Identifying the role of MTHFD1L in prostate cancer progression from genetic analysis and experimental validation.

One-carbon metabolism plays a crucial role in tumorigenesis as it supplies the one-carbon units necessary for nucleotide synthesis, epigenetic regulation, and redox metabolism, ensuring the rapid proliferation of cancer cells. However, their roles in prostate cancer progression remain poorly understood. In this study, we investigated the association between genetic variants in the one-carbon metabolism pathway and clinical outcomes in patients receiving androgen deprivation therapy for prostate cancer. The associations of 130 single-nucleotide polymorphisms located within 14 genes involved in the one-carbon metabolism pathway with cancer-specific survival (CSS), overall survival, and progression-free survival were assessed using Cox regression in 630 patients with prostate cancer. Subsequently, functional studies were performed using prostate cancer cell lines. After adjusting for covariates and multiple testing, MTHFD1L rs2073190 was found to be significantly associated with CSS (P = 0.000184). Further pooled analysis of multiple datasets demonstrated that MTHFD1L was upregulated in prostate cancer and increased MTHFD1L expression was positively correlated with tumor aggressiveness and poor patient prognosis. Functionally, MTHFD1L knockdown suppressed prostate cancer cell proliferation and colony formation. RNA sequencing and pathway analysis revealed that differentially expressed genes were predominantly enriched in the cell cycle pathway. In conclusion, genetic variants in MTHFD1L of one-carbon metabolism may serve as promising predictors, and our findings offer valuable insights into the underlying genetic mechanisms of prostate cancer progression.

Tumor-derived interleukin-1 receptor antagonist exhibits immunosuppressive functions and promotes pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a pernicious disease characterized by an immunosuppressive milieu that is unresponsive to current immunotherapies. Interleukin-1 receptor antagonist (IL-1Ra) is a natural anti-inflammatory cytokine; however, its contribution to cancer pathogenesis and immunosuppression remains elusive. In this research, we investigated the role and mechanism of IL-1Ra in malignant progression of PDA. RESULTS: Through analyzing clinical dataset and examining the pathological tumor tissues and serum samples, we have demonstrated that IL-1Ra expression is elevated in human PDA and positively associated with malignant progression of PDA. To study the biological function of IL-1Ra in tumors, we generated a set of mouse pancreatic cancer cell lines with a knockout (KO) of the Il1rn gene, encoding IL-1Ra, and compared the tumor growth rates in immune-competent and immune-deficient mice. We found that the Il1rn KO cells exhibited greater tumor inhibition in immune-competent mice, highlighting the crucial role of a functional immune system in Il1rn KO-mediated anti-tumor response. Consistently, we found an increase in CD8+ T cells and a decrease in CD11b+Ly6G- immunosuppressive mononuclear population in the tumor microenvironment of Il1rn KO-derived tumors. To monitor the inhibitory effects of IL-1Ra on immune cells, we utilized a luciferase-based reporter CD4+ T cell line and splenocytes, which were derived from transgenic mice expressing ovalbumin-specific T cell receptors in CD8+ T cells, and mice immunized with ovalbumin. We showed that IL-1Ra suppressed T cell receptor signaling and inhibited antigen-specific interferon-γ (IFN-γ) secretion and cytolytic activity in splenocytes. CONCLUSIONS: Our findings illustrate the immunosuppressive properties of the natural anti-inflammatory cytokine IL-1Ra, and provide a rationale for considering IL-1Ra-targeted therapies in the treatment of PDA.

Disruption of CCL2 in Mesenchymal Stem Cells as an Anti-Tumor Approach against Prostate Cancer.

