Enhanced SREBP2-driven cholesterol biosynthesis by PKCλ/ι deficiency in intestinal epithelial cells promotes aggressive serrated tumorigenesis

Abstract

The metabolic and signaling pathways regulating aggressive mesenchymal colorectal cancer (CRC) initiation and progression through the serrated route are largely unknown. Although relatively well characterized as BRAF mutant cancers, their poor response to current targeted therapy, difficult preneoplastic detection, and challenging endoscopic resection make the identification of their metabolic requirements a priority. Here, we demonstrate that the phosphorylation of SCAP by the atypical PKC (aPKC), PKCλ/ι promotes its degradation and inhibits the processing and activation of SREBP2, the master regulator of cholesterol biosynthesis. We show that the upregulation of SREBP2 and cholesterol by reduced aPKC levels is essential for controlling metaplasia and generating the most aggressive cell subpopulation in serrated tumors in mice and humans. Since these alterations are also detected prior to neoplastic transformation, together with the sensitivity of these tumors to cholesterol metabolism inhibitors, our data indicate that targeting cholesterol biosynthesis is a potential mechanism for serrated chemoprevention.

Fig. 1. Loss of atypical PKC causes robust reprogramming of cholesterol metabolism by enhancing biosynthesis.

a–c Gene set enrichment analysis (GSEA) results of transcriptomic data from Prkci^f/fPrkcz^f/f;Villin-Cre mouse small intestinal tumors (n = 3 tumors from three distinct mice) versus WT small intestines (n = 3 tissues from three distinct mice). The top 5 gene sets in compilation H (MsigDB) (a), GSEA plot (b), and GSEA results related to cholesterol metabolism (c) are shown. d RNA-seq data showing the log2-fold changes between Prkci^f/fPrkcz^f/f;Villin-Cre tumors (n = 3 tumors from three distinct mice) and WT tissue (n = 3 tissue from three distinct mice). e, f Inducible deletion of aPKCs in mouse small intestinal epithelial cells (IECs). Prkci^f/fPrkcz^f/f;Villin-CreERT2 or Prkci^f/fPrkcz^f/f; mice were injected intraperitoneally with 6 mg of tamoxifen for 7 days. Schematic (e) and qPCR (f) (n = 4 mice per group). g–i Metabolic tracing with deuterium water (²H₂O). In vivo experiment schematic (g), schematic depicting ²H (red) labeling from deuterated water tracer (²H₂O) and incorporation into newly synthesized cholesterol and fatty acids (h), and newly synthesized lipids (i) in tumoral (n = 3 tumors from three distinct mice) and non-tumoral tissues in the small intestines of Prkci^f/fPrkcz^f/f;Villin-Cre and WT mice (n = 3 tissues from three distinct mice per group). Open circles represent [¹²C]carbon atoms. j Total cholesterol content in the small intestine of Prkci^f/fPrkcz^f/f;Villin-Cre and WT mice (n = 6 mice per group). k Serum total cholesterol levels in Prkci^f/fPrkcz^f/f;Villin-Cre (n = 23) and WT (n = 14) mice. l–p scRNA-seq of mouse small intestinal epithelial cells (n = 3 tissues from three distinct WT mice, n = 1 tissue from one Prkci^f/fPrkcz^f/f;Villin-Cre mouse, and n = 5 tumors from two distinct Prkci^f/fPrkcz^f/f;Villin-Cre mice). l, m Uniform manifold approximation and projection (UMAP) plots of in Prkci^f/fPrkcz^f/f;Villin-Cre mouse small intestinal epithelial cells colored by tissue origins (l) and cell types (m). n UMAP feature plots of epithelial cells colored by the expression of hallmark cholesterol homeostasis gene set. o, p Dot plots of indicated gene signatures against tissue origins (o) or tumor cell subtypes in the tumor compartment (p). Data were presented as mean ± SEM. Wald test in d and two-tailed, unpaired Student’s t-test in f, i, j, and k. Source data are provided as a Source Data file.

