Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Jul 1.
Published in final edited form as: Chemosphere. 2024 May 14;359:142332. doi: 10.1016/j.chemosphere.2024.142332

Perfluorooctanesulfonic acid exposure leads to downregulation of 3-hydroxy-3-methylglutaryl-CoA synthase 2 expression and upregulation of markers associated with intestinal carcinogenesis in mouse intestinal tissues

Josiane Weber Tessmann a, Pan Deng b, Jerika Durham a, Chang Li c, Moumita Banerjee c, Qingding Wang c, Ryan A Goettl d, Daheng He d, Chi Wang d, Eun Y Lee e, B Mark Evers c, Bernhard Hennig f, Yekaterina Y Zaytseva a,c
PMCID: PMC11157449  NIHMSID: NIHMS1996753  PMID: 38754493

Abstract

Perfluorooctanesulfonic acid (PFOS) is a widely recognized environment pollutant known for its high bioaccumulation potential and a long elimination half-life. Several studies have shown that PFOS can alter multiple biological pathways and negatively affect human health. Considering the direct exposure to the gastrointestinal (GI) tract to environmental pollutants, PFOS can potentially disrupt intestinal homeostasis. However, there is limited knowledge about the effect of PFOS exposure on normal intestinal tissues, and its contribution to GI-associated diseases remains to be determined. In this study, we examined the effect of PFOS exposure on the gene expression profile of intestinal tissues of C57BL/6 mice using RNAseq analysis. We found that PFOS exposure in drinking water significantly downregulates mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme, in intestinal tissues of mice. We found that diets containing the soluble fibers inulin and pectin, which are known to be protective against PFOS exposure, were ineffective in reversing the downregulation of HMGCS2 expression in vivo. Analysis of intestinal tissues also demonstrated that PFOS exposure leads to upregulation of proteins implicated in colorectal carcinogenesis, including β-catenin, c-MYC, mTOR and FASN. Consistent with the in vivo results, PFOS exposure leads to downregulation of HMGCS2 in mouse and human normal intestinal organoids in vitro. Furthermore, we show that shRNA-mediated knockdown of HMGCS2 in a human normal intestinal cell line resulted in increased cell proliferation and upregulation of key proliferation-associated proteins such as cyclin D, survivin, ERK1/2 and AKT, along with an increase in lipid accumulation. In summary, our results suggest that PFOS exposure may contribute to pathological changes in normal intestinal cells via downregulation of HMGCS2 expression and upregulation of pro-carcinogenic signaling pathways that may increase the risk of colorectal cancer development.

Keywords: environmental pollutants, perfluorooctanesulfonic acid, gastrointestinal tract, HMGCS2, ketogenesis, colorectal carcinogenesis

Graphical Abstract

graphic file with name nihms-1996753-f0006.jpg

1. Introduction

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a group of synthetic environmental pollutants widely used in industrial applications and consumer products (Gluge et al., 2020). These manufactured chemicals can contaminate air, soil and water during their production and use, and have frequently been detected in biological samples, such as breast milk and human blood (Centers for Disease Control and Prevention, 2022; Consumer Product Safety Commission, 2023). General population exposure mainly occurs through consuming contaminated water and food (Consumer Product Safety Commission, 2023). PFAS are pollutants that have been found globally in surface- and groundwater drinking-water resources (Evich et al., 2022; Sims et al., 2022). According to a study conducted by the U.S. Geological Survey, approximately 45% of drinking water in the US is contaminated by at least one type of PFAS (Smalling et al., 2023). These compounds are made of fluorinated carbon chains and the carbon–fluorine bond is one of the strongest bonds in organic chemistry, PFAS are extremely stable and resistant to environmental and metabolic degradation, resulting in their high bioaccumulation and toxicity potential (Cousins et al., 2020). Long-term exposure to PFAS has been increasingly associated with negative health outcomes, including human development, metabolic disorders, reduced immune responses and an increased risk of cancer (Zeng et al., 2019; Chen et al., 2020; Fenton et al., 2021; U.S. Environmental Protection Agency, 2023).

Perfluorooctanesulfonic acid (PFOS) is one of the most detected PFASs, with an estimated elimination half-life of 3.4 to 4.8 years in humans (Olsen et al., 2007; Li et al., 2018). Sampling of drinking water systems under the Unregulated Contaminant Monitoring Rule, conducted 2013–2015, showed PFOS concentrations ranged from 0.04 μg/L (the minimum reporting level at this time) to 7 μg/L (U.S. Environmental Protection Agency, 2022). In April 2024, EPA announced the final National Primary Drinking Water Regulation which enforced the maximum contaminant level of PFOS to be 4 nanograms per liter (ng/L) in drinking water by 2027 (U.S. Environmental Protection Agency, 2024). Because recent health effects studies show that PFOS can impact human health at exposure levels much lower, the U.S. Environmental Protection Agency currently has established that interim lifetime non cancer health advisory of 0.02 ng/L for PFOS can be applied to both short-term and chronic risk assessment scenarios (U.S. Environmental Protection Agency, 2022). The current studies also suggest that PFOS may be a carcinogen due to its pro-tumorigenic effects reported in multiple cancers, including breast, esophagus, glioblastoma, kidney, liver, lung and prostate (Durham et al., 2023).

Importantly, the gastrointestinal (GI) tract is directly exposed to PFOS, where it can accumulate and impact intestinal homeostasis under both physiological and pathological conditions (Efsa Panel on Contaminants in the Food Chain et al., 2018; Liang et al., 2021). A National Academies of Science, Engineering, and Medicine report provided evidence suggestive of an increased risk of ulcerative colitis associated with PFAS exposure (National Academies of Sciences, 2022). Another study observed that PFOS induces inflammatory bowel disease (IBD)-like injury in rats, marked by inflammatory infiltration into the intestinal tissues (Liang et al., 2021). Consistent with these findings, several studies suggest that PFOS can influence the production of inflammatory cytokines, thereby contributing to chronic inflammation (Office of Environmental Health Hazard Assessment, 2010; Zhang et al., 2023). Notably, individuals with IBD have an elevated risk of colorectal cancer (CRC) development, a phenomenon attributed to chronic inflammation which can initiate and drive tumorigenesis (Lucafo et al., 2021). CRC represents a significant public health concern, especially given its global trend of increasing incidence among the younger population (Zaborowski et al., 2021). Possible causes of early-onset disease include dietary and lifestyle factors, as well as exposure to environmental contaminants (Adigun et al., 2023).

In a prior study, our group analyzed the effects of PFOS in C57BL/6J mice exposed to PFOS in drinking water and fed diets supplemented with cellulose as a control versus soluble fibers inulin and pectin as a potential mitigation strategy for PFOS exposure (Deng et al., 2022). This study demonstrated that PFOS exposure alters a gene transcription profile and lipid metabolism in the mouse livers, and markedly changes the composition of intestinal microbiota. Incorporation of soluble fibers such as inulin and pectin in diets partially normalize the PFOS-induced changes suggesting that a high fiber diet can be a potential mitigation strategy for PFOS exposure (Deng et al., 2022). In the present study, we performed RNAseq analysis on biobanked intestinal tissues to explore the effects of PFOS exposure in drinking water on transcriptional profile of intestinal epithelium. We tested the hypothesis that PFOS exposure disrupts intestinal homeostasis, potentially leading to pathological changes such as inflammation, altered signaling and metabolic pathways that may increase the risk of GI-associated diseases including CRC. Understanding the impact of PFOS exposure on GI pathology can provide valuable insights to guide the development of potential mitigation interventions to reduce the harmful effects of this environmental pollutant.

2. Materials and methods

2.1. Diets and study design

The diet specifications and the study design were described previously by Deng (Deng et al., 2022) and are summarized in Figure 1A. Briefly, wild-type male C57BL/6J mice were fed diets supplemented with cellulose as a control, inulin or pectin. PFOS (heptadecafluorooctanesulfonic acid potassium salt; 77282; Sigma-Aldrich, St. Louis, MO, USA; purity >98%) was mixed with drinking water and given to the animals ad libitum for 7 weeks. The exposure dose of PFOS corresponded to 3 μg/g body weight per day. At the end of the experiment, the samples were collected and stored at −80°C for analysis.

Figure 1. PFOS exposure downregulates HMGCS2 levels.

Figure 1.

