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Identification of acetylated lysine residues by LC-ESI-MS/MS. (A) Immunoprecipitated EGFR from HT29 cells and HCT116 ShSrx cells were stained with coomassie brilliant blue. EGFR bands were extracted form MS analysis. (B) Representative spectrum of K203 acetylation identified by MS.

Sulfiredoxin (SRXN1/Srx) is a multifunction enzyme with a primary antioxidant role of reducing the overoxidized inactive form of peroxiredoxins (Prxs). The function and mechanisms of Srx in cancer development are not well understood. Here, Srx is preferentially expressed in human colorectal cancer cells but not in normal colon epithelial cells. Loss-of-function studies demonstrate that knockdown of Srx in poorly differentiated colorectal cancer cells not only leads to the inhibition of colony formation and cell invasion in vitro, but also reduces tumor xenograft growth and represses metastasis to distal organs in a mouse orthotopic implantation model. Notably, exactly opposite effects were observed in gain-of-function experiments when Srx was ectopically expressed in well-differentiated colorectal cancer cells. Mechanistically, expression of Srx enhances the activation of MAPK signaling through increasing the C-terminal tyrosine phosphorylation levels of EGFR. This function of Srx is mediated through its inhibition of EGFR acetylation at K1037, a novel posttranslational modification of EGFR in human colorectal cancer cells identified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI/MS-MS) proteomic analysis. Furthermore, abolishment of K1037 acetylation in human colorectal cancer cells by site-specific mutagenesis leads to sustained activation of EGFR–MAPK signaling. Combined, these data reveal that Srx promotes colorectal cancer cell invasion and metastasis through a novel mechanism of enhancing EGFR signaling.Implications: Sulfiredoxin is a critical oncogenic protein that can be used as a molecular target to develop therapeutics for patients with metastatic colorectal cancer. Mol Cancer Res; 13(12); 1554–66. ©2015 AACR.

Non-small cell lung cancer (NSCLC) is one of the most aggressive cancer types, and metastasis of cancer cells is the major reason leading to death of patients. Aryl hydrocarbon receptor (AHR) is an important transcription factor involved in the initiation and progression of lung cancer. Polo-like kinase 1 (PLK1), a serine/threonine kinase, is another oncogene that promotes the malignancy of multiple cancer types. Preliminary examination indicates that AHR is a potential substrate of PLK1. Nonetheless, the phosphorylation and interaction of these two factors, as well as the subsequent biological significance in lung cancer remain to be determined. Here we prove that PLK1 phosphorylates AHR at serine 489 (S489) in lung adenocarcinoma (LUAD), a subtype of NSCLC. Overexpressing of exogenous AHR with mutation of phosphomemetic amino acid (S489D), enhances migration and invasion in transwell assays compared to cells harboring AHR that is unable to be phosphorylated (S489A), indicating that phosphorylation of AHR by PLK1 induces the metastatic potential of lung cancer. This is further confirmed in mouse studies using intravenous or subcutaneous inoculation of cancer cells. RNA-seq analyses of cells with S489D or S489A show that type 2 deiodinase (DIO2) is underrepresented in S489A but overexpressed in S489D. DIO2 converts thyroxine (T4) to triiodothyronine (T3), which is the more active form of thyroid hormone and activates downstream effect. Treatment with T3 or T4 promotes the metastasis of LUAD, whereas depletion of DIO2 or treatment with deiodinase inhibitor iopanoic acid disrupts this property. Furthermore, this phenomenon is verified by in vivo mouse experiment using intravenous injection of DIO2-depleted cancer cells. Taken together, these results identify phosphorylation of AHR by PLK1 as a mechanism that leads to progression of LUAD. Citation Format: Chaohao Li, Daheng He, Fengyi Mao, Xinyi Wang, Yanning Hao, Yifan Kong, Christine F. Brainson, Jinghui Liu, Yanquan Zhang, Ruixin Wang, Qiongsi Zhang, Zhiguo Li, Xiongjian Rao, Sai Wu, Chi Wang, Qiou Wei, Jianlin Wang, Xiaoqi Liu. Phosphorylation of AHR by PLK1 promotes metastasis of LUAD via upregulation of DIO2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2578.

Intracellular antioxidant enzymes are critical for maintenance of redox homeostasis, but whether and how they contribute to the malignancy of cancer cells remains poorly understood. Sulfiredoxin (Srx) is a unique oxidoreductase in that it not only restores peroxidase activity of peroxiredoxins (Prxs) but also functions as a pivotal stimulator of oncogenic signaling. We found that abnormally high level of Srx promotes colorectal cancer (CRC) malignancy by stimulating gelatin degradation, invadopodia formation, and cell invasion. Fascin, an actin-bundling protein, was discovered and validated as one of the critical downstream targets of Srx activation. We demonstrated that depletion of Srx in CRC cells leads to upregulation of miR-143-3p, which mediates degradation of fascin mRNA through binding to conserved sites within the 3' untranslated region (UTR). Depletion of fascin in CRC cells recapitulates the effect of Srx loss, and restoration of fascin in Srx-depleted cells by miR-143-3p inhibitor or overexpression rescues defects in cell invasion. Therefore, our data demonstrate that the Srx-miR143-fascin axis plays a key role in promoting the malignancy of human CRC cells. In the future, the Srx-miR143-fascin axis can be used as a functional pathway to evaluate the efficacy of therapeutic drugs or be targeted to develop promising chemotherapeutics for treatment of CRC patients.

