RESUMEN

Intrahepatic cholangiocarcinoma (ICC) is a type of liver cancer associated with poor prognosis and increased mortality; the limited treatment strategy highlights the urgent need for investigation. Traditional Chinese Medicine (TCM), used alone or in combination with other treatments, can enhance therapeutic efficacy, improve life quality of patients and extend overall survival. In total, two rounds of screening of a TCM library of 2,538 active compounds were conducted using a Cell Counting Kit­8 assay and ICC cell lines. Cell proliferation and migration abilities were assessed through colony formation, 5­ethynyl­2'­deoxyuridine, would healing and Transwell assays. The impact of digitoxin (DT) on signaling pathways was initially investigated using RNA sequencing and further validated using reverse transcription­quantitative PCR, western blotting, lectin blotting and flow cytometry. ICC cells stably overexpressing ST6 ß­galactoside α­2,6­sialyltransferase 1 (ST6GAL1) were generated through lentiviral transfection. It was shown that DT emerged as a highly effective anti­ICC candidate from two rounds high­throughput library screening. DT could inhibit the proliferation and migration of ICC cells by suppressing NF­κB activation and reducing nuclear phosphorylated­NF­κB levels, along with diminishing ST6GAL1 mRNA and protein expression. The aforementioned biological effects and signal pathways of DT could be counteracted by overexpressing ST6GAL1 in ICC cells. In conclusion, DT suppressed ICC cell proliferation and migration by targeting the NF­κB/ST6GAL1 signaling axis. The findings of the present study indicated the promising therapeutic effects of DT in managing ICC, offering new avenues for treatment strategies.

Asunto(s)

Neoplasias de los Conductos Biliares , Proliferación Celular , Colangiocarcinoma , Digitoxina , Transducción de Señal , Humanos , Antígenos CD/metabolismo , Antígenos CD/genética , beta-D-Galactósido alfa 2-6-Sialiltransferasa/metabolismo , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/patología , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/patología , Colangiocarcinoma/metabolismo , Colangiocarcinoma/genética , Digitoxina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , FN-kappa B/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

INTRODUCTION: Colorectal cancer is the third most common cause of cancer death. Rectal cancer makes up a third of all colorectal cases. Treatment for locally advanced rectal cancer includes chemoradiation followed by surgery. We have previously identified ST6GAL1 as a cause of resistance to chemoradiation in vitro and hypothesized that it would be correlated with poor response in human derived models and human tissues. METHODS: Five organoid models were created from primary human rectal cancers and ST6GAL1 was knocked down via lentivirus transduction in one model. ST6GAL1 and Cleaved Caspase-3 (CC3) were assessed after chemoradiation via immunostaining. A tissue microarray (TMA) was created from twenty-six patients who underwent chemoradiation and had pre- and post-treatment specimens of rectal adenocarcinoma available at our institution. Immunohistochemistry was performed for ST6GAL1 and percent positive cancer cell staining was assessed and correlation with pathological grade of response was measured. RESULTS: Organoid models were treated with chemoradiation and both ST6GAL1 mRNA and protein significantly increased after treatment. The organoid model targeted with ST6GAL1 knockdown was found to have increased CC3 after treatment. In the tissue microarray, 42 percent of patient samples had an increase in percent tumor cell staining for ST6GAL1 after treatment. Post-treatment percent staining was associated with a worse grade of treatment response (p = 0.01) and increased staining post-treatment compared to pre-treatment was also associated with a worse response (p = 0.01). CONCLUSION: ST6GAL1 is associated with resistance to treatment in human rectal cancer and knockdown in an organoid model abrogated resistance to apoptosis caused by chemoradiation.

Asunto(s)

Quimioradioterapia , Neoplasias del Recto , beta-D-Galactósido alfa 2-6-Sialiltransferasa , Humanos , Antígenos CD , beta-D-Galactósido alfa 2-6-Sialiltransferasa/efectos de los fármacos , beta-D-Galactósido alfa 2-6-Sialiltransferasa/metabolismo , beta-D-Galactósido alfa 2-6-Sialiltransferasa/efectos de la radiación , Estadificación de Neoplasias , Neoplasias del Recto/tratamiento farmacológico , Neoplasias del Recto/genética , Neoplasias del Recto/radioterapia

RESUMEN

Aberrant glycosylation is a hallmark of a cancer cell. One prevalent alteration is an enrichment in α2,6-linked sialylation of N-glycosylated proteins, a modification directed by the ST6GAL1 sialyltransferase. ST6GAL1 is upregulated in many malignancies including ovarian cancer. Prior studies have shown that the addition of α2,6 sialic acid to the epidermal growth factor receptor (EGFR) activates this receptor, although the mechanism was largely unknown. To investigate the role of ST6GAL1 in EGFR activation, ST6GAL1 was overexpressed in the OV4 ovarian cancer line, which lacks endogenous ST6GAL1, or knocked-down in the OVCAR-3 and OVCAR-5 ovarian cancer lines, which have robust ST6GAL1 expression. Cells with high expression of ST6GAL1 displayed increased activation of EGFR and its downstream signaling targets, AKT and NFκB. Using biochemical and microscopy approaches, including total internal reflection fluorescence microscopy, we determined that the α2,6 sialylation of EGFR promoted its dimerization and higher order oligomerization. Additionally, ST6GAL1 activity was found to modulate EGFR trafficking dynamics following EGF-induced receptor activation. Specifically, EGFR sialylation enhanced receptor recycling to the cell surface following activation while simultaneously inhibiting lysosomal degradation. 3D widefield deconvolution microscopy confirmed that in cells with high ST6GAL1 expression, EGFR exhibited greater colocalization with Rab11 recycling endosomes and reduced colocalization with LAMP1-positive lysosomes. Collectively, our findings highlight a novel mechanism by which α2,6 sialylation promotes EGFR signaling by facilitating receptor oligomerization and recycling.

Asunto(s)

Receptores ErbB , beta-D-Galactósido alfa 2-6-Sialiltransferasa , Humanos , beta-D-Galactósido alfa 2-6-Sialiltransferasa/genética , beta-D-Galactósido alfa 2-6-Sialiltransferasa/metabolismo , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Ováricas/fisiopatología , Transducción de Señal , Transporte de Proteínas/genética , Unión Proteica