Elucidating the role of the oral microbiota, Parvimonas micra, in colorectal cancer pathogenesis

Abstract

Alteration of gut microbiome composition has been commonly observed in colorectal cancer (CRC) patients. Next generation 16S-amplicon sequencing studies revealed the abundance of an oral microbiota, Parvimonas micra, in tumor mucosa of CRC patients compared to healthy controls. Nevertheless, the role of P. micra in CRC remains to be investigated. This study was carried out to determine the tumorigenic potential of P. micra in CRC and to explore the regulatory pathways of tumorigenesis using an in vitro cell line model. To do this, an anaerobic human colon cell culture system using the HT-29 cell line was first established and evaluated. HT-29 viability (aerobic, 91.93%; anaerobic, 90.91%) in both aerobic and anaerobic (mimicking the colon mucosal condition) culture systems was found to be comparable. In addition, relative expression of proliferative (Ki-67) and anaerobic markers (HIF-, GLUT1, SLC16A1, SLC16A3, LDHA, CA9) of HT-29, was not significantly different when cultured in either aerobic or anaerobic culture systems. Using this anaerobic culture system, during P. micra-HT-29 interaction assays, P. micra infection was found to increase HT-29 proliferation by 38.45%, with the highest cell migration rate at 24 hr, after a 2-hour infection at a MOI of 100:1 (bacteria: cell) ratio. Furthermore, at 2 hr post-infection, P. micra was also found to significantly induced IL-5, IL-8, CCL20 and CSF2 inflammatory marker expression in HT-29 cells. Using an iTRAQ-LC-MS/MS system, HT-29 proteomic profile during P. micra infection was determined, where 157 and 214 proteins were found to be up- and down-regulated, respectively. P. micra-associated CRC tumorigenesis was found to be accompanied by up-regulation of PSMB4 in ubiquitin-proteasome pathways and down-regulation of CUL1, MCM3 and YWHAH that play important roles in cell cycle, which in turn modulate 22 clinically relevant epithelial-mesenchymal transition (EMT)-markers. In summary, P. micra was found to exacerbate tumorigenesis in the HT-29 CRC cell line via upregulation of inflammatory, ubiquitin-proteosome and EMT pathways. Further study is important to confirm the interplay of this bacteria along with other gut microbiota in an in vivo CRC model.