MTHFD1 Regulates Autophagy to Promote Growth and Metastasis in Colorectal Cancer via the PI3K-AKT-mTOR Signaling Pathway

Abstract

Objectives: Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) is the enzyme with the activities of methylenetetrahydrofolate dehydrogenase, methylenetetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase. Our aim was to elucidate the function of MTHFD1 in colorectal cancer (CRC).

Methods: In vitro assessments of the proliferation, invasion, and migration abilities of CRC cells were conducted using Immunohistochemistry, Transwell invasion assays, Western blot (WB), and Cell counting Kit-8 assays. WB was also utilized to measure autophagy protein levels and PI3K-AKT-mTOR signaling pathway expression. Furthermore, the role of MTHFD1 was evaluated in vivo by using subcutaneous xenograft tumor models and lateral tail vein metastasis models of human CRC in nude mice.

Results: Overexpression of MTHFD1 promoted the abilities of tumorigenesis and metastasis in CRC in vitro and in vivo and reduced autophagy, attributing to the PI3K-AKT-mTOR signaling pathway in CRC cells. In contrast, the down-regulation of MTHFD1 increased autophagy and suppressed their proliferation, migration, and invasion.

Conclusions: MTHFD1 can modulate the PI3K-AKT-mTOR signaling pathway to suppress autophagy and stimulate tumorigenesis and metastasis.

Keywords: MTHFD1; PI3K‐AKT‐mTOR; autophagy; colorectal cancer; invasion; migration; proliferation.

FIGURE 1

MTHFD1 expression levels are higher in CRC tissue than in the adjacent normality tissue. (A) the levels of MTHFD1 mRNA were analyzed by the TIMER database in different tumors and normal tissues. (B) MTHFD1 mRNA expression levels were determined in 36 samples of CRC and corresponding noncancerous tissues by RT‐qPCR. (C) In 91 CRC tissues and corresponding noncancerous tissues, immunohistochemical staining, and detection of MTHFD1 protein were performed. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 2

MTHFD1 knockdown inhibits CRC cell proliferation, migration, and invasion. (A) The efficiency of MTHFD1 knockdown in HCT‐116 and DLD‐1 was accessed by WB. (B) The efficiency of MTHFD1 overexpression in SW480 and DLD‐1 was detected by WB. (C–F) CCK‐8 assays were used to assess cellular proliferation. (G, H) Colony formation assays detected cell proliferation. (I–L) MTHFD1 knockdown was shown to reduce cell migration and invasion in Transwell assays. (M–P) Cell migration abilities were measured by the wound healing assays. All experiments in vitro were carried out in triplicate and were repeated a minimum of three times. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 3

MTHFD1 knockdown modulates tumorigenesis proliferation and metastasis in vivo. (A) Photographs of nude mice were captured 21 days postinjection with either control cells or cells with MTHFD1 knockdown. (B–D) After sacrificing the mice, the tumors were weighed and the volume and growth curve of the tumors were measured. (E, F) The expression levels of MTHFD1 and Ki‐67 proteins of the tumors harvested from the nude mice were measured. (G) Photographs of the lungs of nude mice were taken 2 months postinjection via the lateral tail vein with either control cells or cells with MTHFD1 knockdown. (H, I) H&E staining was performed to visualize metastatic tumor nodules in the lungs of nude mice. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 4

Low MTHFD1 expression can enhance autophagy in CRC cells via the PI3K‐AKT‐mTOR signaling pathway. (A–C) Analysis of the differentially expressed genes associated with the expression of MTHFD1 using GO and KEGG pathways. (D, E) The autophagosomes of HCT‐116 and DLD‐1 cells were observed by TEM. (F–H) The measurement of autophagy‐associated proteins P62, LC3, Beclin1, and ULK1 in CRC cells was performed using WB. (I–K) WB examined relevant PI3K‐AKT‐mTOR signaling pathway protein levels in CRC cells. All experiments in vitro were carried out in triplicate and were repeated at least three times. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 5

MTHFD1 promotes the proliferation, migration, and invasion of CRC cells through the mediation of autophagy. (A, B) The colony assays showed that HCQ enhanced the proliferation potential. (C–F) The Transwell assays and wounding assays showed that HCQ enhanced the migration and invasion potential. (G) The colony assays showed that RAPA suppressed the proliferation of MTHFD1 overexpression cells. (H–K) The Transwell assays and wounding assays showed that RAPA inhibited the migration and invasion of MTHFD1 overexpression cells. All experiments in vitro were carried out in triplicate and were repeated at least three times. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.