The B56γ3-containing protein phosphatase 2A attenuates p70S6K-mediated negative feedback loop to enhance AKT-facilitated epithelial-mesenchymal transition in colorectal cancer.

BACKGROUND: Protein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC). METHODS: Polyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein-protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients. RESULTS: We showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC. CONCLUSIONS: Our finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC. Video Abstract.

Multivalent dipeptidyl peptidase IV fragment-nanogold complex inhibits cancer metastasis by blocking pericellular fibronectin.

Inhibition of cancer metastasis is a fundamental challenge in cancer treatment. We have previously shown that metastasis of cancer cells in the lung is critically promoted by the interaction between the superficial dipeptidyl peptidase IV (DPP IV) expressed on lung endothelial cells and the pericellular polymeric fibronectin (polyFN) of circulating cancer cells. In the present study, we aimed to search for DPP IV fragments with high avidity to polyFN and develop FN-targeted gold nanoparticles (AuNPs) conjugated with DPP IV fragments for treating cancer metastasis. We first identified a DPP IV fragment encompassing amino acids 29-130 of DPP IV, designated DP4A, which contained FN-binding sites and could specifically bind to FN immobilized on gelatin agarose beads. Furthermore, we conjugated maltose binding protein (MBP)-fused DP4A proteins to AuNPs for fabricating a DP4A-AuNP complex and evaluated its FN-targeted activity in vitro and anti-metastatic efficacy in vivo. Our results show that DP4A-AuNP exhibited higher binding avidity to polyFN than DP4A by 9 folds. Furthermore, DP4A-AuNP was more potent than DP4A in inhibiting DPP IV binding to polyFN. In terms of polyFN-targeted effect, DP4A-AuNP interacted with FN-overexpressing cancer cells and was endocytosed into cells 10 to 100 times more efficiently than untargeted MBP-AuNP or PEG-AuNP with no noticeable cytotoxicity. Furthermore, DP4A-AuNP was superior to DP4A in competitive inhibition of cancer cell adhesion to DPP IV. Confocal microscopy analysis revealed that binding of DP4A-AuNP to pericellular FN induced FN clustering without altering its surface expression on cancer cells. Notably, intravenous treatment with DP4A-AuNP significantly reduced metastatic lung tumor nodules and prolonged the survival in the experimental metastatic 4T1 tumor model. Collectively, our findings suggest that the DP4A-AuNP complex with potent FN-targeted effects may have therapeutic potential for prevention and treatment of tumor metastasis to the lung.

Inhibition of lncRNA RPPH1 activity decreases tumor proliferation and metastasis through down-regulation of inflammation-related oncogenes.

OBJECTIVE: Ribonuclease P RNA component H1 (RPPH1) is a long non-coding RNA (lncRNA) associated with cancer progression. Higher RPPH1 expression in breast and cervical cancer samples than that in normal tissues were observed through the lncRNASNP2 database; therefore, silencing RPPH1 expression might be a potential strategy for cancer treatments, even though RPPH1 is also an RNA subunit of ribonuclease P involved in processing transfer RNA (tRNA) precursors and the effect of RPPH1 knockdown is not yet fully understood. METHODS: Differentially expressed genes (DEGs) were identified through RNA sequencing in each shRNA-transfected RPPH1 knockdown MDA-MB-231, RPPH1 knockdown HeLa cell, and respective control cells, then the gene ontology enrichment analysis was performed by IPA and MetaCore database according to these DEGs, with further in vitro experiments validating the effect of RPPH1 silencing in MDA-MB-231 and HeLa cells. RESULTS: Hundreds of down-regulated DEGs were identified in RPPH1 knockdown MDA-MB-231 and HeLa cells while bioinformatics analysis revealed that these genes were involved in pathways related to immune response and cancerogenesis. Compared to mock- and vector-transfected cells, the production of mature tRNAs, cell proliferation and migration capacity were inhibited in RPPH1-silenced HeLa and MDA-MB-231 cells. Additionally, RPPH1 knockdown promoted G1 cell cycle arrest mainly through the down-regulation of cyclin D1, although glycolytic pathways were only affected in RPPH1 knockdown HeLa cells but not MDA-MB-231 cells. CONCLUSION: This study demonstrated that knockdown RPPH1 affected tRNA production, cell proliferation and metabolism. Our findings might provide insight into the role of RPPH1 in tumor development.

