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. 2018 Aug 12;2018:6013728.
doi: 10.1155/2018/6013728. eCollection 2018.

Differential Regulation of Methylation-Regulating Enzymes by Senescent Stromal Cells Drives Colorectal Cancer Cell Response to DNA-Demethylating Epi-Drugs

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Free PMC article

Differential Regulation of Methylation-Regulating Enzymes by Senescent Stromal Cells Drives Colorectal Cancer Cell Response to DNA-Demethylating Epi-Drugs

Khushboo Agrawal et al. Stem Cells Int. .
Free PMC article

Abstract

The advanced-stage colon cancer spreads from primary tumor site to distant organs where the colon-unassociated stromal population provides a favorable niche for the growth of tumor cells. The heterocellular interactions between colon cancer cells and colon-unassociated fibroblasts at distant metastatic sites are important, yet these cell-cell interactions for therapeutic strategies for metastatic colon cancer remain underestimated. Recent studies have shown the therapeutic potential of DNA-demethylating epi-drugs 5-azacytidine (AZA) and 5-aza-2'-deoxycytidine (DAC) for the treatment of solid tumors. While the effects of these epi-drugs alone or in combination with other anticancer therapies are well described, the influence of stromal cells and their secretome on cancer cell response to these agents remain elusive. In this study, we determined the effect of normal and senescent colon-unassociated fibroblasts and their conditioned medium on colorectal cancer (CRC) cell response to AZA and DAC using a cell-based DNA demethylation reporter system. Our data show that fibroblasts accelerate cell proliferation and differentially regulate the expression of DNA methylation-regulating enzymes, enhancing DAC-induced demethylation in CRC cells. In contrast, the conditioned medium from senescent fibroblasts that upregulated NF-κB activity altered deoxycytidine kinase levels in drug-untreated CRC cells and abrogated DAC effect on degradation of DNA methyltransferase 1. Similar to 2D cultures, senescent fibroblasts increased DNA demethylation of CRC cells in coculture spheroids, in addition to increasing the stemness of CRC cells. This study presents the first evidence of the effect of normal and senescent stromal cells and their conditioned medium on DNA demethylation by DAC. The data show an increased activity of DAC in high stromal cell cocultures and suggest the potential of the tumor-stroma ratio in predicting the outcome of DNA-demethylating epigenetic cancer therapy.

Figures

Figure 1
Figure 1
Effect of fibroblasts and their conditioned medium on HCT116 DNA demethylation in 2D cultures. (a) Representative images showing RFP nuclear fluorescence but no EGFP fluorescence in the untreated control, and changes in EGFP fluorescence following DAC treatment in HCT116 monocultures or cocultures with BJ. 20x objective; scale bar: 100 μm. Graphs showing a significant increase in EGFP intensity in HCT116 cocultured with normal nonirradiated (non-IR) BJ and 1-week-irradiated (1-wk-IR) sBJ fibroblasts in comparison to HCT116 monocultures following treatment with different concentrations of DAC and AZA. Data are mean ± SEM, n = 2–4, ∗∗ p < 0.01, p < 0.05 compared to HCT116 monocultures. (b) A significant increase in EGFP intensity of HCT116 cells cocultured with 3-week-irradiated (3-wk-IR) sBJ or sMRC-5 cells following treatment with 1 μM DAC. (c) Schematic diagram of the setup of conditioned medium culture and graph showing no effect of sBJ or sMRC-5 conditioned medium on EGFP intensity following treatment of HCT116 monocultures with 1 μM DAC. Data are mean ± SEM, n = 2–4.
Figure 2
Figure 2
Changes in the expression of DNMT1 and TET1. Representative western blots and densitometry analysis of DNMT1 and TET1 levels in untreated or DAC-treated (a) HCT116 monocultures and cocultures with normal BJ or sBJ and (b) HCT116 monocultures grown in conditioned medium from normal BJ or sBJ cells. DNMT1 and TET1 blots in (a) and (b) are taken from different gels. Data are mean ± SEM, n = 3, ∗∗∗ p < 0.001, p < 0.05.
Figure 3
Figure 3
Coculture effect on HCT116 proliferation and analysis of secretory factors in conditioned medium. (a, b) The effect of normal and senescent fibroblasts (a) and SASP of senescent fibroblasts (b) on the proliferation of HCT116. Data are mean ± SEM, n = 2–4, ∗∗∗ p < 0.001, ∗∗ p < 0.01, p < 0.05 compared to HCT116 monocultures or 0% conditioned medium, one-sample t-test. (c) Viability of nonirradiated and irradiated HCT116 following DAC treatment. Data are mean ± SEM, n = 4, ∗∗∗ p < 0.001, ∗∗ p < 0.01 comparing cell viability with or without conditioned medium, Student's t-test, unpaired. (d) Increased levels of IFN-α, IL-6, IL-8, and MCP-1 in sBJ conditioned medium compared to conditioned medium from normal BJ cells and its effect on NF-κB activity. Data are mean ± SEM, n = 3, ∗∗∗ p < 0.001, ∗∗ p < 0.01 comparing conditioned medium from BJ to sBJ, Student's t-test, unpaired.
Figure 4
Figure 4
Fibroblasts and conditioned medium-induced effects on HCT116 susceptibility to DAC in spheroid cultures and expression of EMT markers. (a) Images of high stromal cell coculture spheroids showing the presence of BJ cells in the spheroid interior. The z-plane heights are indicated on the top of images. HCT116 are nonfluorescent and the exterior of the spheroid is stained blue with Hoechst. 20x objective; scale bar: 50 μm. (b) Increase in EGFP intensity in coculture spheroids of HCT116 and BJ or sBJ cells compared to HCT116 monoculture spheroids. Data are mean ± SEM, n > 20 spheroids per group from 3 independent experiments, ∗∗∗ p < 0.001, p < 0.05, Kruskal-Wallis test with Dunnet's multiple comparisons test. (c) Representative images showing the effect of DAC on HCT116 monoculture spheroids and coculture spheroids of HCT116 and BJ fibroblasts. 20x objective; scale bar: 10 μm. (d) The effect of conditioned medium from normal and sBJ cultures on DAC-induced demethylation of HCT116 monoculture spheroids. Data are mean ± SEM, n > 20 spheroids per group from 3 independent experiments, one-sample t-test. (e) Increase in the size of untreated coculture spheroids of HCT116 and BJ or sBJ compared to HCT116 monoculture spheroids (left) and no effect of DAC treatment on coculture spheroid size (right). Data are mean ± SEM, n > 20 spheroids per group from 3 independent experiments, ∗∗∗ p < 0.001, p < 0.05, Kruskal-Wallis test with Dunnet's multiple comparison test. (f) Representative Western blots showing the induction of vimentin expression in HCT116 sorted from high and low stromal cell coculture spheroids of HCT116 and sBJ.

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