High levels of redox enzymes have been commonly observed in various types of human cancer, although whether and how the enzymes contribute to cancer malignancy and therapeutic resistance have yet to be understood. Peroxiredoxin IV (Prx4) is an antioxidant with bona fide peroxidase and molecular chaperone functions. Here, we report that Prx4 is highly expressed in prostate cancer patient specimens, as well as established prostate cancer cell lines, and that its levels can be further stimulated through the activation of androgen receptor signaling. We used lentivirus-mediated shRNA knockdown and CRISPR-Cas9 based KO techniques to establish Prx4-depleted prostate cancer cells, which showed delayed cell cycle progression, reduced rate of cell proliferation, migration, and invasion compared to control cells. In addition, we used proteome profiler phosphokinase arrays to identify signaling changes in Prx4-depleted cells; we found that loss of Prx4 results in insufficient phosphorylation of both Akt and its downstream kinase GSK3α/β. Moreover, we demonstrate that Prx4-depleted cells are more sensitive to ionizing radiation as they display compromised ability to scavenge reactive oxygen species and increased accumulation of DNA damage. In mouse xenograft models, we show depletion of Prx4 leads to significant suppression of tumor growth, and tumors formed by Prx4-depleted cells respond more effectively to radiation therapy. Our findings suggest that increased levels of Prx4 contribute to the malignancy and radioresistance of prostate cancer through the activation of Akt/GSK3 signaling pathways. Therefore, strategies targeting Prx4 may be utilized to potentially inhibit tumor growth and overcome radioresistance in prostate cancer.

Peroxiredoxin IV (Prx4) is a multifunction enzyme with a primary antioxidant role of reducing free radicals such as hydrogen peroxide and peroxynitrite. Literature review suggests that Prx4 upregulation in colorectal cancer is associated with higher tumor metastasis and shorter survival of patients. However, the function and mechanisms of Prx4 in cancer development are not well understood. The goal of this study is to validate the oncogenic role of Prx4 and to identify potential mechanisms of invasion and metastasis. Western Blot was performed to compare the expression of Prx4 protein in a normal colon cell line and seven colorectal cancer cell lines. shRNA mediated knockdown of Prx4 was performed in cancer cell lines with higher expression of Prx4. Stable vector control and Prx4 knockdown cells were used to perform Scratch Wound healing assay to compare the rate of migration. Transwell Matrigel invasion assay was performed using the same cell lines with 10% FBS containing-medium as chemoattractant. Vector control and Prx4 knockdown HCT116 cells were orthotopically implanted into the cecum wall of immunodeficient mice and humanely euthanized four weeks later. Prx4 expression is upregulated in colorectal cancer cell lines compared to normal colon cell line NCM356. Stable knockdown of Prx4 in HCT116 and RKO cell lines resulted in a significant decrease in migration and invasion rates of cancer cell lines in vitro compared to vector control. Orthotopic implantation of HCT116 vector control and Prx4 knockdown cells into cecum wall resulted in a lower metastasis rate to the liver and the lungs. We are currently conducting mechanistic studies to determine how the loss of Prx4 leads to a decrease in malignancy of colorectal cancer cell lines. Prx4 promotes colorectal cancer cell invasion and metastasis. Our study suggests Prx4 is a critical oncogenic protein that can be used as a therapeutic target for patients. This work was partially supported by the National Institutes of Health R01CA222596, Molecular Mechanisms of Toxicity Training Grant in Toxicology (T32ES07266), and American Cancer Society RSG-16-213-01-TBE. Citation Format: Pratik Thapa, Hong Jiang, Yanning Hao, Na Ding, Aziza Alshahrani, Yekaterina Zaytseva, Qiou Wei. Peroxiredoxin IV promotes progression of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5998.

Correct folding of nascent peptides occurs in the endoplasmic reticulum (ER). It is a complicate process primarily accomplished by the coordination of multiple redox proteins including members of the protein disulfide isomerase (PDI) family. As a critical member of the PDI family, thioredoxin domain containing protein 5 (TXNDC5) assists the folding of newly synthesized peptides to their mature form through series of disulfide bond exchange reactions. Interestingly, TXNDC5 is frequently found overexpressed in specimens of many human diseases including various types of cancer. In this review, we summarized the biochemical function of TXNDC5 in mammalian cells and the recent progress on the understanding of its role and molecular mechanisms in cancer development. Findings of TXNDC5 in the activation of intracellular signaling pathways, stimulation of cell growth & proliferation, facilitation of cell survival and modulation of extracellular matrix to affect cancer cell invasion and metastasis are reviewed. These published studies suggest that strategies of targeting TXNDC5 can be developed as potentially valuable methods for the treatment of certain types of cancer in patients. Keywords: Cancer, Cell signal, Protein disulfide isomerase, Protein folding, Receptor modulation

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo. Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

The peroxiredoxin (Prx) family of proteins functions as major cellular antioxidants that mediate oxidative signaling under physiological conditions as well as scavenge extra hydrogen peroxide in the context of oxidative stress. Prx4 is one of Prx family members which is mainly localized in endoplasmic reticulum (ER), but can be also found in cytosol and extracellular space. In addition to its function as a peroxidase, Prx4 has a specific role in oxidative protein folding in ER. However, the specific function of Prx4 has not been well defined. Previous studies indicate that Prxs are frequently upregulated in various types of human cancer. However, the role of Prx4 in prostate cancer has not been well defined. The purpose of this study is to examine the expression of Prx4 in prostate cancer and to explore its functional significance in cancer radiation resistance and recurrence. Bioinformatic tools were used to evaluate genetic alterations of PRDX4 gene as well as the levels of its transcripts in prostate normal and cancer populations. Kaplan-Meier survival analysis was used to explore the association of Prx4 levels with the prognosis of prostate cancer patients. Western blot and immunohistochemistry were used to evaluate the expression of Prx4 protein in cell lines and patient specimens. Loss of Prx4 in cells was achieved by knock down using lentiviral shRNA or knockout using CRISPR-Cas9 techniques. Cell proliferation, survival, and protein profiler kinase arrays were used to examine the differences between control and Prx4-depleted cells with or without ionizing radiation. We demonstrated that PRDX4 gene is frequently amplified in prostate cancer and the level of its transcript is highly elevated. Patients with Prx4 at higher quartile have significantly reduced probability of survival compared with those in lower quartile. Prostate cancer cells express much higher levels of Prx4 than normal epithelial cells. Moreover, Prx4 is upregulated by the activation of AR-dependent signaling, and depletion of Prx4 sensitizes prostate cancer cells to radiation-induced cell death. Mechanistically, Prx4 contributes to prostate cancer radiation resistance through the activation of PI3K/AKT signaling pathway. A combination of bioinformatic, histochemical, cellular, and molecular methods reveals that Prx4 plays a critical role in prostate cancer cell proliferation, radio-resistance and reoccurrence. Citation Format: Na Ding, Hong Jiang, Pratik Thapa, Yanning Hao, Aziza Alshahrani, Shengming Yang, Vivek M. Rangnekar, Xiaoqi Liu, Qiou Wei. Peroxiredoxin 4 contributes to radiation resistance of prostate cancer cells through the activation of the PI3K/AKT signaling pathway [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-079.