Fig. 2. Loss of PKCλ/ι is sufficient for the dysregulated cholesterol metabolism.

a, b GSEA results of the top 8 gene sets in compilation H for sgPrkci/sgPrkcz versus sgC (a) and sgPrkci versus sgC (b) mouse tumor organoids (MTOs) (n = 3 biological replicates per group). c GSVA results of MTOs. d qPCR of MTOs (n = 3 biological replicates per group). e Immunoblotting and qPCR in sgPRKCI/PRKCZ and sgC HCT116 cells cultured in 5% lipoprotein depleted serum (LPDS) with 5 μM of lovastatin for 24 h (n = 3 biological replicates). f Immunoblotting and qPCR in sgPRKCI/PRKCZ and sgC 293 T cells cultured in 1% LPDS for 24 h (n = 3 biological replicates). g Immunoblotting and qPCR in sgPRKCI and sgC HCT116 cells cultured in 5% LPDS with 5 μM of lovastatin for 24 h (n = 3 biological replicates). h Immunoblotting and qPCR in sgPRKCI and sgC 293 T cells cultured in 1% LPDS for 24 h (n = 6 biological replicates). i Total cholesterol content in sgPRKCI and sgC 293 T cells cultured in 1% LPDS for 40 h (n = 3 biological replicates). j Schematic depicting labeling on cholesterol from [U-¹³C₆]glucose. Closed black circles represent [¹³C]carbon; open circles represent [¹²C]carbon atoms. k Isotopologue distribution of cholesterol from ¹³C glucose in sgPRKCI and sgC 293 T cells cultured in 1% LPDS for 24 h (n = 3 biological replicates). l Relative incorporation of ¹³C into cholesterol from [U-¹³C] glucose and relative cholesterol abundance in sgPRKCI and sgC 293 T cells cultured in 1% LPDS for 24 h (n = 3 biological replicates). m Schematic depicting labeling on palmitate from [U-¹³C₆]glucose. Closed black circles represent [¹³C]carbon; open circles represent [¹²C]carbon atoms. n Newly synthesized palmitate from [U-¹³C₆]glucose in sgPRKCI and sgC 293 T cells cultured in 1% LPDS for 24 h (n = 3 biological replicates). Data were presented as mean ± SEM. One-way ANOVA and post hoc Tukey’s test (d) and Two-tailed, unpaired Student’s t-test (e–i, k, l, n). Source data are provided as a Source Data file.

Fig. 3. SREBP2 processing and translocation is induced by PKCλ/ι deficiency.

a Metabolic map depicting cholesterol biosynthesis pathway showing fold change of SREBP2 target genes from RNAseq data of Prkci^f/fPrkcz^f/f;Villin-Cre mouse tumors (n = 3 tumors from three distinct mice) versus WT small intestines (n = 3 tissue from three distinct mice). b, c Genome browser view at promoter regions (b) and Hypergeometric Optimization of Motif EnRichment (HOMER) analysis for SREBP2 motif (c) in IECs (n = 1 mouse per group). d Immunoblotting of mouse small intestine and tumor samples. Duo: Duodenum, Jej: Jejunum, Ile: Ileum, Tum: Tumor (n = 1 mouse per group). e Immunohistochemistry (IHC) for SREBP2 in mouse small intestine and tumors (n = 3 mice per group). f Immunoblotting of mouse IECs (n = 2 tissues from two distinct WT mice, n = 3 tissues from three distinct Prkcz^f/f;Villin-Cre mice, and n = 2 tissues from two distinct Prkci^f/f;Villin-Cre mice). g, h Inducible deletion of aPKCs in intestinal organoids established from Prkci^f/fPrkcz^f/f;Villin-CreERT2 mouse small intestine. Schematic and qPCR (g) and immunoblotting (h) in organoids treated with 1 μM of 4-hydroxytamoxifen (4OHT) or vehicle for 4 days (n = 3 biological replicates). i Immunoblotting of sgC, sgPRKCI and sgPRKCI/PRKCZ 293 T cells. j, k Immunofluorescence staining of SREBP2 and DAPI (j) and nuclear SREBP2/DAPI ratios (k) in sgC (n = 71 cells examined over 3 independent experiments and sgPRKCI 293 T cells (n = 55 cells examined over 3 independent experiments). l, m Immunofluorescence staining of SREBP2 and DAPI in WT and Prkci^f/fPrkcz^f/f;Villin-Cre mouse small intestine. Representative images (l) and SREBP2/DAPI intensity ratio (m) in each cell nucleus of WT and Prkci^f/fPrkcz^f/f;Villin-Cre mouse small intestinal tumors (WT: n = 214 cells from one mouse, Tumor: n = 442 cells from one tumor). n Dot plot of indicated gene signatures against tumor cell subtypes in the tumor compartment of Prkci^f/fPrkcz^f/f;Villin-Cre mouse small intestine (n = 5 tumors from two distinct Prkci^f/fPrkcz^f/f;Villin-Cre mice). o, p Immunofluorescence staining of SREBP2, ANXA10 and DAPI in Prkci^f/fPrkcz^f/f;Villin-Cre mouse small intestinal tumors. Representative images (o), and ANXA10 intensity in cells with high and low SREBP2 expression (p) (cutoff: top and bottom quartile, n = 127 cells examined over three different tumor areas from one mouse). Data were presented as mean ± SEM. The ratiometric images of SREBP2 and DAPI signal intensities are shown in the intensity-modulated display mode (IMD) according to the color scale in (j) and (l). Wald test in a HOMER hypergeometric test in c and two-tailed, unpaired Student’s t-test in g, k, m and p. Scale bars, 10 μm. Source data are provided as a Source Data file.