BACKGROUND: MSCs are known to secrete abundant CCL2, which plays a crucial role in recruiting TAMs, promoting tumor progression. It is important to know whether disrupting MSC-derived CCL2 affects tumor growth. METHODS: Murine bone marrow-derived MSCs were characterized by their surface markers and differentiation abilities. Proliferation and migration assays were performed in order to evaluate the functions of MSCs on cancer cells. CCL2 expression in MSCs was reduced by small interfering RNA (siRNA) or completely disrupted by CRISPR/Cas9 knockout (KO) approaches. An immune-competent syngeneic murine model of prostate cancer was applied in order to assess the role of tumor cell- and MSC-derived CCL2. The tumor microenvironment was analyzed to monitor the immune profile. RESULTS: We confirmed that tumor cell-derived CCL2 was crucial for tumor growth and MSCs migration. CCL2 KO MSCs inhibited the migration of the monocyte/macrophage but not the proliferation of tumor cells in vitro. However, the mice co-injected with tumor cells and CCL2 KO MSCs exhibited anti-tumor effects when compared with those given tumor cell alone and with control MSCs, partly due to increased infiltration of CD45+CD11b+Ly6G- mononuclear myeloid cells. CONCLUSIONS: Disruption of MSC-derived CCL2 enhances anti-tumor functions in an immune-competent syngeneic mouse model for prostate cancer.

Investigation of Anti-Tumor Effects of an MLK1 Inhibitor in Prostate and Pancreatic Cancers.

It was shown that mixed lineage kinase 1 (MLK1) regulates pancreatic cancer growth; however, its role in prostate cancer remains unclear. We showed that MLK1 is a tumor marker in prostate cancer by analyzing clinical gene expression data and identified a novel MLK1 inhibitor (NSC14465) from the compound library of the National Cancer Institute (NCI) using a MLK1 protein structure. The inhibitory effects of MLK1 were validated by an in vitro kinase assay and by monitoring phosphorylation signaling, and the anti-proliferation function was shown in several prostate and pancreatic cancer cell lines. We also demonstrated anti-tumor ability and prevention of cancer-related weight loss in a syngeneic orthotopic mouse model of pancreatic cancer that mimicked the tumor growth environment in the pancreas. Our results demonstrate that the MLK1 inhibitor is an anti-tumor agent for malignant prostate and pancreatic cancers.

Genetic variants in MAPK10 modify renal cell carcinoma susceptibility and clinical outcomes.

AIMS: The mitogen-activated protein kinase (MAPK) cascades integrate various upstream signals to regulate many cellular functions, including proliferation, differentiation, and survival. Dysregulation of these pathways has been implicated in the occurrence and progression of a variety of cancers. MAIN METHODS: This study aimed to assess the association of 192 single nucleotide polymorphisms in 22 MAPK cascade genes with renal cell carcinoma (RCC) risk and survival in 312 patients and 318 controls. KEY FINDINGS: After multiple testing correction and multivariate analysis, the minor T allele of MAPK10 rs12648265 remained associated with a lower risk of RCC (adjusted odds ratio 0.64, 95% confidence interval 0.50-0.82, P = 0.000426) and metastasis (adjusted hazard ratio 0.50, 95% confidence interval 0.30-0.82, P = 0.006). Presence of the rs12648265 T allele demonstrated a trend towards being associated with increased MAPK10 expression, and meta-analysis of four RCC datasets indicated that high MAPK10 expression is associated with a favourable prognosis. Furthermore, activation of MAPK10 by the potent agonist anisomycin inhibited RCC cell growth in vitro, suggesting an involvement of MAPK10 in RCC progression. SIGNIFICANCE: In conclusion, MAPK10 may be a meaningful biomarker and a potential therapeutic target in RCC.

Roles of Interleukin-1 Receptor Antagonist in Prostate Cancer Progression.

