Epigenomic reprogramming during pancreatic cancer progression links anabolic glucose metabolism to distant metastasis

Abstract

During the progression of pancreatic ductal adenocarcinoma (PDAC), heterogeneous subclonal populations emerge that drive primary tumor growth, regional spread, distant metastasis, and patient death. However, the genetics of metastases largely reflects that of the primary tumor in untreated patients, and PDAC driver mutations are shared by all subclones. This raises the possibility that an epigenetic process might operate during metastasis. Here we report large-scale reprogramming of chromatin modifications during the natural evolution of distant metastasis. Changes were targeted to thousands of large chromatin domains across the genome that collectively specified malignant traits, including euchromatin and large organized chromatin histone H3 lysine 9 (H3K9)-modified (LOCK) heterochromatin. Remarkably, distant metastases co-evolved a dependence on the oxidative branch of the pentose phosphate pathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammed chromatin, malignant gene expression programs, and tumorigenesis. These findings suggest a model whereby linked metabolic-epigenetic programs are selected for enhanced tumorigenic fitness during the evolution of distant metastasis.

Figure 1. Global epigenetic reprogramming during the evolution of distant metastasis

(a) IHC stains against H3K9me2/3 performed on tumor sections as indicated for patient A124 (regional/peritoneal spread), which corresponds to samples detailed in Supplementary Table 1 (founder: A124PrF; subclone: A124PrS; peritoneal metastasis: A124Per). Staining was diffusely positive (brown nuclei) across all tumor sections. (b) IHC and IF stains on tumor sections from patient A125 (distant metastatic spread), which corresponds to samples detailed in Supplementary Table 1 (founder: A125PrF; subclone: A125PrS; liver metastasis: A125Lv1). There was progressive loss of nuclear staining that initiated in primary tumor subclones that seeded metastases (middle panels) with diffuse loss in the liver metastases (scale bars=100μm for IHC, 20μm for IF; IF colors: red=H3K9me2; blue=DAPI; green=vimentin). (c) Western blots on cells lines collected from a peritoneal subclone (Per) and distant (liver, lung) metastatic subclones from same patient (pA38, see Supplementary Fig. 1c for corresponding IHC) also showed reduced levels of heterochromatin modifications with increased acetylation in distant metastatic subclones compared to peritoneal. (d) Densitometry summary of western blot findings for the indicated histone modifications across cell lines from distant metastatic subclones compared to peritoneal carcinomatosis (derived from blots shown in Supplementary Fig. 1e–f, n=8 biological replicates, error bars: s.e.m., *p<0.01 by two-tailed t-tests).

Figure 2. Epigenomic reprogramming of chromatin domains during PDAC subclonal evolution

(a) Representative (left panels) and total summarized (right panels) ChIP-seq experiments revealed loss of H3K9me2 from LOCKs between peritoneal (A38Per) and distant metastatic and primary tumor precursor subclones (others). H3K27me3 remained strong in all subclones. (b) WGBS data on cell lines (A38, A13, top panel) and frozen tissue samples (A124, A125 bottom panels) showed that samples from local regional spread and parental clones (A38Per, A124PrF, A124Per, A125PrF) possessed hypermethylated LOCKs. In contrast, distant metastatic subclones (A125Lv1/2, A13Lg, A38Lv, A38Lg) and their primary tumor subclones (A125PrS, A13Pr1, A13Pr2) showed hypomethylation of DNA across the same LOCK regions. (c) Examples of individual LOCKs displaying hyper- vs. hypomethylation across subclones, as described above. WGBS signals are plotted above (0.5–1.0) and below (0.0–0.5) a midpoint of 0.5 in IGV, which represents the background (basal) level of methylation across all samples. (d) Global levels of H3K36me3, H3K27ac, and DNA methylation within ECDs did not show any consistent differences between subclones. (e) In contrast, distant metastatic subclones and primary tumor subclones (red lines) displayed local reprogramming of H3K36me3 and H3K27ac specifically over DE genes within ECDs, compared to the same DE genic ECD regions from A38Per controls (black lines) compared against each subclone independently.

Figure 3. Reprogrammed chromatin domains encode divergent malignant properties

(a) A38Lg was remarkably resistant to H₂0₂ treatments compared to A38Per. MTT signals reflect cell viability normalized to untreated controls (n=4 technical replicates, error bars: s.d.m., *p<0.03 by two tailed t-tests). (b) Western blots for proteins involved in epithelial and EMT differentiation were differentially expressed between A38Per and A38Lg, as predicted by GO analyses of reprogrammed DE genes from LOCKs. (c) A38Lg was highly resistant to gemcitabine compared to A38Per, as predicted by GO analyses of reprogrammed DE genes from ECDs. MTT signals reflect cell viability normalized to untreated controls (n=4 technical replicates, error bars: s.d.m., *p<0.01 by two tailed t-tests). (d) A38Lg possessed elevated levels of γH2AX by western blot, consistent with activation of DNA repair pathways. (e) Western blots showed that A38Lg lost hyper-phosphorylated ERK and (f) was resistant to ERK targeted therapy, compared to A38Per. MTT signals reflect cell viability, normalized to untreated controls (n=4 technical replicates, error bars: s.d.m., *p<0.03 by two tailed t-tests). (g) A38Lg also lost sensitivity to KRAS knockdown by matrigel 3D tumor forming assays, compared to A38Per (n=4 technical replicates, error bars: s.d.m., *p<0.01 by two tailed t-tests). Scale bars: 400μm.

