Melatonin increases Olaparib sensitivity and suppresses cancer-associated fibroblast infiltration via suppressing the LAMB3-CXCL2 axis in TNBC.

Triple-negative breast cancer (TNBC) is the most malignant breast cancer subtype, characterized with high aggressiveness and a high recurrence rate. Olaparib is the first US Food and Drug Administration-approved poly(ADP ribose) polymerase (PARP) inhibitor (PARPi) to treat breast cancer patients with a germline BRCA1 or BRCA2 mutation. However, resistance to Olaparib treatment restricts the therapeutic effects, and thus novel therapeutics are urgently required. In the present study, we identified that the combination of melatonin and Olaparib synergistically enhanced the sensitivity of TNBC cells. Moreover, melatonin exerted promising antitumor activities in Olaparib-resistant cells, implying the potential for its clinical application. An RNA-sequencing analysis revealed that melatonin treatment downregulated laminin subunit beta 3 (LAMB3) expression. Genetic ablation of LAMB3 significantly increased Olaparib sensitivity, and subsequently suppressed proliferation, epithelial-to-mesenchymal transition (EMT)-related gene expressions, and aggressiveness of breast cancer cells. Accordingly, LAMB3 expression was positively correlated with C-X-C motif chemokine ligand 2 (CXCL2), and they collaboratively promoted cancer-associated fibroblast (CAF) infiltration. An in vivo study demonstrated that combined treatment with melatonin and Olaparib showed enhanced inhibitory efficacy against tumor growth, LAMB3 expression, CXCL2 levels, and CAF infiltration compared to single treatment groups, and combined treatment with melatonin and Olaparib significantly ameliorated the immunosuppressive tumor microenvironment. These findings illustrate a promising therapeutic strategy using melatonin to overcome Olaparib resistance and activate antitumor immunity via attenuating the LAMB3-CXCL2 axis in breast cancer patients.

Differential regulation of Tetraodon nigroviridis Mx gene promoter activity by constitutively-active forms of STAT1, STAT2, and IRF9.

Induction of interferons (IFNs) produces an innate immune response through activation of the JAK-STAT signaling pathway. Type I IFN signaling activates downstream gene expression through the IFN-stimulated gene factor 3 (ISGF3) complex, while type II IFN (IFN-γ) signaling is mediated through active STAT1 protein. The IFN target gene Mx is involved in the defense against viral infection. However, the mechanism by which Tetraodon (pufferfish) Mx is regulated by IFN signaling has not been identified. In this study, we describe the cloning and expression of Tetraodon STAT1, STAT2, and IFN regulatory factor 9 (IRF9). By combining constitutively-active STAT1 (STAT1-JH1) and STAT2 (STA2-JH1) fusion proteins with IRF9, we demonstrate that a constitutively-active ISGF3 complex increases the transcriptional activity of the Tetraodon Mx promoter via direct binding to two IFN-stimulated response element (ISRE) sites. In addition, a constitutively-active TnIRF9-S2C containing a fusion of the C-terminal region of STAT2 and IRF9 also activated the Mx promoter through binding to the ISRE sites. Furthermore, constitutively-active STAT1-JH1 elevates Mx promoter activity through two IFN gamma-activated sequence (GAS) elements. The Mx promoter is also activated by constitutively-active TnIRF9-S2C and STAT1-JH1 protein, as determined using an in vivo luciferase assay. We conclude that the Tetraodon Mx gene is activated via Type I (IFN-1) and Type II (IFN-γ) signaling. These results provide mechanistic insights into the role of IFN signaling in teleosts, and the in vivo luciferase assay may be suitable as a tool for studying induction and regulation by IFNs in teleost fish.

Layer-by-layer assembly of quercetin-loaded zein/γPGA/low-molecular-weight chitosan/fucoidan nanosystem for targeting inflamed blood vessels.

