Antitumor efficacy of dietary spinach in an Apc-mutant rat model
Pirc and wild-type (WT) rats were fed AIN93 control (Ctrl) diet, or AIN93 diet containing 10% w/w freeze-dried baby spinach (SPI), starting at 4 weeks of age (Fig. 1a). No significant treatment-related effects were observed with respect to food consumption and body weight throughout the study (Fig. 1b). By colonoscopy [12,14,18], SPI suppressed adenomatous polyps as early as 12 weeks into the experiment (Fig. 1c), i.e., after 8 weeks of SPI treatment. During this period and at later times, colonoscopy data revealed consistent inhibition of small colon polyps, and significant suppression of large colon polyps after week 20 (Fig. 1d).
When the experiment was terminated, after the rats had reached 30 weeks of age, tumor multiplicity was decreased significantly both in the colon and in the small intestine, and tumor volume also was reduced significantly in the colon by SPI treatment (Fig. 1e). No marked changes were observed histologically, but bromodeoxyuridine (BrdU) labeling hinted at reduced cell proliferation rates by SPI in some regions of the colonic crypt (Additional file 1). Immunohistochemistry and immunoblotting experiments indicated that b-catenin overexpression in colon tumors was unaffected by SPI treatment (Fig. 1f and Additional file 2). Thus, despite the oncogenic driver of the Apc-mutant genetic background, antitumor mechanisms other than b-catenin downregulation were pursued.
The gut microbiota is altered by dietary spinach
We performed 16S rRNA sequencing of the gut microbial community in Pirc and WT rats. For a complete view of the taxonomic and other data, refer to Additional file 3. The observed Operational Taxonomic Units (OTUs, Table 1 in Additional file 3) and Shannon index revealed that a-diversity was unaffected by host genotype, but was increased significantly by SPI treatment in Pirc and WT rats (Fig. 2a, black vs. green symbols). There was no segregation between Pirc and WT rats for weighted UniFrac principal coordinates analysis (PCoA) (Fig. 2b), but a significant separation was observed in unweighted UniFrac PCoA (Fig. 2c). The gut microbiome in both genotypes clustered separately in weighted UniFrac PCoA between Ctrl and SPI groups (Fig. 2b), with a marked shift in unweighted UniFrac PCoA (Fig. 2c). These data are consistent with previous findings indicating that diet plays a dominant role over genetic background with respect to shaping interindividual variations in host-associated microbial communities [19,20].
The Pirc model had a higher abundance of Bacteroidetes and Proteobacteria than WT rats, while Firmicutes, Actinobacteria, and Tenericutes were lower (Fig. 2d), as observed in mouse and human microbiomes [21-24]. These abundances were reversed by SPI intake, independent of host genotype, as evidenced by the increased relative abundance of Firmicutes and decreased Bacteroidetes (Fig. 2e). Similar findings were noted at the Family (Fig. 2f) and genus level (Fig. 2g). For example, in Pirc and WT rats, SPI treatment increased the relative abundance of Lachnospiraceae and decreased Ruminococcaceae (Fig 2f, green bars), and at the genus level SPI ingestion reduced the relative abundance of Bacteroides and Desulfovibrio (Fig. 2g, green bars). These results suggested that SPI consumption reshapes the microbiome composition, reversing the effects of the Apc-mutant background and host genetic predisposition.
Linear discriminant effect size (LEfSe) was used to further analyze the OTU microbiome data (Tables 2-4 in Additional file 3). From the corresponding cladograms (Fig. 3a-c), host genotype and dietary SPI intake both influenced Ruminococcaceae and Lachnospiraceae family members. In response to SPI treatment, LEfSe analyses revealed that Pirc and WT rats shared ~50% commonality among changes at the genus level (Fig. 3d). Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) categorized 328 terms following Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (Table 5 in Additional file 3). Linoleate and ether lipid metabolism were altered significantly in Pirc vs. WT rats, and PICRUSt revealed a marked effect of SPI intake. Thus, after SPI consumption, 85 terms (57 decreased and 28 increased) and 112 terms (71 decreased and 41 increased) were changed in Pirc and WT rats, respectively, and seventy-five terms overlapped between the two genotypes, i.e., 54 decreased and 21 increased (Fig. 3e). Increases in membrane transporters, cell motility, signal transduction, transcription, carbohydrate metabolism, and kinases were among the top 10 terms prioritized by KEGG analysis in Pirc and WT rats given SPI, along with porphyrin and chlorophyll metabolism (Fig. 3f). Decreased terms were related to protein translation, replication/repair, and energy/nucleotide metabolism. Pathway changes included an increase in linoleate and butanoate metabolism, and a decrease in the TCA cycle and pathways in cancer (Fig. 3g).
