MYC Levels Regulate Metastatic Heterogeneity in Pancreatic Adenocarcinoma

Abstract

The degree of metastatic disease varies widely among patients with cancer and affects clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multifluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC)-a tumor type in which most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation of MYC and its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor-associated macrophages, leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC. SIGNIFICANCE: Here, we investigate metastatic variation seen clinically in patients with PDAC and murine PDAC tumors and identify MYC as a major driver of this heterogeneity.This article is highlighted in the In This Issue feature, p. 275.

Figure 1.

Advanced pancreatic tumors exhibit intertumoral differences in their propensity for metastasis. A, CT imaging of human PDAC liver metastasis demonstrating heterogeneity in metastatic burden in stage IV disease. Arrowheads indicate solitary metastasis in the top image and selected metastases in the bottom. B, Density plot and histogram showing the distribution of total (liver and lung) metastases enumerated from CT scans of human stage IV PDAC at the time of diagnosis (n = 55). Values above each histogram bar represent the number of patients in each group. The vertical dotted line (red) represents the cutoff between Met^Low tumors [≤10 metastases (mets)] and Met^High tumors (>10 mets) determined by k-means clustering. C, Quantification of tumor area (based on tumor dimensions from largest cross-sectional plane on imaging) comparing Met^Low and Met^High cases from the cohort in B. D, Overall survival analysis of the cohort in B. E, Top, schematic view of the KPCXY model, showing multiple primary tumors distinguishable by color arising in the pancreas with matched metastases in the liver. Bottom, representative fluorescent stereomicroscopic images showing a YFP⁺ tumor adjoining a CFP⁺ tumor in the pancreas (left) and liver metastases derived from the CFP⁺ tumor in the same animal (right). F, Density plot and histogram showing the distribution of total (liver and lung) metastases enumerated at autopsy of KPCXY mice. Values above each histogram bar represent the number of tumors giving rise to the indicated number of metastases, based on color (n = 85 tumors from 30 KPCXY mice). The vertical dotted line (red) represents the cutoff between Met^Low tumors (≤10 mets, n = 58) and Met^High tumors (>10 mets, n = 28) determined by k-means clustering. G, Quantification of tumor area comparing Met^Low and Met^High tumors from the cohort in F. Statistical analysis by Student unpaired t test with P values indicated (ns, not significant). Box-and-whisker plots in C and G indicate mean and interquartile range. Scale bar for E, 1 mm.

Figure 2.

SCNA analysis confirms fluorescence-based lineage relationships and reveals genetic heterogeneity in paired primary pancreatic tumors and liver metastases. A, Schematic representation of KPCXY pancreatic tumor and matching liver metastases with multiregion sampling for copy-number sequence analysis. B, Representative genome-wide copy-number profiles of Met^High (CFP⁺ fluorescence) and Met^Low (YFP⁺ fluorescence) tumors from mouse 832 (m832) as depicted in Fig. 1E. Gray shading denotes alterations that are unique to the Met^High (CFP⁺) tumor. The y-axis illustrates normalized read count values (low ratio), which are directly proportional to genome copy number at a given chromosomal location. The copy-number profiles are centered around a mean of 1 with gains and deletions called for segments with values higher and lower than the mean, respectively (Methods). C, Representative genome-wide copy-number profiles of three subsampled tissue regions of the Met^High (CFP⁺) primary tumor from m832. Gray shading denotes alterations that are found heterogeneously from multiregion sequencing of the primary tumor. D, Genome-wide heat map with hierarchical clustering based on copy-number alterations of matched primary and metastatic samples profiled from m832. E, Representative genome-wide copy-number profiles of fluorescently matched primary and metastatic tissue from two profiled mice (m832, left; m836, right), illustrating the shared clonal genetic lineage. F, Zoomed-in chromosomal views of copy-number alterations with distinguishing breakpoint patterns supporting shared genetic lineage. Panels are ordered as in E.

