Vitamin D Induces Global Gene Transcription in Human Corneal Epithelial Cells: Implications for Corneal Inflammation

Purpose: Our previous studies show that human corneal epithelial cells (HCEC) have a functional vitamin D receptor (VDR) and respond to vitamin D by dampening TLR-induced inflammation. Here, we further examined the timing of the cytokine response to combined vitamin D–TLR treatment and used genome-wide microarray analysis to examine the effect of vitamin D on corneal gene expression. Methods: Telomerase-immortalized HCEC (hTCEpi) were stimulated with polyinosinic-polycytidylic acid (poly[I:C]) and 1,25-dihydroxyvitamin D3 (1,25D3) for 2 to 24 hours and interleukin (IL)-8 expression was examined by quantitative (q)PCR and ELISA. Telomerase-immortalized HCEC and SV40-HCEC were treated with 1,25D3 and used in genome-wide microarray analysis. Expression of target genes was validated using qPCR in both cell lines and primary HCEC. For confirmation of IκBα protein, hTCEpi were treated with 1,25D3 for 24 hours and cell lysates used in an ELISA. Results: Treatment with 1,25D3 increased poly(I:C)-induced IL-8 mRNA and protein expression after 2 to 6 hours. However, when cells were pretreated with 1,25D3 for 24 hours, 1,25D3 decreased cytokine expression. For microarray analysis, 308 genes were differentially expressed by 1,25D3 treatment in hTCEpi, and 69 genes in SV40s. Quantitative (q)PCR confirmed the vitamin D–mediated upregulation of target genes, including nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα). In addition to increased transcript levels, IκBα protein was increased by 28% following 24 hours of vitamin D treatment. Conclusions: Microarray analysis demonstrates that vitamin D regulates numerous genes in HCEC and influences TLR signaling through upregulation of IκBα. These findings are important in dissecting the role of vitamin D at the ocular surface and highlight the need for further research into the functions of vitamin D and its influence on corneal gene expression

Deubiquitinase UCHL1 Maintains Protein Homeostasis through the PSMA7–APEH–Proteasome Axis in High-grade Serous Ovarian Carcinoma

High-grade serous ovarian cancer (HGSOC) is characterized by chromosomal instability, DNA damage, oxidative stress, and high metabolic demand that exacerbate misfolded, unfolded, and damaged protein burden resulting in increased proteotoxicity. However, the underlying mechanisms that maintain protein homeostasis to promote HGSOC growth remain poorly understood. This study reports that the neuronal deubiquitinating enzyme, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), is overexpressed in HGSOC and maintains protein homeostasis. UCHL1 expression was markedly increased in HGSOC patient tumors and serous tubal intraepithelial carcinoma (HGSOC precursor lesions). High UCHL1 levels correlated with higher tumor grade and poor patient survival. UCHL1 inhibition reduced HGSOC cell proliferation and invasion, as well as significantly decreased the in vivo metastatic growth of ovarian cancer xenografts. Transcriptional profiling of UCHL1-silenced HGSOC cells revealed downregulation of genes implicated with proteasome activity along with upregulation of endoplasmic reticulum stress–induced genes. Reduced expression of proteasome subunit alpha 7 (PSMA7) and acylaminoacyl peptide hydrolase (APEH), upon silencing of UCHL1, resulted in a significant decrease in proteasome activity, impaired protein degradation, and abrogated HGSOC growth. Furthermore, the accumulation of polyubiquitinated proteins in the UCHL1-silenced cells led to attenuation of mTORC1 activity and protein synthesis, and induction of terminal unfolded protein response. Collectively, these results indicate that UCHL1 promotes HGSOC growth by mediating protein homeostasis through the PSMA7–APEH–proteasome axis.This study identifies the novel links in the proteostasis network to target protein homeostasis in HGSOC and recognizes the potential of inhibiting UCHL1 and APEH to sensitize cancer cells to proteotoxic stress in solid tumors

Deciphering the Molecular Mechanisms of Estrogen Signaling in Gastrointestinal Tumors