Open in a new tab

(A) Schematic of experimental design: seven-week-old male C57BL/6 mice were placed on an irradiated diet supplemented with cellulose (control), inulin or pectin. PFOS exposure occurred through daily oral ingestion of 3 μg/g body weight via drinking water. The mice were maintained on their respective diets for a 7-week period. (B) Volcano plot of differentially expressed genes (DEGs) between mice exposed to PFOS and the control (cellulose) group. Red and green dots represent upregulated and downregulated genes, respectively. (C) mRNA expression levels of HMGCS2 and VEGFR, normalized to GAPDH in intestinal tissues, were measured by qRT-PCR. Data are presented as mean ± SD (**p<0.01). (D) Venn diagram displaying overlapping genes identified in the intestines of mice exposed to PFOS and fed with different diets. (E) Western blot and (F) densitometric quantification analysis of HMGCS2 protein levels on intestinal tissues of mice exposed to PFOS and treated with cellulose (control), inulin- or pectin-supplemented diets. (G) Western blot ,densitometric quantification analysis of HMGCS2 protein levels, and representative images of wild-type mouse intestinal organoids treated with 1 μg/mL of PFOS for 15 days, (H) human intestinal organoids treated with 1 μg/mL of PFOS for 30 days, and (I) normal human colon primary cells treated with 1 μg/mL of PFOS for 6 months (n=3). The levels of HMGCS2 expression were determined based on normalization of each band to β-actin (Scale bar: 40 μm).

2.2. RNA isolation and RNA-sequencing

Intestinal tissues were preserved at −80°C and total RNA from mice exposed to PFOS and fed diets supplemented with cellulose, inulin or pectin were isolated using a RNeasy Plus kit (#74134, Qiagen, Germantown, MD, USA). mRNA samples from 4 mice were used for RNAseq analysis of control (cellulose), PFOS (cellulose), pectin and pectin/PFOS groups. mRNA samples from 3 mice were used for the inulin and inulin/PFOS groups due to unsatisfactory mRNA quality in one sample. Library preparation, sequencing and bioinformatics analysis were performed by Beijing Genomics Institute (Cambridge, MA, USA, https://www.bgi.com/global/). DNBSEQ platform was used for sequencing. FASTQ data first went through data quality control process to remove low-quality reads, reads with adaptor sequences, and reads with high levels of N base using SOAPnuke (Chen et al., 2018). HISAT (Kim et al., 2015) was used to align the clean reads to the reference genome, and Bowtie2 (Langmead and Salzberg, 2012) was used to align the clean reads to the reference genes. The average mapping ratio with reference genome was 97.13%; with gene is 81.84%; 18472 genes were identified. Reference Genome Version: GCF_000001635.27_GRCm39 (NCBI). The gene expression quantification was obtained based on mapping results with RSEM (Li and Dewey, 2011). The differential gene expression analysis was performed using DESeq2 software (Love et al., 2014). Bioinformatics analysis was also verified by the University of Kentucky Markey Cancer Center’s Biostatistics and Bioinformatics Shared Resource Facility. The data presented in this publication have been deposited in NCBI’s Gene Expression Omnibus and are assessable through GEO Series accession number GSE248402 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE248402).

2.3. Real-time reverse transcription–polymerase chain reaction (RT-PCR) analysis

cDNA from total RNA isolation was synthesized using a high-capacity cDNA reverse transcription kit (#4368814, Applied Biosystems, Bedford, MA, USA). RT-PCR reactions were performed using a TaqMan Gene Expression Master Mix (#4369016, Applied Biosystems) TaqMan® MGB probes, FAM™ dye-labeled for mouse HMGCS2 (Mn00550050_m1), VEGFR (Mn0122242_m1) and GAPDH (Mn99999915_g1) were purchased from Thermo Fisher (Waltham, MA, USA), and used following the instructions provided by the manufacturer. mRNA expression levels were measured in a StepOnePlus Real Time PCR system (Applied Biosystems).

2.4. Organoid isolation and treatment

Mouse intestinal organoids were isolated as previously described (Li et al., 2023). Human organoids were isolated following the protocol of Sato et al. (Sato et al., 2009) and cultivated in IntestiCult-SF Organoid Growth Medium (StemCell #100–0340). The organoid cultures were treated with 1 μg/mL of PFOS for 15 days (mouse) or 30 days (human) and then collected for protein analysis.

2.5. Cell culture and transfection

The normal human colon primary (NHCP) cell line was obtained from Celprogen, Torrance, CA, USA (#36037–08) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin–streptomycin. The cells were cultivated in a humidified atmosphere containing 5% CO2 at 37°C. The cells were exposed to a concentration of 1 μg/mL of PFOS over a 6-month period, with PFOS being replenished during every medium change. Upon reaching 80% confluence, the cells were trypsinized and split at a 1:10 ratio. Stable HMGCS2 knockdown NHCP cells were established using Mission lentiviral transduction particles purchased from Sigma-Aldrich (TRCN0000045859 [shRNA#1] and TRCN0000429288 [shRNA#2]). pLKO.1-puro non-mammalian shRNA was used as a non-target control (NTC). Cells were transfected with 8 μg/mL of polybrene and selected with 10 μg/mL of puromycin. HMGCS2 knockdown was confirmed by western blotting.

2.6. Tissue collection

Normal and CRC tissues were collected from consented patients with stage II–IV CRC who had undergone surgery at the University of Kentucky Medical Center (IRB # 52094, PI: Zaytseva) and biobanked in Dr. Zaytseva’s laboratory. Frozen tissues were homogenized using cold stainless-steel beads in Cell Lysis Buffer (#9803, Cell Signaling, Danvers, MA, USA) supplemented with protease inhibitor cocktail (#11836170001, Roche, Indianapolis, IN, USA) and 1 mM PMSF (#93482, Sigma, St. Louis, MO, USA) and stored at −80C for analysis..

2.7. Protein extraction and western blot analysis

Human and mouse tissues, organoids and NHCP cells were lysed using Cell Lysis Buffer (#9803, Cell Signaling, Danvers, MA, USA) supplemented with protease inhibitor cocktail (#11836170001, Roche, Indianapolis, IN, USA) and 1 mM PMSF (#93482, Sigma, St. Louis, MO, USA). Equal amounts of protein were separated on SDS-PAGE and transferred onto PVDF membranes, followed by blocking with 5% non-fat dry milk. The membranes were probed with the following primary antibodies: HMGCS2 (#ab137043), FASN (#3180), p-mTOR (#2971), c-Myc (#5605), total β-catenin (#610154), active β-catenin (#7270), survivin (#2808), cyclin D1 (#ab134175), total Erk1/2 (#4695), phospho-Erk1/2 (#4376), total AKT (#2920), phospho-AKT (#4056), CD36 (#ab133625) and β-actin (#A5441). Then, membranes were incubated with the appropriate secondary antibodies (rabbit #7074 or mouse #7076) and protein bands were detected with Immobilon Western Chemiluminescent HRP substrate (#WBKLS0500, Sigma). The quantification of band intensities was performed using ImageJ software.

2.8. Cell viability assay

NHCP cells were seeded onto 96-well plates at a concentration of 1×103 cells per well, followed by incubation at 37°C for 6 days. Then, 100 μL of medium containing 10 μL of PrestoBlue cell viability reagent (#A13262, Thermo Fisher) was added to the wells and incubated for 2 h at 37°C and protected from light. The fluorescence was measured at 560 nm excitation and 590 nm emission using a microplate reader (Varioskan LUX, Thermo Fisher).

2.9. Confocal microscopy

NHCP cells were seeded on an 8-well coverslip μ-slide (#80807, Ibidi, Fitchburg, WI, USA) for 24 h. Subsequently, the cells were washed with PBS and incubated with the fluorescent fatty acid analog BODIPY FL (#D3822, Thermo Fisher) and Hoescht 33342 (#62249, Thermo Fisher) for 30 min at 37°C. Afterwards, cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min. Images were captured using a Nikon A1 Confocal Microscope.

2.10. CRC Tissue microarray

The immunoreactivity score of HMGCS2 expression was assessed in CRC tumors and adjacent normal tissue collected from patients diagnosed with stage I-IV who had surgery at UK Chandler Medical Center (Tumor Microarray ID BH 15991A, n = 55 normal, n = 16 stage I, n = 9 stage II, n = 10 stage III and n = 5 stage IV). The score was conducted blindly by a GI pathologist. Immunoreactivity score was calculated by multiplying values for staining intensity (0 = no staining; 1 = weak; 2 = moderate and 3 = strong staining) with values for the percentage of positive cells (0 = no positive cells; 1 = 0–10%; 2 = 11–50%; 3 = 51–100% positive cells).