Sulfiredoxin (SRXN1/Srx) is a multifunction enzyme with a primary antioxidant role of reducing the overoxidized inactive form of peroxiredoxins (Prxs). The function and mechanisms of Srx in cancer development are not well understood. Here, Srx is preferentially expressed in human colorectal cancer cells but not in normal colon epithelial cells. Loss-of-function studies demonstrate that knockdown of Srx in poorly differentiated colorectal cancer cells not only leads to the inhibition of colony formation and cell invasion in vitro, but also reduces tumor xenograft growth and represses metastasis to distal organs in a mouse orthotopic implantation model. Notably, exactly opposite effects were observed in gain-of-function experiments when Srx was ectopically expressed in well-differentiated colorectal cancer cells. Mechanistically, expression of Srx enhances the activation of MAPK signaling through increasing the C-terminal tyrosine phosphorylation levels of EGFR. This function of Srx is mediated through its inhibition of EGFR acetylation at K1037, a novel posttranslational modification of EGFR in human colorectal cancer cells identified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI/MS-MS) proteomic analysis. Furthermore, abolishment of K1037 acetylation in human colorectal cancer cells by site-specific mutagenesis leads to sustained activation of EGFR–MAPK signaling. Combined, these data reveal that Srx promotes colorectal cancer cell invasion and metastasis through a novel mechanism of enhancing EGFR signaling. Implications: Sulfiredoxin is a critical oncogenic protein that can be used as a molecular target to develop therapeutics for patients with metastatic colorectal cancer. Mol Cancer Res; 13(12); 1554–66. ©2015 AACR.

Sulfiredoxin (Srx) reduces hyperoxidized 2-cysteine–containing peroxiredoxins (Prxs) and protects cells against oxidative stress. Previous studies have shown that Srx is highly expressed in primary specimens of lung cancer patients and plays a pivotal role in lung tumorigenesis and cancer progression. However, the oncogenic mechanisms of Srx in cancer are incompletely understood. In this study, we found that Srx knockdown sensitizes lung cancer cells to endoplasmic reticulum (ER) stress–induced cell death. Through MS analysis, we determined that Srx forms a complex with the ER-resident protein thioredoxin domain–containing protein 5 (TXNDC5). Using reciprocal co-immunoprecipitation, immunofluorescence imaging, subcellular fractionation, and domain-mapping assays with site-specific mutagenesis and purified recombinant proteins, we further characterized the Srx–TXNDC5 interaction. In response to ER stress but not to oxidative stress, Srx exhibits an increased association with TXNDC5, facilitating the retention of Srx in the ER. Of note, TXNDC5 knockdown in lung cancer cells inhibited cell proliferation and repressed anchorage-independent colony formation and migration, but increased cell invasion and activation of mitogen-activated protein kinases. Using immunohistochemical staining, we demonstrate that TXNDC5 is highly expressed in patient-derived lung cancer specimens. Bioinformatics analysis of publicly available data sets revealed that those with high Srx levels have significantly shorter survival and that those with high TXNDC5 levels have longer survival. We conclude that the cellular levels of Srx and TXNDC5 may be useful as biomarkers to predict the survival of individuals with lung cancer.

Summary Peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3) are two major antioxidant proteins that play a critical role in maintaining redox homeostasis for tumor progression. Here, we identify the prototypical pyranonaphthoquinone natural product frenolicin B (FB) as a selective inhibitor of Prx1 and Grx3 through covalent modification of active-site cysteines. FB-targeted inhibition of Prx1 and Grx3 results in a decrease in cellular glutathione levels, an increase of reactive oxygen species (ROS), and concomitant inhibition of cancer cell growth, largely by activating the peroxisome-bound tuberous sclerosis complex to inhibit mTORC1/4E-BP1 signaling axis. FB structure-activity relationship studies reveal a positive correlation between inhibition of 4E-BP1 phosphorylation, ROS-mediated cancer cell cytotoxicity, and suppression of tumor growth in vivo. These findings establish FB as the most potent Prx1/Grx3 inhibitor reported to date and also notably highlight 4E-BP1 phosphorylation status as a potential predictive marker in response to ROS-based therapies in cancer.

