Lactate-mediated epigenetic reprogramming regulates formation of human pancreatic cancer-associated fibroblasts

  1. Tushar D Bhagat
  2. Dagny Von Ahrens
  3. Meelad Dawlaty
  4. Yiyu Zou
  5. Joelle Baddour
  6. Abhinav Achreja
  7. Hongyun Zhao
  8. Lifeng Yang
  9. Brijesh Patel
  10. Changsoo Kwak
  11. Gaurav S Choudhary
  12. Shanisha Gordon-Mitchell
  13. Srinivas Aluri
  14. Sanchari Bhattacharyya
  15. Srabani Sahu
  16. Prafulla Bhagat
  17. Yiting Yu
  18. Matthias Bartenstein
  19. Orsi Giricz
  20. Masako Suzuki
  21. Davendra Sohal
  22. Sonal Gupta
  23. Paola A Guerrero
  24. Surinder Batra
  25. Michael Goggins
  26. Ulrich Steidl
  27. John Greally
  28. Beamon Agarwal
  29. Kith Pradhan
  30. Debabrata Banerjee
  31. Deepak Nagrath  Is a corresponding author
  32. Anirban Maitra  Is a corresponding author
  33. Amit Verma  Is a corresponding author
  1. Albert Einstein College of Medicine, Montefiore Medical Center, United States
  2. University of Michigan, United States
  3. Rutgers University, United States
  4. UT MD Anderson Cancer Center, United States
  5. Cleveland Clinic, United States
  6. University of Nebraska Medical Center, United States
  7. Johns Hopkins, United States
  8. GenomeRxUs LLC, United States
8 figures, 1 table and 3 additional files

Figures

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 …

https://doi.org/10.7554/eLife.50663.002
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 …

https://doi.org/10.7554/eLife.50663.003
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 …

https://doi.org/10.7554/eLife.50663.004
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) …

https://doi.org/10.7554/eLife.50663.005
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 …

https://doi.org/10.7554/eLife.50663.006
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 …

https://doi.org/10.7554/eLife.50663.007
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 …

https://doi.org/10.7554/eLife.50663.008
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 …

https://doi.org/10.7554/eLife.50663.010
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) …

https://doi.org/10.7554/eLife.50663.011
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
RankNameP-valueq-value (Benjamini)# Target Sequences with Motif% of Targets Sequences with Motif# Background Sequences with Motif% of Background Sequences with MotifMotif
1Atf3(bZIP)/GBM-ATF3-ChIP-Seq(GSE33912)/Homer1.00E-0703447.86%2593.15.81%
2AP-1(bZIP)/ThioMac-PU.1-ChIP-Seq(GSE21512)/Homer1.00E-0603838.75%2995.76.71%
3Jun-AP1(bZIP)/K562-cJun-ChIP-Seq(GSE31477)/Homer1.00E-050.00031122.56%722.11.62%
4BMAL1(bHLH)/Liver-Bmal1-ChIP-Seq(GSE39860)/Homer1.00E-050.000385719.57%756716.95%
5Fosl2(bZIP)/3T3L1-Fosl2-ChIP-Seq(GSE56872)/Homer1.00E-050.00031493.40%1040.82.33%
6BATF(bZIP)/Th17-BATF-ChIP-Seq(GSE39756)/Homer1.00E-050.00033227.35%2570.85.76%
7Fra1(bZIP)/BT549-Fra1-ChIP-Seq(GSE46166)/Homer1.00E-040.00062666.07%2089.94.68%
8Smad2(MAD)/ES-SMAD2-ChIP-Seq(GSE29422)/Homer1.00E-040.001165214.89%5725.212.83%
9NPAS2(bHLH)/Liver-NPAS2-ChIP-Seq(GSE39860)/Homer1.00E-040.002654312.40%4734.110.61%
10Smad4(MAD)/ESC-SMAD4-ChIP-Seq(GSE29422)/Homer1.00E-030.00365514.96%5819.313.04%
11Smad3(MAD)/NPC-Smad3-ChIP-Seq(GSE36673)/Homer1.00E-030.0035123828.27%1153325.84%
12Usf2(bHLH)/C2C12-Usf2-ChIP-Seq(GSE36030)/Homer1.00E-030.00422004.57%1574.53.53%
13HIF2a(bHLH)/785_O-HIF2a-ChIP-Seq(GSE34871)/Homer1.00E-030.00661784.06%1393.53.12%
14CEBP:CEBP(bZIP)/MEF-Chop-ChIP-Seq(GSE35681)/Homer1.00E-030.01411072.44%789.31.77%
15MafA(bZIP)/Islet-MafA-ChIP-Seq(GSE30298)/Homer1.00E-020.0233157.19%2709.16.07%
16Bach2(bZIP)/OCILy7-Bach2-ChIP-Seq(GSE44420)/Homer1.00E-020.023861.96%622.71.40%
17Brachyury(T-box)/Mesoendoderm-Brachyury-ChIP-exo(GSE54963)/Homer1.00E-020.03261343.06%1056.72.37%
18USF1(bHLH)/GM12878-Usf1-ChIP-Seq(GSE32465)/Homer1.00E-020.03871904.34%1574.43.53%
19n-Myc(bHLH)/mES-nMyc-ChIP-Seq(GSE11431)/Homer1.00E-020.05112726.21%2354.15.27%
20Fli1(ETS)/CD8-FLI-ChIP-Seq(GSE20898)/Homer1.00E-020.052843910.03%3953.78.86%

Additional files

Supplementary file 1

Pathways that are overexpressed and demethylated in pancreatic cancer associated fibroblasts.

https://doi.org/10.7554/eLife.50663.012
Supplementary file 2

Common sets of genes epigenetically altered in primary and de novo generated CAFs.

https://doi.org/10.7554/eLife.50663.013
Supplementary file 3

Differentially expressed transcripts after CXCR knockdown in CAF cells.

https://doi.org/10.7554/eLife.50663.014

Download links