Chitosan acts as a versatile carrier in polymeric nanoparticle (NP) for diverse drug administration routes. Delivery of antioxidants, such as quercetin (Qu) showcases potent antioxidant and anti-inflammatory properties for reduction of various cardiovascular diseases, but low water solubility limits uptake. To address this, we developed a novel layer-by-layer zein/gamma-polyglutamic acid (γPGA)/low-molecular-weight chitosan (LC)/fucoidan NP for encapsulating Qu and targeting inflamed vessel endothelial cells. We used zein (Z) and γPGA (r) to encapsulate Qu (Qu-Zr NP) exhibited notably higher encapsulation efficiency compared to zein alone. Qu-Zr NP coated with LC (Qu-ZrLC2 NP) shows a lower particle size (193.2 ± 2.9 nm), and a higher zeta potential value (35.2 ± 0.4 mV) by zeta potential and transmission electron microscopy analysis. After coating Qu-ZrLC2 NP with fucoidan, Qu-ZrLC2Fa NP presented particle size (225.16 ± 0.92 nm), zeta potential (-25.66 ± 0.51 mV) and maintained antioxidant activity. Further analysis revealed that Qu-ZrLC2Fa NP were targeted and taken up by HUVEC cells and EA.hy926 endothelial cells. Notably, we observed Qu-ZrLC2Fa NP targeting zebrafish vessels and isoproterenol-induced inflamed vessels of rat. Our layer-by-layer formulated zein/γPGA/LC/fucoidan NP show promise as a targeted delivery system for water-insoluble drugs. Qu-ZrLC2Fa NP exhibit potential as an anti-inflammatory therapeutic for blood vessels.

Arginine Methylation of DDX3 by PRMT1 Mediates Mitochondrial Homeostasis to Promote Breast Cancer Metastasis.

Dysregulated mitochondrial dynamics and metabolism play important roles in tumorigenesis. Metastasizing tumor cells predominantly utilize mitochondrial metabolism, and regulators of metabolic reprogramming may provide reliable biomarkers for diagnosing cancer metastasis. Here, we identified a type I arginine methyltransferase-DEAD-box polypeptide 3, X-linked (PRMT1-DDX3) axis that promotes breast cancer metastasis by coordinating mitochondrial biogenesis and mitophagy to ensure mitochondrial quality control. Mechanistically, PRMT1 induces arginine methylation of DDX3, which enhances its protein stability and prevents proteasomal degradation. DDX3 mediates mitochondrial homeostasis by translocating to mitochondria where it facilitates phosphatase and tensin homology-induced kinase 1 translation in response to mitochondrial stress. Inhibition of DDX3 suppresses mitochondrial biogenesis and mitophagy, resulting in diminished cancer stemness and metastatic properties. Overall, this study uncovers a mechanism by which the PRMT1-DDX3 axis regulates mitochondrial homeostasis to support breast cancer metastasis, suggesting strategies for targeting metabolic vulnerabilities to treat metastatic breast cancer. Significance: DDX3 is stabilized by PRMT1-mediated arginine methylation and coordinates mitophagy and mitochondrial biogenesis by upregulating PINK1 to facilitate breast cancer progression.

A low-molecular-weight chitosan fluorometric-based assay for evaluating antiangiogenic drugs.

Low-molecular-weight chitosan (LMWCS) damaged cell membranes in zebrafish showed its possibility to release reporter proteins for detection. In this study, we developed a simple fluorometric-based assay for the evaluation of clinical antiangiogenic drugs using LMWCS and Tg(fli1:EGFP) transgenic zebrafish, which expressed green-fluorescence protein (GFP) in the endothelial cells of blood vessel. In vitro stable and transiently transfected cell lines was released luciferase and green fluorescent protein (GFP) for intensity evaluation upon LMWCS fluorometric-based assay. In vivo Tg(fli1:EGFP) transgenic zebrafish was also released GFP from endothelial cells of blood vessels and show an increase of fluorescent intensity upon LMWCS fluorometric-based assay. Treatment with the clinical antiangiogenic drug sorafenib and analyzed by LMWCS fluorometric-based assay showed significantly reduction of angiogenesis. Furthermore, treatment with 2 µM sorafenib showed a significant reduction in angiogenesis of the intersegmental vein (ISV) and dorsal longitudinal anastomotic vessels (DLAV) in Tg(fli1:EGFP) transgenic zebrafish. Fluorescence intensity reduction from 2 µM sorafenib was used as a factor in the LMWCS fluorescence-based assay for relative antiangiogenic evaluation. Relative angiogenesis evaluation of the clinical drugs axitinib, cabozantinib, and regorafenib showed a significant reduction. Collectively, this study provided a simple, convenient, and rapid LMWCS fluorometric-based assay for evaluating angiogenic drugs using transgenic zebrafish.