2.11. Statistical analysis

The Biostatistics and Bioinformatics Shared Resource Facility assisted with TCGA and tumor microarray data analysis and validation of RNAseq analysis. Statistical significance between the control and PFOS treated groups was analyzed using t-test. Comparisons between the NTC and HMGCS2sh groups were performed using one-way ANOVA and Dunnett’s post-test. Differences in densitometric analysis between normal and CRC tissues were assessed using a paired t-test. Comparations of immunoreactive scores were conducted using a linear mixed model. Statistical analyses were performed using GraphPad Prism (version 9.4.1).

3. Results

3.1. PFOS exposure downregulates HMGCS2 expression in intestinal tissues of mice, mouse and human organoid models, and normal human intestinal cells

Mice were administered diets incorporating different fibers: cellulose (control, insoluble), inulin (soluble) or pectin (soluble) and subjected to PFOS exposure in drinking water for a period of 7 weeks (Deng et al., 2022). (Figure 1A). The RNAseq analysis of intestinal epithelium revealed that expression of HMGCS2, a rate-limiting ketogenic enzyme in the synthesis of ketone bodies, is significantly downregulated in mice exposed to PFOS as compared to a control group (Figure 1B). In contrast, expression of vascular endothelial growth factor receptor (VEGFR) and alcohol dehydrogenase 6A (ADH6A) are significantly upregulated (Figure 1B). qRT-PCR analysis further confirmed that HMGCS2 is downregulated and VEGFR is upregulated in mouse intestinal tissues due to PFOS exposure (Figure 1C). We excluded ADH6A from confirmational analysis since this gene is mouse specific. The further analysis, as demonstrated by Venn diagram showing numbers of overlapping and non-overlapping genes differentially expressed among three different diet groups, revealed that HMGCS2 is the only gene displaying differential expression due to PFOS exposure across all three groups (Figure 1D). Western blot analysis on intestinal tissues confirmed that HMGCS2 is downregulated at the protein level in PFOS exposed samples across all diet groups (Figure 1E–F). To further support that reduction of HMGCS2 expression is due to PFOS exposure, wild type mouse normal intestinal organoids were exposed to 1 μg/mL of PFOS in complete culture medium, and western blot analysis was performed after a 15-day exposure period. The results on organoids established from two different mice demonstrate that PFOS exposure significantly reduces expression of HMGCS2 (Figure 1G). Moreover, PFOS exposure also decreases the level of HMGCS2 in human normal intestinal organoids treated with PFOS for 30 days (Figure 1H) and in normal human colon primary (NHCP) cell line treated with PFOS for 6 months (Figure I). The morphology of the cells shows no apparent differences between the control and PFOS groups in mouse organoids (Figure 1G), in human organoids (Figure 1H) and in NHCP cells (Figure 1I), suggesting the absence of toxicity associated with PFOS treatment. Together, these results demonstrated that PFOS exposure decreases expression of HMGCS2 in intestinal cells.

3.2. PFOS exposure induces changes in gene expression and upregulates the levels of proteins associated with carcinogenesis in intestinal tissues of mice

The analysis of RNAseq data resulted in identification of three genes which are significantly changed in the control (cellulose) group due to PFOS exposure; however, PFOS exposure led to significant changes in multiple genes in the inulin and pectin diet groups (Figure 1D). Therefore, to identify which pathways are dysregulated by PFOS, we performed KEGG analysis on RNAseq data from the inulin and pectin groups. We have identified signal transduction, cancer, lipid metabolism and immune pathways as the top pathways significantly altered by PFOS exposure in both the pectin and inulin groups (Figure 2A). To assess the impact of PFOS exposure independent of dietary influences, we examined key proteins within the signaling pathways identified in the KEGG analysis in the control and PFOS-treated group on cellulose diet. Interestingly, we found that PFOS upregulates fatty acid synthase (FASN), a key enzyme in de novo lipid synthesis and a well-established oncogenic protein associated with CRC (Zaytseva et al., 2012; Zaytseva, 2021; Drury et al., 2022) (Figure 2B–C). Moreover, we found that PFOS exposure induces the expression of other pro-oncogenic proteins linked to CRC, including β-catenin, c-Myc, p-mTOR, as well as proteins involved in cellular proliferation, such as cyclin D1 and AKT (Nguyen et al., 2020; Huang and Yang, 2022). In contrast, the levels of pERK seem to be lower in the PFOS group (Figure 2B–C).

Figure 2. PFOS exposure upregulates expression of proteins associated with intestinal carcinogenesis.

Figure 2.

Open in a new tab

(A) KEGG pathway classification in mice exposed to PFOS and kept on inulin or pectin diets. (B-C) Western blot and densitometric quantification analysis of mouse intestinal tissues. Control (cellulose) versus PFOS (cellulose) groups.

3.3. HMGCS2 knockdown in a normal primary colon cell line results in enhanced proliferation and lipid accumulation

To investigate the functional role of HMGCS2 in NHCP cells we used shRNA-mediated knockdown of HMGCS2. As shown in Figure 3A, knockdown of HMGCS2 leads to cells with significantly increased cell viability. Consistent with our finding in intestinal tissues of mice demonstrating that PFOS-mediated decrease in HMGCS2 expression is associated with an increase in Cyclin D, survivin, AKT and mTOR upregulation and activation, the proteins well known to drive cellular proliferation (Figure 2B) and knockdown of HMGCS2 also upregulate Cyclin D and survivin expression and activity of AKT and mTOR (Figure 3B–C). In addition, we observed activation of pERK1/2 in HMGCS2 knockdown cells. While FASN expression remained unchanged in these cells, we observed upregulation of CD36 expression, a fatty acid transporter (Figure 3B–C), and an increase in lipid accumulation, as shown by BODIPY staining (Figure 3D).

Figure 3. HMGCS2 knockdown increases cell proliferation and lipid accumulation.

Figure 3.

Open in a new tab

(A) Cell viability of NTC and HMGCS2 knockdown normal human colon primary cells measured by Presto Blue assay. Data are presented as mean ± SD. The one-way ANOVA followed by Dunnett’s post-test was used to analyze statistical significance (**p≤0.01, ***p≤0.001). (B-C) Western blot and densitometric quantification analysis for HMGCS2 and proliferation / carcinogenic markers. At least 3 replicates were performed for each protein (D) Cells were stained with BODIPY FL (green) and Hoescht (blue) before being processed for confocal analysis (Scale bar: 25 μm).

3.4. Colorectal cancer is associated with downregulation of HMGCS2 expression

To assess HMGCS2 expression in human specimens and the role of this protein in disease, we utilized public databases. Analysis of The Cancer Genome Atlas data shows that HMGCS2 mRNA expression is lower in CRC tissues compared to normal mucosa (Figure 4A). Moreover, according to the Protein Atlas data, the decrease of expression of HMGCS2 is notably associated with significantly lower five-year survival rates in patients with colon cancer (Figure 4B). To validate these results, we assessed the expression of HMGCS2 in matched normal mucosa and CRC tissues in 15 randomly selected cases and showed that HMGCS2 is downregulated in CRC compared to adjacent normal tissues (Figures 4C–D). Additionally, the expression of HMGCS2 protein was analyzed in a larger number of CRC patients and adjacent normal mucose through tumor microarray staining. We observed a decrease in HMGCS2 levels corresponding to the tumor stage, with the stages III and IV exhibiting lower levels of HMGCS2 expression as compared to normal mucosa (Figure 4E–F). Interestingly, in some cases we observed a partial loss of HMGCS2 expression in different parts of the tumor as pointed by the arrows in tumor tissues from case #1582 (stage IV) (Figure 4F).

Figure 4. HMGCS2 mRNA and protein levels are downregulated in CRC.

Figure 4.

Open in a new tab

(A) HMGCS2 mRNA expression in colorectal cancer patient samples from the TCGA dataset, comprising 22 normal tissues and 215 tumor samples. (B) 5-year survival analysis of patients with colorectal cancer based on HMGCS2 protein levels (source proteinatlas.org). (C) Western blot analysis and (D) densitometric quantification of HMGCS2 protein levels in 15 randomly selected CRC cases. Matched normal mucosa (N) and tumor tissues (T). β-actin was used as an endogenous loading control. (E) Distribuition of HMGCS2 immunoreactivity score was analyzed in tumor tissues from patients diagnosed with CRC stage I-IV. The linear mixed model was used to anlyse statistical signifcance. (F) Representative images of HMGCS2 expression in matched normal and tumor tissues.