Lung cancer is the leading cause of cancer death worldwide. Cigarette smoking and exposure to chemical carcinogens are among the risk factors of lung tumorigenesis. In this study, we found that cigarette smoke condensate and urethane significantly stimulated the expression of sulfiredoxin (Srx) at the transcript and protein levels in cultured normal lung epithelial cells, and such stimulation was mediated through the activation of nuclear related factor 2 (Nrf2). To study the role of Srx in lung cancer development in vivo, mice with Srx wildtype, heterozygous or knockout genotype were subjected to the same protocol of urethane treatment to induce lung tumors. By comparing tumor multiplicity and volume between groups of mice with different genotype, we found that Srx knockout mice had a significantly lower number and smaller size of lung tumors. Mechanistically, we demonstrated that loss of Srx led to a decrease of tumor cell proliferation as well as an increase of tumor cell apoptosis. These data suggest that Srx may have an oncogenic role that contributes to the development of lung cancer in smokers or urethane-exposed human subjects.

Redox signaling is a critical component of cell signaling pathways that are involved in the regulation of cell growth, metabolism, hormone signaling, immune regulation and variety of other physiological functions. Peroxiredoxin (Prx) is a family of thiol-based peroxidase that acts as a regulator of redox signaling. Members of Prx family can act as antioxidants and chaperones. Sulfiredoxin (Srx) is an antioxidant protein that exclusively reduces over-oxidized typical 2-Cys Prx. Srx has different affinities for individual Prx and it also catalyzes the deglutathionylation of variety of substrates. Individual component of the Srx-Prx system plays critical role in carcinogenesis by modulating cell signaling pathways involved in cell proliferation, migration and metastasis. Expression levels of individual component of the Srx-Prx axis have been correlated with patient survival outcome in multiple cancer types. This review will summarize the molecular basis of differences in the affinity of Srx for individual Prx and the role of individual component of the Srx-Prx system in tumor progression and metastasis. This enhanced understanding of molecular aspects of Srx-Prx interaction and its role in cell signal transduction will help define the Srx-Prx system as a future therapeutic target in human cancer.

Sulfiredoxin (Srx) is multifunction enzyme with a primary antioxidant role of reducing the overoxidized inactive form of peroxiredoxins (Prxs). In contrast to the well-studied biochemical function, the biological significance of Srx in human diseases including cancer has not been well studied. Our previous studies show that it plays an important role in tumorigenesis and malignant progression of human skin, lung and colorectal cancers. Fascin homologue 1 (FSCN1) is an actin-binding protein that is critical for cancer cell invasion and metastasis. The purpose of this study is to understand the molecular mechanism of Srx on the invasion and metastasis of human colorectal cancer cells through the regulation of FSCN1. The effects of Srx on the migration and invasion of human colon cancer HCT116 cells were evaluated by wound healing and matrigel invasion assay, respectively. Actin stress fiber was visualized by F-actin fluorescence staining. Western blotting and reverse transcription & real-time PCR were used to examine the protein and RNA levels of FSCN1 in HCT116 cells. The effect of Srx on FSCN1 promoter and 3’ UTR were determined using luciferase reporter construct. We demonstrated that (1) knockdown of Srx suppressed HCT116 colony formation and cell invasion in the matrigel invasion assay; (2) knockdown of Srx inhibited FSCN1 expression in HCT116 cells at both RNA and protein level; (3) the inhibition is the result of decreased FSCN1 mRNA stability; (5) Srx stabilizes FSCN1 mRNA 3’ UTR mediated reporter expression; (4) knockdown of Srx reduced actin stress-fiber formation; (5) Srx and Prx4 synchronically regulates FASN1 expression. Therefore, we concluded that Srx promotes the invasion and metastasis of human colon cancer cells through the upregulation of FSCN1. Citation Format: Hong Jiang, Qi Ying, B. Mark Evers, Qiou Wei. Sulfiredoxin promotes the invasion of human colorectal cancer cells through the activation of fascin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 177.

Mammalian cells develop various transmembrane receptors including the family of receptor tyrosine kinases (RTKs) to respond to extracellular stimuli. Epidermal growth factor receptor (EGFR) is the first identified member of the RTKs and it plays a pivotal role in colorectal cancer by stimulating cell proliferation and survival. Binding of extracellular ligands, mainly EGF, leads to the dimerization of EGFR that triggers the activation of downstream mitogen activated protein kinases. In addition to the amount of ligand binding, the strength and duration of EGFR signaling are also affected by the posttranslational modifications of the receptor itself, such as phosphorylation, ubiquitination, acetylation, etc. In the previous study, we demonstrated that Sulfiredoxin (Srx) enhances the EGFR signaling in colorectal cancer cells through the inhibition of receptor acetylation at lysine residue 1061. However, the molecular mechanism by which Srx regulates the level of EGFR acetylation is still unclear. In this study, we investigated (1) the mechanism of EGFR acetylation and its regulation by Srx; (2) the functional consequence of EGFR acetylation on EGF-induced receptor dimerization, recycling, trafficking and degradation. Human colon cancer cell lines were cultured and maintained in standard conditions. Western blotting, immunoprecipitation and Mass spectrometry were used to identify cysteine oxidation of HDAC6 and characterize enzymes that are involved in the acetylation of EGFR. Stable cells that overexpress or loss of target genes, such as Srx, HDAC6, EGFR and/or mutants, were established using the 3rd generation lentiviral ShRNA infection or the CRISPR-Cas9 system. The activity of histone deacetylases was determined using commercial kit in the presence/absence of pan- or isoform-specific chemical inhibitors of HDACs. Upon EGF stimulation, the dimerization, recycling, trafficking and degradation of EGFR were demonstrated through a combination of immunoblotting, flow-cytometry and immunofluorescent imaging. We demonstrated that HDAC6 plays a central role in the regulation of EGFR acetylation through its interaction with EGFR. Depletion of Srx in human colon cancer cells increases the levels of intracellular reactive oxygen species, leading to inhibition of HDAC6 activity through regulating the redox state of cysteines 407/417. Depletion of HDAC6 not only leads to increased level of EGFR acetylation but also suppresses the binding between EGFR and its adaptor proteins. EGF-induced receptor dimerization and recycling are altered in HDAC6 knockout cells, result in inhibition of EGFR downstream signaling. Taken together, this study indicates that Srx enhances the strength and duration of EGF signaling through the ROS-HDAC6-EGFR axis in human colon cancer cells. Citation Format: Qi Ying, Hong Jiang, B. Mark Evers, Qiou Wei. Histone deacetylase 6 plays a critical role in sulfiredoxin-mediated activation of the epidermal growth factor receptor signaling in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2644.