Identification and characterization of alternative promoters of zebrafish Rtn-4/Nogo genes in cultured cells and zebrafish embryos.

In mammals, the Nogo family consists of Nogo-A, Nogo-B and Nogo-C. However, there are three Rtn-4/Nogo-related transcripts were identified in zebrafish. In addition to the common C-terminal region, the N-terminal regions of Rtn4-n/Nogo-C1, Rtn4-m/Nogo-C2 and Rtn4-l/Nogo-B, respectively, contain 9, 25 and 132 amino acid residues. In this study, we isolated the 5'-upstream region of each gene from a BAC clone and demonstrated that the putative promoter regions, P1-P3, are functional in cultured cells and zebrafish embryos. A transgenic zebrafish Tg(Nogo-B:GFP) line was generated using P1 promoter region to drive green fluorescent protein (GFP) expression through Tol2-mediated transgenesis. This line recapitulates the endogenous expression pattern of Rtn4-l/Nogo-B mRNA in the brain, brachial arches, eyes, muscle, liver and intestines. In contrast, GFP expressions by P2 and P3 promoters were localized to skeletal muscles of zebrafish embryos. Several GATA and E-box motifs are found in these promoter regions. Using morpholino knockdown experiments, GATA4 and GATA6 were involved in the control of P1 promoter activity in the liver and intestine, while Myf5 and MyoD for the control of P1 and P3 promoter activities in muscles. These data demonstrate that zebrafish Rtn4/Nogo transcripts might be generated by coupling mechanisms of alternative first exons and alternative promoter usage.

The ADAM9/UBN2/AKR1C3 axis promotes resistance to androgen-deprivation in prostate cancer.

Metastatic and castration-resistant disease is a fatal manifestation of prostate cancer (PCa). The mechanism through which resistance to androgen deprivation in PCa is developed remains largely unknown. Our understanding of the tumor microenvironment (TME) and key signaling pathways between tumors and their TME is currently changing in light of the generation of new knowledge with regard to cancer progression. A disintegrin and metalloproteinase domain-containing protein 9 (ADAM9) is a membranous bridge forming cell-cell and cell-matrix connections that regulate tumor aggressiveness and metastasis. However, it is not known whether ADAM9 expressed in the TME contributes to the CRPC phenotype. In this study, we aimed to investigate the expression patterns of ADAM9 in prostate cancer-associated fibroblasts (CAFs). We also intended to elucidate the effects of both stromal cell- and cancer cell-derived ADAM9 on the progression of CRPC and the implicated molecular pathways. By using both clinical specimens and cell lines, we herein showed that unlike the membrane anchored ADAM9 overexpressed by both PCa cells and prostate CAFs, the secreted isoform of ADAM9 (sADAM9) was strongly detected in CAFs, but rarely in tumor cells, and that could be a serum marker for PCa patients. We demonstrated that functionally sADAM9 are characterized as chemoattractant for the directed movement of androgen-independent PCa cells through integrin downstream FAK/AKT pathway, supporting that elevated sADAM9 by prostate CAFs could be responsible for the promotion of CRPC metastasis. Moreover, by stimulating PCa cells with sADAM9, we found that ubinuclein-2 (UBN2) expression was increased. A positive correlation of ADAM9 and UBN2 expression was observed in androgen receptor-expressing PCa cell lines and further confirmed in clinical PCa specimens. Using a genetic modification approach, we identified UBN2 as a downstream target gene of ADAM9 that is critical for the survival of androgen-dependent PCa cells in response to androgen deprivation, through the induction and effect of the aldo-keto reductase family 1 member C3 (AKR1C3). Collectively, our results reveal a novel action of ADAM9 on the transition of androgen-dependent PCa cells into an androgen-independent manner through the UBN2/AKR1C3 axis; the aforementioned action could contribute to the clinically-observed acquired androgen-deprivation therapy resistance.

