Faculty Bibliography

Ras proteins are integral to the mediation of signaling cascades to downstream effectors, regulating a multitude of cellular processes. Mutations within Ras and its associated signaling pathways are implicated in various human pathologies, including inflammatory disorders and malignancies. The immune checkpoint proteins, programmed cell death protein 1 (PD-1) and its ligands PD-L1, along with Indoleamine 2,3-dioxygenase-1 (IDO1), are pivotal in facilitating tumor immune escape. While the influence of oncogenic Ras on PD-L1 expression is extensively documented, the regulatory role of KRas in IDO1 expression remains inadequately understood. In the current study, we demonstrate that IDO1 and PD-L1 expressions are differentially regulated in KRas-mutant cancers. Treatment with the KRas^G12C-specific inhibitor, ARS-1620, significantly increased IDO1 expression, which inversely correlated with PD-L1 expression in the KRas^G12C-mutant H358 cell line. Notably, IDO1 expression was slightly diminished in KRas-mutant patients with lung and pancreatic ductal adenocarcinomas. Experimental data revealed that IFN-γ induces IDO1 expression; however, this induction is attenuated in the presence of constitutively active KRas. These findings suggest that KRas signaling negatively regulates IDO1 expression while enhancing PD-L1 expression. Moreover, the induction of IDO1 expression following KRas inhibition appears to operate independently of the MAPK pathway. Our results propose that concurrent targeting of KRas and IDO1 could potentiate therapeutic efficacy in KRas-mutant cancers, overcoming resistance to immune checkpoint blockade.

While EZH2 enzymatic activity is well-known, emerging evidence suggests that EZH2 can exert functions in a methyltransferase-independent manner. In this study, we have uncovered a novel mechanism by which EZH2 positively regulates the expression of SKP2, a critical protein involved in cell cycle progression. We demonstrate that depletion of EZH2 significantly reduces SKP2 protein levels in several cell types, while treatment with EPZ-6438, an EZH2 enzymatic inhibitor, has no effect on SKP2 protein levels. Consistently, EZH2 depletion leads to cell cycle arrest, accompanied by elevated expression of CIP/KIP family proteins, including p21, p27, and p57, whereas EPZ-6438 treatment does not modulate their levels. We also provide evidence that EZH2 knockdown, but not enzymatic inhibition, suppresses SKP2 mRNA expression, underscoring the transcriptional regulation of SKP2 by EZH2 in a methyltransferase-independent manner. Supporting this, analysis of the Cancer Genome Atlas database reveals a close association between EZH2 and SKP2 expression in human malignancies. Moreover, EZH2 depletion but not enzymatic inhibition positively regulates the expression of major epithelial-mesenchymal transition (EMT) regulators, such as ZEB1 and SNAIL1, in transformed cells. Our findings shed light on a novel mechanism by which EZH2 exerts regulatory effects on cell proliferation and differentiation through its methyltransferase-independent function, specifically by modulating SKP2 expression.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is pivotal in development, metabolic homeostasis, and immune responses. While recent research has highlighted AhR's significant role in modulating oxidative stress responses, its mechanistic relationship with ferroptosis-an iron-dependent, non-apoptotic cell death-remains to be fully elucidated. In our study, we discovered that AhR plays a crucial role in ferroptosis, in part by transcriptionally regulating the expression of the solute carrier family 7 member 11 (SLC7A11). Our findings indicate that both pharmacological inactivation and genetic ablation of AhR markedly enhance erastin-induced ferroptosis. This enhancement is achieved by suppressing SLC7A11, leading to increased lipid peroxidation. We also obtained evidence of post-translational modifications of SLC7A11 during ferroptosis. Additionally, we observed that indole 3-pyruvate (I3P), an endogenous ligand of AhR, protects cells from ferroptosis through an AhR-dependent mechanism. Based on these insights, we propose that AhR transcriptionally regulates the expression of SLC family genes, which in turn play a pivotal role in mediating ferroptosis. This underscores AhR's essential role in suppressing lipid oxidation and ensuring cell survival under oxidative stress.

The Aryl Hydrocarbon Receptor (AhR) is a ligand-activated transcriptional factor pivotal in responding to environmental stress and maintaining cellular homeostasis. Exposure to specific xenobiotics or industrial compounds in the environment activates AhR and its subsequent signaling, inducing oxidative stress and related toxicity. Past research has also identified and characterized several classes of endogenous ligands, particularly some tryptophan (Trp) metabolic/catabolic products, that act as AhR agonists, influencing a variety of physiological and pathological states, including the modulation of immune responses and cell death. Heavy metals, being non-essential elements in the human body, are generally perceived as toxic and hazardous, originating either naturally or from industrial activities. Emerging evidence indicates that heavy metals significantly influence AhR activation and its downstream signaling. This review consolidates current knowledge on the modulation of the AhR signaling pathway by heavy metals, explores the consequences of co-exposure to AhR ligands and heavy metals, and investigates the interplay between oxidative stress and AhR activation, focusing on the regulation of immune responses and ferroptosis.

Chronic environmental exposure to toxic metal(loid)s significantly contributes to human cancer development and progression. It is estimated that approximately 90% of cancer deaths are a result of metastasis of malignant cells, which is initiated by epithelial-mesenchymal transition (EMT) during early carcinogenesis. EMT is regulated by many families of genes and microRNAs (miRNAs) that control signaling pathways for cell survival, death, and/or differentiation. Recent mechanistic studies have shown that toxic metal(loid)s alter the expression of miRNAs responsible for regulating the expression of genes involved in EMT. Altered miRNA expressions have the potential to be biomarkers for predicting survival and responses to treatment in cancers. Significantly, miRNAs can be developed as therapeutic targets for cancer patients in the clinic. In this mini review, we summarize key findings from recent studies that highlight chemical-miRNA-gene interactions leading to the perturbation of EMT after exposure to toxic metal(loid)s including arsenic, cadmium, nickel, and chromium.

