Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts
- Albert Einstein College of Medicine, Montefiore Medical Center, United States
- University of Michigan, United States
- Rutgers University, United States
- UT MD Anderson Cancer Center, United States
- Cleveland Clinic, United States
- University of Nebraska Medical Center, United States
- Johns Hopkins, United States
- GenomeRxUs LLC, United States
Figures
Figure 1
Widespread epigenetic and transcriptomic alterations are seen in pancreatic cancer associated fibroblasts.
(A) Unsupervised clustering of cytosine methylation profiles from seven primary patient-derived pancreatic cancer associated fibroblasts (CAFs) and four healthy controls (Hst: Hepatic stellate …
Figure 2 with 1 supplement
CXCR4 is demethylated and overexpresed in CAFs and increases pancreatic cancer invasiveness.
(A) A set of 120 transcripts are commonly hypomethylated in primary patient-derived CAFs and de novo generated CAFs and includes CXCR4. (B,C) The CXCR4 promoter is demethylated in primary …
Figure 2—figure supplement 1
Inhibition of CXCR4 reduces PDAC cell invasion in de novo generated CAFs.
(A) CXCR4 siRNAs lead to knockdown of protein in CAFs when compared to control siRNAs. Western blot and image intensity is shown. (B) CXCR4 knockdown in dn-CAFs leads to abrogation of the increased …
Figure 3 with 2 supplements
Increased 5hmC is seen in de novo CAFs generated from MSCs and is regulated by a paracrine lactate mediated metabolic flux.
(A) Mapping of carbon atoms transition using U-13C3-lactate. Mass isotopomer distribution (MID) shows uptake of labelled lactate by MSCs cultured in α-MEM leading to the generation of α-KG (M2 αKG) …
Figure 3—figure supplement 1
Metabolite tracking in CAF cells.
(A) Mass Isotopologue Distribution of Alanine from U-13C-Lactate tracing experiments indicate that lactate-derived carbon contributes significantly to de novo alanine synthesis. These experiments …
Figure 3—figure supplement 2
Lactate increases TET activity in CAFs.
(A) Conditioned media from ctrl and LDH siRNA mediated knockdown PANC-1 (PDAC) cells was collected. MSCs were exposed to control conditioned media (ctrl), or LDH knockdown conditioned and used to …
Figure 4
5hmC gains are seen during MSC to CAF conversion and occur at gene associated with cancer associated fibroblasts.
(A) Genome wide 5hmC analysis was done by OXBS in MSCs and converted CAFs. 5hmC gains are depicted as percentage gained (CAF/MSC) and were seen to occur throughout the genome (B) 5hmC gains in CAFs …
Figure 5
Increased 5hmC and CXCR4 expression is seen in primary human PDAC and murine KPC PDAC tumors.
(A, B) 5hmC (A) and CXCR4 (B) immunohistochemical staining was done on human PDAC TMAs and grading of intensity of stain in the tumor stromal CAFs was estimated. (C) Most CAF like cells in PDAC …
Figure 6
TET2 inactivation in MSCs leads to reduced tumor growth and inefficient CAF conversion in vivo.
(A) Wildtype and TET2 KO MSCs were exposed to conditioned media from KPC PDAC cells for 14 days. TET2 KO MSCs demonstrated decreased fibroblastic appearance when compared to wildtype controls. (B,C) …
Author response image 1
Mass Isotopologue Distribution of Alanine from U-13C-Lactate tracing experiments indicate that lactate-derived carbon contributes significantly to de novo alanine synthesis.
Author response image 2
Tables
Table 1
Transcription factor sites that are enriched around 5hmC gains during CAF conversion.