BACKGROUND: Inflammation is known to promote tumor formation and progression; however, we found a natural anti-inflammatory factor, interleukin (IL)-1 receptor antagonist (IL1RN), in a mouse transgenic adenocarcinoma of the mouse prostate (TRAMP)-C1-derived tumor microenvironment (TME). We sought to characterize the functions of the IL1RN-secreting cells in the TME. METHODS: We compared tumors collected from two syngeneic mouse models and isolated tumor-infiltrating leukocytes (TILs) with different cluster of differentiation 11b (CD11b) statuses. We examined the proliferation functions of the TILs and the IL1RN using several approaches, including a colony-formation assay and DNA synthesis levels. RESULTS: We demonstrated that CD11b-deficient TILs (TILs/CD11b-) secreted the IL1RN and promoted proliferation by analyzing conditioned media. In addition to mouse TRAMP-C1, proliferation functions of the IL1RN were confirmed in several human castration-resistant prostate cancer (CRPC) cell lines and one normal epithelial cell line. The androgen-sensitive lymph node carcinoma of the prostate (LNCaP) cell line showed cytotoxic responses to IL1ß treatment and androgen-dependent regulation of IL-1 receptor type 1 (IL1R1), while the C4-2 CRPC cell line did not. IL1RN rescued LNCaP cells from the cytotoxic effects of IL1ß/IL1R1 signaling. CONCLUSIONS: Our results support TILs/CD11b- cells being able to protect androgen-dependent cells from inflammatory damage and promote the malignant progression of prostate cancers partly through the IL1RN in the TME.

Tumor-infiltrating Leukocytes Suppress Local Inflammation Via Interleukin-1 Receptor Antagonist in a Syngeneic Prostate Cancer Model.

BACKGROUND: Several lines of evidence have demonstrated the tumor-promoting function of inflammation. Since many chemokines are important in coordinating immune cells during inflammation, monitoring intratumoral chemokines provides a way to study the tumor microenvironment. METHODS: To identify tumorigenic chemokines, we compared two syngeneic mouse prostate cancer cell lines by an antibody array and quantitative reverse-transcription polymerase chain reaction (RT-PCR). The tumor microenvironment was analyzed by monitoring gene expressions in mouse tumor tissues, primary cells, and tumor-infiltrating leukocytes (TILs). RESULT: We identified a group of pro-inflammatory chemokines associated with a tumorigenic transgenic adenocarcinoma mouse prostate (TRAMP)-C1 cell line. In the tumor microenvironment, the TILs secrete a natural anti-inflammatory factor, interleukin-1 receptor antagonist (IL1RN), which inhibits the functions of pro-inflammatory molecules and likely accounts for tumor type-specific anti-inflammation functions. CONCLUSION: Our results support that tumor cells recruit TILs by pro-inflammatory chemokines to establish an IL1RN-mediated anti-inflammatory environment in the syngeneic prostate cancer model.

A Synergistic Anti-Cancer Effect of Troglitazone and Lovastatin in a Human Anaplastic Thyroid Cancer Cell Line and in a Mouse Xenograft Model.

Anaplastic thyroid cancer (ATC) is a malignant subtype of thyroid cancers and its mechanism of development remains inconclusive. Importantly, there is no effective strategy for treatment since ATC is not responsive to conventional therapies, including radioactive iodine therapy and thyroid-stimulating hormone suppression. Here, we report that a combinational approach consisting of drugs designed for targeting lipid metabolism, lovastatin (an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGCR) and troglitazone (an agonist of peroxisome proliferator-activated receptor gamma, PPARγ), exhibits anti-proliferation in cell culture systems and leads to tumor regression in a mouse xenograft model. The composition contains a sub-lethal concentration of both drugs and exhibits low toxicity to certain types of normal cells. Our results support a hypothesis that the inhibitory effect of the combination is partly through a cell cycle arrest at G0/G1 phase, as evidenced by the induction of cyclin-dependent kinase inhibitors, p21cip and p27kip, and the reduction of hyperphosphorylated retinoblastoma protein (pp-Rb)-E2F1 signaling. Therefore, targeting two pathways involved in lipid metabolism may provide a new direction for treating ATC.

Androgen deprivation therapy-induced epithelial-mesenchymal transition of prostate cancer through downregulating SPDEF and activating CCL2.