Curcumin-induced antitumor effects on triple-negative breast cancer patient-derived xenograft tumor mice through inhibiting salt-induced kinase-3 protein.

This study demonstrated for the first time that curcumin effectively inhibits the growth of triple-negative breast cancer (TNBC) tumors by inhibiting the expression of salt-induced kinase-3 (SIK3) protein in patient-derived xenografted tumor mice (TNBC-PDX). For TNBC patients, chemotherapy is the only option for postoperative adjuvant treatment. In this study, we detected the SIK3 mRNA expression in paired-breast cancer tissues by qPCR analysis. The results revealed that SIK3 mRNA expression was significantly higher in tumor tissues when compared to the normal adjacent tissues (73.25 times, n = 183). Thus, it is proposed for the first time that the antitumor effect induced by curcumin by targeting SIK3 can be used as a novel strategy for the therapy of TNBC tumors. In vitro mechanism studies have shown that curcumin (>25 µM) inhibits the SIK3-mediated cyclin D upregulation, thereby inhibiting the G1/S cell cycle and arresting TNBC (MDA-MB-231) cancer cell growth. The SIK3 overexpression was associated with increased mesenchymal markers (i.e., Vimentin, α-SMA, MMP3, and Twist) during epithelial-mesenchymal transition (EMT). Our results demonstrated that curcumin inhibits the SIK3-mediated EMT, effectively attenuating the tumor migration. For clinical indications, dietary nutrients (such as curcumin) as an adjuvant to chemotherapy should be helpful to TNBC patients because the current trend is to shrink the tumor with preoperative chemotherapy and then perform surgery. In addition, from the perspective of chemoprevention, curcumin has excellent clinical application value.

Arginine is an epigenetic regulator targeting TEAD4 to modulate OXPHOS in prostate cancer cells.

Arginine plays diverse roles in cellular physiology. As a semi-essential amino acid, arginine deprivation has been used to target cancers with arginine synthesis deficiency. Arginine-deprived cancer cells exhibit mitochondrial dysfunction, transcriptional reprogramming and eventual cell death. In this study, we show in prostate cancer cells that arginine acts as an epigenetic regulator to modulate histone acetylation, leading to global upregulation of nuclear-encoded oxidative phosphorylation (OXPHOS) genes. TEAD4 is retained in the nucleus by arginine, enhancing its recruitment to the promoter/enhancer regions of OXPHOS genes and mediating coordinated upregulation in a YAP1-independent but mTOR-dependent manner. Arginine also activates the expression of lysine acetyl-transferases and increases overall levels of acetylated histones and acetyl-CoA, facilitating TEAD4 recruitment. Silencing of TEAD4 suppresses OXPHOS functions and prostate cancer cell growth in vitro and in vivo. Given the strong correlation of TEAD4 expression and prostate carcinogenesis, targeting TEAD4 may be beneficially used to enhance arginine-deprivation therapy and prostate cancer therapy.

Arginine starvation elicits chromatin leakage and cGAS-STING activation via epigenetic silencing of metabolic and DNA-repair genes.

