Roger Garcia

Introduction. Colorectal cancer (CRC) ranks third in incidence and second in mortality among all cancers globally¹. Among patients diagnosed with CRC, 40% present with recurrence after treatment². The gut microbiota can modulate CRC cellular mechanisms that promote resistance; specific host-pathogen interactions driving such resistance are under investigation³. Fusobacterium nucleatum is a Gram-negative anaerobic bacillus associated with tissue and fecal samples of CRC patients, as well as poorer patient prognosis and cancer recurrence after chemotherapy⁴. These findings warrant further investigation into the mechanisms driving this association, as well as potential improvements to CRC diagnosis and treatments that can result from such work. Methods. HCT116 CRC cell lines were co-cultured with F. nucleatum. Changes in expression of ATG7 and ULK1 were measured by RT-qPCR. Autophagosome formation was measured with transmission electron microscopy. Apoptotic response of CRC cells to chemotherapy was measured by flow cytometry. In xenograft experiments, four-week-old male BALB/c nude mice were housed under specific pathogen-free conditions. HCT116 cells were injected subcutaneously into the right axilla. Recombinant adenovirus vectors were constructed with miR-18a* and miR-4802 and were used to infect mice. To evaluate diagnostics, F. nucleatum abundance was used in combination with fecal immunochemical test (FIT) and evaluated using samples from 104 patients with CRC. For autophagy inhibitor combination chemotherapy evaluation, 24 patients were evaluated for safety and efficacy. Results. CRC cell co-culture with F. nucleatum increased expression of autophagy-related genes and increased autophagosome formation. When treated with oxaliplatin or 5-fluorouracil, F. nucleatum promoted resistance as measured by apoptotic response to therapy. When an autophagy inhibitor was added, F. nucleatum-induced resistance was eliminated. Transfection of CRC cells with miRNA-18a* or miRNA-4802 mimics eliminated F. nucleatum-induced resistance, while transfection of cells with the corresponding miRNA inhibitors recapitulated resistance, suggesting that F. nucleatum induces autophagy and chemoresistance by downregulating these two miRNA molecules. siRNA-mediated TLR4 knockout eliminated chemoresistance, indicating that TLR4 activation is essential for F. nucleatum-induced chemoresistance³. After diagnostic evaluation, sensitivity of FIT for detection of all CRC stages was optimized by combination with F. nucleatum abundance measurement⁵. Phase I clinical studies suggested that combination chemotherapy involving hydroxychloroquine, an autophagy inhibitor, and vorinostat enhanced CASP3 cleavage, suggesting enhanced apoptosis⁶. Conclusion. F. nucleatum promotes CRC chemoresistance by activating TLR4, downregulating miR-18a* and miR-4802, enhancing autophagic activity and evading apoptosis. Discovery of this relationship opens a path to translational applications that can improve current CRC diagnostics and treatment, including FIT and autophagy inhibitor combination chemotherapy.

1. Wong SH, Yu J. Gut microbiota in colorectal cancer: mechanisms of action and clinical applications. Nat Rev Gastroenterol Hepatol. 2019;16(11):690-704. doi:10.1038/s41575-019-0209-8
2. Biller LH, Schrag D. Diagnosis and Treatment of Metastatic Colorectal Cancer: A Review. JAMA. 2021;325(7):669. doi:10.1001/jama.2021.0106
3. Yu T, Guo F, Yu Y, et al. Fusobacterium nucleatum Promotes Chemoresistance to Colorectal Cancer by Modulating Autophagy. Cell. 2017;170(3):548-563.e16. doi:10.1016/j.cell.2017.07.008
4. Brennan CA, Garrett WS. Fusobacterium nucleatum — symbiont, opportunist and oncobacterium. Nature Reviews Microbiology. 2019;17(3):156-166. doi:10.1038/s41579-018-0129-6
5. Wong SH, Kwong TNY, Chow T-C, et al. Quantitation of faecal Fusobacterium improves faecal immunochemical test in detecting advanced colorectal neoplasia. Gut. 2017;66(8):1441-1448. doi:10.1136/gutjnl-2016-312766
6. Mahalingam D, Mita M, Sarantopoulos J, et al. Combined autophagy and HDAC inhibition. Autophagy. 2014;10(8):1403-1414. doi:10.4161/auto.29231