Fig. 4. Dysregulated cholesterol metabolism is associated with low aPKC expression in human serrated tumors.

a, b Enrichment scores (ES) of GSVA on hallmark cholesterol homeostasis (a) and fatty acid metabolism in human CRC samples (b) in GSE79462 (SSL: n = 7, TA: n = 9), GSE76987 (Normal: n = 10, Adenoma: n = 10, SSL: n = 21) and GSE4045 (Serrated: n = 8, Conventional: n = 29). c Kaplan–Meier curves for 5-year overall survival of proximal and distal CRC patients in the TCGA COADREAD dataset (Proximal: n = 205, Distal: n = 305). d GSVA enrichment scores of indicated gene sets in proximal (n = 213) and distal (n = 318) CRC patients in the TCGA. e aPKC expression in proximal and distal CRC patients in the TCGA (Proximal: n = 213, Distal: n = 318). f GSVA enrichment scores on hallmark cholesterol in CRC patients with high and low PRKCI expression (n = 148). g GSVA enrichment scores on Hallmark cholesterol in CRC patients stratified by PRKCI expression and primary tumor location (n = 61, 49, 66, 85). h GSEA for TFMC high and low CRC patients in the TCGA (n = 148). i GSVA enrichment scores on hallmark cholesterol in iCMS2 and iCMS3 CRC patients in the TCGA (iCMS2: n = 298, iCMS3: n = 232). j–m UMAP plot colored by Prkci^f/fPrkcz^f/f;Villin-Cre tumor cell subtypes (j), UMAP feature plot colored by the expression of Hallmark cholesterol homeostasis (k), violin plot (l), and pie chart of the relative distribution of tumor cell subtypes (m) expressing the Hallmark cholesterol homeostasis in tumor epithelial cells (GSE132465, n = 23 patient samples). n, o Immunoblotting (n) and qPCR (o) in WCM#10272 and WCM#3 human colorectal cancer organoids (n = 3 biological replicates). p Representative images of immunofluorescence staining for ANXA10, SREBP2, aPKCs, and DAPI in conventional and serrated human CRCs in the tissue microarray (TMA, n = 468 patient samples). Data were presented as mean ± SEM. Box and whiskers graphs indicate the median and the 25^th and 75^th percentiles, with minimum and maximum values at the extremes of the whiskers. Brown-Forsythe and Welch ANOVA tests and Dunnett post hoc test (a, b), two-tailed Mann–Whitney test (a, b, d–g, i), log-rank test (c), one-way ANOVA and post hoc Tukey’s test (g), two-tailed, unpaired Student’s t-test (l, o). Scale bars, 50 μm. Source data are provided as a Source Data file.