R-spondin proteins are newly identified secreted molecules that activate beta-catenin signaling. However, the mechanism of R-spondin action and its relationship with Wnt signaling remain unclear. Here we show that human R-spondin1 (hRspo1) is a high affinity ligand for the Wnt co-receptor LRP6 (K(d) = 1.2 nm). hRspo1 induces glycogen synthase kinase 3-dependent phosphorylation and activation of LRP6. DKK1, an LRP6 antagonist, inhibits hRspo1-induced LRP6 phosphorylation. We further demonstrate that hRspo1 synergizes with Frizzled5 in Xenopus axis induction assays and induces the phosphorylation of Dishevelled, a cytoplasmic component downstream of Frizzled function. Our study reveals interesting similarity and distinction between Wnt and R-spondin signaling.

The homeodomain-containing protein Nkx2.2 is critical for the development of oligodendrocyte lineage cells, but the target genes of Nkx2.2 regulation have not been identified. In the present study, we found that the myelin basic protein gene is one of the genes that is regulated by Nkx2.2. Expression of Nkx2.2 represses the expression of myelin basic protein in oligodendrocyte progenitors. Two regulatory elements in the myelin basic protein promoter were identified and found to interact with Nkx2.2 in vitro. Despite their sequence divergence, both sites were involved in the Nkx2.2-mediated repression of the myelin basic protein promoter. Binding of Nkx2.2 also blocked and disrupted the binding of the transcriptional activator Puralpha to the myelin basic protein promoter. Additionally Nkx2.2 recruited a histone deacetylase 1-mSin3A complex to the myelin basic protein promoter. We also found that the transcription factor Sp1 was able to compete off the binding of Nkx2.2 to its consensus binding site in vitro and reversed the repressive effect of Nkx2.2 in vivo. Our data revealed a novel role for Nkx2.2 in preventing the precocious expression of myelin basic protein in immature oligodendrocytes. Based on this study and our previous reports, a model for myelin basic protein gene control is proposed.

Myelin basic protein (MBP) is one of the major components of the myelin sheath that insulates axons. In the central nervous system, MBP is synthesized by differentiating oligodendrocytes. The expression of MBP in oligodendrocytes is regulated mainly at the transcriptional level. The Sp1 family of transcription factors has been shown to be important in the regulation of many genes. Binding of Sp1 to the GC box in the proximal MBP promoter has been shown to be indispensable for the activation of MBP gene expression. Previous results from our laboratory have shown that the increase in p27 K i p 1 that accompanies oligodendrocyte differentiation is paralleled by an increase in Sp1. We also have shown that the increase in MBP expression resulting from elevated p27 K i p 1 levels is mediated through Sp1 and that this effect occurs specifically in oligodendrocytes. In this study, we found that increased expression of p27 K i p 1 together with the nervous-system-specific transcription factor Sox10 can activate the MBP promoter even in nonoligodendrocyte cells. This indicates that Sox10 confers cell type specificity on the expression of MBP. Both Sp1 and Sox10 can enhance MBP promoter activity when expressed alone. Cotransfection of plasmids encoding Sp1 and Sox10 induces increased activation of the MBP promoter over expression of either transcription factor alone. This effect is not limited to oligodendrocyte cell lines, in that Sp1 and Sox10 can also synergistically activate the MBP promoter when expressed in NIH3T3 fibroblasts. Mutation of the Sp1 binding sites in the MBP promoter eliminates Sox10 stimulated activation, suggesting that the MBP promoter is activated, at least in part, through protein-protein interactions between Sp1 and Sox10.

Expression of myelin basic protein in differentiating oligodendrocytes is mainly regulated at the transcriptional level. To better understand the regulation of myelin basic protein gene expression in mammalian cells, we cloned and characterized the rat myelin basic protein promoter by a genome walking technique. Extensive sequence homology has been found among mouse, rat and human MBP promoters. Alignment of the proximal core promoter of mouse and rat reveals highly conserved cis-elements that are important for regulating myelin basic protein gene transcription. One major transcription start site along with two minor sites have been identified in both mouse and rat myelin basic protein gene promoters using RNA ligase-mediated rapid amplification of 5' cDNA ends. The amplified rat myelin basic protein promoter was cloned into a luciferase reporter construct. Transient transfection experiments show that both mouse and rat myelin basic protein promoters yield increased expression when oligodendrocytes differentiate. The sequence and characterization of the rat MBP promoter provide a useful tool to investigate MBP gene regulation in mammalian cells.

p27(Kip1) levels increase in many cells as they leave the cell cycle and begin to differentiate. The increase in p27(Kip1) levels generally precedes the expression of differentiation-specific genes. Previous studies from our laboratory showed that the overexpression of p27(Kip1) enhances myelin basic protein (MBP) promoter activity. This activation is specific to p27(Kip1). Additionally, inhibition of cyclin-dependent kinase activity alone is not sufficient to increase MBP expression. In this study, we focused on understanding how p27(Kip1) can activate gene transcription by using the MBP gene in oligodendrocytes as a model. We show that the enhancement of MBP promoter activity by p27(Kip1) is mediated by a proximal region of the MBP promoter that contains a conserved GC box binding sequence. This sequence binds transcription factors Sp1 and Sp3. Increased expression of p27(Kip1) increases the level of Sp1 promoter binding to the GC box but does not change the level of Sp3 binding. The binding of Sp1 to this element activates the MBP promoter. p27(Kip1) leads to increased Sp1 binding through a decrease in Sp1 protein turnover. Enhancement of MBP promoter activity by an increase in the level of p27(Kip1) involves a novel mechanism that is mediated through the stabilization and binding of transcription factor Sp1.