Spinach consumption impacts key genes associated with pathogenesis
RNA-sequencing (RNA-seq) mapped 17,378 transcripts in colonic tissues from Pirc and WT rats, and principal component analysis (PCA) completely segregated tumor tissues from normal tissues (Fig. 4a). Tumor development had a more marked effect on overall transcriptome levels than host genetics (Pirc vs. WT rats) and SPI consumption. There were 261 differentially expressed genes (DEGs) identified between Pirc and WT normal-looking tissues, half of which (138 genes) overlapped with 2180 DEGs associated with tumor development (Fig. 4b). Heatmaps of all DEGs showed a distinct tumor feature when compared to Pirc and WT normal-looking tissues (Fig. 4c, PCT vs. PCN and PCT vs. WCN).
Geneset Enrichment Analysis (GSEA) combined with HALLMARK identified nine pathways altered significantly in Pirc normal-looking colon compared to WT normal colon (Fig. 4d, upper panel), indicating differences at the level of host genetics. Five of these pathways were further altered in colon tumors (Fig. 4d, lower panel, underlined), i.e., Inflammatory Response, Allograft Rejection, TNFa signaling through NF-kB, Adipogenesis, and Myogenesis. As expected in the Apc-mutant background, Wnt/b-catenin signaling was upregulated in Pirc colon tumors (Fig. 4d, red font). Immunoblotting corroborated that b-catenin overexpression was associated with poly(ADP-ribose)polymerase (PARP) cleavage, increased cyclin D1, and decreased p53 in Pirc tumors compared to adjacent normal and WT normal colonic tissues (Additional file 2).
Other pathways of note were related to cell cycle changes, immune response, oxidative stress, and metabolism (Fig. 4d). Using RT-qPCR for validation, genes upregulated significantly in Pirc colon tumors vs. Pirc adjacent normal-looking colon included Cxcl6, Serpine1, and Il-1b, whereas Hspb8, Tpm2 and Fhl1 were downregulated significantly (Fig. 4e). Compared to adjacent normal colon, upregulation of Defa6 and Bcl3 and downregulation of App, Myh11, and Myl9 indicated changes in tight junctions and anti-microbial activity in Pirc colon tumors (Fig. 4f).
We also mapped 559 microRNAs (miRNAs) via small RNA-seq, which segregated tumor vs. normal-looking colon (Fig. 4g). Similar to the mRNA profiles (Fig. 4c), miRNAs had a distinct tumor feature as compared to Pirc and WT normal-looking tissues (Fig. 4h, PCT vs. PCN and PCT vs. WCN). There were 115 differentially expressed miRNAs (DEmiRs) associated with tumor formation (Fig. 4h and Table 6 in Additional file 3). We combined TargetScan with RNA-seq and small RNA-seq datasets to identify miRNA-RNA pairs most altered in the Apc-mutant background (Fig. 4i). Validation by qPCR corroborated significant downregulation in Pirc colon tumors of miR-215, miR-143, and mir-145 compared with adjacent normal-looking colonic mucosa (Fig. 4j). Other candidates, such as mir-146b, mir-34a, and mir-21, did not reach statistical significance.
Attention shifted next to SPI effects on predicted targets (Fig. 5 and Additional file 4). Compared to the AIN basal diet control group, SPI consumption altered 4 genes in common among the 101 DEGs in WT rats and 80 DEGs in Pirc normal colon (Fig. 5a). GSEA indicated significant downregulation of cell cycle-related pathways and upregulation of immune-related pathways in Pirc and WT rats fed SPI, with five pathways in common among the genotypes (Fig. 5b). The latter pathways included TNFa Signaling through NFkB, Hypoxia, Epithelial Mesenchymal Transition, Apoptosis, and KRAS Signaling. After SPI consumption, 2945 DEGs were identified in Pirc colon tumors compared to adjacent normal colon, and 1754 of the DEGs also were detected in tumors vs. adjacent normal colon from rats given control diet (Fig. 5c). Among the pathways most strongly implicated were IFN-a and IFN-g for tumors from SPI-fed rats compared to rats given control diet.
