Gut Microbiota Dysbiosis Accelerates Prostate Cancer Progression Through Increased LPCAT1 Expression and Enhanced DNA Repair Pathways

Abstract

Gut microbiota dysbiosis is related to cancer development and progression. Our previous study showed that Ruminococcus was more abundant in CRPC (Castration-resistant prostate cancer) than HSPC (Hormone-sensitive prostate cancer) individuals. Here, we determined the potential mechanism of microbiota dysbiosis in prostate cancer (PCa) progression. Metagenomics was used to verify the gut microbial discrepancies between CRPC and HSPC individuals. Fecal microbiota transplantation (FMT) was performed by transferring the fecal suspension of CRPC or HSPC individuals to TRAMP mice. Afterwards, the mice's prostate histopathology and gut microbiota composition were determined. Since Ruminococcus was demonstrated to correlate with phospholipid metabolism, we used lipidomics to examine the mice's fecal lipid profiles. The expression of LPCAT1 the key enzyme for phospholipid remodeling in mice prostate was also examined. Meanwhile, both microbial functions prediction and LPCAT1 GSEA analysis (Gene Set Enrichment Analysis) indicated DNA repair pathways, we further determined the expressions of RAD51 and DNA-PKcs in mice prostate. The results showed that gut Ruminococcus was significantly more abundant in CRPC individuals. FMT using CRPC feces accelerated mice's PCa progression and increased their gut Ruminococcus abundance. Majority of fecal lipids including lysophosphatidylcholine and phosphatidylcholine were upregulated in CRPC FMT treated mice, accompanied with enhanced expressions of LPCAT1, RAD51, and DNA-PKcs in mice prostate. We reported an abundant colonization of Ruminococcus in the gut of CRPC individuals and mice receiving their fecal suspensions, and revealed the promotive capability of Ruminococcus in PCa progression via upregulating LPCAT1 and DNA repair protein expressions. The bacterium and its downstream pathways may become the targets of therapies for PCa in the future.

Keywords: DNA repair; LPCAT1; Ruminococcus; glycerophospholipid; microbiota dysbiosis.

Figure 1

Structure of gut microbial communities in PCa patients. Taxonomic hierarchy tree using GraPhlAn tool displayed the overall structure of gut microbial communities in PCa patients from phylum to species (outer to inner circle). The node size represented the relative abundance of the taxon. The top 20 taxa were annotated.

Figure 2

Differentiated microbial phylotypes between CRPC and HSPC individuals. Metagenomics identified 44 differentiated microbial phylotypes between the two cohorts with LEfSe analysis (LDA>2, P<0.05), Ruminococcus and three subordinate species were more abundant in CRPC individuals.

Figure 3

Histopathological images of mice prostate after FMT. FMT with CRPC feces significantly accelerated mice’s PCa progression. The bar represents 100 µm.

Figure 4

FMT with CRPC feces increased mice’s gut Ruminococcus. (A) 16s rRNA sequencing identified increased gut Ruminococcus in CRPC FMT treated mice. (B) Relative abundance of Ruminococcus between two groups of mice after FMT. The bars represents mean ± SD.

Figure 5

Fecal lipid profiles of mice after FMT. (A) Lipidomics showed that the levels of most lipid classes including glycerophospholipids were increased in mice after FMT with CRPC feces. The blue bars indicated the number of lipids within each lipid class that increased in CRPC FMT treated mice., while the greed bars indicated the number of lipids that decreased. (B) On the species level, 17 species were significantly different between the two groups, 16:0 LPC and 18:2 LPC were significantly increased in CRPC FMT treated mice.

Figure 6

Differentiated microbial functions and LPCAT1 GSEA analysis. (A) PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) identified 11 microbial KEGG pathways that were different between CRPC and HSPC individuals, non-homologous end-joining pathway (Red Bar) was enhanced in CRPC cohort. The bar represented CRPC/HSPC ratio in relative abundance of certain KEGG pathways. (B) GSEA analysis of LPCAT1 indicated positive correlation between LPCAT1 amplification status and DNA repair pathways. Cancer Genome Atlas (TCGA) data regarding prostate cancer patients was used. Through comparison between mRNA expression and LPCAT1, patients were separated into LPCAT1 high-expression group and LPCAT1 low-expression group. GSEA (Software GSEA_4.1.0.) was performed to identify enriched signaling pathways associated with LPCAT1 expression, gene sets with p value < 0.05, false discovery rate (FDR) < 0.25 were considered significant.

Figure 7

Overexpressed LPCAT1 and DNA repair proteins in mice prostate after FMT with CRPC feces. Both western blots (A) and immunohistochemistry (B) showed that the expression of LPCAT1 was enhanced in mice prostate after FMT with CRPC feces. (C) The expressions of RAD51 and DNA-PKcs were enhanced in mice prostate after FMT with CRPC feces.

Figure 8

Downregulation of LPCAT1 suppressed PCa cell proliferation and migration (A) LPCAT1, DNA-PKcs, RAD51 were highly expressed in PCa cell lines (PC3, DU145, LnCaP) when compared to RWPE-1 (normal prostate epithelial cell). (B) Inhibition of LPCAT1 downregulated DNA-PKcs and RAD51 expressions in PC-3 cells. (C, D) Cloning formation and migration assays showed that downregulation of LPCAT1 suppressed PC-3 cell proliferation and migration. Data are presented as mean ± SD. ^### P < .001.