Members of the Peroxiredoxin (Prx) family are major cellular antioxidants that scavenge hydrogen peroxide and play essential roles in oxidative stress and cell signaling. 2-Cys Prxs, including Prx1, 2, 3 and 4, have been indicated in multiple oncogenic signaling pathways and thus may contribute to various processes of cancer development. The significance of 2-Cys Prxs in lung cancer development and their biological function in signal transduction have not been fully investigated. In this study we analyzed the expression of 2-Cys Prxs in lung cancer, and examined their levels of expression in a variety of cell lines established from human lung normal or cancer tissues. We found that 2-Cys Prxs, in particular, Prx1 and Prx4, were preferentially expressed in cell lines derived from human lung cancer. Through isoform specific knockdown of individual Prx, we demonstrated that Prx1 and Prx4 (but not Prx3) were required for human lung cancer A549 cells to form soft agar colony and to invade through matrigel in culture. Knockdown of Prx1 or Prx4 significantly reduced the activation of c-Jun and repressed the AP-1 mediated promoter activity. In mouse xenograft models, knockdown of Prx4 in A549 cells reduced subcutaneous tumor growth and blocked metastasis formation initiated through tail vein injection. Moreover, overexpression of Prx1 or Prx4 further enhanced the malignancy of A549 cells both in culture and in mouse xenografts in vivo. These data provide an in-depth understanding of the contribution of Prx1 and Prx4 to lung cancer development and are of importance for future development of therapeutic methods that targeting 2-Cys Prxs.

Sulfiredoxin (Srx), the exclusive enzyme that reduces the hyperoxidized inactive form of peroxiredoxins (Prxs), has been found highly expressed in several types of human skin cancer. To determine whether Srx contributed to skin tumorigenesis in vivo, Srx null mice were generated on an FVB background. Mouse skin tumorigenesis was induced by a 7,12-dimethylbenz[α]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) protocol. We found that the number, volume and size of papillomas in Srx(-/-) mice were significantly fewer compared with either wild-type (Wt) or heterozygous (Het) siblings. Histopathological analysis revealed more apoptotic cells in tumors from Srx(-/-) mice. Mechanistic studies in cell culture revealed that Srx was stimulated by TPA in a redox-independent manner. This effect was mediated transcriptionally through the activation of mitogen-activated protein kinase and Jun-N-terminal kinase. We also demonstrated that Srx was capable of reducing hyperoxidized Prxs to facilitate cell survival under oxidative stress conditions. These findings suggested that loss of Srx protected mice, at least partially, from DMBA/TPA-induced skin tumorigenesis. Therefore, Srx has an oncogenic role in skin tumorigenesis and targeting Srx may provide novel strategies for skin cancer prevention or treatment.

Oxidative stress is known to cause tumorigenesis through induction of DNA and lipid damage. It also promotes cancer progression through a largely unknown mechanism. Sulfiredoxin (Srx) is a novel oxidative stress-induced antioxidant protein whose function in tumorigenesis and cancer progression has not been well studied. We report that Srx is highly expressed in human lung cancer. Knockdown of Srx reduces anchorage-independent colony formation, cell migration, and invasion of human lung cancer cells. Srx preferentially interacts with Peroxiredoxin (Prx) IV relative to other Prxs due to its intrinsic higher binding affinity. Knockdown of Prx IV recapitulates the phenotypic changes of depleting Srx. Disruption or enhancement of the Srx–Prx IV axis leads respectively to reduction or acceleration of tumor growth and metastasis formation in vivo. Through identification and validation of the downstream mediators we unraveled the Srx-mediated signaling network that traverses AP-1–activating and other phosphokinase signaling cascades. Our work reveals that the Srx–Prx IV axis is critical for lung cancer maintenance and metastasis, suggesting that targeting the Srx–Prx IV axis may provide unique effective strategies for cancer prevention and treatment.

Previous studies have shown that a dominant negative form of c-Jun (TAM67) suppresses mouse skin carcinogenesis both in vitro and in vivo . The current study identifies Sulfiredoxin (Srx) as a unique target of activator protein-1 (AP-1) activation and TAM67 inhibition. Manipulation of Srx levels by ShRNA or over-expression demonstrates that Srx is critical for redox homeostasis through reducing hyperoxidized peroxiredoxins. In JB6 cells, knockdown of Srx abolishes tumor promoter-induced transformation and enhances cell sensitivity to oxidative stress. Knockdown of Srx also impairs c-Jun phosphorylation, implicating a role for Srx in the feedback regulation of AP-1 activity. Screening of patient tissues by tissue microarray reveals elevated Srx expression in several types of human skin cancers. Our study indicates that Srx is a functionally significant target of AP-1 blockade that may have value in cancer prevention or treatment.

Our previous studies have found that expression of p27 in oligodendrocytes enhances myelin basic protein (MBP) gene expression through a mechanism that involves the transcription factor Sp1. In this study we show that this activation only requires the N-terminal 45 amino acids of p27 containing a functional cyclin-binding motif. In an effort to identify other cofactors that are involved in the p27-mediated activation of MBP gene expression, a yeast two-hybrid assay was performed using an N-terminal truncated p27 and a mouse embryo cDNA library. Galectin-4 was found to interact with p27 in the yeast two-hybrid assay. This novel interaction was also confirmed using a glutathione-S-transferase interaction assay and immunoprecipitation assays. Expression of galectin-4 in primary oligodendrocytes was confirmed by western blot. Additionally, the MBP promoter could be activated by expression of galectin-4 in CG4 oligodendrocytes, similar to the effects of increased p27 levels. We also show that Sp1 and galectin-4 interact in cells, while a complex of all three proteins could not be found. We conclude that galectin-4 is involved in the p27-mediated activation of the MBP gene, possibly through modulation of the glycosylation status of the transcription factor Sp1.