Figure 3.

The Met^High phenotype is associated with focal, high-amplitude Myc amplifications and elevated expression. A, Schematic representation of focal amplifications identified in profiled primary tumors. Vertical gray line denotes the location of amplicon and likely driver gene. B, Zoomed-in schematic representation of three identified Myc amplicons in Met^High tumors illustrating the focal and high-amplitude nature of the event (left). Each event (amplicon) is illustrated by a different colored segment line. The shared amplified region between the different amplicons is denoted by the chromosomal cytoband top of panel and illustrated in a UCSC Genome Browser view (right) with RefSeq genes, including Myc, illustrated. C, Box-and-whisker plot showing Myc mRNA levels in Met^High tumors (n = 7) and paired metastases (n = 34) compared with Met^Low tumors (n = 13). FPKM, fragments per kilobase of exon per million. D, Volcano plot illustrating genes meeting cutoffs for differential expression [log fold change (logFC) >1, P_adj. < 0.05] between Met^High and Met^Low tumors (n = 20 tumors used in the comparison). Genes upregulated in Met^High tumors are highlighted in green, and genes upregulated in Met^Low tumors are highlighted in red. E, Top 10 hallmark gene sets identified as enriched in Met^High tumors compared with Met^Low tumors using all differentially expressed genes (DEG; P_adj. < 0.05). F, Top five transcription factor (TF) binding sites enriched in DEGs in Met^High tumors compared with Met^Low tumors (P_adj. < 0.05) identified by Metacore prediction software. G, Heat map showing unsupervised clustering of DEGs (logFC >1, P_adj. < 0.05) between Met^High and Met^Low tumors (n = 20) and their association with PDAC transcriptional subtypes previously reported by Collisson and colleagues (42), Moffitt and colleagues (15), and Bailey and colleagues (16). ADEX, aberrantly differentiated endocrine exocrine; QM-PDA, quasi-mesenchymal-pancreatic ductal adenocarcinoma. H, Kaplan–Meier analysis showing overall survival of patients with PDAC in the TCGA cohort stratified into those with a Met^High signature (red line) versus those with a Met^Low signature (green line). Signature based on DEGs with absolute logFC >0.58 and P_adj. < 0.05 (736 up- and 1,036 downregulated genes). Statistical analysis in C was performed by Wilcoxon test (*, P = 3.9 × 10⁻⁴; **, P = 5.3 × 10⁻⁵). Box and whiskers represent median mRNA expression and interquartile range. Statistical analysis in H was performed by log-rank test.

Figure 4.

MYC regulates metastasis by enhancing tumor cell intravasation. A, Bar graph showing Myc mRNA levels in cell lines derived from Met^High and Met^Low tumors, normalized to Gapdh (n = 6 Met^High and n = 5 Met^Low cell lines). B, Western blot showing corresponding MYC protein levels in cell lines derived from Met^High and Met^Low tumors shown in A. C, Representative fluorescent images of primary tumors and associated liver and lung metastases following orthotopic transplantation of the cell lines in A and B into NOD.SCID mice. The bar graph shows the total number of metastases (liver and lung) counted following orthotopic transplantation of five Met^Low cell lines or five Met^High cell lines (pooled data from n = 49 mice in total). D, Representative fluorescent images of primary tumors, liver metastases, and lung metastases following orthotopic transplantation of Met^Low cell lines that were stably transduced with either a Myc_OE or an empty vector (EV) construct. The bar graph shows the total number of metastases (liver and lung) counted following orthotopic transplantations of Myc_OE or EV cells. Data were pooled from four independent Met^Low lines transduced with either the Myc_OE or EV construct transplanted into 12 NOD.SCID (for the Myc_OE cells) or 10 NOD.SCID mice (for the EV cells). E, Quantification of CTCs in arterial blood derived from the orthotopic tumors depicted in C (n = 27 mice examined) and D (n = 12 mice examined). F, Representative fluorescent images of lung metastases following tail vein injection of cell lines derived from the Met^Low and Met^High primary tumor clones. The bar graph shows the total number of lung metastases counted following tail vein injection of five Met^Low cell lines or five Met^High cell lines (pooled data from n = 36 mice in total). Statistical analysis by Student unpaired t test with significance indicated (*, P = 0.0152; **, P = 0.013; ***, P = 0.0008; ns, not significant). Error bars indicate SEM (C–F). Scale bar, 1 mm (C, D, and F).

