Faculty Bibliography

Most transcripts from human genomes are non-coding RNAs (ncRNAs) that are not translated into proteins. ncRNAs are divided into long (lncRNAs) and small non-coding RNAs (sncRNAs). LncRNAs regulate their target genes both transcriptionally and post-transcriptionally through interactions with proteins, RNAs, and DNAs. Maternally expressed gene 3 (MEG3), a lncRNA, functions as a tumor suppressor. MEG3 regulates cell proliferation, cell cycle, apoptosis, hypoxia, autophagy, and many other processes involved in tumor development. MEG3 is downregulated in various cancer cell lines and primary human cancers. Heavy metals, such as hexavalent chromium (Cr(VI)), arsenic, nickel, and cadmium, are confirmed human carcinogens. The exposure of cells to these metals causes a variety of cancers. Among them, lung cancer is the one that can be induced by exposure to all of these metals. In vitro studies have demonstrated that the chronic exposure of normal human bronchial epithelial cells (BEAS-2B) to these metals can cause malignant cell transformation. Metal-transformed cells have the capability to cause an increase in cell proliferation, resistance to apoptosis, elevated migration and invasion, and properties of cancer stem-like cells. Studies have revealed that MEG is downregulated in Cr(VI)-transformed cells, nickel-transformed cells, and cadmium (Cd)-transformed cells. The forced expression of MEG3 reduces the migration and invasion of Cr(VI)-transformed cells through the downregulation of the neuronal precursor of developmentally downregulated protein 9 (NEDD9). MEG3 suppresses the malignant cell transformation of nickel-transformed cells. The overexpression of MEG3 decreases Bcl-xL, causing reduced apoptosis resistance in Cd-transformed cells. This paper reviews the current knowledge of lncRNA MEG3 in metal carcinogenesis.

Cadmium (Cd) is a heavy metal found in cigarette smoke, as well as in air and drinking water due to agricultural and industrial activities, and it poses a health risk to the general population. Prolonged low-dose Cd exposure via inhalation or ingestion causes lung and kidney cancers in humans and in animal models. While high doses of Cd exposure are correlated with the occupational setting and are cytotoxic, low doses of Cd are mainly correlated with exposure in the general population and induce carcinogenesis. The mechanism by which Cd-exposed cells overcome calcium chelation and induce malignant transformation remains unclear. This study examines how cells exposed to low doses of Cd survive loss of E-cadherin cell-cell adhesion via activation of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription 5 (STAT5), which work to upregulate genes associated with survival and proliferation. To demonstrate the role of Cd in EGFR/STAT5 activation, we exposed two epithelial cell lines, BEAS-2B and HEK293, to two different doses (0.4 µM and 1.6 µM) of Cadmium chloride hemipentahydrate (CdCl₂·2.5H₂O) that are environmentally relevant to levels of Cd found in food and cigarettes for 24 h (hours) and 9 weeks (wks). When comparing cells treated with Cd with control cells, the Cd treated cells exhibited faster proliferation; therefore, we studied activation of EGFR via the STAT5 pathway using immunofluorescence (IF) for protein expression and localization and, in addition, RT-qPCR to examine changes in EGFR/STAT5 inducible genes. Our results showed an increase in EGFR and phosphorylated EGFR (p-EGFR) protein, with 1.6 µM of Cadmium having the highest expression at both 24-hour (hr) and 9-week (wk) exposures. Moreover, the IF analysis also demonstrated an increase of STAT5 and phosphorylated STAT5 (pSTAT5) in both short-term and long-term exposure, with 0.4 µM having the highest expression at 24 h. Finally, via Western blot analysis, we showed that there was a dose-dependent decrease in E-cadherin protein expression and increased N-cadherin in cells treated with low doses of Cd. These data demonstrate that epithelial cells can overcome Cd-mediated toxicity via activation of EGFR pathway to induce cell proliferation and survival and promote epithelial to mesenchymal transition.

Canonical histone messenger RNAs (mRNAs) are transcribed during S phase and do not terminate with a poly(A) tail at the 3' end. Instead, the histone mRNAs display a stem-loop structure at their 3-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. We previously demonstrated that exposure to arsenic, an environmental carcinogen, induces polyadenylation of canonical histone H3.1 mRNA, causing transformation of human cells in vitro. Arsenic decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Similarly, we also reported that nickel and arsenic have similar effects on canonical histone mRNA transcription and translation. Most recently, we further demonstrated that bisphenols' exposure increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. This facilitates the abnormal stability of at least one canonical histone isoform (H3.1), and also increases H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic, nickel and bisphenols exposure may contribute to metal and bisphenol-induced carcinogenesis.

Arsenic ranks at the top among all toxic metals and poses a serious threat to human health. Inorganic arsenite and arsenate compounds have been classified as human carcinogens in various types of cancers. Maternally expressed gene 3 (MEG3), a tumor suppressor that is commonly lost in cancer, was investigated in this study for its role in the migration and invasion of arsenic-transformed cells. Our results showed that MEG3 was downregulated in both arsenic-transformed cells (As-T) and cells treated with low doses of arsenic for three months (As-treated). The analysis using TCGA dataset revealed that MEG3 expression was significantly reduced in the tumor tissues from human lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) compared to normal lung tissues. The results from the methylation-specific PCR (MSP) assay demonstrated enhanced methylation in the MEG3 promoters in both As-T and As-treated cells, indicating that increased methylation of the MEG3 promoter caused MEG3 downregulation in these cells. Moreover, As-T cells displayed increased migration and invasion and higher levels of NAD(P)H quinone dehydrogenase 1 (NQO1) and fascin actin-bundling protein 1 (FSCN1). Consistently, the results from immunohistochemistry staining showed that both NQO1 and FSCN1 are highly expressed in human lung squamous cell carcinoma tissues compared to those in normal lungs. Knockdown of MEG3 in normal BEAS-2B cells also led to increased migration and invasion, along with elevated levels of NQO1 and FSCN1. The negative regulation of MEG3 on FSCN1 was restored by NQO1 overexpression in both As-T and BEAS-2B cells. The results from immunoprecipitation assays confirmed the direct binding of NQO1 to FSCN1. Overexpression of NQO1 increased migration and invasion abilities in BEAS-2B cells, while knockdown of NQO1 by its shRNA reduced these two hallmarks of cancer. Interestingly, the reduced migration and invasion by NQO1 knockdown were restored by FSCN1. Collectively, the loss of MEG3 upregulated NQO1, which in turn stabilized FSCN1 protein through its direct binding, resulting in elevated migration and invasion in arsenic-transformed cells.

Emerging evidence suggests that extracellular vesicles (EVs), which represent a crucial mode of intercellular communication, play important roles in cancer progression by transferring oncogenic materials. Nickel (Ni) has been identified as a human group I carcinogen; however, the underlying mechanisms governing Ni-induced carcinogenesis are still being elucidated. Here, we present data demonstrating that Ni exposure generates EVs that contribute to Ni-mediated carcinogenesis and cancer progression. Human bronchial epithelial (BEAS-2B) cells and human embryonic kidney-293 (HEK293) cells were chronically exposed to Ni to generate Ni-treated cells (Ni-6W), Ni-transformed BEAS-2B cells (Ni-3) and Ni-transformed HEK293 cells (HNi-4). The signatures of EVs isolated from Ni-6W, Ni-3, HNi-4, BEAS-2B, and HEK293 were analyzed. Compared to their respective untreated cells, Ni-6W, Ni-3, and HNi-4 released more EVs. This change in EV release coincided with increased transcription of the EV biogenesis markers CD82, CD63, and flotillin-1 (FLOT). Additionally, EVs from Ni-transformed cells had enriched protein and RNA, a phenotype also observed in other studies characterizing EVs from cancer cells. Interestingly, both epithelial cells and human umbilical vein endothelial (HUVEC) cells showed a preference for taking up Ni-altered EVs compared to EVs released from the untreated cells. Moreover, these Ni-altered EVs induced inflammatory responses in both epithelial and endothelial cells and increased the expression of coagulation markers in endothelial cells. Prolonged treatment of Ni-alerted EVs for two weeks induced the epithelial-to-mesenchymal transition (EMT) in BEAS-2B cells. This study is the first to characterize the effect of Ni on EVs and suggests the potential role of EVs in Ni-induced cancer progression.

Stress contributes to the development of many human diseases, including cancer. Based on the source of stress, it can be divided into external stress, such as environmental carcinogens, chemicals, and radiation, and internal stress, like endoplasmic reticulum (ER) stress, hypoxia, and oxidative stress. Nuclear Protein 1 (NUPR1, p8 or Com-1) is a small, highly basic transcriptional regulator that participates in regulating a variety of cellular processes including DNA repair, ER stress, oxidative stress response, cell cycle, autophagy, apoptosis, ferroptosis and chromatin remodeling. A large number of studies have reported that NUPR1 expression can be stimulated rapidly in response to various stresses. Thus, NUPR1 is also known as a stress-response gene. Since the role of NUPR1 in breast cancer was identified in 1999, an increasing number of studies sought to reveal its function in cancer. High expression of NUPR1 has been identified in oral squamous cell carcinoma, breast cancer, lung cancer, multiple myeloma, liver cancer and renal cancer. In this review, we summarize current studies of NUPR1 in response to multiple external stressors and internal stressors, and its role in mediating stressors to cause different cell signaling responses. In addition, this review discusses the function of NUPR1 in carcinogenesis, tumorigenesis, metastasis, and cancer therapy. Thus, this review gives a comprehensive insight into the role of NUPR1 in mediating signals from stress to different cell responses, and this process plays a role in the development of cancer.

The stem-cell-like behavior of cancer cells plays a central role in tumor heterogeneity and invasion and correlates closely with drug resistance and unfavorable clinical outcomes. However, the molecular underpinnings of cancer cell stemness remain incompletely defined. Here, we show that SNHG1, a long non-coding RNA that is over-expressed in ~95% of human muscle-invasive bladder cancers (MIBCs), induces stem-cell-like sphere formation and the invasion of cultured bladder cancer cells by upregulating Rho GTPase, Rac1. We further show that SNHG1 binds to DNA methylation transferase 3A protein (DNMT3A), and tethers DNMT3A to the promoter of miR-129-2, thus hyper-methylating and repressing miR-129-2-5p transcription. The reduced binding of miR-129-2 to the 3'-UTR of Rac1 mRNA leads to the stabilization of Rac1 mRNA and increased levels of Rac1 protein, which then stimulates MIBC cell sphere formation and invasion. Analysis of the Human Protein Atlas shows that a high expression of Rac1 is strongly associated with poor survival in patients with MIBC. Our data strongly suggest that the SNHG1/DNMT3A/miR-129-2-5p/Rac1 effector pathway drives stem-cell-like and invasive behaviors in MIBC, a deadly form of bladder cancer. Targeting this pathway, alone or in combination with platinum-based therapy, may reduce chemoresistance and improve longer-term outcomes in MIBC patients.

Chronic exposure of human bronchial epithelial BEAS-2B cells to hexavalent chromium (Cr(VI)) causes malignant cell transformation. These transformed cells exhibit increases in migration and invasion. Neuronal precursor of developmentally downregulated protein 9 (NEDD9) is upregulated in Cr(VI)-transformed cells compared to that of passage-matched normal BEAS-2B cells. Knockdown of NEDD9 by its shRNA reduced invasion and migration of Cr(VI)-transformed cells. Maternally expressed gene 3 (MEG3), a long noncoding RNA, was lost and microRNA 145 (miR-145) was upregulated in Cr(VI)-transformed cells. MEG3 was bound to miR-145 and this binding reduced its activity. Overexpression of MEG3 or inhibition of miR-145 decreased invasion and migration of Cr(VI)-transformed cells. Overexpression of MEG3 was able to decrease miR-145 level and NEDD9 protein level in Cr(VI)-transformed cells. Ectopic expression of MEG3 was also shown to reduce β-catenin activation. Inhibition of miR-145 in Cr(VI)-transformed cells decreased Slug, an important transcription factor that regulates epithelial-to-mesenchymal transition (EMT). Inhibition of miR-145 was found to increase MEG3 in Cr(VI)-transformed cells. Further studies showed that mutation of MEG3 at the binding site for miR-145 did not change NEDD9 and failed to decrease invasion and migration. The present study demonstrated that loss of MEG3 and upregulation of miR-145 elevated NEDD9, resulting in activation of β-catenin and further upregulation of EMT, leading to increased invasion and migration of Cr(VI)-transformed cells.

First responders (FR) exposed to the World Trade Center (WTC) Ground Zero air over the first week after the 9/11 disaster have an increased heart disease incidence compared to unexposed FR and the general population. To test if WTC dusts were causative agents, rats were exposed to WTC dusts (under isoflurane [ISO] anesthesia) 2 h/day on 2 consecutive days; controls received air/ISO or air only. Hearts were collected 1, 30, 240, and 360 d post-exposure, left ventricle total RNA was extracted, and transcription profiles were obtained. The data showed that differentially expressed genes (DEG) for WTC vs. ISO rats did not reach any significance with a false discovery rate (FDR) < 0.05 at days 1, 30, and 240, indicating that the dusts did not impart effects beyond any from ISO. However, at day 360, 14 DEG with a low FDR were identified, reflecting potential long-term effects from WTC dust alone, and the majority of these DEG have been implicated as having an impact on heart functions. Furthermore, the functional gene set enrichment analysis (GSEA) data at day 360 showed that WTC dust could potentially impact the myocardial energy metabolism via PPAR signaling and heart valve development. This is the first study showing that WTC dust could significantly affect some genes that are associated with the heart/CV system, in the long term. Even > 20 years after the 9/11 disaster, this has potentially important implications for those FR exposed repeatedly at Ground Zero over the first week after the buildings collapsed.