The chemokine CC motif ligand 2 (CCL2) is important in recruiting tumor-associated macrophages and is involved in the development of castration-resistance prostate cancer (CRPC) after androgen-deprivation therapy (ADT); however, the underlying mechanism remains unclear. We found that inactivation of the androgen receptor (AR) reduces a transcriptional repressor (SAM pointed domain-containing ETS transcription factor, SPDEF) of CCL2, which mediates epithelial-to-mesenchymal transition (EMT) of prostate tumor cells. Cell lines derived from a prostate-specific Pten/Trp53-null mouse and capable of a spontaneous EMT were utilized for identification of CCL2, and showed that reduced SPDEF expression was associated with an elevated CCL2-activated EMT. AR signaling inhibits CCL2 through a SPDEF-mediated mechanism in that the SPDEF recognizes the CCL2 promoter and transcriptionally represses its activity. Ectopically expressed SPDEF reduced the EMT and rescued expression of CCL2 in SPDEF-expressing cells, which induced the EMT and promotes malignant functions of prostate cancer cells. In tissues from prostate cancer patients with ADT, low SPDEF levels were correlated with high CCL2 expression compared to patients without ADT. We present a novel mechanism that contributes to the EMT and metastatic phenotype observed in a subset of ADT-resistant prostate cancer, where the CCL2 is stimulated through the inactivated of AR-mediated SPDEF.

Disruption of ETV6 leads to TWIST1-dependent progression and resistance to epidermal growth factor receptor tyrosine kinase inhibitors in prostate cancer.

BACKGROUND: ETS variant gene 6 (ETV6) is a putative tumor suppressor and repressed by epidermal growth factor receptor (EGFR) signaling in prostate cancer. Since EGFR antagonists seem ineffective in castration-resistant prostate cancer (CRPC), we aim to study the role of ETV6 in the development of drug resistance. METHODS: Etv6 target gene was validated by ChIP and promoter reporter assays. Correlation of ETV6 and TWIST1 was analyzed in human clinical datasets and tissue samples. Migration, invasion, and metastasis assays were used to measure the cellular responses after perturbation of ETV6 -TWIST1 axis. Proliferation and tumor growth in xenograft model were performed to evaluate the drug sensitivities of EGFR-tyrosine kinase inhibitors (TKIs). RESULTS: ETV6 inhibits TWIST1 expression and disruption of ETV6 promotes TWIST1-dependent malignant phenotypes. Importantly, ETV6 is required to the anti-proliferation effects of EGFR-TKIs, partly due to the inhibitory function of ETV6 on TWIST1. We also found that EGFR-RAS signaling is tightly controlled by ETV6, supporting its role in TKI sensitivity. CONCLUSIONS: Our study demonstrates that disruption of ETV6 contributes to EGFR-TKI resistance, which is likely due to derepression of TWIST1 and activation of EGFR-RAS signaling. Our results implicate ETV6 as a potential marker for predicting efficacy of an EGFR-targeted anticancer approach. Combination treatment of TWIST1 inhibitors could sensitize the anti-proliferation effects of EGFR-TKIs.

Simvastatin Inhibits Cell Proliferation and Migration in Human Anaplastic Thyroid Cancer.

Malignant human anaplastic thyroid cancer (ATC) is pertinacious to conventional therapies. The present study investigated the anti-cancer activity of simvastatin and its underlying regulatory mechanism in cultured ATC cells. Simvastatin (0-20 µM) concentration-dependently reduced cell viability and relative colony formation. Depletions of mevalonate (MEV) and geranylgeranyl pyrophosphate (GGpp) by simvastatin induced G1 arrest and increased apoptotic cell populations at the sub-G1 phase. Adding MEV and GGpp prevented the simvastatin-inhibited cell proliferation. Immunoblotting analysis illustrated that simvastatin diminished the activation of RhoA and Rac1 protein, and this effect was prevented by pre-treatment with MEV and GGpp. Simvastatin increased the levels of p21cip and p27kip proteins and reduced the levels of hyperphosphorylated-Rb, E2F1 and CCND1 proteins. Adding GGpp abolished the simvastatin-increased levels of p27kip protein, and the GGpp-caused effect was abolished by Skp2 inhibition. Introduction of Cyr61 siRNA into ATC cells prevented the epidermal growth factor (EGF)-enhanced cell migration. The EGF-induced increases of Cyr61 protein expression and cell migration were prevented by simvastatin. Taken together, these results suggest that simvastatin induced ATC proliferation inhibition through the deactivation of RhoA/Rac1 protein and overexpression of p21cip and p27kip, and migration inhibition through the abrogation of Cyr61 protein expression.