We also considered two scenarios for the antitumor efficacy: (1) genes up- or downregulated in colon tumors relative to adjacent normal colon that were reversed by SPI in Pirc normal colon, and (2) genes that were normalized in colon tumors from rats given SPI compared with colon tumors from Pirc rats given control diet. The first scenario would implicate primary prevention of colonic aberrant crypt foci or microadenomas, before they advanced to later stages. These genes included Serpine1, Itga6,Duoxa2, Tcf7l1, Plcd1 and Slc30a10 (Fig. 5d). Comparing tumor to tumor in scenario 2, Ccl21 and Klf7 were normalized by SPI ingestion, relative to basal diet (Fig. 5e).
In terms of miRNAs, among 66 DEmiRs in colon tumors from SPI-fed rats, 41 DEmiRs similarly were detected in colon tumors from animals on Ctrl diet (Fig. 5f). After investigating RNA-miRNA pairs and validating as before (Fig 4j), colon tumors had loss of miR-145 with increased Serpine1 and gain of mir-34a with reduced Klf4 (Fig. 5g and Table 7 in Additional file 3). A negative correlation for mir-145/Serpine1 was maintained after SPI consumption, whereas the mir-34a/Klf4 trend was reversed by SPI treatment (Fig. 5g).
Crosstalk between microbiome and host transcriptome responses
Integrating antitumor outcomes (Fig. 1) with a-diversity (Fig. 2), we observed a significant inverse association for tumor multiplicity (Fig. 6a, left panel) but not tumor volume (Fig. 6a, right panel). Tumor multiplicity was inversely correlated with three unclassified Bacteroidales families (Fig. 6b and Table 8 in Additional file 3). At the genus level, one unclassified Lachnospiraceae and one unclassified Ruminococcaceae genus were negatively correlated with tumor multiplicity, whereas one other unclassified Ruminococcaceae genus was positively correlated (Fig. 6c). Metagenome prediction in relation to tumor multiplicity outcomes found significant inverse correlations for butanoate metabolism and calcium signaling, and positive associations for peptidases and pathways in cancer (Fig. 6d).
We also compared microbiome and host gene expression changes based on the transcriptomic data (Fig. 6e). Significant positive correlations were noted for Lachnospiraceae (Unc0396i) and the efflux transporter Slc30a10, Bacteroidales (Unc00krl) and Ruminococcaceae (Unc01k4o) and the phospholipase C family member Plcd1, and Ruminococcaceae (Unide781 and Unc00vst) and the serine protease inhibitor Serpine1. Negative correlations were detected for Ruminococcaceae (Unide781 and Unc00vst) and the transcription factor Tcf7l1.
Metabolomic corroboration of mechanistic leads
To validate correlations from the microbiome and transcriptome studies, metabolomics was performed on adenomatous colon polyps and normal colon tissues obtained from Pirc rats at 30 weeks (Fig. 1a). As predicted from the microbiome data (Fig. 3g), among the fifty-one metabolites identified (Table 9 in Additional file 3) several were associated with fatty acid metabolism, the TCA cycle, and pathways in cancer (Fig. 7). Linoleate and its downstream metabolites from the 15-lipoxygenase-1 (15-LOX-1) pathway exert proapoptotic antitumor mechanisms in CRC [25-27]; notably, lower levels of these metabolites in Pirc colon tumors tended to be normalized in adenomatous polyps following SPI treatment, comparable to the levels detected in normal-looking Pirc colon ±SPI (Fig. 7a). Similar trends were observed for 2-aceto-2-hydroxybutanoate, which was increased significantly in colon tumors after SPI treatment (Fig. 7b). On the other hand, L-glutamate and N-acetylneuraminate were detected at higher levels in colon tumors, and SPI treatment reduced these metabolites in adenomatous polyps, comparable to the levels observed in normal-looking Pirc colon ±SPI (Figs. 7c and 7d). Key intermediates are discussed further below.