Fig. 5. SCAP stability is regulated by PKCλ/ι-mediated phosphorylation.

a Schematic of SREBP2 translocation, cleavage, and activation. b, c Immunofluorescence staining of FLAG, RCAS1, and DAPI in 293 T cells transfected with scramble small interfering RNA (siRNA) (siC) or siPRKCI cultured in media containing 10% FBS or 5% LPDS for 24 h. Representative images (b) and the Pearson correlation coefficients (c) between FLAG and RCAS1 signal intensities (siC-FBS:n = 37, siPRKCI-FBS:n = 38, siC-LPDS:n = 39, siPRKCI-LPDS: n = 39 cells examined over 3 independent experiments). d Immunoblotting of cell lysates and GFP-tagged immunoprecipitants of 293 T cells transfected with indicated plasmids. e In vitro phosphorylation assay of GFP-tagged SCAP by recombinant PKCλ/ι with ATPγS followed by p-nitrobenzyl mesylate (PNBM) alkylation and immunoblotting for the indicated proteins. f Immunoblotting of cell lysates of sgPRKCI and sgC 293 T cells transfected with FLAG-SCAP. g, h In vitro phosphorylation assay of FLAG-tagged SCAP WT (SCAP^WT) or the triple mutant (SCAP^AAA) by recombinant PKCλ/ι. Immunoblotting (g) and ThioP/FLAG-SCAP intensity ratio (h) (n = 3 biological replicates). i, j Pulse-chase assay with 50 μg/mL of cycloheximide (CHX) in 293 T cells transfected with SCAP^WT or SCAP^AAA. Immunoblotting (i) and quantification of SCAP intensities normalized to actin (j, n = 3 biological replicates). Data were presented as mean ± SEM. Two-tailed, unpaired Student’s t-test (c, h, j). Scale bars, 10 μm. Source data are provided as a Source Data file.

Fig. 6. PKCλ/ι-mediated phosphorylation promotes SCAP degradation by ubiquitination through TRC8.

a Immunoblotting of cell lysate and GFP-tagged immunoprecipitates of 293 T cells transfected with indicated plasmids. b Volcano plot of biotinylated proteins in PKCλ/ι-BioID2 versus Empty-BioID2 in 293 T cells (n = 3 biological replicates). c Immunoblotting of cell lysate and FLAG-tagged immunoprecipitates of 293 T cells transfected with indicated plasmids. d, e Pulse-chase assay with 50 μg/mL of CHX in 293 T cells transfected with siC or siTRC8. Immunoblotting (d) and quantification of SCAP intensities normalized to actin (e) (n = 3 biological replicates). f Immunoblotting of 293 T cells transfected and cultured in media containing 5% LPDS for 16 h. g Immunoblotting of nuclear fraction of 293 T cells and cultured in media containing 5% LPDS for 16 h. h, i Immunofluorescence staining of SREBP2 and DAPI in 293 T cells stably expressing SCAP^WT and SCAP^AAA cultured in media containing 5% LPDS for 24 h. Representative images (h) and nuclear SREBP2/DAPI ratios (i) (SCAP^WT: n = 82, SCAP^AAA: n = 65 cells examined over 3 independent experiments). j, k Immunofluorescence staining of SREBP2 and DAPI in 293 T cells transfected with siC or siTRC8 cultured in media containing 5% LPDS for 24 h. Representative images (j) and nuclear SREBP2/DAPI ratios (k) (siC: n = 98, siTRC8: n = 110 cells examined over 3 independent experiments). Data were presented as mean ± SEM. Two-tailed, unpaired Student’s t-test. Scale bars, 10 μm. Source data are provided as a Source Data file.