Colorectal and pancreatic cancers are predominant gastrointestinal (GI) tumors with estimated 90,970 deaths in the United Sates in 2016, representing ~ 60% of the total GI tumors related-mortalities. Several investigations, including meta-analysis, preclinical, and in vitro studies have established the protective role of estrogens and related receptors against GI tumors. The main estrogen nuclear receptor in the colon is estrogen receptor beta (ERβ/ESR2). During colon cancer progression, ERβ expression is considerably reduced. Re-expressing ERβ in colon cancer cell line (SW480) induces significant changes in miRnome. miR-205 is among the upregulated genes which directly targets the oncogene PROX1. In vivo studies demonstrated that both ERβ and miR-205 exert anti-metastatic effects. SW620, a highly metastasized human colorectal cancer cell line was used for further analysis. This cell line dose not express any ERα or ERβ, and ERβ was introduced using lentiviral. Here we show that 17β-estradiol (E2) has an ERβ-dependent as well as ERβ-independent effects suggesting possible role of alternative receptors in estrogen signaling in colonic epithelium such as G-protein coupled estrogen receptor 1 (GPER1). In response to E2 or GPER1-selective agonist G1 treatments, several oncogenic long non-coding RNAs (lncRNAs) show downregulation, including MALAT1, NEAT1, ZEB1-AS1 and HOTAIR. Several of these lncRNAs are involved in epithelial-to-mesenchymal transition (EMT) and tumor metastasis. Collectively, our data demonstrate that estrogen singling in the colon has anti-metastatic effects by modulating the expression of the cancer-related noncoding RNAs. EMT transition is frequently linked to a chemoresistance phenotype, which is a common phenomenon in pancreatic cancer. Genistein, a phytoestrogen, has a chemoenhancing effect when it combined with gemcitabine in pancreatic cancer cell lines (PANC1 and MiaPaCa2). Using RNA-seq, genistein induces expression of genes that are related to apoptosis, calcium signaling, and endoplasmic reticulum stress, which can all be linked to enhanced GPER1 activity. Genistein treatment also reduces the expression of several inflammation related genes including MUC1. We demonstrate that G1 treatment similarly reduces proliferation, but dose not fully mimic the apoptotic features of genistein. Collectively, these data indicate that using an estrogenic compound, such as genistein, may enhance the anti-proliferative and apoptotic effects of chemotherapeutic agents such as gemcitabine.Biology and Biochemistry, Department o

Genome-wide effects of MELK-inhibitor in triple-negative breast cancer cells indicate context-dependent response with p53 as a key determinant

Triple-negative breast cancer (TNBC) is an aggressive, highly recurrent breast cancer subtype, affecting approximately one-fifth of all breast cancer patients. Subpopulations of treatment-resistant cancer stem cells within the tumors are considered to contribute to disease recurrence. A potential druggable target for such cells is the maternal embryonic leucine-zipper kinase (MELK). MELK expression is upregulated in mammary stem cells and in undifferentiated cancers, where it correlates with poor prognosis and potentially mediates treatment resistance. Several MELK inhibitors have been developed, of which one, OTSSP167, is currently in clinical trials. In order to better understand how MELK and its inhibition influence TNBC, we verified its anti-proliferative and apoptotic effects in claudin-low TNBC cell lines MDA-MB-231 and SUM-159 using MTS assays and/or trypan blue viability assays together with analysis of PARP cleavage. Then, using microarrays, we explored which genes were affected by OTSSP167. We demonstrate that different sets of genes are regulated in MDA-MB-231 and SUM-159, but in both cell lines genes involved in cell cycle, mitosis and protein metabolism and folding were regulated. We identified p53 (TP53) as a potential upstream regulator of the regulated genes. Using western blot we found that OTSSP167 downregulates mutant p53 in all tested TNBC cell lines (MDA-MB-231, SUM-159, and BT-549), but upregulates wild-type p53 in the luminal A subtype MCF-7 cell line. We propose that OTSSP167 might have context-dependent or off-target effects, but that one consistent mechanism of action could involve the destabilization of mutant p53.QC 20170315</p

Differentially expressed genes identified by microarrays.

<p>A) Graphical representation of significantly regulated genes in MDA-MB-231 compared with SUM-159 cells after treatment with 45 nM OTS167 for 72h. Genes were considered regulated if p<0.05 and M > |0.4|. B) FOXM1 was identified as an enriched transcriptional regulator of the differentially expressed genes. FOXM1 total protein level was confirmed to decrease significantly upon OTS167 treatments. Cells were treated with different concentrations of OTS167 for 48h, and 20nM of OTS167 was sufficient to decrease FOXM1 protein levels in MDA-MB-231 and 45 nM in SUM-159 cells. Actin used a loading control, and densitometric data for relative band density is noted below.</p

qPCR confirms gene expression changes identified in the microarrays.

<p>(A) MDA-MB-231, (B) SUM-159, and (C) BT-549 after 48h and 72h treatments of 45 nM and 4.5 nM OTS167. Significance was calculated using the Student’s two-tailed t-test: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Data for MDA-MB-231 consists of three experiments, data for SUM-159 represents one experiment and data for BT-549 represents two experiments. All experiments were performed in triplicates.</p

Biological processes enriched among OTS167 upregulated genes.

<p>Biological processes enriched among OTS167 upregulated genes.</p

Biological processes enriched among OTS167 downregulated genes.

<p>Biological processes enriched among OTS167 downregulated genes.</p

OTS167 reduces transcript and protein levels of mutant p53.

<p>(A) qPCR reveals that mutant p53 transcript is significantly downregulated in MDA-MB-231, BT-549 and SUM-159 after 48h and 72h (45 nM) OTS167 treatment. (B) Western blots show that mutant p53 protein is significantly reduced in MDA-MB-231, BT-549 and SUM-159, but WT p53 is increased in MCF-7 (48h, 45 nM OTS167, densitometric data from three experiments). (C) p53 protein reduction is independent of cell density (MDA-MB-231, 96h, 45nM OTS167). Significance was calculated using a Student’s two-tailed t-test: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).</p