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Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis
Published on: November 26, 2015
A reciprocal role of prostate cancer on stromal DNA damage
J Banerjee1, R Mishra2, X Li1
1Department of Urology, Vanderbilt University, Nashville, TN, USA.
Abstract:
DNA damage found in prostate cancer-associated fibroblasts (CAF) promotes tumor progression. In the absence of somatic mutations in CAF, epigenetic changes dictate how stromal coevolution is mediated in tumors. Seventy percent of prostate cancer patients lose expression of transforming growth factor-beta type II receptor (TGFBR2) in the stromal compartment (n=77, P-value=0.0001), similar to the rate of glutathione S-transferase P1 (GSTP1) silencing. Xenografting of human prostate cancer epithelia, LNCaP, resulted in the epigenetic Tgfbr2 silencing of host mouse prostatic fibroblasts. Stromal Tgfbr2 promoter hypermethylation, initiated by LNCaP cells, was found to be dependent on interleukin 6 expression, based on neutralizing antibody studies. We further found that pharmacologic and transgenic knockout of TGF-β responsiveness in prostatic fibroblasts induced Gstp1 promoter methylation. It is known that TGF-β promotes DNA stability, however, the mechanism is not well understood. Both prostatic human CAF and mouse transgenic knockout of Tgbr2 had elevated DNA methyltransferase I (DNMT1) activity and histone H3 lysine 9 trimethylation (H3K9me3) to suggest greater promoter methylation. Interestingly, the conditional knockout of Tgfbr2 in mouse prostatic fibroblasts, in modeling epigenetic silencing of Tgfbr2, had greater epigenetic gene silencing of multiple DNA damage repair and oxidative stress response genes, based on promoter methylation array analysis. Homologous gene silencing was validated by reverse transcriptase (RT)-PCR in mouse and human prostatic CAF. Not surprisingly, DNA damage repair gene silencing in the prostatic stromal cells corresponded with the presence of DNA damage. Restoring the expression of the epigenetically silenced genes in wild-type fibroblasts with radiation-induced DNA damage reduced tumor progression. Tumor progression was inhibited even when epigenetic silencing was reversed in the Tgfbr2-knockout prostatic fibroblasts. Taken together, fibroblastic epigenetic changes causative of DNA damage, initiated by association with cancer epithelia, is a dominant mediator of tumor progression over TGF-β responsiveness.
Insights
Prostate cancer-associated fibroblasts (CAF) with DNA damage promote tumor growth. Epigenetic changes in CAF, not mutations, drive this, leading to gene silencing and increased DNA damage, accelerating cancer progression.
Area of Science:
- Oncology
- Epigenetics
- Cancer Biology
Background:
- Prostate cancer-associated fibroblasts (CAF) contribute to tumor progression through DNA damage.
- Epigenetic alterations, rather than somatic mutations, mediate stromal coevolution in tumors.
- Loss of transforming growth factor-beta type II receptor (TGFBR2) expression in stromal cells is common in prostate cancer.
Purpose of the Study:
- To investigate the role of epigenetic changes in CAF-driven prostate tumor progression.
- To elucidate the mechanism by which cancer epithelia induce epigenetic silencing in fibroblasts.
- To determine the impact of TGF-β pathway alterations on DNA damage and repair in stromal cells.
Main Methods:
- Xenografting of human prostate cancer cells (LNCaP) with mouse fibroblasts.
- Analysis of Tgfbr2 and Gstp1 promoter methylation.
- Pharmacologic and genetic manipulation of TGF-β responsiveness in fibroblasts.
- Measurement of DNA methyltransferase I (DNMT1) activity and histone modifications (H3K9me3).
- Promoter methylation array analysis of DNA damage repair and oxidative stress response genes.
- Validation of gene silencing using RT-PCR.
- Experiments restoring gene expression in fibroblasts.
Main Results:
- LNCaP cells induced epigenetic silencing of Tgfbr2 in mouse fibroblasts via interleukin 6-dependent promoter hypermethylation.
- Pharmacologic or genetic inhibition of TGF-β responsiveness in fibroblasts led to Gstp1 promoter methylation.
- Fibroblasts with silenced Tgfbr2 exhibited increased DNMT1 activity and H3K9me3, suggesting enhanced promoter methylation.
- Epigenetic silencing of Tgfbr2 in fibroblasts resulted in broader silencing of DNA damage repair and oxidative stress genes.
- Silencing of these genes in stromal cells correlated with increased DNA damage.
- Restoring gene expression in fibroblasts reduced tumor progression, even in Tgfbr2-knockout models.
Conclusions:
- Epigenetic modifications in fibroblasts, initiated by cancer epithelia, are a primary driver of prostate tumor progression.
- Fibroblastic DNA damage, resulting from epigenetic changes, plays a dominant role over TGF-β responsiveness in tumor advancement.
- Targeting fibroblastic epigenetic alterations offers a potential therapeutic strategy for prostate cancer.
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