Sex steroid-induced DNA methylation changes and inflammation response in prostate cancer.

BACKGROUND: Sex steroid hormones have been reported to induce inflammation causing dysregulation of cytokines in prostate cancer cells. However, the underlying epigenetic mechanism has not well been studied. The objective of this study was to evaluate the effect of sex steroid hormones on epigenetic DNA methylation changes in prostate cancer cells using a signature PCR methylation array panel that correspond to 96 genes with biological function in the human inflammatory and autoimmune signals in prostate cancer. Of the 96-gene panel, 32 genes showed at least 10% differentially methylation level in response to hormonal treatment when compared to untreated cells. Genes that were hypomethylated included CXCL12, CXCL5, CCL25, IL1F8, IL13RAI, STAT5A, CXCR4 and TLR5; and genes that were hypermethylated included ELA2, TOLLIP, LAG3, CD276 and MALT1. Quantitative RT-PCR analysis of select genes represented in a cytokine expression array panel showed inverse association between DNA methylation and gene expression for TOLLIP, CXCL5, CCL18 and IL5 genes and treatment of prostate cancer cells with 5'-aza-2'-deoxycytidine with or without trichostatin A induced up-regulation of TOLLIP expression. Further analysis of relative gene expression of matched prostate cancer tissues when compared to benign tissues from individual patients with prostate cancer showed increased and significant expression for CCL18 (2.6-fold; p<0.001), a modest yet significant increase in IL5 expression (1.17-fold; p=0.015), and a modest increase in CXCL5 expression (1.4-fold; p=0.25). In conclusion, our studies demonstrate that sex steroid hormones can induce aberrant gene expression via differential methylation changes in prostate carcinogenesis.

Knockdown of estrogen receptor-α induces autophagy and inhibits antiestrogen-mediated unfolded protein response activation, promoting ROS-induced breast cancer cell death.

Approximately 70% of all newly diagnosed breast cancers express estrogen receptor (ER)-α. Although inhibiting ER action using targeted therapies such as fulvestrant (ICI) is often effective, later emergence of antiestrogen resistance limits clinical use. We used antiestrogen-sensitive and -resistant cells to determine the effect of antiestrogens/ERα on regulating autophagy and unfolded protein response (UPR) signaling. Knockdown of ERα significantly increased the sensitivity of LCC1 cells (sensitive) and also resensitized LCC9 cells (resistant) to antiestrogen drugs. Interestingly, ERα knockdown, but not ICI, reduced nuclear factor (erythroid-derived 2)-like (NRF)-2 (UPR-induced antioxidant protein) and increased cytosolic kelch-like ECH-associated protein (KEAP)-1 (NRF2 inhibitor), consistent with the observed increase in ROS production. Furthermore, autophagy induction by antiestrogens was prosurvival but did not prevent ERα knockdown-mediated death. We built a novel mathematical model to elucidate the interactions among UPR, autophagy, ER signaling, and ROS regulation of breast cancer cell survival. The experimentally validated mathematical model explains the counterintuitive result that knocking down the main target of ICI (ERα) increased the effectiveness of ICI. Specifically, the model indicated that ERα is no longer present in excess and that the effect on proliferation from further reductions in its level by ICI cannot be compensated for by increased autophagy. The stimulation of signaling that can confer resistance suggests that combining autophagy or UPR inhibitors with antiestrogens would reduce the development of resistance in some breast cancers.

Endoplasmic Reticulum Stress Protein GRP78 Modulates Lipid Metabolism to Control Drug Sensitivity and Antitumor Immunity in Breast Cancer.

The unfolded protein response is an endoplasmic reticulum stress pathway mediated by the protein chaperone glucose regulated-protein 78 (GRP78). Metabolic analysis of breast cancer cells shows that GRP78 silencing increases the intracellular concentrations of essential polyunsaturated fats, including linoleic acid. Accumulation of fatty acids is due to an inhibition of mitochondrial fatty acid transport, resulting in a reduction of fatty acid oxidation. These data suggest a novel role of GRP78-mediating cellular metabolism. We validated the effect of GRP78-regulated metabolite changes by treating tumor-bearing mice with tamoxifen and/or linoleic acid. Tumors treated with linoleic acid plus tamoxifen exhibited reduced tumor area and tumor weight. Inhibition of either GRP78 or linoleic acid treatment increased MCP-1 serum levels, decreased CD47 expression, and increased macrophage infiltration, suggesting a novel role for GRP78 in regulating innate immunity. GRP78 control of fatty acid oxidation may represent a new homeostatic function for GRP78. Cancer Res; 76(19); 5657-70. ©2016 AACR.

Targeting GRP78 and antiestrogen resistance in breast cancer.

Breast cancer is the most prevalent cancer in women, with over 200,000 new cases diagnosed each year. Over 70% of breast cancers express the estrogen receptor-α, and drugs targeting these receptors such as tamoxifen or Faslodex(®) often fail to cure these patients. Many estrogen receptor-positive tumors lose drug sensitivity, making endocrine resistance a major clinical problem. Recently, investigation into the molecular mechanisms of endocrine resistance has highlighted a causative role of the unfolded protein response in antiestrogen resistance. In particular, the master regulator of the unfolded protein response, GRP78, was observed to be elevated in endocrine-resistant breast cancer and directly affected antiestrogen therapy responsiveness. GRP78 was found to impact many different cellular processes that may affect breast cancer survival. Recently, various compounds have been reported to affect GRP78 activity and it may be advantageous to combine these drugs with antiestrogens to overcome endocrine therapy resistance.

Modelling the effect of GRP78 on anti-oestrogen sensitivity and resistance in breast cancer.

Understanding the origins of resistance to anti-oestrogen drugs is of critical importance to many breast cancer patients. Recent experiments show that knockdown of GRP78, a key gene in the unfolded protein response (UPR), can re-sensitize resistant cells to anti-oestrogens, and overexpression of GRP78 in sensitive cells can cause them to become resistant. These results appear to arise from the operation and interaction of three cellular systems: the UPR, autophagy and apoptosis. To determine whether our current mechanistic understanding of these systems is sufficient to explain the experimental results, we built a mathematical model of the three systems and their interactions. We show that the model is capable of reproducing previously published experimental results and some new data gathered specifically for this paper. The model provides us with a tool to better understand the interactions that bring about anti-oestrogen resistance and the effects of GRP78 on both sensitive and resistant breast cancer cells.