4. Discussion

PFOS is one of the most commonly detected chemicals among the PFAS group of contaminants known as “forever chemicals” (Wee and Aris, 2023). Although increasing evidence has demonstrated that PFOS exposure may be harmful to human health, there is currently limited available information regarding its impact on the GI tract. In this current study, for the first time, we report that exposure to PFOS in drinking water leads to downregulation of HMGCS2 expression in the intestinal tissues of wild-type mice. Notably, the incorporation of soluble fibers, inulin and pectin, into the diet has no effect on the PFOS-induced downregulation of HMGCS2 protein levels. Additionally, we show that PFOS exposure in drinking water leads to upregulation of proteins associated with colorectal carcinogenesis in the intestinal tissues of these mice. These findings provide a novel insight to how PFOS exposure is linked to adverse health outcomes in the context of GI pathology. Furthermore, this study demonstrates that HMGCS2 plays an important role in regulating cellular proliferation and lipid homeostasis in human normal colon cells in vitro. Finally, we show that HMGCS2 protein levels are downregulated in human CRC compared to normal adjacent tissues. These data collectively suggest an important role of PFOS-mediated downregulation of HMGCS2 expression in pathological alterations of the intestinal epithelium. A summary of our findings is shown in Figure 5.

Figure 5. The potential mechanisms of how PFOS exposure by drinking water alters intestinal homeostasis.

Figure 5.

Open in a new tab

PFOS exposure in drinking water leads to downregulation of HMGCS2 and upregulation of FASN, β-catenin, c-MYC, mTOR, cyclin-D1 and AKT protein levels. Furthermore, loss of HMGCS2 results in increased cell proliferation and upregulation of CD36, mTOR, cyclin-D1, AKT, ERK and survivin, along with an increase in lipid accumulation. Further studies are warranted to determine if PFOS exposure can increase the risk of disease-associated GI pathology.

The mitochondrial enzyme HMGCS2 is the rate-limiting step in the production of ketone bodies (KBs) (Hwang et al., 2022). Among KBs, β-hydroxybutyrate (BHB) predominates as the most abundant within the circulating KB pool (Dabek et al., 2020). HMGCS2 is primarily expressed in the liver but it is also found in extrahepatic tissues, including colon (Camarero et al., 2006; Nishitani et al., 2022), where it exerts a significant role in maintaining intestinal homeostasis. For instance, HMGCS2 sustains intestinal stem cell numbers and lineage-balanced differentiation. Loss of HMGCS2 compromises intestinal stemness and regeneration after injury, a condition partially restored by the exogenous administration of BHB (Cheng et al., 2019). Another study showed that TNFα inhibits BHB production by reducing HMGCS2 expression in intestinal cells. Overexpression of HMGCS2 attenuates TNFα-induced apoptosis and inflammatory responses (Kim et al., 2021). Consistent with these findings, HMGCS2 downregulation is observed in intestinal pathological conditions such as IBD (Kim et al., 2021; Martin-Adrados et al., 2023) and CRC (Zou et al., 2019). Moreover, KBs exhibit anti-inflammatory, antioxidant and anti-cancer properties in CRC (Rojas-Morales et al., 2020; Dmitrieva-Posocco et al., 2022; Hwang et al., 2022). On the other hand, PFOS has been linked to the induction of chronic inflammation, immunosuppression, oxidative stress and cellular proliferation (Sarah Elmore and Karin Ricker, 2021; Zhang et al., 2023). Previous reports suggest PFOS involvement in ulcerative colitis and IBD conditions (Liang et al., 2021; National Academies of Sciences, 2022), which are recognized risk factors for CRC development (Lucafo et al., 2021). These findings suggest a possible mechanistic link between PFOS-induced downregulation of HMGCS2 and GI pathology.

The biological effects induced by PFOS have been suggested to be mediated through activation of nuclear receptors, including peroxisome proliferator activated receptor alpha (PPARα), constitutive androstane receptor (CAR) and pregnane X receptor (PXR) (Sarah Elmore and Karin Ricker, 2021). Notably, PPARα positively regulates HMGCS2 gene expression (Grabacka et al., 2016). Interestingly, activation of PXR or CAR has been observed to suppress HMGCS2 expression (Nakamura et al., 2007; Shizu et al., 2020; Shizu et al., 2021). The proposed mechanism suggests that both PXR and CAR inhibit PPARα-dependent gene expression by competing for recruitment of the transcription coactivator PGC1α (Shizu et al., 2020; Shizu et al., 2021). In addition, several studies have provided evidence indicating that PFOS can increase the activity of estrogen receptors (Sarah Elmore and Karin Ricker, 2021), which potentiate the induction of c-MYC (Mukherjee and Conrad, 2005; Rehman et al., 2022). Also, a study found that HMGCS2 is a direct target of c-MYC, leading to the repression of HMGCS2 transcriptional activity in intestinal cells (Camarero et al., 2006). In line with these observations, our study shows upregulation of c-MYC protein levels in the intestinal tissues of PFOS-exposed mice. Collectively, these findings contribute to the understanding of potential mechanisms through which PFOS may mediate the downregulation of HMGCS2.

Our results demonstrate that the dietary supplement containing the soluble fibers inulin and pectin has no effect on the PFOS-induced downregulation of HMGCS2. Short-chain fatty acids constitute the primary end products of dietary fiber fermentation (den Besten et al., 2013). The major short-chain fatty acids formed by the gut bacteria are acetate, propionate and butyrate (Nogal et al., 2021). In contrast to pectin, inulin increases the relative production of propionate and butyrate over the production of acetate (den Besten et al., 2013). Butyrate, a key energy source for colonocytes, possesses anti-inflammatory properties and enhances the epithelial barrier in intestinal cells, thereby maintaining the epithelial homeostasis (Parada Venegas et al., 2019; Salvi and Cowles, 2021). A depletion of butyrate-producing bacteria and lower intestinal concentrations of butyrate have been observed in IBD patients (Marchesi et al., 2007; Huda-Faujan et al., 2010; Machiels et al., 2014). Butyrate undergoes β-oxidation to form Acetyl-CoA, subsequently used in the citric acid cycle or serving as the substrate for HMGCS2 in the production of KBs (Helenius et al., 2015). As demonstrated by Xu et al., butyrate can regulate the activity of HMGCS2 and the KB levels. Administration of sodium butyrate effectively increases blood BHB levels in mice. Further analysis reveals that sodium butyrate activates HMGCS2, without altering protein levels, through SIRT-5-mediated desuccinylation for KB production (Xu et al., 2023). Additionally, a recent study showed that BHB reduces the proliferation of colonic crypt cells and potently suppresses intestinal tumor growth (Dmitrieva-Posocco et al., 2022). In light of these findings, the downregulation of HMGCS2 induced by PFOS may decrease the potential beneficial effects of diets supplemented with inulin and pectin.

Our study demonstrates that PFOS exposure alters cancer, lipid metabolism and immune system signaling pathways in wild-type mouse intestinal tissues. Moreover, PFOS upregulates protein levels of β-catenin, c-MYC, mTOR and FASN, suggesting the involvement of PFOS in the activation of proteins associated with GI carcinogenesis. In CRC, the frequent hyperactivation of the Wnt signaling pathway is a pivotal initiating and driving event (Schatoff et al., 2017). The central effector in the Wnt canonical pathway is β-catenin. Upon activation, β-catenin is translocated to the nucleus, where it activates Wnt target genes, including c-MYC and CCND1 (gene encoding cyclin D1) (Schatoff et al., 2017). Activation of these genes contributes to the dysregulation of cell proliferation and cell cycle control, two crucial aspects implicated in cancer development and progression (Wang et al., 2008; Montalto and De Amicis, 2020). In addition, activation of Wnt/β-catenin signaling decreased protein and mRNA levels of HMGCS2 and BHB production in CRC cells and wild-type intestinal organoids (Kim et al., 2019).

mTOR is also a well-known pathway contributing to the initiation and progression of CRC (Alqurashi et al., 2013). Multiple signals activate the mTOR pathway, such as the classical upstream activator AKT (Lagasse, 2013; Wang and Zhang, 2014). The oncogenic activation of PI3K/AKT/mTOR signaling has been suggested to stimulate de novo lipogenesis via upregulation of SREBP1, a master transcriptional regulator of lipogenic enzymes such as FASN (Porstmann et al., 2008; Caron et al., 2015; Yi et al., 2018). Furthermore, Wang et al. reported that PI3K/AKT/mTOR signaling cooperates with HMGCS2 in the regulation of intestinal cell differentiation. The authors demonstrated that inhibition of mTOR increases HMGCS2 protein levels and BHB production, contributing to intestinal cell differentiation. Conversely, an increase in ketogenesis inhibits mTOR signaling and induces differentiation (Wang et al., 2017).