Rationale: One of the most common metabolic defects in cancers is the deficiency in arginine synthesis, which has been exploited therapeutically. Yet, challenges remain, and the mechanisms of arginine-starvation induced killing are largely unclear. Here, we sought to demonstrate the underlying mechanisms by which arginine starvation-induced cell death and to develop a dietary arginine-restriction xenograft model to study the in vivo effects. Methods: Multiple castration-resistant prostate cancer cell lines were treated with arginine starvation followed by comprehensive analysis of microarray, RNA-seq and ChIP-seq were to identify the molecular and epigenetic pathways affected by arginine starvation. Metabolomics and Seahorse Flux analyses were used to determine the metabolic profiles. A dietary arginine-restriction xenograft mouse model was developed to assess the effects of arginine starvation on tumor growth and inflammatory responses. Results: We showed that arginine starvation coordinately and epigenetically suppressed gene expressions, including those involved in oxidative phosphorylation and DNA repair, resulting in DNA damage, chromatin-leakage and cGAS-STING activation, accompanied by the upregulation of type I interferon response. We further demonstrated that arginine starvation-caused depletion of α-ketoglutarate and inactivation of histone demethylases are the underlying causes of epigenetic silencing. Significantly, our dietary arginine-restriction model showed that arginine starvation suppressed prostate cancer growth in vivo, with evidence of enhanced interferon responses and recruitment of immune cells. Conclusions: Arginine-starvation induces tumor cell killing by metabolite depletion and epigenetic silencing of metabolic genes, leading to DNA damage and chromatin leakage. The resulting cGAS-STING activation may further enhance these killing effects.

Genome-wide CRISPR/Cas9 knockout screening uncovers a novel inflammatory pathway critical for resistance to arginine-deprivation therapy.

Arginine synthesis deficiency due to the suppressed expression of ASS1 (argininosuccinate synthetase 1) represents one of the most frequently occurring metabolic defects of tumor cells. Arginine-deprivation therapy has gained increasing attention in recent years. One challenge of ADI-PEG20 (pegylated ADI) therapy is the development of drug resistance caused by restoration of ASS1 expression and other factors. The goal of this work is to identify novel factors conferring therapy resistance. Methods: Multiple, independently derived ADI-resistant clones including derivatives of breast (MDA-MB-231 and BT-549) and prostate (PC3, CWR22Rv1, and DU145) cancer cells were developed. RNA-seq and RT-PCR were used to identify genes upregulated in the resistant clones. Unbiased genome-wide CRISPR/Cas9 knockout screening was used to identify genes whose absence confers sensitivity to these cells. shRNA and CRISPR/Cas9 knockout as well as overexpression approaches were used to validate the functions of the resistant genes both in vitro and in xenograft models. The signal pathways were verified by western blotting and cytokine release. Results: Based on unbiased CRISPR/Cas9 knockout screening and RNA-seq analyses of independently derived ADI-resistant (ADIR) clones, aberrant activation of the TREM1/CCL2 axis in addition to ASS1 expression was consistently identified as the resistant factors. Unlike ADIR, MDA-MB-231 overexpressing ASS1 cells achieved only moderate ADI resistance both in vitro and in vivo, and overexpression of ASS1 alone does not activate the TREM1/CCL2 axis. These data suggested that upregulation of TREM1 is an independent factor in the development of strong resistance, which is accompanied by activation of the AKT/mTOR/STAT3/CCL2 pathway and contributes to cell survival and overcoming the tumor suppressive effects of ASS1 overexpression. Importantly, knockdown of TREM1 or CCL2 significantly sensitized ADIR toward ADI. Similar results were obtained in BT-549 breast cancer cell line as well as castration-resistant prostate cancer cells. The present study sheds light on the detailed mechanisms of resistance to arginine-deprivation therapy and uncovers novel targets to overcome resistance. Conclusion: We uncovered TREM1/CCL2 activation, in addition to restored ASS1 expression, as a key pathway involved in full ADI-resistance in breast and prostate cancer models.

Expression and characterization of a constitutively active STAT6 from Tetraodon.

