Hypomethylating therapy in an aggressive stroma-rich model of pancreatic carcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that resists current treatments. To test epigenetic therapy against this cancer, we used the DNA demethylating drug 5-aza-2'-deoxycytidine (DAC) in an aggressive mouse model of stromal rich PDAC (KPC-Brca1 mice). In untreated tumors, we found globally decreased 5-methyl-cytosine (5-mC) in malignant epithelial cells and in cancer-associated myofibroblasts (CAF), along with increased amounts of 5-hydroxymethyl-cytosine (5-HmC) in CAFs, in progression from pancreatic intraepithelial neoplasia to PDAC. DAC further reduced DNA methylation and slowed PDAC progression, markedly extending survival in an early-treatment protocol and significantly though transiently inhibiting tumor growth when initiated later, without adverse side effects. Escaping tumors contained areas of sarcomatoid transformation with disappearance of CAFs. Mixing-allografting experiments and proliferation indices showed that DAC efficacy was due to inhibition of both the malignant epithelial cells and the CAFs. Expression profiling and immunohistochemistry highlighted DAC induction of STAT1 in the tumors, and DAC plus IFN-γ produced an additive antiproliferative effect on PDAC cells. DAC induced strong expression of the testis antigen deleted in azoospermia-like (DAZL) in CAFs. These data show that DAC is effective against PDAC in vivo and provide a rationale for future studies combining hypomethylating agents with cytokines and immunotherapy.

Figure 1. Survival and histopathology in the KPC-Brca1 rapid-onset model of PDAC

A, Kaplan-Meier survival curve for Kras^LSL-G12D; p53^LSL-R270H/+; Pdx1-cre; Brca1^flex2/flex2 (KPC-Brca1) mice compared to Kras^LSL-G12D; p53^LSL-R270H/+; Pdx1-cre (KPC) mice. The KPC-Brca1 model results in a significantly faster progression of cancer with a T₅₀ of 88 days (n=32) compared to a T₅₀ of 150 days in KPC mice (n=28; p<0.001). B, PanIN lesion consisting of proliferating p53^R270H-positive dysplastic epithelial cells, surrounded by a dense cuff of reactive non-neoplastic stromal myofibroblasts, which are p53-negative. C–E, Progression from early PanIN to late PanIN and invasive adenocarcinoma is associated with an accumulation of ASMA-positive cancer-associated myofibroblasts (CAFs; asterisks).

Figure 2. Stage-specific and cell type-specific levels of genomic 5mC and 5HmC and DNMT1 in stromal and epithelial cells during tumor progression

A, The section of early PanIN and adjacent histologically normal pancreatic parenchyma was subjected to triple-IF with antibodies recognizing vimentin (blue), 5mC (green) and 5HmC (red). Most of the proliferating epithelial cells in the early PanIN lesion (asterisk) stain strongly for 5mC and only weakly for 5HmC. The 5mC signal is partly punctate in mouse cell nuclei, presumably reflecting concentrations of heterochromatin, while the 5HmC signal is more diffuse. The reactive stromal myofibroblasts (arrows) are primarily staining for 5mC but are starting to accumulate 5HmC. B, Late PanIN lesion in which the majority of vimentin-positive CAFs (arrows) show a stronger IF signal for 5HmC than for 5mC. There is a moderate reduction in the average 5mC IF signal in the proliferating dysplastic epithelial cells of the PanIN lesions (asterisk) but, contrasting with the stromal cells, this is not accompanied by a strong increase in 5HmC. C, In PDAC there is reduced 5mC signal intensity in the malignant epithelial cells (quantitative IF measurements in Suppl. Fig. S1), with only a weak accumulation of 5HmC, while the proliferating CAFs show even less staining for 5mC, contrasting with a very strong signal for 5HmC.

Figure 3. Low doses of DAC inhibit tumor PDAC tumor progression in KPC-Brca1 mice

A, Results of the initial pilot experiment in which DAC or PBS control injections were started at 3 weeks of age, with necropsies performed at a single time point after 5 weeks of treatment. Pancreatic weight is mostly accounted for by tumor mass (see part B). B, Gross photographs of representative pancreata from the pilot experiment. C, Levels of 5mC in genomic DNA measured by the cytosine incorporation assay in tumors from the experiments in which mice were treated with DAC or PBS vehicle control for 6–7 weeks. D, Kaplan-Meier survival curves showing the highly significant anti-tumor effects of DAC when started either at 3 weeks (all mice with PanIN at the start of therapy; few with invasive lesions) or at 8 weeks of age (all mice with invasive PDAC at the start of therapy). E, Reduction in proliferation index (% Ki67-positive cells per high power field in 10 fields in 7 cases) by DAC in both CAFs and malignant epithelial cells.

Figure 4. Tumor reconstitution-allografting experiment and reduced promoter CpG methylation of the STAT1 gene in PDAC cells after DAC treatment

A, Short term cultures of pancreatic CAFs or PDAC epithelial cells from the KPC-Brca1 tumors were pre-treated with DAC or grown without the drug, followed by mixing of the two types of cells and allografting into the flanks of nude mice. Mice were sacrificed after 3 weeks and the tumor diameters were measured. As indicated by comparing condition (A) to condition (B), the presence of CAFs promotes tumor growth. As indicated by comparing condition (B) to condition (C), pre-treating the carcinoma cells alone significantly reduces the growth of the tumor allografts. As indicated by comparing condition (D) to condition (E), treating the CAFs as well before mixing them with the malignant epithelial cells leads to the greatest net anti-tumor effect. B, Bisulfite sequencing of genomic DNA from PDAC tumor cells exposed to 0 or 0.5 μM DAC in short-term culture, and whole PDAC tumors from mice receiving PBS vehicle control or DAC shows partial demethylation of a potential regulatory region overlapping a block of histone H3K4m1 chromatin modification (from ENCODE/LICR fetal liver ChIP-Seq data) extending from approximately 0.6 to 1kb upstream of the STAT1 first exon. The bar graph summarizes the bisulfite sequencing data (% methylated CpG’s in amplicon a) for the indicated number of in vivo-treated tumors analyzed. Bisulfite sequencing of the two amplicons (b, c) overlapping the STAT1 CpG island (green) showed nearly complete lack of CpG methylation in both DAC-treated and untreated tumor samples (data not shown).

Figure 5. DAC produces strong increases of STAT1 in PDAC epithelial cells and DAZL in CAFs in the KPC-Brca1 tumors in vivo

A, Immunostaining for STAT1 and STAT2 proteins in PDAC tumors from mice that received DAC, or PBS vehicle control shows that DAC induces both proteins, with particularly strong activation of STAT1 expression. B, IHC and IF showing strong induction of the testis antigen DAZL in the stromal myofibroblasts of the DAC-treated tumors.

Figure 6. DAC and the JAK-STAT activator interferon-γ have an additive anti-proliferative effect on PDAC cells

Primary PDAC epithelial cells isolated from the KPC-Brca1 mice and grown in short-term culture were treated for 4 days with the indicated single agents or combinations of low-dose DAC (0.5 μM) and interferon-γ (100 ng/ml) as described in Methods, followed by MTT assays for relative numbers of viable cells. The results show an additive anti-proliferative effect of DAC plus interferon-γ.