Reactive oxygen species (ROS) are by-products of metabolism of oxygen and they play an important role in normal homeostasis and cell signaling, as well as in the initiation of diseases including cancer when their production is upregulated. Thus, it is imperative to understand the cellular and molecular basis by which ROS impact on various biological and pathological processes. In this report, we show that human keratinocyte cell line (HaCaT) treated with hydrogen peroxide displayed an increased activity of AhR, leading to enhanced expression of its downstream targets including cytochrome P450 genes. Intriguingly, preincubation of the complete culture medium with hydrogen peroxide accelerated AhR activation and its downstream signaling. Subsequent mass spectrometric analysis reveals that the oxidant elicits the production of oxindole, a tryptophan catabolic product. We further demonstrate that 2-oxindole (a major form of oxindole) is capable of activating AhR, strongly suggesting that ROS may exert a significant impact on AhR signaling. Consistent with this, we also observe that hexavalent chromium [Cr(VI)], a heavy metal known to generate ROS in vivo, enhances AhR protein levels, as well as stimulates expression of CYP1A2 in an AhR-dependent manner. Significantly, we show that hydrogen peroxide and 2-oxindole induce expression of IDO1 and PD-L1, two immune checkpoint proteins. Given the role of IDO1 and PD-L1 in mediating T cell activity and/or differentiation, we postulate that ROS in the tumor microenvironment may play a crucial role in immune suppression via perturbing AhR signaling.

Given that COVID-19 continues to wreak havoc around the world, it is imperative to search for a conserved region involved in viral infection so that effective vaccines can be developed to prevent the virus from rapid mutations. We have established a twelve-fragment library of recombinant proteins covering the entire region of spike protein of both SARS-CoV-2 and SARS-CoV from Escherichia coli. IgGs from murine antisera specifically against 6 spike protein fragments of SARS-CoV-2 were produced, purified, and characterized. We found that one specific IgG against the fusion process region, named COVID19-SF5, serologically cross-reacted with all twelve S-protein fragments. COVID19-SF5, with amino acid sequences from 880 to 1084, specifically bound to VERO-E6 and BEAS-2B cells, with K_d values of 449.1 ± 21.41 and 381.9 ± 31.53 nM, and IC₅₀ values of 761.2 ± 28.2 nM and 862.4 ± 32.1 nM, respectively. In addition, COVID19-SF5 greatly enhanced binding of the full-length CHO cell-derived spike protein to the host cells in a concentration-dependent manner. Furthermore, COVID19-SF5 and its IgGs inhibited the infection of the host cells by pseudovirus. The combined data from our studies reveal that COVID19-SF5, a novel cell-binding fragment, may contain a common region(s) for mediating viral binding during infection. Our studies also provide valuable insights into how virus variants may evade host immune recognition. Significantly, the observation that the IgGs against COVID19-SF5 possesses cross reactivity to all other fragments of S protein, suggesting that it is possible to develop universal neutralizing monoclonal antibodies to curb rapid mutations of COVID-19.

Ras proteins are small GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, and differentiation. Mutations or deregulated activities of Ras are frequently the driving force for oncogenic transformation and tumorigenesis. Posttranslational modifications play a crucial role in mediating the stability, activity, or subcellular localization/trafficking of numerous cellular regulators including Ras proteins. A series of recent studies reveal that Ras proteins are also regulated by sumoylation. All three Ras protein isoforms (HRas, KRas, and NRas) are modified by SUMO3. The conserved lysine42 appears to be the primary site for mediating sumoylation. Expression of KRas^V12/R42 mutants compromised the activation of the Raf/MEK/ERK signaling axis, leading to a reduced rate of cell migration and invasion in vitro in multiple cell lines. Moreover, treatment of transformed pancreatic cells with a SUMO E2 inhibitor blocks cell migration in a concentration-dependent manner, which is associated with a reduced level of both KRas sumoylation and expression of mesenchymal cell markers. Furthermore, mouse xenograft experiments reveal that expression of a SUMO-resistant mutant appears to suppress tumor development in vivo. Combined, these studies indicate that sumoylation functions as an important mechanism in mediating the roles of Ras in cell proliferation, differentiation, and malignant transformation and that the SUMO-modification system of Ras oncoproteins can be explored as a new druggable target for various human malignancies.

Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anti-cancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activity or mediates its downstream signaling. To this end, we used a combination of affinity-pulldown and mass spectrometry to search for proteins that are physically associated with KRas. One of the top hits was Radil, a gene product with a Ras-association (RA) domain. Radil is known to be a downstream effector of Rap1, inhibiting RhoA signaling to regulate cell adhesion and migration. We demonstrate that Radil interacted with all three isoforms of Ras including HRas, NRas, and KRas, although it exhibited the strongest interaction with KRas. Moreover, Radil interacts with GTP-bound Ras more efficiently, suggesting a possibility that Radil may be involved in Ras activation. Supporting this, ectopic expression of Radil led to transient activation of MEK and ERK; Radil knockdown resulted in weakened activation of Ras downstream signaling components, which was coupled with decreased cell proliferation and invasion, and reduced expression of mesenchymal cell markers. Moreover, Radil knockdown greatly reduced the number of adhesion foci and depolymerized actin filaments, molecular processes that facilitate cancer cell migration. Taken together, our current studies strongly suggest that Radil is an important player for regulating Ras signaling, cell adhesion, and the epithelial-mesenchymal transition, and may provide new directions for Ras-related anti-cancer drug development.