Previous studies have reported that PFOS can induce metabolic perturbation and disrupt lipid metabolism in liver of mice (Deng et al., 2022). Lipid metabolism has a profound impact on intestinal inflammation as well as CRC initiation and progression (Zaytseva, 2021; Andersen, 2022). FASN, a key enzyme involved in the de novo biogenesis of fatty acids, has been linked to the development of IBD (Matsuo et al., 2014) and is a well-established hallmark of cancer (Vanauberg et al., 2023). Moreover, upregulation of FASN is associated with CRC cell proliferation, metastatic potential and poor survival of CRC patients (Zaytseva et al., 2012; Lu et al., 2019). In a previous study, it was reported that loss of HMGCS2 enhances lipogenesis and FASN expression in hepatocellular carcinoma (Wang et al., 2020). In contrast, our data show that knockdown of HMGCS2 in normal intestinal cells does not alter FASN protein levels. Instead, HMGCS2 knockdown promotes the overexpression of CD36 and accumulation of lipid droplets. Fatty acids are obtained either through endogenous synthesis or by exogenous uptake via cell surface receptors, such as CD36 (Zaytseva, 2021; Guerrero-Rodriguez et al., 2022). Fatty acids are incorporated into triglycerides, and excessive triglycerides, along with other lipid types like cholesterol, are stored in lipid droplets (Zaytseva et al., 2015; Zaytseva, 2021). Notably, accumulation of lipid droplets has been reported in CRC compared to normal colonic tissues (Zaytseva et al., 2015; Salita et al., 2022). Taken together, our results suggest that PFOS-induced upregulation of FASN is not mediated by HMGCS2. However, HMGCS2 seems to play a significant role in lipid metabolism, partially through the upregulation of CD36. Further studies are needed to better understand the mechanism of PFOS-induced lipid alterations in normal intestinal and cancer cells.

In the present study, we demonstrated that PFOS exposure and subsequent HMGCS2 downregulation induce upregulation of proteins associated with cellular proliferation, such as cyclin D and AKT in intestinal tissues of mice. Interestingly, we observed a similar upregulation of these proteins in human normal colon cells with HMGCS2 knockdown, and a decrease in HMGCS2 expression was accompanied by an increase in cell viability. These findings are consistent with other studies demonstrating that loss of HMGCS2 protein promotes proliferation and triggers upregulation of cyclin D expression (Wan et al., 2019; Wang et al., 2019; Mao et al., 2023). Together, these data suggest that PFOS-induced downregulation of HMGCS2 may contribute to the regulation of cellular proliferation.

Finally, our results are in accordance with previous studies that demonstrated lower HMGCS2 protein levels in CRC compared to normal mucosa and lower expression of HMGCS2 is associated with lower survival rate of CRC patients (Zou et al., 2019; Wei et al., 2022). We also demonstrated a trend of downregulation in HMGCS2 with the progression of tumor stages, particularly evident in stages III and IV where significantly lower levels of HMGCS2 expression were observed. In published studies, downregulation of HMGCS2 has been linked to advanced stages of hepatocellular carcinoma and poorly differentiated CRC, suggesting that HMGCS2 expression decreases with progression of cancer to more advanced stages and is associated with aggressiveness of disease (Camarero et al., 2006; Wang et al., 2019). Our data suggest that PFOS-mediated downregulation of HMGCS2 and upregulation of oncogenic proteins may be a crucial step in disrupting intestinal homeostasis and contributing to disease initiation.

In summary, this study is the first to demonstrate that PFOS exposure downregulates HMGCS2 levels in intestinal cells. Although incorporating inulin or pectin supplemented diets had no impact on the PFOS-induced downregulation of HMGCS2, diet-derived soluble fibers may still be protective against pathologies of carcinogenesis either directly or indirectly through mechanisms other than down-regulation of HMGCS2 (Song et al., 2015; Sivaprakasam et al., 2016; Vernia et al., 2021). Furthermore, PFOS induces the upregulation of oncogenic proteins associated with CRC and increases the expression of proliferation-associated proteins, suggesting the potential role of PFOS in promoting GI pathological changes and increasing risk of CRC. Overall, these findings provide novel insights into the mechanisms by which PFOS can contribute to the pathogenesis of the GI tract.

Highlights.

  • PFOS exposure downregulates HMGCS2 protein expression in normal intestinal tissues

  • Inulin and pectin diets do not alleviate PFOS-induced HMGCS2 downregulation

  • PFOS exposure upregulates pathways and proteins associated with colon carcinogenesis

  • Loss of HMGCS2 increases proliferation and lipid accumulation in normal colon cells

  • Downregulation of HMGCS2 is observed in CRC and is associated with lower survival

Acknowledgements:

University of Kentucky Markey Cancer Center’s Biospecimen Procurement and Translational Pathology Shared Resource Facility (SRF) assisted with surgery specimen collection from CRC patients and IHC staining. The Biostatistics and Bioinformatics SRF provided support on RNA-seq data storage, quality control, analysis, and depositing in NCBI’s Gene Expression Omnibus and on the TCGA data analysis. SRFs are supported by National Cancer Institute grant P30 CA177558. Special thank you to Markey Cancer Center’s Research Communications Office and Donna Gilbreath for assisting with preparation of this manuscript.

Funding:

This work is supported by National Institute of Environmental Health Sciences grant P42 ES007380 (University of Kentucky Superfund Research Center, Project 1-YZ), National Cancer Institute grant R01 CA249734 (YZ), and National Natural Science Foundation of China 82373942 (PD). The publishing fee will be paid by the National Institute of Environmental Health Sciences grant P42 ES007380.

Abbreviations

ADH6A

alcohol dehydrogenase 6A

BHB

β-hydroxybutyrate

CAR

constitutive androstane receptor

CRC

colorectal cancer

FASN

fatty acid synthase

GI

gastrointestinal

HMGCS2

3-hydroxy-3-methylglutaryl-CoA synthase 2

IBD

inflammatory bowel disease

KBs

ketone bodies

NHCP

normal human colon primary

PFAS

perfluoroalkyl and polyfluoroalkyl substances

PFOS

perfluorooctanesulfonic acid

PPARα

peroxisome proliferator activated receptor alpha

PXR

pregnane X receptor

RT-PCR

real-time reverse transcription–polymerase chain reaction

VEGFR

vascular endothelial growth factor receptor

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflicts of Interest: The authors declare no conflicts of interest.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