Tumor Progression, Microenvironments, and Therapeutics.

Tumor malignancy starts from transformation [...].

Alpha-Mangostin Reduces Pericellular Fibronectin on Suspended Tumor Cells and Therapeutically, but Not Prophylactically, Suppresses Distant Metastasis.

Major cancer deaths can be ascribed to distant metastasis to which the assembly of pericellular fibronectin (periFN) on suspended tumor cells (STCs) in the bloodstream that facilitate endothelial attachment can lead. Even though mangosteen pericarps (MP) extracts and the major component α-mangostin (α-MG) exhibit potent cancer chemopreventive properties, whether they can prophylactically and therapeutically be used as dietary nutraceuticals to prevent distant metastasis by suppressing periFN assembly on STCs within the circulation remains obscure. Immunofluorescence staining, MTT assays, flow cytometric assays, immunoblotting, and experimental metastasis mouse models were used to detect the effects of MP extracts or α-MG on periFN on STCs, tumor cell proliferation and apoptosis, the AKT activity, and tumor lung metastasis. The periFN assembly on STCs was significantly diminished upon treatments of STCs with either α-MG or MP extracts in a dose-dependent manner without inhibiting cell proliferation and viability due to increased AKT activity. Pretreatment of STCs with α-MG appeared to suppress tumor lung metastasis and prolong mouse survival rates. Oral gavage with MP extracts could therapeutically, but not prophylactically, prevent lung metastasis of STCs. We concluded that MP extracts or the major component α-MG may therapeutically serve as a potent anti-metastatic nutraceutical.

Targeting protumor factor chitinase-3-like-1 secreted by Rab37 vesicles for cancer immunotherapy.

Background: Chitinase 3-like-1 (CHI3L1) is a secretion glycoprotein associated with the immunosuppressive tumor microenvironment (TME). The secretory mode of CHI3L1 makes it a promising target for cancer treatment. We have previously reported that Rab37 small GTPase mediates secretion of IL-6 in macrophages to promote cancer progression, whereas the roles of Rab37 in the intracellular trafficking and exocytosis of CHI3L1 are unclear. Methods: We examined the concentration of CHI3L1 in the culture medium of splenocytes and bone marrow derived macrophages (BMDMs) from wild-type or Rab37 knockout mice, and macrophage or T cell lines expressing wild type, active GTP-bound or inactive GDP-bound Rab37. Vesicle isolation, total internal reflection fluorescence microscopy, and real-time confocal microscopy were conducted. We developed polyclonal neutralizing-CHI3L1 antibodies (nCHI3L1 Abs) to validate the therapeutic efficacy in orthotopic lung, pancreas and colon cancer allograft models. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37 and CHI3L1, and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from cancer patients. Results: We demonstrate a novel secretion mode of CHI3L1 mediated by the small GTPase Rab37 in T cells and macrophages. Rab37 mediated CHI3L1 intracellular vesicle trafficking and exocytosis in a GTP-dependent manner, which is abolished in the splenocytes and BMDMs from Rab37 knockout mice and attenuated in macrophage or T cell lines expressing the inactive Rab37. The secreted CHI3L1 activated AKT, ß-catenin and NF-κB signal pathways in cancer cells and macrophages to foster a protumor TME characterized by activating M2 macrophages and increasing the population of regulatory T cells. Our developed nCHI3L1 Abs showed the dual properties of reducing tumor growth/metastases and eliciting an immunostimulatory TME in syngeneic orthotopic lung, pancreas and colon tumor models. Clinically, high plasma level or intratumoral expression of CHI3L1 correlated with poor survival in 161 lung cancer, 155 pancreatic cancer and 180 colon cancer patients. Conclusions: These results provide the first evidence that Rab37 mediates CHI3L1 secretion in immune cells and highlight nCHI3L1 Abs that can simultaneously target both cancer cells and tumor microenvironment.