To investigate the inhibition property of a novel antifungal polypeptide APS in vitro and its possible antifungal mechanism.The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of APS against different fungi were detected by microdilution test. And the voltaic change of planar lipid bilayer was observed during APS treatment.APS at low MIC and MBC was shown to have effects on phytopathogenic fungi tested except aspergillus fumigatus, the minimum values of MIC and MBC being 1.43 micrograms/ml and 1.78 micrograms/ml respectively. After APS was put in, the transmembrane current could be recorded.APS has strong inhibitory effects on the germination of spores of several phytopathogenic fungi. Its antifungal mechanism is likely to form persistent pores on the cell membrane, thus causing membrane permeation and the death of fungi.

Sulfiredoxin (Srx) is the enzyme that reduces the overoxidized inactive form of peroxiredoxins (Prxs). To study the function of Srx in carcinogenesis in vivo, we tested whether loss of Srx protects mice from cancer development. Srx null mice were generated and colon carcinogenesis was induced by an azoxymethane (AOM) and dextran sulfate sodium (DSS) protocol. Compared to either wildtype or heterozygotes, Srx-/- mice had significantly reduced rates in both tumor multiplicity and volume. Mechanistic studies reveal that loss of Srx did not alter tumor cell proliferation; however, increased apoptosis and decreased inflammatory cell infiltration were obvious in tumors from Srx null mice compared to those from wildtype control. In addition to the AOM/DSS model, examination of Srx expression in human reveals a tissue-specific expression pattern. Srx expression was also demonstrated in tumors from colorectal cancer patients and the levels of expression were associated with patients’ clinic stage. These data provide the first in vivo evidence that loss of Srx renders mice resistance to AOM/DSS-induced colon carcinogenesis, suggesting that Srx has a critical oncogenic role in cancer development, and Srx may be used as a marker for human colon cancer pathogenicity. Citation Format: Qiou Wei, Hong Jiang, Alyson Baker, Lisa K. Dodge, Matthieu Gerard, Matthew R. Young, Michel B. Toledano, Nancy H. Colburn. Sulfiredoxin is required for AOM/DSS induced mouse colon carcinogenesis and invasiveness of human colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2727. doi:10.1158/1538-7445.AM2013-2727 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Programmed-cell-death-4 (Pdcd4) is a novel protein that functions as a transformation suppressor. Pdcd4 deficiency renders mice susceptible to increased tumor multiplicity in initiation-promotion skin carcinogenesis. On the contrary transgenic expression of Pdcd4 in mice inhibits DMBA-TPA induced skin tumorigenesis and tumor progression. Pdcd4 interacts with translation initiation factors eIF4A and eIF4G to inhibit translation in an mRNA-specific fashion and consequently blocks pro-oncogenic events such as activation of activator protein-1 (AP-1)-dependent transcription, anchorage-independent transformation and invasion. Pdcd4 expression decreases during carcinogenesis in several human cancer sites including colon, esophagus, lung, pancreas and brain and its activation appears to be important in the mechanism by which some cancer therapeutic drugs work. While Pdcd4 is not mutationally inactivated, its expression is post translationally regulated. Pdcd4 expression is decreased during carcinogenesis by mechanisms involving microRNA-21 (miR21) inhibition of translation and by S6 kinase-dependent proteasome degradation. Loss of Pdcd4 has been shown to affect transcription of TIMP-2, MAP4K1, LOX and u-PAR. Although the tumor suppressor Pdcd4 acts directly on the translation initiation complex, translational targets of Pdcd4 have yet to be identified. In order to identify and characterize functionally significant translational targets of tumor suppressor Pdcd4 when it inhibits translation during tumor promotion, tumorigenesis and invasion of malignant tumors, we knocked down Pdcd4 in T47D breast cancer cells. Loss of Pdcd4 increases cell migration and invasion as well as activity of luciferase reporters of AP-1-dependent transcription and translation initiation. To identify translational targets of Pdcd4, translationally active mRNA species were fractionated by sucrose gradient. Polyribosome bound mRNA fractions were pooled and analyzed for mRNA levels by microarray. Knockdown of Pdcd4 produced more than 2 fold increases in 14 mRNAs in the translationally active polyribosomal fractions compared to the control. The polysome shifts in Pdcd4 translational targets are being confirmed by quantitative RT-PCR and Western blot analysis of protein. Pdcd4 targets recently confirmed by quantitative RT-PCR are PPARGC1B and delta p73, both known to be oncogenic, as well as zinc finger protein ZNF281. The functional significance of these targets is being determined. Discovery of the mRNAs selectively targeted by Pdcd4 will facilitate the elucidation of the mechanism by which Pdcd4 suppresses tumorigenesis and may provide additional targets for intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3079. doi:10.1158/1538-7445.AM2011-3079

