Involvement of Met receptor pathway in the aggressive behavior of colorectal cancer cells induced by PTHrP

NOVOA DÍAZ MARÍA BELEN; CARRIERE PEDRO; GIGOLA GRACIELA; ZWENGER ARIEL; CALVO NATALIA; GENTILI CLAUDIA

WORLD JOURNAL OF GASTROENTEROLOGY

W J G PRESS

Lugar: Beijing; Año: 2022 vol. 28 p. 3177 - 3200

1007-9327

BACKGROUNDParathyroid hormone-related peptide (PTHrP) plays a key role in the development and progression of many tumors. We found in HCT116 cells from colorectal cancer (CRC) that the binding of PTHrP to its receptor (PTHR1) activates events associated with an aggressive phenotype. In HCT116 cells xenografts, PTHrP modulates molecular markers expressions linked to tumor progression. Empirical evidence suggests that Met receptor is involved in the development and evolution of CRC. Based on these data, we hypothesize that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.AIMTo elucidate the relationship between PTHR1, PTHrP and Met in CRC models. METHODSFor in vitro assays, HCT116 and Caco-2 cells from human CRC were incubated in the absence or presence of PTHrP (1-34) (10-8 M). Where indicated, cells were pre-incubated with specific kinase inhibitors or Dimethylsulfoxide (DMSO), the vehicle of the inhibitors. The protein levels were evaluated by Western Blot (WB) technique. Real-time PCR reaction (RT-qPCR) was carried out to determine the changes in gene expression. Wound Healing Assay and morphological monitoring were performed to evaluate cell migration and changes related to the epithelial-mesenchymal transition (EMT), respectively. The number of HCT116 viable cells was counted by Trypan Blue dye exclusion test to evaluate the effects of Irinotecan (CPT-11), Oxaliplatin (OXA) or Doxorubicin (DOXO) with or without PTHrP.For in vivo tests, HCT116 cells xenografts on 6-week-old male, N: NIH (S) _nu mice received a daily intratumoral injection of PTHrP (40 µg / kg) in 100 µl phosphate buffered saline (PBS) or the vehicle suspension (PBS), as a control during 20 days. Humanitarian slaughter was carried out and the tumors were removed, weighed and fixed in a 4% formaldehyde solution for subsequent treatment by immunoassays. To evaluate the expression of molecular markers on human tumor samples, we studied 23 specimens obtained from CRC patients which were received at the Hospital Interzonal de Graves y Agudos Dr. José Penna (in the city of Bahía Blanca, province of Buenos Aires, Argentina) and at the Hospital Provincial de Neuquén (in the city of Neuquén, province of Neuquén, Argentina) from January 1990 to December 2007. 7 cases with normal colorectal tissues were assigned to control group. Tumor tissue samples and clinical histories of patients were analyzed. Paraffin-embedded blocks from primary tumors were reviewed by Hematoxylin-Eosin staining technique, subsequently representative histological samples were selected of each patient. From each paraffin block, tumor sections were stained for immunohistochemical detection.The statistical significance of differences was analyzed using proper statistical analysis. The results were considered statistically significant at p < 0.05. RESULTSBy WB analysis and using total Met antibody we found that PTHrP regulates Met expression in HCT116 cells but not in Caco-2 cells. In HCT116 cells, Met protein levels increased at 30 minutes (p