIP-Seq we used an anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells exhibiting a high endogenous ZEB2 expression. The ChIP-Seq targets were validated using ChIP-qPCR, whereas ZEB2-dependent expression of target genes was assessed by RT-qPCR and Western blotting in shRNA-mediated ZEB2 silenced SNU398 cells and doxycycline-induced ZEB2 overexpressing colorectal carcinoma DLD1 cells. Changes in target gene expression were also assessed using primary human tumor cDNA arrays in conjunction with RT-qPCR. Additional differential expression and correlation analyses were performed using expO and Human Protein Atlas datasets. RESULTS: Over 500 ChIP-Seq positive genes were annotated, and intervals related to these genes were found to include the ZEB2 binding motif CACCTG according to TOMTOM motif analysis in the MEME Suite database. Assessment of ZEB2-dependent expression of target genes in ZEB2-silenced SNU398 cells and ZEB2-induced DLD1 cells revealed that the GALNT3 gene serves as a ZEB2 target with the highest, but inversely correlated, expression level. Remarkably, GALNT3 also exhibited the highest enrichment in the ChIP-qPCR validation assays. Through the analyses of primary tumor cDNA arrays and expO datasets a significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 expression were detected in most of the tumors. We also explored ZEB2 and GALNT3 protein expression using the Human Protein Atlas dataset and, again, observed an inverse correlation in all analyzed tumor types, except malignant melanoma. In contrast to a generally negative or weak ZEB2 expression, we found that most tumor tissues exhibited a strong or moderate GALNT3 expression. CONCLUSIONS: Our observation that ZEB2 negatively regulates a GalNAc-transferase (GALNT3) that is involved in O-glycosylation adds another layer of complexity to the role of ZEB2 in cancer progression and metastasis. Proteins glycosylated by GALNT3 may be exploited as novel diagnostics and/or therapeutic targets.