In this paper, we report the cloning and characterization of the STAT6 gene from the pufferfish, Tetraodon nigroviridis. The TnSTAT6 gene is composed of 20 exons and 19 introns. The exon-intron organization of this gene is similar to that of HsSTAT6 except for the exons encoding the C-terminal transactivation domain. The full-length complementary (c)DNA of TnSTAT6 encodes a 794-amino acid protein that is 31% identical to human STAT6. We generated a constitutively active TnSTAT6-JH1 by fusing the kinase domain of carp JAK1 to the C-terminal end of TnSTAT6 and demonstrated that the fusion protein has specific DNA-binding ability and can activate a reporter construct carrying multiple copies of mammalian IL-4-response elements. Interestingly, TnSTAT6-JH1 associated with and phosphorylated TnSTAT6 on Tyr661. Mutation of this residue, Y661W, in TnSTAT6 abolished its association with TnSTAT6-JH1. This is consistent with the importance of the corresponding Tyr641 of HsSTAT6 in tyrosine phosphorylation and dimer formation. On the other hand, treatment of mammalian IL-4 did not induce tyrosine phosphorylation of wild-type TnSTAT6, suggesting that both the divergent N-terminal domain and coiled-coiled domain of TnSTAT6 may affect the interaction of TnSTAT6 with mammalian IL-4 receptor complexes.

Expression and characterization of the JAK kinase and STAT protein from brine shrimp, Artemia franciscana.

In this study, we isolated and characterized both JAK and STAT genes from Artemia, Artemia franciscana. Although AfJAK showed only 19% identity (33% similarity) to the Drosophila Hop protein, AfJAK contained the characteristic JAK homology domain (JH domain) from JH1 to JH7. On the other hand, AfSTAT showed higher identity (30%) to Drosophila STAT (STAT92E). The low identities of AfJAK and AfSTAT to Drosophila Hop and STAT92E suggest that JAK and STAT proteins are unique in each different species of invertebrate. RT-PCR analysis showed that both AfJAK and AfSTAT transcripts were ubiquitously expressed in the embryo, which is similar to the expression patterns of Drosophila Hop and STAT92E mRNAs during development. In addition, we generated a constitutively active form of AfSTAT by fusing the JH1 domain of AfJAK to the C-terminal end of AfSTAT. This fusion protein, AfSTAT-HA-JH1, autophosphorylated on its tyrosine residue and was able to bind to specific DNA motifs including the STAT-binding motifs in the Drosophila Raf promoter. Both AfJAK and AfSTAT proteins elicited the transactivation potential toward the fly Raf promoter in Sf9 cells. However, tyrosine phosphorylation of AfSTAT was not detected, which is consistent with the cellular localization analysis that most AfSTAT proteins were in the cytoplasm. Our results demonstrate that both JAK and STAT are present in the genome of Artemia, which can serve as the basis for further investigations to explore the role of the JAK/STAT signal pathway in the development and immune response of brine shrimp.

TESC Promotes TGF-α/EGFR-FOXM1-Mediated Tumor Progression in Cholangiocarcinoma.

Cholangiocarcinoma is a relatively uncommon but highly lethal malignancy. Improving outcomes in patients depends on earlier diagnosis and appropriate treatment; however, no satisfactory diagnostic biomarkers or targeted therapies are currently available. To address this shortcoming, we analyzed the transcriptomic datasets of cholangiocarcinoma from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and found that TESC is highly expressed in cholangiocarcinoma. Elevated cellular levels of TESC are correlated with larger tumor size and predict a poor survival outcome for patients. Knockdown of TESC via RNA interference suppresses tumor growth. RNA-sequencing analysis showed that silencing of TESC decreases the level of FOXM1, leading to cell cycle arrest. Correlation analysis revealed that the cellular level of TESC is correlated with that of FOXM1 in cholangiocarcinoma patients. We further observed that upon TGF-α induction, TESC is upregulated through the EGFR-STAT3 pathway and mediates TGF-α-induced tumor cell proliferation. In vivo experiments revealed that knockdown of TESC significantly attenuates tumor cell growth. Therefore, our data provide novel insight into TESC-mediated oncogenesis and reveal that TESC is a potential biomarker or serves as a therapeutic target for cholangiocarcinoma.

Characterization and toxicology evaluation of low molecular weight chitosan on zebrafish.

