FABP5 suppresses colorectal cancer progression via mTOR-mediated autophagy by decreasing FASN expression

Abstract

Lipid metabolism plays an important role in the occurrence and development of cancer, in particular, digestive system tumors such as colon cancer. Here, we investigated the role of the fatty acid-binding protein 5 (FABP5) in colorectal cancer (CRC). We observed marked down-regulation of FABP5 in CRC. Data from functional assays revealed inhibitory effects of FABP5 on cell proliferation, colony formation, migration, invasion as well as tumor growth in vivo. In terms of mechanistic insights, FABP5 interacted with fatty acid synthase (FASN) and activated the ubiquitin proteasome pathway, leading to a decrease in FASN expression and lipid accumulation, moreover, suppressing mTOR signaling and facilitating cell autophagy. Orlistat, a FASN inhibitor, exerted anti-cancer effects both in vivo and in vitro. Furthermore, the upstream RNA demethylase ALKBH5 positively regulated FABP5 expression via an m⁶A-independent mechanism. Overall, our collective findings offer valuable insights into the critical role of the ALKBH5/FABP5/FASN/mTOR axis in tumor progression and uncover a potential mechanism linking lipid metabolism to development of CRC, providing novel therapeutic targets for future interventions.

Keywords: FASN; Autophagy; Colorectal cancer; FABP5; Lipid metabolism; N6-methyladenosine; Orlistat.

Figure 1

FABP5 is silenced in CRC. A) FABP5 protein levels in ten cancers from the CPATC database. (B) FABP5 was down-regulated in CRC compared to normal tissues in the TCGA database(p=2.3e-1). (C) Low expression of FABP5 was associated with shorter survival probability in CRC cases from the TCGA database. (D) Protein expression of FABP5 in CRC from the CPATC database(p=7.73e-13). (E) Representative immunohistochemical staining of FABP5 in CRC tissues from patients, magnification: ×73. (F) Relative average optical density values in 90 CRC and paired normal tissues. (*P < 0.05)

Figure 2

Over-expression of FABP5 suppresses cell proliferation, migration and invasion. Western blot analysis of alterations in protein levels of FABP5 in over-expressing and control CRC cells. (B, C) Over-expression of FABP5 inhibited proliferation of HCT116 (B) and SW620 (C) cells, determined via the CCK8 assay. (D, E) Up-regulation of FABP5 induced a decrease in the number of colonies. (F, G) EdU assay showed suppression of proliferation in both cell types under conditions of FABP5 over-expression, magnification: ×200. (H-J) FABP5 suppressed migration and invasion of HCT116 and SW620 cells, magnification: ×100. (**P < 0.01, ***P < 0.001)

Figure 3

Knockdown of FABP5 promotes malignant biological behaviors of CRC cells. (A) Western blot analysis of the protein levels of FABP5 in knockdown and control CRC cells. (B, C) CCK-8 data showing that silencing of FABP5 promoted proliferation of HCT116 (B) and SW620 (C) cells. (D, E) Down-regulation of FABP5 induced an increase in the number of colonies. (F, G) Down-regulation of FABP5 accelerated proliferation of HCT116 and SW620 cells, as observed with the EdU assay, magnification: ×200. (H-J) Silence of FABP5 facilitated cell migration and invasion of both cell types, magnification: × 100. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

FABP5 functions as a tumor suppressor via interacting with FASN. (A) Co-IP of FABP5 and FASN in HCT116 cells. (B) Western blot analysis of FASN levels in FABP5 over-expressing and silenced HCT116 and SW620 cells. (C) Western blot analysis of FASN in FABP5 over-expressing and depleted HCT116 cells subjected to CHX (10 µmol/L) and MG132 (10 μmol/L) treatment. (D) Co-IP analysis of FASN and ubiquitin in FABP5 over-expression HCT116 cells treated with MG132 (10 μmol/L) for 6 h. (E) Western blot showing FASN knockdown efficiency in HCT116 cells. (F) Silencing of FASN inhibited proliferation of HCT116 cells. (G, H) Down-regulation of FASN induced a decrease in colony number and proliferation of cells. (I) Silencing of FASN suppressed cell migration and invasion, magnification: ×100. (J-L) Cell proliferation was significantly decreased upon treatment with orlistat (50 µmol/L for 24 h), as observed with CCK-8 (J), colony formation (K) and EdU assays (L), magnification: ×200. (M) Orlistat inhibited migration and invasion of HCT116 cells, magnification: ×100. (**P < 0.01, ***P < 0.001)

Figure 5

Orlistat restores CRC cell malignant behaviors induced by FABP5 down-regulation. (A) Western blot showing FABP5 and FASN protein levels under conditions of FABP5 silencing and 50 μmol/L orlistat treatment for 24 h. (B-F) CCK-8, colony formation and EdU assays (Magnification: ×200) showing cell proliferation under conditions of FABP5 knockdown and orlistat treatment. (G, H) Orlistat reverses CRC cell migration and invasion (Magnification: ×100) induced by FABP5 silencing. (I-K) Nile red staining (Magnification: ×200) in FABP5/FASN altered cells. (***P < 0.001)

Figure 6

FABP5 interacts with FASN to promote cell autophagy via mTOR. (A-D) Enrichment analysis of transcriptome and lipid metabolomes. (E, F) Western blot analysis of LC3 and Beclin1 in FABP5 over-expression or down-regulation and FABP5 down-regulation+orlistat treatment groups. (G, H) Western blot analysis of PI3K/AKT/mTOR in FABP5 over-expression or down-regulation and FABP5 +orlistat treatment groups.

Figure 7

FABP5 suppresses tumor growth via regulation of FASN in vivo. (A-C) Primary tumor samples obtained from mice subcutaneously injected with HCT116 cells transfected with FABP5 over-expression and control cell groups (A). Relative tumor volumes (B) and weights (C) at the endpoint (n = 5). (D-F) Primary tumor samples were obtained from mice subcutaneously injected with HCT116 cells transfected with FASN knockdown and control group (D). Relative tumor volumes (E) and weights (F) measured at the endpoint (n = 5). (G-H) Primary tumor samples obtained from mice subcutaneously injected with HCT116 cells with FABP5 silencing, FABP5 silencing plus orlistat treatment, and control groups (G). Relative tumor volumes (H) and weights (I) measured at the endpoint (n = 5). (J-L) Representative immunohistochemistry images showing expression of FABP5 and FASN in xenograft tumor tissues, magnification: ×73. (**P < 0.01, ***P < 0.001)

Figure 8

ALKBH5 positively regulates FABP5 to exert anti-cancer effects. (A) Western blots showing positive regulation of FABP5 by ALKBH5 via down-regulation of m6A writers (METTL3, METTL14, WTAP) and erasers (FTO, ALKBH5). (B) Knockdown efficiency of m6A molecules assessed via RT-PCR. (C) Western blot showing FABP5 and FASN expression under conditions of ALKBH5 up-regulation and down-regulation. (D, E) Efficiency of ALKBH5 over-expression assessed via RT-PCR. (F) ALKBH5, FABP5 and FASN protein levels evaluated under conditions of ALKBH5 over-expression, ALKBH5 over-expression with FABP5 knockdown and control in HCT116 cells. (G-M) FABP5 knockdown reversed the decrease in cell proliferation, colony formation, migration and invasion induced by ALKBH5 in CCK-8 (G), colony formation (H, I), EdU (J, K; Magnification: ×200) and transwell assays (L, M; Magnification: ×100). (**P < 0.01, ***P < 0.001)