Loss of SPDEF and gain of TGFBI activity after androgen deprivation therapy promote EMT and bone metastasis of prostate cancer.

Androgen deprivation therapy (ADT) targeting the androgen receptor (AR) is a standard therapeutic regimen for treating prostate cancer. However, most tumors progress to metastatic castration-resistant prostate cancer after ADT. We identified the type 1, 2, and 4 collagen-binding protein transforming growth factor-ß (TGFß)-induced protein (TGFBI) as an important factor in the epithelial-to-mesenchymal transition (EMT) and malignant progression of prostate cancer. In prostate cancer cell lines, AR signaling stimulated the activity of the transcription factor SPDEF, which repressed the expression of TGFBI ADT, AR antagonism, or overexpression of TGFBI inhibited the activity of SPDEF and enhanced the proliferation rates of prostate cancer cells. Knockdown of TGFBI suppressed migration and proliferation in cultured cells and reduced prostate tumor growth and brain and bone metastasis in xenograft models, extending the survival of tumor-bearing mice. Analysis of prostate tissue samples collected before and after ADT from the same patients showed that ADT reduced the nuclear abundance of SPDEF and increased the production of TGFBI. Our findings suggest that induction of TGFBI promotes prostate cancer growth and metastasis and can be caused by dysregulation or therapeutic inhibition of AR signaling.

Epidermal growth factor receptor signaling promotes metastatic prostate cancer through microRNA-96-mediated downregulation of the tumor suppressor ETV6.

It has been suggested that ETV6 serves as a tumor suppressor; however, its molecular regulation and cellular functions remain unclear. We used prostate cancer as a model system and demonstrated a molecular mechanism in which ETV6 can be regulated by epidermal growth factor receptor (EGFR) signaling through microRNA-96 (miR-96)-mediated downregulation. In addition, EGFR acts as a transcriptional coactivator that binds to the promoter of primary miR-96 and transcriptionally regulates miR-96 levels. We analyzed two sets of clinical prostate cancer samples, confirmed association patterns that were consistent with the EGFR-miR-96-ETV6 signaling model and demonstrated that the reduced ETV6 levels were associated with malignant prostate cancer. Based on results derived from multiple approaches, we identified the biological functions of ETV6 as a tumor suppressor that inhibits proliferation and metastasis in prostate cancer. We present a molecular mechanism in which EGFR activation leads to the induction of miR-96 expression and suppression of ETV6, which contributes to prostate cancer progression.

DNA methyltransferase DNMT3A associates with viral proteins and impacts HSV-1 infection.

Viral infections can alter the cellular epigenetic landscape, through modulation of either DNA methylation profiles or chromatin remodeling enzymes and histone modifications. These changes can act to promote viral replication or host defense. Herpes simplex virus type 1 (HSV-1) is a prominent human pathogen, which relies on interactions with host factors for efficient replication and spread. Nevertheless, the knowledge regarding its modulation of epigenetic factors remains limited. Here, we used fluorescently-labeled viruses in conjunction with immunoaffinity purification and MS to study virus-virus and virus-host protein interactions during HSV-1 infection in primary human fibroblasts. We identified interactions among viral capsid and tegument proteins, detecting phosphorylation of the capsid protein VP26 at sites within its UL37-binding domain, and an acetylation within the major capsid protein VP5. Interestingly, we found a nuclear association between viral capsid proteins and the de novo DNA methyltransferase DNA (cytosine-5)-methyltransferase 3A (DNMT3A), which we confirmed by reciprocal isolations and microscopy. We show that drug-induced inhibition of DNA methyltransferase activity, as well as siRNA- and shRNA-mediated DNMT3A knockdowns trigger reductions in virus titers. Altogether, our results highlight a functional association of viral proteins with the mammalian DNA methyltransferase machinery, pointing to DNMT3A as a host factor required for effective HSV-1 infection.