Figure 5.

MYC recruits prometastatic macrophages to the tumor microenvironment. A, Representative immunofluorescence images (top) and quantification (bottom) of T cells (CD3⁺), neutrophils (antineutrophil antibody⁺), and macrophages (F4/80⁺) in primary KPCXY tumors categorized as Met^Low or Met^High, with quantification below (n = 3 mice for each subgroup and four to five random fields of view analyzed). B, Representative immunofluorescence images (left) and quantification (right) of macrophages that have migrated across a transwell filter following coculture with Met^High or Met^Low tumor cells (n = 2 Met^Low and n = 2 Met^High cell lines used; three replicates per cell line with three 20× images taken per transwell; each dot represents quantification of an independent image). C, Quantification of tumor-infiltrating macrophages (as a percentage of total CD45⁺ cells) in Met^Low or Met^High subcutaneous tumors assessed by flow cytometry (n = 5 Met^High cell lines and n = 3 Met^Low cell lines; two NOD.SCID mice examined per cell line with two tumors per mouse; each dot represents an independent tumor). D, Quantification of tumor-infiltrating macrophages (as a percentage of total CD45⁺ cells) in Myc_OE or control (EV) subcutaneous tumors assessed by flow cytometry (n = 2 Myc_OE cell lines and n = 2 EV cell lines; two NOD.SCID mice examined per cell line with two tumors per mouse; each dot represents an independent tumor). E and F, Representative immunofluorescence images (left) and quantification (right) of Arg1⁺ (E) and CD206⁺ (F) TAMs in primary KPCXY tumors categorized as Met^Low or Met^High (n = 3 mice for each subgroup and four to five random fields of view analyzed). G and H, Quantification of Arg1⁺ (G) and CD206⁺ (H) TAMs in primary MYC_OE or control (EV) orthotopic tumors assessd by immunflourescence staining (n = 2 Myc_OE cell lines and n = 2 EV cell lines; two NOD.SCID mice examined per cell line; four to five random fields of view analyzed). I, Quantification of tumor cell intravasation from an iTEM assay. MYC_OE– or EV-transduced tumor cells were cultured in transwell filters seeded with an endothelial cell monolayer in the presence or absence of macrophages (see Methods). Tumor cells that traversed the endothelial layer were quantified and normalized to the EV control in the absence of macrophages for each of two Met^Low tumor lines. J, Schematic outline of the macrophage depletion experiment. Mice were orthotopically implanted with Myc_OE cells (n = 2 independent cell lines), and after 10 days, tumor-bearing animals were treated with a combination of colony-stimulating factor receptor inhibitor (CSFRi; GW2580) and liposomal clodronate (CLD) or vehicle. Metastases were quantified 14 days later. K, Quantification of total metastases (liver and lung) following the macrophage depletion strategy outlined in J (n = 6 control mice and n = 7 GW2580 + CLD mice; each dot represents an independent mouse). Statistical analysis (A–H and K) by Student unpaired t test with significance indicated (*, P < 0.05; **, P < 0.005; ***, P < 0.0001; ns, not significant); statistical analysis (I) by two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars indicate SEM. Scale bars, 10 μm (A, E, and F) and 50 μm (B).

Figure 6.