Converged Rab37/IL-6 trafficking and STAT3/PD-1 transcription axes elicit an immunosuppressive lung tumor microenvironment.

Background: Increased IL-6 level, M2 macrophages and PD-1+CD8+ T cells in tumor microenvironments (TME) have been identified to correlate with resistance to checkpoint blockade immunotherapy, yet the mechanism remains poorly understood. Rab small GTPase-mediated trafficking of cytokines is critical in immuno-modulation. We have previously reported dysregulation of Rab37 in lung cancer cells, whereas the roles of Rab37 in tumor-infiltrating immune cells and cancer immunotherapy are unclear. Methods: The tumor growth of the syngeneic mouse allograft in wild type or Rab37 knockout mice was analyzed. Imaging analyses and vesicle isolation were conducted to determine Rab37-mediated IL-6 secretion. STAT3 binding sites at PD-1 promoter in T cells were identified by chromatin immunoprecipitation assay. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37, IL-6 and PD-1 and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from lung cancer patients. Results: We revealed that Rab37 regulates the secretion of IL-6 in a GTPase-dependent manner in macrophages to trigger M2 polarization. Macrophage-derived IL-6 promotes STAT3-dependent PD-1 mRNA expression in CD8+ T cells. Clinically, tumors with high stromal Rab37 and IL-6 expression coincide with tumor infiltrating M2-macrophages and PD1+CD8+ T cells that predicts poor prognosis in lung cancer patients. In addition, lung cancer patients with an increase in plasma IL-6 level are found to be associated with immunotherapeutic resistance. Importantly, combined blockade of IL-6 and CTLA-4 improves survival of tumor-bearing mice by reducing infiltration of PD1+CD8+ T cells and M2 macrophages in TME. Conclusions: Rab37/IL-6 trafficking pathway links with IL-6/STAT3/PD-1 transcription regulation to foster an immunosuppressive TME and combined IL-6/CTLA-4 blockade therapy exerts potent anti-tumor efficacy.

The Fibronectin Expression Determines the Distinct Progressions of Malignant Gliomas via Transforming Growth Factor-Beta Pathway.

Due to the increasing incidence of malignant gliomas, particularly glioblastoma multiforme (GBM), a simple and reliable GBM diagnosis is needed to screen early the death-threaten patients. This study aimed to identify a protein that can be used to discriminate GBM from low-grade astrocytoma and elucidate further that it has a functional role during malignant glioma progressions. To identify proteins that display low or no expression in low-grade astrocytoma but elevated levels in GBM, glycoprotein fibronectin (FN) was particularly examined according to the mining of the Human Protein Atlas. Web-based open megadata minings revealed that FN was mainly mutated in the cBio Cancer Genomic Portal but dominantly overexpressed in the ONCOMINE (a cancer microarray database and integrated data-mining platform) in distinct tumor types. Furthermore, numerous different cancer patients with high FN indeed exhibited a poor prognosis in the PrognoScan mining, indicating that FN involves in tumor malignancy. To investigate further the significance of FN expression in glioma progression, tumor specimens from five malignant gliomas with recurrences that received at least two surgeries were enrolled and examined. The immunohistochemical staining showed that FN expression indeed determined the distinct progressions of malignant gliomas. Furthermore, the expression of vimentin (VIM), a mesenchymal protein that is strongly expressed in malignant cancers, was similar to the FN pattern. Moreover, the level of epithelial-mesenchymal transition (EMT) inducer transforming growth factor-beta (TGF-ß) was almost recapitulated with the FN expression. Together, this study identifies a protein FN that can be used to diagnose GBM from low-grade astrocytoma; moreover, its expression functionally determines the malignant glioma progressions via TGF-ß-induced EMT pathway.

MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is widely prevalent in Taiwan, and high metastatic spread of ESCC leads to poor survival rate. Fibronectin (FN) assembly on the cell membrane may induce ESCC mobility. MicroRNAs (MiRNAs) are abundant in and participate in tumorigenesis in many cancers. However, the role of MiRNA in FN assembly-related ESCC mobility remains unexplored. METHODS: We divided ESCC CE81T cells into high-FN assembly (CE81FN+) and low-FN assembly (CE81FN-) groups by flow cytometry. MiRNA microarray analysis identified miR-146a expression as the most down-regulated miRNA in comparison of CE81FN+ and CE81FN- cells. RESULTS: Cell proliferation and migration were decreased when CE81FN+ cells overexpressed transgenic miR-146a compared to the parental cells, indicating an inverse correlation between low miR-146a expression and high proliferation as well as motility of FN assembly ESCC cells. Furthermore, vimentin is the target gene of miR-146a involved in ESCC tumorigenesis. MiR-146a suppressed cell proliferation, migration and invasion of CE81FN+ cells through the inhibition of vimentin expression, as confirmed by real-time PCR, Western blotting and Transwell™ assay. Analysis of one hundred and thirty-six paired ESCC patient specimens revealed that low miR-146a and high vimentin levels were frequently detected in tumor, and that the former was associated with late tumor stages (III and IV). Notably, either low miR-146a expression or high vimentin level was significantly associated with poor overall survival rate among ESCC patients. CONCLUSIONS: This is the first report to link FN assembly in the cell membrane with miR-146a, vimentin and ESCC tumorigenesis both in vitro and in ESCC patients.

Depleting RhoA/Stress Fiber-Organized Fibronectin Matrices on Tumor Cells Non-Autonomously Aggravates Fibroblast-Driven Tumor Cell Growth.

Fibronectin (FN) expressed by tumor cells has been known to be tumor suppressive but the pericellular FN (periFN) assembled on circulating tumor cells appears to evidently promote distant metastasis. Whereas the regulation of periFN assembly in suspended cells has currently been under investigation, how it is regulated in adherent tumor cells and the role of periFN in primary tumor growth remain elusive. Techniques of RNAi, plasmid transfections, immunoblotting, fluorescence/immunohistochemistry staining, cell proliferation assays, and primary tumor growth in C57BL6 mice and Fischer 344 rats were employed in this study. We found that endogenously synthesized FN in adherent tumor cells was required for periFN assembly which was aligned by RhoA-organized actin stress fiber (SF). Depleting periFN on adherent tumor cells congruently promoted in vivo tumor growth but surprisingly did not autonomously impact on in vitro tumor cell proliferation and apoptosis, suggestive of a non-autonomous role of periFN in in vivo tumor growth. We showed that the proliferative ability of shFN-expressing tumor cells was higher than shScramble cells did in the presence of fibroblasts. Altogether, these results suggested that depriving RhoA/SF-regulated periFN matrices non-autonomously promotes fibroblast-mediated tumor cell growth.

Pterostilbene Sensitizes Cisplatin-Resistant Human Bladder Cancer Cells with Oncogenic HRAS.

Analysis of various public databases revealed that HRAS gene mutation frequency and mRNA expression are higher in bladder urothelial carcinoma. Further analysis revealed the roles of oncogenic HRAS, autophagy, and cell senescence signaling in bladder cancer cells sensitized to the anticancer drug cisplatin using the phytochemical pterostilbene. A T24 cell line with the oncogenic HRAS was chosen for further experiments. Indeed, coadministration of pterostilbene increased stronger cytotoxicity on T24 cells compared to HRAS wild-type E7 cells, which was paralleled by neither elevated apoptosis nor induced cell cycle arrest, but rather a marked elevation of autophagy and cell senescence in T24 cells. Pterostilbene-induced autophagy in T24 cells was paralleled by inhibition of class I PI3K/mTOR/p70S6K as well as activation of MEK/ERK (a RAS target) and class III PI3K pathways. Pterostilbene-induced cell senescence on T24 cells was paralleled by increased pan-RAS and decreased phospho-RB expression. Coadministration of PI3K class III inhibitor 3-methyladenine or MEK inhibitor U0126 suppressed pterostilbene-induced autophagy and reversed pterostilbene-enhanced cytotoxicity, but did not affect pterostilbene-elevated cell senescence in T24 cells. Animal study data confirmed that pterostilbene enhanced cytotoxicity of cisplatin plus gemcitabine. These results suggest a therapeutic application of pterostilbene in cisplatin-resistant bladder cancer with oncogenic HRAS.