Sirtuin 7 plays a role in ribosome biogenesis and protein synthesis.

It has been shown that SIRT7 regulates rDNA transcription and that reduced SIRT7 levels inhibit tumor growth. This anti-tumor effect could be due to reduced Pol I activity and perturbed ribosome biogenesis. In this study, using pulse labeling with RNA and amino acid analogs, we found that SIRT7 knockdown efficiently suppressed both RNA and protein synthesis. Surprisingly, SIRT7 knockdown preferentially inhibited protein synthesis over rDNA transcription, whereas the levels of both were reduced to similar extents following Pol I knockdown. Using an affinity purification mass spectrometry approach and functional analyses of the resulting SIRT7 interactome, we identified and validated SIRT7 interactions with proteins involved in ribosomal biogenesis. Indeed, SIRT7 co-fractionated with monoribosomes within a sucrose gradient. Using reciprocal isolations, we determined that SIRT7 interacts specifically with mTOR and GTF3C1, a component of the Pol III transcription factor TFIIIC2 complex. Further studies found that SIRT7 knockdown triggered an increase in the levels of LC3B-II, an autophagosome marker, suggesting a link between SIRT7 and the mTOR pathway. Additionally, we provide several lines of evidence that SIRT7 plays a role in modulating Pol III function. Immunoaffinity purification of SIRT7-GFP from a nuclear fraction demonstrated specific SIRT7 interaction with five out of six components of the TFIIIC2 complex, but not with the TFIIIA or TFIIIB complex, the former of which is required for Pol III-dependent transcription of tRNA genes. ChIP assays showed SIRT7 localization to the Pol III targeting genes, and SIRT7 knockdown triggered a reduction in tRNA levels. Taken together, these data suggest that SIRT7 may regulate Pol III transcription through mTOR and the TFIIIC2 complex. We propose that SIRT7 is involved in multiple pathways involved in ribosome biogenesis, and we hypothesize that its down-regulation may contribute to an antitumor effect, partly through the inhibition of protein synthesis.

Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription.

Among mammalian sirtuins, SIRT7 is the only enzyme residing in nucleoli where ribosomal DNA is transcribed. Recent reports established that SIRT7 associates with RNA Pol I machinery and is required for rDNA transcription. Although defined by its homology to the yeast histone deacetylase Sir2, current knowledge suggests that SIRT7 itself has little to no deacetylase activity. Because only two SIRT7 interactions have been thus far described: RNA Pol I and upstream binding factor, identification of proteins and complexes associating with SIRT7 is critical to understanding its functions. Here, we present the first characterization of SIRT7 interaction networks. We have systematically investigated protein interactions of three EGFP-tagged SIRT7 constructs: wild type, a point mutation affecting rDNA transcription, and a deletion mutant lacking the predicted coiled-coil domain. A combinatorial proteomics and bioinformatics approach was used to integrate gene ontology classifications, functional protein networks, and normalized abundances of proteins co-isolated with SIRT7. The resulting refined proteomic data set confirmed SIRT7 interactions with RNA Pol I and upstream binding factor and highlighted association with factors involved in RNA Pol I- and II-dependent transcriptional processes and several nucleolus-localized chromatin remodeling complexes. Particularly enriched were members of the B-WICH complex, such as Mybbp1a, WSTF, and SNF2h. Prominent interactions were validated by a selected reaction monitoring-like approach using metabolic labeling with stable isotopes, confocal microscopy, reciprocal immunoaffinity precipitation, and co-isolation with endogenous SIRT7. To extend the current knowledge of mechanisms involved in SIRT7-dependent regulation of rDNA transcription, we showed that small interfering RNA-mediated SIRT7 knockdown leads to reduced levels of RNA Pol I protein, but not messenger RNA, which was confirmed in diverse cell types. The down-regulation of RNA Pol I protein levels placed in the context of SIRT7 interaction networks led us to propose that SIRT7 plays a crucial role in connecting the function of chromatin remodeling complexes to RNA Pol I machinery during transcription.

Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription.

Among mammalian sirtuins, SIRT7 is the only enzyme residing in nucleoli where ribosomal DNA is transcribed. Recent reports established that SIRT7 associates with RNA Pol I machinery and is required for rDNA transcription. Although defined by its homology to the yeast histone deacetylase Sir2, current knowledge suggests that SIRT7 itself has little to no deacetylase activity. Because only two SIRT7 interactions have been thus far described: RNA Pol I and upstream binding factor, identification of proteins and complexes associating with SIRT7 is critical to understanding its functions. Here, we present the first characterization of SIRT7 interaction networks. We have systematically investigated protein interactions of three EGFP-tagged SIRT7 constructs: wild type, a point mutation affecting rDNA transcription, and a deletion mutant lacking the predicted coiled-coil domain. A combinatorial proteomics and bioinformatics approach was used to integrate gene ontology classifications, functional protein networks, and normalized abundances of proteins co-isolated with SIRT7. The resulting refined proteomic data set confirmed SIRT7 interactions with RNA Pol I and upstream binding factor and highlighted association with factors involved in RNA Pol I- and II-dependent transcriptional processes and several nucleolus-localized chromatin remodeling complexes. Particularly enriched were members of the B-WICH complex, such as Mybbp1a, WSTF, and SNF2h. Prominent interactions were validated by a selected reaction monitoring-like approach using metabolic labeling with stable isotopes, confocal microscopy, reciprocal immunoaffinity precipitation, and co-isolation with endogenous SIRT7. To extend the current knowledge of mechanisms involved in SIRT7-dependent regulation of rDNA transcription, we showed that small interfering RNA-mediated SIRT7 knockdown leads to reduced levels of RNA Pol I protein, but not messenger RNA, which was confirmed in diverse cell types. The down-regulation of RNA Pol I protein levels placed in the context of SIRT7 interaction networks led us to propose that SIRT7 plays a crucial role in connecting the function of chromatin remodeling complexes to RNA Pol I machinery during transcription.

Dietary isothiocyanate-induced apoptosis via thiol modification of DNA topoisomerase IIα.

Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.

Genistein induces topoisomerase IIbeta- and proteasome-mediated DNA sequence rearrangements: Implications in infant leukemia.

Genistein is a bioflavonoid enriched in soy products. However, high levels of maternal soy consumption have been linked to the development of infant leukemia ALL and AML. The majority of infant leukemia is linked to mixed lineage leukemia gene (MLL) translocations. Previous studies have implicated topoisomerase II (Top2) in genistein-induced infant leukemia. In order to understand the roles of the two Top2 isozymes in and the molecular mechanism for genistein-induced infant leukemia, we carried out studies in vitro using purified recombinant human Top2 isozymes, as well as studies in cultured mouse myeloid progenitor cells (32Dc13) and Top2beta knockout mouse embryonic fibroblasts (MEFs). First, we showed that genistein efficiently induced both Top2alpha and Top2beta cleavage complexes in the purified system as well as in cultured mouse cells. Second, genistein induced proteasomal degradation of Top2beta in 32Dc13 cells. Third, the genistein-induced DNA double-strand break (DSB) signal, gamma-H2AX, was dependent on the Top2beta isozyme and proteasome activity. Fourth, the requirement for Top2beta and proteasome activity was mirrored in genistein-induced DNA sequence rearrangements, as monitored by a DNA integration assay. Together, our results suggest a model in which genistein-induced Top2beta cleavage complexes are processed by proteasome, leading to the exposure of otherwise Top2beta-concealed DSBs and subsequent chromosome rearrangements, and implicate a major role of Top2beta and proteasome in genistein-induced infant leukemia.