Chitosan is suggested as no or low toxicity and biocompatible biomaterial. Digestion of chitosan to reduce molecular weight and formulate nanoparticle was generally used to improve efficiency for DNA or protein delivery. However, the toxicity of low-molecular-weight chitosan (LMWCS) towards freshwater fishes has not been well evaluated. Here, we reported the toxic mechanism of LMWCS using zebrafish (Danio rerio) liver (ZFL) cell line, zebrafish larvae, and adult fish. LMWCS rapidly induced cytotoxicity of ZFL cells and death of zebrafish. Cell membrane damaged by LMWCS reduced cell viability. Damaged membrane of epithelial cell in zebrafish larvae induced breakage of the yolk. Adult fish exhibited hypoxia before death due to multiple damages induced by LMWCS. Although the toxicity of LMWCS was revealed in zebrafish model, the toxicity was only present in pH < 7 and easy be neutralized by other negative ions. Collectively, these data improved a new understanding of LMWCS properties.

Reduction in MnSOD promotes the migration and invasion of squamous carcinoma cells.

Reactive oxygen species (ROS) homeostasis is maintained at a higher level in cancer cells, which promotes tumorigenesis. Oxidative stress induced by anticancer drugs may further increase ROS to promote apoptosis, but can also enhance the metastasis of cancer cells. The effects of ROS homeostasis on cancer cells remain to be fully elucidated. In the present study, the effect of a reduction in manganese superoxide dismutase (MnSOD) on the migration and invasion of A431 cells was investigated. Our previous micro­assay data revealed that the mRNA expression of MnSOD was higher in the invasive A431­III cell line compared with that in the parental A431 cell line (A431­P). In the present study, high protein levels of MnSOD and H2O2 production were observed in A431­III cells; however, catalase protein levels were significantly lower in A431­III cells compared with those in the A431­P cell line. The knockdown of MnSOD increased H2O2 levels, enzyme activity, the mRNA levels of matrix metalloproteinase­1, ­2 and ­9, and the migratory and invasive abilities of the cells. Inducing a reduction in H2O2 using diphenyleneiodonium (DPI) and N­acetyl­l­cysteine decreased the migratory abilities of the cell lines, and DPI attenuated the migratory ability that had been increased by MnSOD small interfering RNA knockdown. Luteolin (Lu) and quercetin (Qu) increased the expression of catalase and reduced H2O2 levels, but without an observed change in the protein levels of MnSOD. Taken together, these data suggest that reduced MnSOD may induce ROS imbalance in cells and promote the metastatic ability of cancer cells. Lu and Qu may attenuate these processes and may be promising potential anticancer agents.

Metastatic Colorectal Cancer Rewrites Metabolic Program Through a Glut3-YAP-dependent Signaling Circuit.

Rationale: Cancer cells reprogram cellular metabolism to fulfill their needs for rapid growth and metastasis. However, the mechanism controlling this reprogramming is poorly understood. We searched for upregulated signaling in metastatic colorectal cancer and investigated the mechanism by which Glut3 promotes tumor metastasis. Methods: We compared RNA levels and glycolytic capacity in primary and metastatic colon cancer. The expression and association of Glut3 with clinical prognosis in colon cancer tissues was determined by immunohistochemistry. Glut3 gain-of-function and loss-of-function were established using colon cancer HCT116, HT29, and metastatic 116-LM cells, and tumor invasiveness and stemness properties were evaluated. Metabolomic profiles were analyzed by GC/MS and CE-TOF/MS. The metastatic burden in mice fed a high-fat sucrose diet was assessed by intravenous inoculation with Glut3 knockdown 116-LM cells. Results: Upregulation of glycolytic genes and glycolytic capacity was detected in metastatic colorectal cancer cells. Specifically, Glut3 overexpression was associated with metastasis and poor survival in colorectal cancer patients. Mechanistically, Glut3 promoted invasiveness and stemness in a Yes-associated protein (YAP)-dependent manner. Activation of YAP in turn transactivated Glut3 and regulated a group of glycolytic genes. Interestingly, the expression and phosphorylation of PKM2 were concomitantly upregulated in metastatic colorectal cancer, and it was found to interact with YAP and enhance the expression of Glut3. Importantly, a high-fat high-sucrose diet promoted tumor metastasis, whereas the inhibition of either Glut3 or YAP effectively reduced the metastatic burden. Conclusion: Activation of the Glut3-YAP signaling pathway acts as a master activator to reprogram cancer metabolism and thereby promotes metastasis. Our findings reveal the importance of metabolic reprogramming in supporting cancer metastasis as well as possible therapeutic targets.

KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC.

During the evolution into castration or therapy resistance, prostate cancer cells reprogram the androgen responses to cope with the diminishing level of androgens, and undergo metabolic adaption to the nutritionally deprived and hypoxia conditions. AR (androgen receptor) and PKM2 (pyruvate kinase M2) have key roles in these processes. We report in this study, KDM8/JMJD5, a histone lysine demethylase/dioxygnase, exhibits a novel property as a dual coactivator of AR and PKM2 and as such, it is a potent inducer of castration and therapy resistance. Previously, we showed that KDM8 is involved in the regulation of cell cycle and tumor metabolism in breast cancer cells. Its role in prostate cancer has not been explored. Here, we show that KDM8's oncogenic properties in prostate cancer come from its direct interaction (1) with AR to affect androgen response and (2) with PKM2 to regulate tumor metabolism. The interaction with AR leads to the elevated expression of androgen response genes in androgen-deprived conditions. They include ANCCA/ATAD2 and EZH2, which are directly targeted by KDM8 and involved in sustaining the survival of the cells under hormone-deprived conditions. Notably, in enzalutamide-resistant cells, the expressions of both KDM8 and EZH2 are further elevated, so are neuroendocrine markers. Consequently, EZH2 inhibitors or KDM8 knockdown both resensitize the cells toward enzalutamide. In the cytosol, KDM8 associates with PKM2, the gatekeeper of pyruvate flux and translocates PKM2 into the nucleus, where the KDM8/PKM2 complex serves as a coactivator of HIF-1α to upregulate glycolytic genes. Using shRNA knockdown, we validate KDM8's functions as a regulator for both androgen-responsive and metabolic genes. KDM8 thus presents itself as an ideal therapeutic target for metabolic adaptation and castration-resistance of prostate cancer cells.

Expression and characterization of two STAT isoforms from Sf9 cells.

In invertebrates, the JAK-STAT signaling pathway is involved in the anti-bacterial response and is part of an anti-viral response in Drosophila. In this study, we show that two STAT transcripts are generated by alternative splicing and encode two isoforms of Sf-STAT with different C-terminal ends. These two isoforms were produced and purified using the recombinant baculovirus technology. Both purified isoforms showed similar DNA-binding activity and displayed weak but significant transactivation potential toward a Drosophila promoter that contained a STAT-binding motif. No significant activation of the Sf-STAT protein in Sf9 cells was found by infection with baculovirus AcMNPV.

Flavonoids Luteolin and Quercetin Inhibit RPS19 and contributes to metastasis of cancer cells through c-Myc reduction.

Flavonoids luteolin and quercetin can inhibit growth and metastasis of cancer cells. In our previous report, luteolin and quercetin was shown to block Akt/mTOR/c-Myc signaling. Here, we found luteolin and quercetin reduced protein level and transactivation activity of RPS19 in A431-III cells, which is isolated from parental A431 (A431-P) cell line. Further investigation the inhibitory mechanism of luteolin and quercetin on RPS19, we found c-Myc binding sites on RPS19 promoter. The Akt inhibitor LY294002, mTOR inhibitor rapamycin and c-Myc inhibitor 10058-F4 significantly suppressed RPS19 expression and transactivation activities. Overexpression and knockdown of c-Myc in cancer cells show RPS19 expression was regulated by c-Myc. Furthermore, Knockdown and overexpression of RPS19 was used to analyze of the function of RPS19 in cancer cells. The epithelial-mesenchymal transition (EMT) markers and metastasis abilities of cancer cells were also regulated by RPS19. These data suggest that luteolin and quercetin might inhibit metastasis of cancer cells by blocking Akt/mTOR/c-Myc signaling pathway to suppress RPS19-activated EMT signaling.