Fibronectin in Cancer: Friend or Foe.

The role of fibronectin (FN) in tumorigenesis and malignant progression has been highly controversial. Cancerous FN plays a tumor-suppressive role, whereas it is pro-metastatic and associated with poor prognosis. Interestingly, FN matrix deposited in the tumor microenvironments (TMEs) promotes tumor progression but is paradoxically related to a better prognosis. Here, we justify how FN impacts tumor transformation and subsequently metastatic progression. Next, we try to reconcile and rationalize the seemingly conflicting roles of FN in cancer and TMEs. Finally, we propose future perspectives for potential FN-based therapeutic strategies.

VAMP8, a vesicle-SNARE required for RAB37-mediated exocytosis, possesses a tumor metastasis suppressor function.

We previously identified a metastasis suppressor RAB37 small GTPase that regulated exocytosis of tissue inhibitor of metalloproteinases 1 (TIMP1) to suppress lung cancer metastasis. Here, we show that vesicle-associated membrane protein 8 (VAMP8), a v-SNARE (vesicle soluble N-ethylmaleimide-sensitive factor activating protein receptor), interacts with RAB37 and drives the secretion of TIMP1 to inhibit tumor metastases. Confocal and total internal reflection fluorescence microscopic images demonstrated that VAMP8 co-localized with RAB37 and facilitated trafficking of RAB37-TIMP1 vesicles. Reconstitution experiments using tail-vein injection and lung-to-lung metastasis in mice showed that VAMP8 was essential for RAB37-regulated vesicle trafficking of TIMP1 to suppress cancer metastasis. Lung cancer patients with low VAMP8 showed distant metastasis, poor overall survival and progression-free survival. Importantly, multivariate Cox regression analysis indicated that patients with low VAMP8/low RAB37 expression profile showed significantly high risk of death (hazard ratio = 3.42, P < 0.001) even after adjusting for tumor metastasis parameter. Our findings reveal that VAMP8 is a novel v-SNARE crucial for RAB37-mediated exocytic transport of TIMP1 to suppress lung tumor metastasis. VAMP8 possesses a tumor metastasis suppressor function with a prognostic value in lung cancer.

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 µM of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

The decrease of glycolytic enzyme hexokinase 1 accelerates tumor malignancy via deregulating energy metabolism but sensitizes cancer cells to 2-deoxyglucose inhibition.

Malignant tumors often display an aberrant energy metabolism that relies primarily on glycolysis to produce adenosine triphosphate (ATP) the so-called Warburg effect or aerobic glycolysis. Thus, the elucidation of this energetic alteration in malignant tumors is important in the search for more effective therapeutics against malignant cancers, the most deadly human disease. To investigate whether attenuated glycolytic activity modulates tumor progression, the effects of silencing the first and rate-limiting glycolytic enzyme hexokinase (HK) isozymes HK1 and HK2 were examined. There was an inverse correlation between the expression of HK1 and HK2 in human cancer cells. In cervical carcinoma cells, the HK1 but not HK2 knockdown induced a phenotypic change characteristic of epithelial-mesenchymal transition, which accelerated tumor growth and metastasis both in vitro and in vivo analyses. Notably, the silencing of HK1 disrupted aerobic respiration and increased glycolysis, but it had no effect on ATP generation. These metabolic changes were associated with higher HK2 and lactate dehydrogenase 1 expression but a lower citrate synthase level. Particularly, the HK1 knockdown induced aberrant energy metabolism that was almost recapitulated by HK2 overexpression. Moreover, the HK1-silenced cells showed strong glucose-dependent growth and 2-deoxyglucose (2-DG) induced cell proliferation inhibition. These results clearly indicate that the silencing of HK1, but not HK2, alters energy metabolism and induces an EMT phenotype, which enhances tumor malignancy, but increases the susceptibility of cancer cells to 2-DG inhibition. In addition, this work also suggests that the glycolytic inhibitors should be used only to treat cancers with elevated glycolytic activity.