https://doi.org/10.7554/eLife.50663.009| Rank | Name | P-value | q-value (Benjamini) | # Target Sequences with Motif | % of Targets Sequences with Motif | # Background Sequences with Motif | % of Background Sequences with Motif | Motif |
|---|---|---|---|---|---|---|---|---|
| 1 | Atf3(bZIP)/GBM-ATF3-ChIP-Seq(GSE33912)/Homer | 1.00E-07 | 0 | 344 | 7.86% | 2593.1 | 5.81% |  |
| 2 | AP-1(bZIP)/ThioMac-PU.1-ChIP-Seq(GSE21512)/Homer | 1.00E-06 | 0 | 383 | 8.75% | 2995.7 | 6.71% |  |
| 3 | Jun-AP1(bZIP)/K562-cJun-ChIP-Seq(GSE31477)/Homer | 1.00E-05 | 0.0003 | 112 | 2.56% | 722.1 | 1.62% |  |
| 4 | BMAL1(bHLH)/Liver-Bmal1-ChIP-Seq(GSE39860)/Homer | 1.00E-05 | 0.0003 | 857 | 19.57% | 7567 | 16.95% |  |
| 5 | Fosl2(bZIP)/3T3L1-Fosl2-ChIP-Seq(GSE56872)/Homer | 1.00E-05 | 0.0003 | 149 | 3.40% | 1040.8 | 2.33% |  |
| 6 | BATF(bZIP)/Th17-BATF-ChIP-Seq(GSE39756)/Homer | 1.00E-05 | 0.0003 | 322 | 7.35% | 2570.8 | 5.76% |  |
| 7 | Fra1(bZIP)/BT549-Fra1-ChIP-Seq(GSE46166)/Homer | 1.00E-04 | 0.0006 | 266 | 6.07% | 2089.9 | 4.68% |  |
| 8 | Smad2(MAD)/ES-SMAD2-ChIP-Seq(GSE29422)/Homer | 1.00E-04 | 0.0011 | 652 | 14.89% | 5725.2 | 12.83% |  |
| 9 | NPAS2(bHLH)/Liver-NPAS2-ChIP-Seq(GSE39860)/Homer | 1.00E-04 | 0.0026 | 543 | 12.40% | 4734.1 | 10.61% |  |
| 10 | Smad4(MAD)/ESC-SMAD4-ChIP-Seq(GSE29422)/Homer | 1.00E-03 | 0.003 | 655 | 14.96% | 5819.3 | 13.04% |  |
| 11 | Smad3(MAD)/NPC-Smad3-ChIP-Seq(GSE36673)/Homer | 1.00E-03 | 0.0035 | 1238 | 28.27% | 11533 | 25.84% |  |
| 12 | Usf2(bHLH)/C2C12-Usf2-ChIP-Seq(GSE36030)/Homer | 1.00E-03 | 0.0042 | 200 | 4.57% | 1574.5 | 3.53% |  |
| 13 | HIF2a(bHLH)/785_O-HIF2a-ChIP-Seq(GSE34871)/Homer | 1.00E-03 | 0.0066 | 178 | 4.06% | 1393.5 | 3.12% |  |
| 14 | CEBP:CEBP(bZIP)/MEF-Chop-ChIP-Seq(GSE35681)/Homer | 1.00E-03 | 0.0141 | 107 | 2.44% | 789.3 | 1.77% |  |
| 15 | MafA(bZIP)/Islet-MafA-ChIP-Seq(GSE30298)/Homer | 1.00E-02 | 0.023 | 315 | 7.19% | 2709.1 | 6.07% |  |
| 16 | Bach2(bZIP)/OCILy7-Bach2-ChIP-Seq(GSE44420)/Homer | 1.00E-02 | 0.023 | 86 | 1.96% | 622.7 | 1.40% |  |
| 17 | Brachyury(T-box)/Mesoendoderm-Brachyury-ChIP-exo(GSE54963)/Homer | 1.00E-02 | 0.0326 | 134 | 3.06% | 1056.7 | 2.37% |  |
| 18 | USF1(bHLH)/GM12878-Usf1-ChIP-Seq(GSE32465)/Homer | 1.00E-02 | 0.0387 | 190 | 4.34% | 1574.4 | 3.53% |  |
| 19 | n-Myc(bHLH)/mES-nMyc-ChIP-Seq(GSE11431)/Homer | 1.00E-02 | 0.0511 | 272 | 6.21% | 2354.1 | 5.27% |  |
| 20 | Fli1(ETS)/CD8-FLI-ChIP-Seq(GSE20898)/Homer | 1.00E-02 | 0.0528 | 439 | 10.03% | 3953.7 | 8.86% |  |
Additional files
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Supplementary file 1
Pathways that are overexpressed and demethylated in pancreatic cancer associated fibroblasts.
- https://doi.org/10.7554/eLife.50663.012
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Supplementary file 2
Common sets of genes epigenetically altered in primary and de novo generated CAFs.
- https://doi.org/10.7554/eLife.50663.013
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Supplementary file 3
Differentially expressed transcripts after CXCR knockdown in CAF cells.
- https://doi.org/10.7554/eLife.50663.014