  1. Adigun AO, Adebile TM, Okoye C, Ogundipe TI, Ajekigbe OR, Mbaezue RN, Okobi OE, 2023. Causes and Prevention of Early-Onset Colorectal Cancer. Cureus 15, e45095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alqurashi N, Gopalan V, Smith RA, Lam AK, 2013. Clinical impacts of mammalian target of rapamycin expression in human colorectal cancers. Hum Pathol 44, 2089–2096. [DOI] [PubMed] [Google Scholar]
  3. Andersen CJ, 2022. Lipid Metabolism in Inflammation and Immune Function. Nutrients 14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Camarero N, Mascaro C, Mayordomo C, Vilardell F, Haro D, Marrero PF, 2006. Ketogenic HMGCS2 Is a c-Myc target gene expressed in differentiated cells of human colonic epithelium and down-regulated in colon cancer. Mol Cancer Res 4, 645–653. [DOI] [PubMed] [Google Scholar]
  5. Caron A, Richard D, Laplante M, 2015. The Roles of mTOR Complexes in Lipid Metabolism. Annu Rev Nutr 35, 321–348. [DOI] [PubMed] [Google Scholar]
  6. Centers for Disease Control and Prevention, 2022. Per- and Polyfluorinated Substances (PFAS) Factsheet. National Biomonitoring Program, Washington, DC. [Google Scholar]
  7. Chen Y, Chen Y, Shi C, Huang Z, Zhang Y, Li S, Li Y, Ye J, Yu C, Li Z, Zhang X, Wang J, Yang H, Fang L, Chen Q, 2018. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. Gigascience 7, 1–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen Z, Yang T, Walker DI, Thomas DC, Qiu C, Chatzi L, Alderete TL, Kim JS, Conti DV, Breton CV, Liang D, Hauser ER, Jones DP, Gilliland FD, 2020. Dysregulated lipid and fatty acid metabolism link perfluoroalkyl substances exposure and impaired glucose metabolism in young adults. Environ Int 145, 106091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng CW, Biton M, Haber AL, Gunduz N, Eng G, Gaynor LT, Tripathi S, Calibasi-Kocal G, Rickelt S, Butty VL, Moreno-Serrano M, Iqbal AM, Bauer-Rowe KE, Imada S, Ulutas MS, Mylonas C, Whary MT, Levine SS, Basbinar Y, Hynes RO, Mino-Kenudson M, Deshpande V, Boyer LA, Fox JG, Terranova C, Rai K, Piwnica-Worms H, Mihaylova MM, Regev A, Yilmaz OH, 2019. Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet. Cell 178, 1115–1131 e1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Consumer Product Safety Commission, 2023. Characterizing PFAS Chemistries, Sources, Uses, and Regulatory Trends in U.S. and International Markets. Rockville, MD, p. 168. [Google Scholar]
  11. Cousins IT, DeWitt JC, Gluge J, Goldenman G, Herzke D, Lohmann R, Ng CA, Scheringer M, Wang Z, 2020. The high persistence of PFAS is sufficient for their management as a chemical class. Environ Sci Process Impacts 22, 2307–2312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dabek A, Wojtala M, Pirola L, Balcerczyk A, 2020. Modulation of Cellular Biochemistry, Epigenetics and Metabolomics by Ketone Bodies. Implications of the Ketogenic Diet in the Physiology of the Organism and Pathological States. Nutrients 12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. den Besten G, van Eunen K, Groen AK, Venema K, Reijngoud DJ, Bakker BM, 2013. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. J Lipid Res 54, 2325–2340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Deng P, Durham J, Liu J, Zhang X, Wang C, Li D, Gwag T, Ma M, Hennig B, 2022. Metabolomic, Lipidomic, Transcriptomic, and Metagenomic Analyses in Mice Exposed to PFOS and Fed Soluble and Insoluble Dietary Fibers. Environ Health Perspect 130, 117003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dmitrieva-Posocco O, Wong AC, Lundgren P, Golos AM, Descamps HC, Dohnalova L, Cramer Z, Tian Y, Yueh B, Eskiocak O, Egervari G, Lan Y, Liu J, Fan J, Kim J, Madhu B, Schneider KM, Khoziainova S, Andreeva N, Wang Q, Li N, Furth EE, Bailis W, Kelsen JR, Hamilton KE, Kaestner KH, Berger SL, Epstein JA, Jain R, Li M, Beyaz S, Lengner CJ, Katona BW, Grivennikov SI, Thaiss CA, Levy M, 2022. beta-Hydroxybutyrate suppresses colorectal cancer. Nature 605, 160–165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Drury J, Young LEA, Scott TL, Kelson CO, He D, Liu J, Wu Y, Wang C, Weiss HL, Fan T, Gentry MS, Sun R, Zaytseva YY, 2022. Tissue-Specific Downregulation of Fatty Acid Synthase Suppresses Intestinal Adenoma Formation via Coordinated Reprograming of Transcriptome and Metabolism in the Mouse Model of Apc-Driven Colorectal Cancer. Int J Mol Sci 23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Durham J, Tessmann JW, Deng P, Hennig B, Zaytseva YY, 2023. The role of perfluorooctane sulfonic acid (PFOS) exposure in inflammation of intestinal tissues and intestinal carcinogenesis. Front Toxicol 5, 1244457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Efsa Panel on Contaminants in the Food Chain, Knutsen HK, Alexander J, Barregard L, Bignami M, Bruschweiler B, Ceccatelli S, Cottrill B, Dinovi M, Edler L, Grasl-Kraupp B, Hogstrand C, Hoogenboom LR, Nebbia CS, Oswald IP, Petersen A, Rose M, Roudot AC, Vleminckx C, Vollmer G, Wallace H, Bodin L, Cravedi JP, Halldorsson TI, Haug LS, Johansson N, van Loveren H, Gergelova P, Mackay K, Levorato S, van Manen M, Schwerdtle T, 2018. Risk to human health related to the presence of perfluorooctane sulfonic acid and perfluorooctanoic acid in food. EFSA J 16, e05194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Evich MG, Davis MJB, McCord JP, Acrey B, Awkerman JA, Knappe DRU, Lindstrom AB, Speth TF, Tebes-Stevens C, Strynar MJ, Wang Z, Weber EJ, Henderson WM, Washington JW, 2022. Per- and polyfluoroalkyl substances in the environment. Science 375, eabg9065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fenton SE, Ducatman A, Boobis A, DeWitt JC, Lau C, Ng C, Smith JS, Roberts SM, 2021. Per- and Polyfluoroalkyl Substance Toxicity and Human Health Review: Current State of Knowledge and Strategies for Informing Future Research. Environ Toxicol Chem 40, 606–630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gluge J, Scheringer M, Cousins IT, DeWitt JC, Goldenman G, Herzke D, Lohmann R, Ng CA, Trier X, Wang Z, 2020. An overview of the uses of per- and polyfluoroalkyl substances (PFAS). Environ Sci Process Impacts 22, 2345–2373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grabacka M, Pierzchalska M, Dean M, Reiss K, 2016. Regulation of Ketone Body Metabolism and the Role of PPARalpha. Int J Mol Sci 17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Guerrero-Rodriguez SL, Mata-Cruz C, Perez-Tapia SM, Velasco-Velazquez MA, 2022. Role of CD36 in cancer progression, stemness, and targeting. Front Cell Dev Biol 10, 1079076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Helenius TO, Misiorek JO, Nystrom JH, Fortelius LE, Habtezion A, Liao J, Asghar MN, Zhang H, Azhar S, Omary MB, Toivola DM, 2015. Keratin 8 absence downregulates colonocyte HMGCS2 and modulates colonic ketogenesis and energy metabolism. Mol Biol Cell 26, 2298–2310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Huang Z, Yang M, 2022. Molecular Network of Colorectal Cancer and Current Therapeutic Options. Front Oncol 12, 852927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Huda-Faujan N, Abdulamir AS, Fatimah AB, Anas OM, Shuhaimi M, Yazid AM, Loong YY, 2010. The impact of the level of the intestinal short chain Fatty acids in inflammatory bowel disease patients versus healthy subjects. Open Biochem J 4, 53–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hwang CY, Choe W, Yoon KS, Ha J, Kim SS, Yeo EJ, Kang I, 2022. Molecular Mechanisms for Ketone Body Metabolism, Signaling Functions, and Therapeutic Potential in Cancer. Nutrients 14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kim D, Langmead B, Salzberg SL, 2015. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12, 357–360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kim JT, Li C, Weiss HL, Zhou Y, Liu C, Wang Q, Evers BM, 2019. Regulation of Ketogenic Enzyme HMGCS2 by Wnt/beta-catenin/PPARgamma Pathway in Intestinal Cells. Cells 8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kim JT, Napier DL, Kim J, Li C, Lee EY, Weiss HL, Wang Q, Evers BM, 2021. Ketogenesis alleviates TNFalpha-induced apoptosis and inflammatory responses in intestinal cells. Free Radic Biol Med 172, 90–100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lagasse M.G.F.a.E., 2013. mTOR pathway in colorectal cancer: an update. Oncotarget 5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Langmead B, Salzberg SL, 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357–359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Li B, Dewey CN, 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Li C, Zhou Y, Wei R, Napier DL, Sengoku T, Alstott MC, Liu J, Wang C, Zaytseva YY, Weiss HL, Wang Q, Evers BM, 2023. Glycolytic Regulation of Intestinal Stem Cell Self-Renewal and Differentiation. Cell Mol Gastroenterol Hepatol 15, 931–947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Li Y, Fletcher T, Mucs D, Scott K, Lindh CH, Tallving P, Jakobsson K, 2018. Half-lives of PFOS, PFHxS and PFOA after end of exposure to contaminated drinking water. Occup Environ Med 75, 46–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Liang H, Yang M, Zeng C, Wu W, Zhao L, Wang Y, 2021. Perfluorooctane sulfonate exerts inflammatory bowel disease-like intestinal injury in rats. PeerJ 9, e10644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Love MI, Huber W, Anders S, 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lu T, Sun L, Wang Z, Zhang Y, He Z, Xu C, 2019. Fatty acid synthase enhances colorectal cancer cell proliferation and metastasis via regulating AMPK/mTOR pathway. Onco Targets Ther 12, 3339–3347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lucafo M, Curci D, Franzin M, Decorti G, Stocco G, 2021. Inflammatory Bowel Disease and Risk of Colorectal Cancer: An Overview From Pathophysiology to Pharmacological Prevention. Front Pharmacol 12, 772101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Machiels K, Joossens M, Sabino J, De Preter V, Arijs I, Eeckhaut V, Ballet V, Claes K, Van Immerseel F, Verbeke K, Ferrante M, Verhaegen J, Rutgeerts P, Vermeire S, 2014. A decrease of the butyrate-producing species Roseburia hominis and Faecalibacterium prausnitzii defines dysbiosis in patients with ulcerative colitis. Gut 63, 1275–1283. [DOI] [PubMed] [Google Scholar]
  41. Mao H, Wang R, Shao F, Zhao M, Tian D, Xia H, Zhao Y, 2023. HMGCS2 serves as a potential biomarker for inhibition of renal clear cell carcinoma growth. Sci Rep 13, 14629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Marchesi JR, Holmes E, Khan F, Kochhar S, Scanlan P, Shanahan F, Wilson ID, Wang Y, 2007. Rapid and noninvasive metabonomic characterization of inflammatory bowel disease. J Proteome Res 6, 546–551. [DOI] [PubMed] [Google Scholar]
  43. Martin-Adrados B, Wculek SK, Fernandez-Bravo S, Torres-Ruiz R, Valle-Noguera A, Gomez-Sanchez MJ, Hernandez-Walias JC, Ferreira FM, Corraliza AM, Sancho D, Esteban V, Rodriguez-Perales S, Cruz-Adalia A, Nakaya HI, Salas A, Bernardo D, Campos-Martin Y, Martinez-Zamorano E, Munoz-Lopez D, Gomez Del Moral M, Cubero FJ, Blumberg RS, Martinez-Naves E, 2023. Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress. Front Immunol 14, 1185517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Matsuo S, Yang WL, Aziz M, Kameoka S, Wang P, 2014. Fatty acid synthase inhibitor C75 ameliorates experimental colitis. Mol Med 20, 1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Montalto FI, De Amicis F, 2020. Cyclin D1 in Cancer: A Molecular Connection for Cell Cycle Control, Adhesion and Invasion in Tumor and Stroma. Cells 9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Mukherjee S, Conrad SE, 2005. c-Myc suppresses p21WAF1/CIP1 expression during estrogen signaling and antiestrogen resistance in human breast cancer cells. J Biol Chem 280, 17617–17625. [DOI] [PubMed] [Google Scholar]
  47. Nakamura K, Moore R, Negishi M, Sueyoshi T, 2007. Nuclear pregnane X receptor cross-talk with FoxA2 to mediate drug-induced regulation of lipid metabolism in fasting mouse liver. J Biol Chem 282, 9768–9776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. National Academies of Sciences, E., and Medicine,, 2022. Guidance on PFAS Exposure, Testing, and Clinical Follow-Up. The National Academies Press, Washington (DC). [PubMed] [Google Scholar]
  49. Nguyen LH, Goel A, Chung DC, 2020. Pathways of Colorectal Carcinogenesis. Gastroenterology 158, 291–302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Nishitani S, Fukuhara A, Tomita I, Kume S, Shin J, Okuno Y, Otsuki M, Maegawa H, Shimomura I, 2022. Ketone body 3-hydroxybutyrate enhances adipocyte function. Sci Rep 12, 10080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Nogal A, Valdes AM, Menni C, 2021. The role of short-chain fatty acids in the interplay between gut microbiota and diet in cardio-metabolic health. Gut Microbes 13, 1–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Office of Environmental Health Hazard Assessment, 2010. Perfluorooctane Sulfonic Acid (PFOS) and Its Salts and Transformation and Degradation Precursors. Sacramento, CA. [Google Scholar]
  53. Olsen GW, Burris JM, Ehresman DJ, Froehlich JW, Seacat AM, Butenhoff JL, Zobel LR, 2007. Half-life of serum elimination of perfluorooctanesulfonate,perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers. Environ Health Perspect 115, 1298–1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Parada Venegas D, De la Fuente MK, Landskron G, Gonzalez MJ, Quera R, Dijkstra G, Harmsen HJM, Faber KN, Hermoso MA, 2019. Short Chain Fatty Acids (SCFAs)-Mediated Gut Epithelial and Immune Regulation and Its Relevance for Inflammatory Bowel Diseases. Front Immunol 10, 277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Porstmann T, Santos CR, Griffiths B, Cully M, Wu M, Leevers S, Griffiths JR, Chung YL, Schulze A, 2008. SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth. Cell Metab 8, 224–236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Rehman N, Khan S, Manzoor S, Abubakar M, Sami R, Alharthy SA, Baty RS, Jafri I, Alsubhi NH, Qari SH, Shahid R, 2022. Estrogen Induces c-myc Transcription by Binding to Upstream ERE Element in Promoter. Applied Sciences 12. [Google Scholar]
  57. Rojas-Morales P, Pedraza-Chaverri J, Tapia E, 2020. Ketone bodies, stress response, and redox homeostasis. Redox Biol 29, 101395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Salita T, Rustam YH, Mouradov D, Sieber OM, Reid GE, 2022. Reprogrammed Lipid Metabolism and the Lipid-Associated Hallmarks of Colorectal Cancer. Cancers (Basel) 14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Salvi PS, Cowles RA, 2021. Butyrate and the Intestinal Epithelium: Modulation of Proliferation and Inflammation in Homeostasis and Disease. Cells 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Elmore Sarah, M. M, Guha Neela, Osborne Gwendolyn, Hsieh Jennifer C.Y., , Ricker Karin, L. K, Schmitz Rose, Marder M. Elizabeth, Tsai Feng C., 2021. Evidence on the Carcinogenicity of Perfluorooctane Sulfonic Acid (PFOS) and Its Salts and Transformation and Degradatio.
  61. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H, 2009. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265. [DOI] [PubMed] [Google Scholar]
  62. Schatoff EM, Leach BI, Dow LE, 2017. Wnt Signaling and Colorectal Cancer. Curr Colorectal Cancer Rep 13, 101–110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Shizu R, Ezaki K, Sato T, Sugawara A, Hosaka T, Sasaki T, Yoshinari K, 2021. PXR Suppresses PPARalpha-Dependent HMGCS2 Gene Transcription by Inhibiting the Interaction between PPARalpha and PGC1alpha. Cells 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Shizu R, Otsuka Y, Ezaki K, Ishii C, Arakawa S, Amaike Y, Abe T, Hosaka T, Sasaki T, Kanno Y, Miyata M, Yamazoe Y, Yoshinari K, 2020. Antiepileptic Drug-Activated Constitutive Androstane Receptor Inhibits Peroxisome Proliferator-Activated Receptor alpha and Peroxisome Proliferator-Activated Receptor gamma Coactivator 1alpha-Dependent Gene Expression to Increase Blood Triglyceride Levels. Mol Pharmacol 98, 634–647. [DOI] [PubMed] [Google Scholar]
  65. Sims JL, Stroski KM, Kim S, Killeen G, Ehalt R, Simcik MF, Brooks BW, 2022. Global occurrence and probabilistic environmental health hazard assessment of per- and polyfluoroalkyl substances (PFASs) in groundwater and surface waters. Sci Total Environ 816, 151535. [DOI] [PubMed] [Google Scholar]