MYC acts through CXCL3 and MIF to promote macrophage recruitment and metastasis. A, Expression of selected cytokines/chemokines in human PDAC. Samples from the COMPASS cohort (enriched for tumor cells by laser capture microdissection) were stratified into MYC-high and MYC-low groups based on RNA-seq (n = 373) and assessed for the expression of five chemokines/cytokines identified as significantly upregulated in Met^High versus Met^Low tumors (Supplementary Fig. S8A). FPKM, fragments per kilobase of exon per million. B, Relative expression of Mif and Cxcl3 in control or Myc knockdown [short hairpin RNA (shRNA)] Met^High cell line 850_Met^High_4. Data are representative of two independent Myc shRNAs (n = 3 biological replicates). C, Bar graph showing fold increase in Cxcl3 and Mif mRNA levels comparing Myc_OE to EV control cell lines. Data are representative of two independent cell lines (n = 3 biological replicates). D, Quantification of total F4/80⁺ tumor-infiltrating macrophages by immunoflourescence in cell lines that were stably transduced with either a Cxcl3 or Mif overexpression construct (Cxcl3_OE and Mif_OE, respectively) or empty vector (EV), with n = 4 tumors examined from each group with four to five random fields of view analyzed. E, Quantification of total metastases (liver and lung) following orthotopic transplantation of EV, Cxcl3_OE, or Mif_OE orthotopic tumors from D. Data were pooled from two independent Met^Low lines transduced with the Cxcl3_OE, Mif_OE, or EV construct transplanted into five NOD.SCID mice (for each cell line). Each dot represents an independent animal. F, Quantification of macrophages that migrated across a transwell filter following coculture with 832 Myc_OE tumor cells treated with either a Cxcr2 inhibitor (AZD5069) or a MIF inhibitor (ISO-1). Data are representative of two independent experiments, including three replicates with four to five 20× images taken per transwell. G, Schematic outline of the CXCR2 and MIF inhibitor experiment. Mice were orthotopically implanted with 832 Myc_OE cells and after 10 days were treated with a CXCR2 inhibitor (AZD5069), MIF inhibitor (ISO-1), combination (AZD5069 + ISO-1), or vehicle. Metastases and macrophages were quantified 14 days later. H and I, Quantification of F4/80⁺ (H) and CD206 (I) macrophages in orthtotopic tumors following the CXCR2 and MIF strategy outlined in G (n = 4 tumors per group; four to five random fields of view analyzed; each dot represents an independent animal). J, Quantification of total metastases (liver and lung) following the CXCR2 and MIF strategy outlined in G (n = 4 control mice, n = 4 AZD5069 mice, n = 4 ISO-1 mice, and n = 4 AZD5069 + ISO-1 mice; each dot represents an independent animal). Statistical analysis by Student t test with significance indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.007; ****, P < 0.0005; *****, P < 0.0001; ns, not significant). Error bars indicate SEM.

Figure 7.

MYC amplification and enhanced transcriptional activity are associated with metastasis in human PDAC. A, Bar graph showing the relative frequencies of MYC amplifications in primary PDAC tumors and metastases from the COMPASS cohort. B, Representative plot of chromosome 8 from a metastatic tumor with MYC amplification. Orientation of breakpoint junctions from intrachromosomal rearrangements indicated by TH, HT, HH, and TT, where T = tail (3′ end of fragment) and H = head (5′ end of fragment). C, Box-and-whisker plot showing MYC mRNA levels (fragments per kilobase of exon per million, FPKM) in primary PDAC tumors and metastases. D, Representative genome-wide absolute copy-number plots of single cells retrieved from a primary (top) and its matched metastasis (bottom) illustrating acquisition of focal MYC amplification in the metastatic lesion. E, Heat-map depiction of cancer single cells (SC) sequenced from a matched primary PDAC and its liver metastasis. Color codes indicate absolute copy number in single cells. Top bar plot depicts tissue site from where single cells were retrieved. F, GSEA of tumors with a major imbalance of mutant KRAS (compared with those with no major imbalance) in the COMPASS cohort. Box-and-whisker plot in C indicates mean and interquartile range.