Fig. 7. Loss PKCλ/ι of renders cells addicted to cholesterol biosynthesis.

a Relative growth of sgC and sgPRKCI 293 T cells cultured in media with 5% LPDS for 4 days (n = 3 biological replicates). b Relative growth of sgC and sgPRKCI HCT116 cells cultured in media with 5% LPDS for 4 days (n = 4 biological replicates). c, d Dose response curves (c) and IC50 (d) of atorvastatin in sgPRKCI and sgC HCT116 cells (n = 6 biological replicates per group). e Growth curves of sgC and sgPRKCI HCT116 cells treated with vehicle or indicated concentration of atorvastatin (n = 4 biological replicates). f Schematic of cholesterol biosynthesis pathway and feedback response caused by statin treatment. g qPCR of sgPRKCI 293 T cells treated with 10 μM of atorvastatin and/or 5 μM of dipyridamole for 16 h (-/-, + /-: n = 6 biological replicates, +/+: n = 5 biological replicates). h qPCR of sgPRKCI HCT116 cells treated with 10 μM of atorvastatin and/or 5 μM of dipyridamole for 16 h (n = 6 biological replicates). i Dose-response curves of atorvastatin with vehicle or indicated concentration of dipyridamole in sgPRKCI HCT116 cells (n = 5 biological replicates per group). j Relative cell growth of human CRC organoids treated with vehicle, 10 μM of atorvastatin, or 10 μM of dipyridamole for 4 days (WCM#10272: n = 6 biological replicates, WCM#3: n = 5 biological replicates). Data were presented as mean ± SEM. Two-tailed, unpaired Student’s t-test (a, b), Two-tailed, extra sum-of squares F test (d), one-way ANOVA and post hoc Tukey’s test (e, g, h, j). Source data are provided as a Source Data file.

Fig. 8. Tumor suppressive effects of cholesterol inhibition in aPKC-deficient serrated tumors.

a–d Prkci^f/fPrkcz^f/f;Villin-Cre mice fed with regular chow or 1.25% cholesterol-supplemented diet for 3 months. Experimental design (a), serum total cholesterol levels (b Chow:n = 6 mice, Cholesterol:n = 5 mice), macroscopic images (c), total tumor number, and tumor load (d) Chow:n = 6 mice, Cholesterol:n = 6 mice). Red lines denote macroscopic tumors in (c). e–l Prkci^f/fPrkcz^f/f;Villin-Cre mice treated with vehicle (n = 9) or 50 mg/kg of atorvastatin orally and 120 mg/kg of dipyridamole intraperitoneally (A/D) (n = 6) daily for 4 weeks. Experimental design (e), immunoblotting of small intestines (f), serum total cholesterol levels (g) macroscopic images (h), H&E staining of tumors (i), total tumor number and tumor load (j), cancer incidence (k), and numbers of sessile serrated lesions (SSL) and carcinomas (l). Red lines denote macroscopic tumors in h and microscopic tumor areas in (i). m, n Representative images of Ki67, ANXA10, and Cleaved Caspase 3 (CC3), staining (m) and their quantification (n) (Ki67, vehicle: n = 10 fields examined from 3 mice, A/D: n = 7 fields examined from 2 mice; ANXA10, vehicle: n = 7 fields examined from 2 mice, A/D: n = 7 fields examined from 2 mice; CC3, vehicle: n = 10 fields examined from 2 mice, A/D: n = 14 fields examined from 2 mice). Red arrows denote positive cells for CC3. o Representative images of immunofluorescence staining for SREBP2, ANXA10, and DAPI in vehicle and A/D treated tumors. p Graphical scheme of PKCλ/ι mediated cholesterol metabolic reprogramming in the serrated tumorigenesis. In the physiological state, PKCλ/ι directly phosphorylates SCAP promoting degradation through a ubiquitination cascade regulated by TRC8. Upon the loss of PKCλ/ι the subsequently increased SCAP stability results in the enhanced activation of SREBP2, the master regulator of cholesterol biosynthesis, to promote serrated CRC development. Data were presented as mean ± SEM. Two-tailed, unpaired Student’s t-test (b, d, g, j, l, n), two-tailed chi-square test (k). Scale bars, 5 mm (c, h), 1 mm (i), 50 μm (m, o). Source data are provided as a Source Data file.