Figure 4. Hyperactive glucose metabolism and 6PG depletion in distant metastatic subclones

(a) MTT assays performed on equal numbers of viable, growth-arrested cells from the indicated subclones showed elevated signal (oxidoreductase activity) across distant metastatic subclones, compared to HPDE and regional PDACs (n=4 technical replicates, error bars: s.d.m., *p<10⁻⁵ by two tailed t-tests). (b) Normalized cell counts for the indicated samples incubated with (+) or without (−) 10mM glucose and treated with 1mM H₂0₂ as indicated (+,−) showed that HPDE cells were sensitive to H₂0₂ under either glucose condition (as expected), whereas regional PDAC samples were resistant to H₂0₂ irrespective of glucose availability (n=3 technical replicates, error bars: s.d.m). (c) In contrast, distant metastatic subclones were sensitive to H₂0₂ when glucose was not present in the media (n=3 technical replicates, error bars: s.d.m, *p<0.001 by two tailed t-tests). (d) Simplified schematic of ¹³C-(1,2)-labeled glucose flow through glycolysis and the PPP. Glucose that enters the oxidative branch of the PPP has one labeled carbon cleaved during conversion of 6PG to Ru5P (m+1), whereas glucose that travels through glycolysis retains both labeled carbons (m+2). Note that cross-talk allows glucose with either labeling pattern to re-enter the other pathway and incorporate. (e) LC-MS for nucleotides and lactate showed that these downstream metabolites acquired greatly elevated ¹³C-1,2 labels from glucose in A38Lg (n=3 biological replicates, error bars: s.d.m., *p<0.01 by two tailed t-tests). (f) Steady state LC-HRMS measurements for 6PG showed either complete (ND: not detected) or near complete loss of metabolite across distant metastases and their precursors.

Figure 5. PGD-dependence in distant metastatic subclones

(a) Western blots against indicated histone modifications performed on peritoneal (A38Per) and distant metastatic (A38Lg) subclones from the same patient showed that global levels of reprogrammed H3K9me2 and acetylation in A38Lg were reversed by removal of glucose from the media (left panel), PGD RNAi (middle panel), and 6AN treatments (right panel). (b) Western blots on A38Lg indicated that PGD knockdown by RNAi did not perturb expression of other PPP components or KRAS. (c) PGD RNAi did not affect the ability of HPDE cells or regional PDAC samples to form tumors in 3D matrigel assays (representative photomicrographs shown with quantified numbers of tumors/well, n=4 technical replicates, error bars: s.d.m.). (d) In contrast, PGD RNAi significantly reduced tumor formation across distant metastatic subclones (n=4 technical replicates, error bars: s.d.m., *p<0.01 by two tailed t-tests). Scale bars: 400μm.

Figure 6. Reversal of reprogrammed chromatin and tumorigenic potential by 6AN

(a) Densitometry summary of western blots shown in Supplementary Fig. 12a (n=8 biological replicates, error bars: s.e.m., *p≤0.01 by two tailed t-tests). 6AN selectively reversed reprogrammed H3K9me2 and H3K9/27ac. (b) Densitometry summary of western blots shown in Supplementary Fig. 12b (n=6 biological replicates, error bars: s.e.m.). 6AN had minimal effects on histone modifications across normal (HPDE, fibroblast) or regional PDAC samples. (c) 6AN ablated tumor formation in 3D matrigel assays (n=4 technical replicates, error bars: s.d.m., *p<0.01 by two tailed t-tests). Scale bars: 400μm. (d) 6AN also blocked the ability of distant metastatic subclones to form tumors when injected into 3D organotypic stromal cultures (n=3 technical replicates, error bars: s.d.m., *p<0.05 by two tailed t-tests). Scale bars: 200μm.

Figure 7. CDH2 and TOP2B are epigenetic targets of 6AN that selectively regulate tumorigenic potential

(a) Real-time RT-qPCR (top panel) and ChIP-qPCR for H3K9me2 and H3K27ac showed that 6AN had minimal effect on CDH2 mRNA expression or epigenetic state (H3K9me2/H3K27ac) over the CDH2 locus in the peritoneal subclone. Numbers along the x-axis of ChIP panels correspond to coordinates of the ChIP-PCR primer positions across the 1.4Mb CDH2 locus (Chr18q 27Mb:primer position). (b) In contrast, RT-PCR (top panel) showed that the distant metastatic subclone (A38Lg) from the same patient over-expressed CDH2 relative to A38Per (compare y-axis values), and that expression was repressed by 6AN (n=4 technical PCR replicates from two biological replicate experiments, error bars: s.d.m., *p=0.002 by two tailed t-tests). ChIP assays further showed that 6AN induced enrichment of H3K9me2 across the 1.4Mb CDH2 locus with corresponding reductions of H3K27ac directly over the genic region (n=2 biological replicates, error bars: s.e.m.). (c) RNAi against both CDH2 and TOP2B (Supplementary Fig. 15c–d) selectively blocked 3D tumor formation in distant metastatic and precursor subclones that over-expressed these genes by RNA-seq, with no effect on A38Per or HPDE cells (n=4 technical replicates, error bars: s.d.m., *p<0.01 by two tailed t-tests). Scale bars: 400μm.