Oxidative stress is known to cause tumorigenesis through induction of DNA and lipid damage. It also promotes tumor progression through a largely unknown mechanism. Sulfiredoxin (Srx) is an oxidation-induced antioxidant protein that catalyzes the reduction of hyperoxidized peroxiredoxins (Prxs) to the reduced form to restore their peroxidase activity. Our previous study demonstrates that Srx is a novel target of activator protein-1 (AP-1) activation and TAM67 (a dominant negative form of c-Jun) inhibition. However, the role of Srx in cell signaling and cancer development has not been well-studied. In this study, our goal is to investigate the function and molecular mechanism of Srx in human lung cancer. Our current work demonstrates an important oncogenic role of Srx in cancer maintenance and progression through the Srx-Prx IV axis and the modulation of specific phosphokinase signaling cascades. First, tissue microarray was used to evaluate Srx expression in human primary lung cancer tissues. We found that Srx is highly expressed in lung squamous cell carcinoma and adenocarcinoma, but not in the normal lung. By knockdown of Srx in human lung cancer A549 cells, we discovered that Srx is required for anchorage independent colony formation, cell migration and invasion of A549 cells. Second, using immunoprecipitation and mass spectrometry, we identified proteins that interact with Srx. Surprisingly, we found that Prx IV was the predominant protein that interacts with Srx. Using purified recombinant proteins and surface plasmon resonance techniques, we demonstrated that Srx has a higher binding affinity to Prx IV than to other Prxs. Third, we found that knockdown of Prx IV, but not other Prxs, recapitulated the phenotypic outcomes of depleting Srx in A549 cells. In addition, when injected into SCID mice, disruption or enhancement of the Srx-Prx IV axis leads respectively to reduction or acceleration of mouse tumor growth and lung metastasis formation. Furthermore, proteome profiler phosphokinase assay revealed that activation of specific phosphokinase signaling cascades, including AP-1, CREB and MAPK signaling, was dependent on the integrity of the Srx-Prx IV axis. In summary, our findings demonstrate a novel oncogenic role of the Srx-Prx IV axis in human lung cancer, and suggest that targeting the Srx-Prx IV axis may provide novel effective strategies for cancer prevention and treatment. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A19.

Sulfiredoxin (Srx) is a novel antioxidant protein that has been shown to reduce the hyper oxidized Peroxiredoxins (Prxs). Our previous study has demonstrated that Srx is required for tumor promoter induced cell transformation and is highly expressed in certain types of human skin cancers (Wei et al PNAS 2008). In this study, our goal is to further examine the expression of Srx in a broad range of human tumors and to explore its functional significance and molecular mechanisms in human cancer development. We found that Srx is highly expressed in several types of human lung cancers and its level is correlated with cancer malignancy and metastasis. Using human lung cancer A549 cell in culture, we found that knockdown of Srx significantly reduces anchorage independent growth in soft agar, cell migration in a wound-healing assay and cell invasiveness in a matrigel invasion assay. In an effort to discover downstream mediators of Srx-signaling pathway, we used affinity-based pull down and mass spectrometry methods to identify proteins that interact with Srx. To our surprise, Prx IV was found to be the most abundant protein that interacts with Srx in at least three different cell lines and the preferential interaction occurs independently of its expression levels. To characterize the Srx-Prx IV interaction, bacterial expressed recombinant Srx, Prx I, II, III and IV were individually purified. In an in vitro binding assay using Surface Plasmon Resonance technique, we confirmed that purified Srx has a much higher binding affinity to Prx IV than to Prx I, II or III. With a lentiviral based shRNA technology, each individual Prx was specifically knocked down in A549 cells. The phenotypic changes of these cells were compared to the Srx knockdown cells. We found that knockdown of Prx IV, but not other Prxs, was sufficient to recapitulate the attenuation of tumor phenotype and progression of Srx knockdown cells. In addition, we also confirmed that Prx IV undergoes rapid hyperoxidation in the presence of oxidative stress and is preferentially reduced by Srx in A549 cells. Overall our data indicate that Srx is highly expressed in human lung cancers and Srx-Prx IV axis is critical for human lung cancer cell maintenance, migration and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1481.

B83 Previous studies from others and our lab have shown that activator protein 1 (AP-1) plays a pivotal role in tumorigenesis in vitro and in vivo. Expression of a dominant negative form c-Jun, TAM67, dramatically inhibits tumor promoter induced skin carcinogenesis. In this study, our goals are to identify target genes that are specifically induced by tumor promoter and inhibited by TAM67, and to investigate their functional significances in tumor promotion and progression. First, we demonstrated that Sulfiredoxin (Srx) is a novel target of AP-1 activation and TAM67 inhibition. In mouse epidermis, expression of Srx was upregulated by tumor promoter and inhibited by TAM67 as revealed by DNA microarray. This was further confirmed in mouse epithelial-derived JB6 cells by RT-PCR, Western blotting, luciferase reporter and chromatin immunoprecipitation assays at transcript and protein levels. Next, we demonstrated that Srx is important for maintaining of cellular redox homeostasis through reduction of overoxidized Peroxiredoxins (Prxs). Knockdown of Srx led to a much slower reduction rate of overoxidized Prxs, whereas overexpression of Srx resulted in an increased reduction rate of overoxidized Prxs. Then we found that knockdown of Srx dramatically inhibited tumor promoter induced neoplastic transformation as indicated by anchorage-independent growth in soft agar. This effect was not due to the inhibition of cell proliferation as depletion of Srx stimulated cell proliferation under adherent conditions. Depletion of Srx also led to impaired AP-1 activation and increased cell death under oxidative conditions, which indicated that Srx might be involved in the regulation of AP-1 activity through a novel positive feedback loop. Finally, we examined the expression profiling of Srx in normal skin and tissues from patients with various skin diseases by tissue microarray. We found strong positive expression of Srx in skin malignant tumors. In summary, we identified Srx as a novel target of tumor promoter induced AP-1 activation and is inhibited by TAM67. We demonstrated that Srx is important for maintaining of cellular redox balance and is required for tumor promoter induced neoplastic transformation. We also found strong expression of Srx in human skin malignant tumors. Therefore, we propose that Srx can be used as a marker for skin malignancy, and manipulation of Srx expression may be of interest in cancer prevention and treatment. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B83.