  66. Sivaprakasam S, Prasad PD, Singh N, 2016. Benefits of short-chain fatty acids and their receptors in inflammation and carcinogenesis. Pharmacol Ther 164, 144–151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Smalling KL, Romanok KM, Bradley PM, Morriss MC, Gray JL, Kanagy LK, Gordon SE, Williams BM, Breitmeyer SE, Jones DK, DeCicco LA, Eagles-Smith CA, Wagner T, 2023. Per- and polyfluoroalkyl substances (PFAS) in United States tapwater: Comparison of underserved private-well and public-supply exposures and associated health implications. Environ Int 178, 108033. [DOI] [PubMed] [Google Scholar]
  68. Song M, Garrett WS, Chan AT, 2015. Nutrients, foods, and colorectal cancer prevention. Gastroenterology 148, 1244–1260 e1216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. U.S. Environmental Protection Agency, 2022. Interim Drinking Water Health Advisory: Perfluorooctane Sulfonic Acid (PFOS) CASRN 1763-23-1. Washington, DC. [Google Scholar]
  70. U.S. Environmental Protection Agency, 2023. Our Current Understanding of the Human Health and Environmental Risks of PFAS. Washington, DC. [Google Scholar]
  71. U.S. Environmental Protection Agency, 2024. Safe Drinking Water Act: Per- and Polyfluoroalkyl Substances (PFAS)–Final PFAS National Primary Drinking Water Regulation. Washington, DC. [Google Scholar]
  72. Vanauberg D, Schulz C, Lefebvre T, 2023. Involvement of the pro-oncogenic enzyme fatty acid synthase in the hallmarks of cancer: a promising target in anti-cancer therapies. Oncogenesis 12, 16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Vernia F, Longo S, Stefanelli G, Viscido A, Latella G, 2021. Dietary Factors Modulating Colorectal Carcinogenesis. Nutrients 13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Wan S, Xi M, Zhao HB, Hua W, Liu YL, Zhou YL, Zhuo YJ, Liu ZZ, Cai ZD, Wan YP, Zhong WD, 2019. HMGCS2 functions as a tumor suppressor and has a prognostic impact in prostate cancer. Pathol Res Pract 215, 152464. [DOI] [PubMed] [Google Scholar]
  75. Wang H, Mannava S, Grachtchouk V, Zhuang D, Soengas MS, Gudkov AV, Prochownik EV, Nikiforov MA, 2008. c-Myc depletion inhibits proliferation of human tumor cells at various stages of the cell cycle. Oncogene 27, 1905–1915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Wang Q, Zhou Y, Rychahou P, Fan TW, Lane AN, Weiss HL, Evers BM, 2017. Ketogenesis contributes to intestinal cell differentiation. Cell Death Differ 24, 458–468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Wang XW, Zhang YJ, 2014. Targeting mTOR network in colorectal cancer therapy. World J Gastroenterol 20, 4178–4188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Wang YH, Liu CL, Chiu WC, Twu YC, Liao YJ, 2019. HMGCS2 Mediates Ketone Production and Regulates the Proliferation and Metastasis of Hepatocellular Carcinoma. Cancers (Basel) 11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Wang YH, Suk FM, Liao YJ, 2020. Loss of HMGCS2 Enhances Lipogenesis and Attenuates the Protective Effect of the Ketogenic Diet in Liver Cancer. Cancers (Basel) 12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Wee SY, Aris AZ, 2023. Revisiting the “forever chemicals”, PFOA and PFOS exposure in drinking water. npj Clean Water 6. [Google Scholar]
  81. Wei R, Zhou Y, Li C, Rychahou P, Zhang S, Titlow WB, Bauman G, Wu Y, Liu J, Wang C, Weiss HL, Evers BM, Wang Q, 2022. Ketogenesis Attenuates KLF5-Dependent Production of CXCL12 to Overcome the Immunosuppressive Tumor Microenvironment in Colorectal Cancer. Cancer Res 82, 1575–1588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Xu Y, Ye X, Zhou Y, Cao X, Peng S, Peng Y, Zhang X, Sun Y, Jiang H, Huang W, Lian H, Yang J, Li J, Ye J, 2023. Sodium butyrate activates HMGCS2 to promote ketone body production through SIRT5-mediated desuccinylation. Front Med 17, 339–351. [DOI] [PubMed] [Google Scholar]
  83. Yi M, Li J, Chen S, Cai J, Ban Y, Peng Q, Zhou Y, Zeng Z, Peng S, Li X, Xiong W, Li G, Xiang B, 2018. Emerging role of lipid metabolism alterations in Cancer stem cells. J Exp Clin Cancer Res 37, 118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Zaborowski AM, Abdile A, Adamina M, Aigner F, d’Allens L, Allmer C, Alvarez A, Anula R, Andric M, Atallah S, Bach S, Bala M, Barussaud M, Bausys A, Bebington B, Beggs A, Bellolio F, Bennett MR, Berdinskikh A, Bevan V, Biondo S, Bislenghi G, Bludau M, Boutall A, Brouwer N, Brown C, Bruns C, Buchanan DD, Buchwald P, Burger JWA, Burlov N, Campanelli M, Capdepont M, Carvello M, Chew HH, Christoforidis D, Clark D, Climent M, Cologne KG, Contreras T, Croner R, Daniels IR, Dapri G, Davies J, Delrio P, Denost Q, Deutsch M, Dias A, D’Hoore A, Drozdov E, Duek D, Dunlop M, Dziki A, Edmundson A, Efetov S, El-Hussuna A, Elliot B, Emile S, Espin E, Evans M, Faes S, Faiz O, Fleming F, Foppa C, Fowler G, Frasson M, Figueiredo N, Forgan T, Frizelle F, Gadaev S, Gellona J, Glyn T, Gong J, Goran B, Greenwood E, Guren MG, Guillon S, Gutlic I, Hahnloser D, Hampel H, Hanly A, Hasegawa H, Iversen LH, Hill A, Hill J, Hoch J, Hoffmeister M, Hompes R, Hurtado L, Iaquinandi F, Imbrasaite U, Islam R, Jafari MD, Kanemitsu Y, Karachun A, Karimuddin AA, Keller DS, Kelly J, Kennelly R, Khrykov G, Kocian P, Koh C, Kok N, Knight KA, Knol J, Kontovounisios C, Korner H, Krivokapic Z, Kronberger I, Kroon HM, Kryzauskas M, Kural S, Kusters M, Lakkis Z, Lankov T, Larson D, Lazar G, Lee KY, Lee SH, Lefevre JH, Lepisto A, Lieu C, Loi L, Lynch C, Maillou-Martinaud H, Maroli A, Martin S, Martling A, Matzel KE, Mayol J, McDermott F, Meurette G, Millan M, Mitteregger M, Moiseenko A, Monson JRT, Morarasu S, Moritani K, Moslein G, Munini M, Nahas C, Nahas S, Negoi I, Novikova A, Ocares M, Okabayashi K, Olkina A, Onate-Ocana L, Otero J, Ozen C, Pace U, Sao Juliao GP, Panaiotti L, Panis Y, Papamichael D, Park J, Patel S, Patron Uriburu JC, Pera M, Perez RO, Petrov A, Pfeffer F, Phang PT, Poskus T, Pringle H, Proud D, Raguz I, Rama N, Rasheed S, Raval MJ, Rega D, Reissfelder C, Reyes Meneses JC, Ris F, Riss S, Rodriguez-Zentner H, Roxburgh CS, Saklani A, Salido AJ, Sammour T, Saraste D, Schneider M, Seishima R, Sekulic A, Seppala T, Sheahan K, Shine R, Shlomina A, Sica GS, Singnomklao T, Siragusa L, Smart N, Solis A, Spinelli A, Staiger RD, Stamos MJ, Steele S, Sunderland M, Tan KK, Tanis PJ, Tekkis P, Teklay B, Tengku S, Jimenez-Toscano M, Tsarkov P, Turina M, Ulrich A, Vailati BB, van Harten M, Verhoef C, Warrier S, Wexner S, de Wilt H, Weinberg BA, Wells C, Wolthuis A, Xynos E, You N, Zakharenko A, Zeballos J, Winter DC, 2021. Characteristics of Early-Onset vs Late-Onset Colorectal Cancer: A Review. JAMA Surg 156, 865–874. [DOI] [PubMed] [Google Scholar]
  85. Zaytseva Y, 2021. Lipid Metabolism as a Targetable Metabolic Vulnerability in Colorectal Cancer. Cancers (Basel) 13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  86. Zaytseva YY, Harris JW, Mitov MI, Kim JT, Butterfield DA, Lee EY, Weiss HL, Gao T, Evers BM, 2015. Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration. Oncotarget 6, 18891–18904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Zaytseva YY, Rychahou PG, Gulhati P, Elliott VA, Mustain WC, O’Connor K, Morris AJ, Sunkara M, Weiss HL, Lee EY, Evers BM, 2012. Inhibition of fatty acid synthase attenuates CD44-associated signaling and reduces metastasis in colorectal cancer. Cancer Res 72, 1504–1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Zeng Z, Song B, Xiao R, Zeng G, Gong J, Chen M, Xu P, Zhang P, Shen M, Yi H, 2019. Assessing the human health risks of perfluorooctane sulfonate by in vivo and in vitro studies. Environ Int 126, 598–610. [DOI] [PubMed] [Google Scholar]
  89. Zhang L, Louie A, Rigutto G, Guo H, Zhao Y, Ahn S, Dahlberg S, Sholinbeck M, Smith MT, 2023. A systematic evidence map of chronic inflammation and immunosuppression related to per- and polyfluoroalkyl substance (PFAS) exposure. Environ Res 220, 115188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  90. Zou K, Hu Y, Li M, Wang H, Zhang Y, Huang L, Xie Y, Li S, Dai X, Xu W, Ke Z, Gong S, Wang Y, 2019. Potential Role of HMGCS2 in Tumor Angiogenesis in Colorectal Cancer and Its Potential Use as a Diagnostic Marker. Can J Gastroenterol Hepatol 2019, 8348967. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES