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.

Chronic exposure of human bronchial epithelial BEAS-2B cells to hexavalent chromium [Cr(VI)] causes malignant cell transformation. Sirtuin-3 (SIRT3) regulates mitochondrial adaptive response to stress, such as metabolic reprogramming and antioxidant defense mechanisms. In Cr(VI)-transformed cells, SIRT3 was upregulated and mitochondrial adenosine triphosphate (ATP) production and proton leak were reduced. Knockdown of SIRT3 by its shRNA further decreased mitochondrial ATP production, proton leak, mitochondrial mass, and mitochondrial membrane potential, indicating that SIRT3 positively regulates mitochondrial oxidative phosphorylation and maintenance of mitochondrial integrity. Mitophagy is critical to maintain proper cellular functions. In Cr(VI)-transformed cells expressions of Pink 1 and Parkin, two mitophagy proteins, were elevated, and mitophagy remained similar as that in passage-matched normal BEAS-2B cells, indicating that in -Cr(VI)-transformed cells mitophagy is suppressed. Knockdown of SIRT3 induced mitophagy, suggesting that SIRT3 plays an important role in mitophagy suppression of Cr(VI)-transformed cells. In Cr(VI)-transformed cells, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was constitutively activated, and protein levels of p62 and p-p62Ser349 were elevated. Knockdown of SIRT3 or treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) decreased the binding of p-p62Ser349 to Keap1, resulting in increased binding of Keap1 to Nrf2 and consequently reduced Nrf2 activation. The results from CHIP assay showed that in Cr(VI)-transformed cells binding of Nrf2 to antioxidant response element (ARE) of SIRT3 gene promoter was dramatically increased. Knockdown of SIRT3 suppressed cell proliferation and tumorigenesis of Cr(VI)-transformed cells. Overexpression of SIRT3 in normal BEAS-2B cells exhibited mitophagy suppression phenotype and increased cell proliferation and tumorigenesis. The present study demonstrated that upregulation of SIRT3 causes mitophagy suppression and plays an important role in cell survival and tumorigenesis of Cr(VI)-transformed cells.

Environmental and occupational exposures to cadmium increase the risk of various cancers, including lung cancer. The carcinogenic mechanism of cadmium, including its prevention remains to be investigated. Using fluorescence and electron spin resonance spin trapping, the present study shows that in immortalized lung cells (BEAS-2BR cells), exposure cadmium generated reactive oxygen species (ROS). Through ROS generation, cadmium increased the protein level of TNF-α, which activated NF-κB and its target protein COX-2, creating an inflammatory microenvironment. As measured by anchorage-independent colony formation assay, cadmium induced malignant cell transformation. Inhibition of ROS by antioxidants inhibited transformation, showing that ROS were important in the mechanism of this process. The inflammatory microenvironment created by cadmium may also contribute to the mechanism of the transformation. Using tandem fluorescence protein mCherry-GFP-LC3 construct, the present study shows that cadmium-transformed cells had a property of autophagy deficiency, resulting in accumulation of autophagosomes and increased p62. This protein upregulated Nrf2, which also upregulated p62 through positive feed-back mechanism. Constitutive Nrf2 activation increased its downstream anti-apoptotic proteins, Bcl-2 and Bcl-xl, resulting in apoptosis resistance. In untransformed BEAS-2BR cells, sulforaphane, a natural compound, increased autophagy, activated Nrf2, and decreased ROS. In cadmium-transformed BEAS-2BR cells, sulforaphane restored autophagy, decreased Nrf2, and decreased apoptosis resistance. In untransformed cells, this sulforaphane induced inducible Nrf2 to decrease ROS and possibly malignant cell transformation. In cadmium-transformed cells, it decreased constitutive Nrf2 and reduced apoptosis resistance. The dual roles of sulforaphane make this natural compound a valuable agent for prevention against cadmium-induced carcinogenesis.

Cr(VI)-containing compounds are well-established lung carcinogens. Chronic exposure of the normal human epithelial cells is able to induce malignant cell transformation, the first stage of metal carcinogenesis. These Cr(VI)-transformed cells exhibit increased level of antioxidants, reduced capacity of generating reactive oxygen species (ROS), and development of apoptosis resistance, promoting tumorigenesis of Cr(VI)-transformed cells, the second stage of metal carcinogenesis. The mechanism of Cr(VI) induced carcinogenesis is still under investigation. Recent studies indicate that ROS play a positive role in the first stage while a negative role in the second stage. Transformed cells adapt metabolism to support tumor initiation and progression. Altered metabolic activities directly participate in the process of cell transformation or support a large requirement for nucleotides, amino acids, and lipids for tumor growth. In malignantly Cr(VI)-transformed cells, mitochondrial oxidative phosphorylation is defective, and pentose phosphate pathway, glycolysis, and glutaminolysis are upregulated. These metabolic reprogramming supports rapid cell proliferation and contributes to tumorigenesis of Cr(VI)-transformed cells. This article summarizes the current progress in the studies of metabolic reprogramming and Cr(VI) carcinogenesis with emphasis on the metabolic enzymes and oxidative stress related major oncogenic pathways.

Hexavalent chromium [Cr(VI)] is a lung carcinogen and its complete mechanism of action remains to be investigated. Metabolic reprogramming of key energy metabolism pathways (e.g., increased anaerobic glycolysis in the presence of oxygen or "Warburg effect", dysregulated mitochondrial function, and lipogenesis) are important to cancer cell and tumor survival and growth. In our current understanding of Cr(VI)-induced carcinogenesis, the role for metabolic reprogramming remains unclear. In this study, we treated human lung epithelial cells (BEAS-2B) with Cr(VI) for 6 months and obtained malignantly transformed cells from an isolated colony grown in soft agar. We also used Cr(VI)-transformed cells from two other human lung cell lines (BEP2D and WTHBF-6 cells). Overall, we found that all the Cr(VI)-transformed cells had no changes in their mitochondrial respiratory functions (measured by the Seahorse Analyzer) compared with passaged-matched control cells. Using a xenograft tumor growth model, we generated tumors from these transformed cells in Nude mice. Using cells obtained from the xenograft tumor tissues, we observed that these cells had decreased maximal mitochondrial respiration, spare respiratory capacity, and coupling efficiency. These results provide evidence that, although mitochondrial dysfunction does not occur during Cr(VI)-induced transformation of lung cells, it does occur during tumor development.

Hexavalent chromium [Cr(VI)] is widely used in various industrial processes and has been recognized as a carcinogen. As the first line of host defense system, the immune system can be a primary target of Cr(VI). T cell population represents a major arm of the immune system that plays a critical role in host anti-tumor immunity. Dysfunction of T cells, such as exhaustion under the persistent presence of tumor antigen, compromise host anti-tumor immunity resulting in oncogenesis. Previous studies have shown Cr(VI) exposure alters the phenotype of human peripheral blood lymphocytes. However, the mechanism of the alteration and whether such an alteration in immunity affects immunosurveillance and promotes carcinogenicity are not clear. Using a culture of mouse splenic T cells as an in vitro model system, the present study assessed the effects of Cr(VI) on T cells, as the first step in our investigation of the mechanism underlying Cr(VI)-inhibited immunosurveillance and carcinogenesis. Our results showed that Cr(VI) decreased the viability of CD4⁺ and CD8⁺ T cells and inhibited their activation, proliferation, cytokine release and cytolytic function.

Heavy metals, such as arsenic, chromium, cadmium, nickel, mercury, and uranium are known to cause many human diseases and health complications after occupational or environmental exposure. Consequently, metals are environmental health concerns. This manuscript is an overview of the 9th Conference on Metal Toxicity and Carcinogenesis held in October 2016 in Lexington, Kentucky. Since 2000, this biennial meeting brings together experts in the field to discuss current and prospective research in an effort to advance research pertaining to metal toxicity and carcinogenesis. In this review we summarize the major topics discussed and provide insight regarding current research in the field and an account of the direction in which the field is progressing.

Hexavalent chromium (Cr(VI)) compounds are confirmed human carcinogens for lung cancer. Our previous studies has demonstrated that chronic exposure of human bronchial epithelial BEAS-2B cells to low dose of Cr(VI) causes malignant cell transformation. The acquisition of cancer stem cell-like properties is involved in the initiation of cancers. The present study has observed that a small population of cancer stem-like cells (BEAS-2B-Cr-CSC) exists in the Cr(VI)-transformed cells (BEAS-2B-Cr). Those BEAS-2B-Cr-CSC exhibit extremely reduced capability of generating reactive oxygen species (ROS) and apoptosis resistance. BEAS-2B-Cr-CSC are metabolic inactive as evidenced by reductions in oxygen consumption, glucose uptake, ATP production, and lactate production. Most importantly, BEAS-2B-Cr-CSC are more tumorigenic with high levels of cell self-renewal genes, Notch1 and p21. Further study has found that fructose-1,6-bisphosphatase (FBP1), an rate-limiting enzyme driving glyconeogenesis, was lost in BEAS-2B-Cr-CSC. Forced expression of FBP1 in BEAS-2B-Cr-CSC restored ROS generation, resulting in increased apoptosis, leading to inhibition of tumorigenesis. In summary, the present study suggests that loss of FBP1 is a critical event in tumorigenesis of Cr(VI)-transformed cells.