FK866 inhibits colorectal cancer metastasis by reducing NAD+ levels in cancer-associated fibroblasts

Background: Extraintestinal metastasis is the main therapeutic challenge for colorectal Cancer, the third most common Cancer worldwide. Various components of the tumor microenvironment, especially cancer-associated fibroblasts (CAFs), play important roles in tumor metastasis. NAMPT is often overexpressed in tumor tissues and is associated with poorer prognosis. However, the specific roles of NAMPT as well as NAD⁺ in tumor metastasis are relatively unknown. Therefore, we investigated the role of NAMPT and related NAD⁺ metabolism in cancer-associated fibroblasts mediated colorectal Cancer metastasis.

Objective: This study sought to explore the molecular mechanism of FK866 in CAFs cell and colorectal Cancer proliferation and metastasis.

Methods: The expression of NAMPT in clinical tissues were detected by immunohistochemically analysis. To investigate the role of NAMPT and NAD⁺ in the interactions between Cancer cells and cancer-associated fibroblasts in tumor microenvironment, we isolated CAFs from normal and Cancer tissues of clinical colorectal Cancer patients. CAFs were treated with different concentrations of FK866, inhibitor of NAMPT, then the NAD⁺ content was detected using kits, the expression of CAFs activity and stemness indexes was assessed by Western blot and immunofluorescence. The secreted factors of these cells were analyzed by cellular inflammatory factor microarrays. The migration of SW480 after co-cultured with FK866-treated CAFs was detected by Transwell. Finally, high-throughput Sequencing was performed to identify the proteins that are associated with the effect of altered NAD⁺ in CAFs on the migration of Cancer cells.

Results: NAMPT expression is significantly higher in colorectal Cancer tissues, especially in metastatic Cancer patients, than that in normal tissues. Inhibition of NAMPT by FK866 in CAFs decreases the expression of activity indicators (α-SMA, PDGFRβ), stemness indicators (BMI-1, OCT4), inflammatory factors and chemokines. Meanwhile, FK866 treatment inhibits the migration ability of SW480 cells co-cultured with CAFs. Finally, high-throughput Sequencing reveals that PITX3 are down-regulated after NAD⁺ reduction in CAFs, which could be reversed by adding NAM, a raw material for NAD⁺ synthesis.

Conclusion: Inhibition of the NAMPT-mediated NAD⁺ synthesis by FK866 may decrease the activation and stemness of CAFs, reduce the secretion of inflammatory and chemokines by suppressing the expression of PITX3, resulting in the suppression of colorectal Cancer metastasis.

Cancer-associated fibroblasts; Colorectal cancer; Metastasis; NAD+; NAMPT; PITX3.