Glycine N-methyltransferase inhibits aristolochic acid nephropathy by increasing CYP3A44 and decreasing NQO1 expression in female mouse hepatocytes.

Plants containing aristolochic acids (AA) are nephrotoxins. Glycine N-methyltransferase (GNMT) acts to bind environmental toxins such as benzo(a)pyrene and aflatoxin B1, translocate into nucleus, and alter hepatic metabolism. This study aims to determine the role of GNMT in AA-induced nephropathy. We established an AA nephropathy mouse model and found that AA type I (AAI)-induced nephropathy at a lower concentration in male than in female mice, implying sex differences in AAI resistance. Microarray analysis and AAI-treated mouse models showed that GNMT moderately reduced AAI-induced nephropathy by lowering the upregulated level of NQO1 in male, but significantly improved the nephropathy additionally by increasing Cyp3A44/3A41 in female. The protective effects of GNMT were absent in female GNMT knockout mice, in which re-expression of hepatic GNMT significantly decreased AAI-induced nephropathy. Mechanism-wise, AAI enhanced GNMT nuclear translocation, resulting in GNMT interaction with the promoter region of the genes encoding Nrf2 and CAR/PXR, the transcription factors for NQO1 and CYP3A44/3A41, respectively. Unlike the preference for Nrf2/NQO1 transcriptions at lower levels of GNMT, overexpression of GNMT preferred CAR/PXR/CYP3A44/3A41 transcriptions and alleviated kidney injury upon AAI treatment. In summary, hepatic GNMT protected mice from AAI nephropathy by enhancing CAR/PXR/CYP3A44/3A41 transcriptions and reducing Nrf2/NQO1 transcriptions.

Decreased succinate dehydrogenase B in human hepatocellular carcinoma accelerates tumor malignancy by inducing the Warburg effect.

Changes in TCA cycle enzymes or respiratory activity are possible mechanisms of aerobic glycolysis that contributes to tumor progression. To clarify whether the decrease of succinate dehydrogenase B (SDHB) alters energy metabolism, induces the Warburg effect and results in tumor malignancy, SDHB expression was examined and modulated in hepatocellular carcinoma (HCC) tissues and cells, respectively. SDHB level was often decreased in malignant HCC cells and tissues. Furthermore, the reduced SDHB expression was associated with advanced tumor stage and poor survival rate. Moreover, silencing of SDHB altered energy metabolism switched from aerobic respiration to glycolysis, resulted in the Warburg effect, and enhanced cell proliferation and motility. In contrast, the SDHB overexpression deregulated bioenergetic metabolism and decreased cell growth and migration. In mouse xenograft models, subcutaneous implantation and tail vein injection with SDHB knockdown cells resulted in a larger tumor volume and accelerated cancer metastasis, respectively. A mutation or decrease in SDHB induced the switch from aerobic respiration to glycolysis. This metabolic alteration was associated with tumor cell dedifferentiation, proliferation, motility and overall patient survival in HCC.

Apoptotic and Nonapoptotic Activities of Pterostilbene against Cancer.

Cancer is a major cause of death. The outcomes of current therapeutic strategies against cancer often ironically lead to even increased mortality due to the subsequent drug resistance and to metastatic recurrence. Alternative medicines are thus urgently needed. Cumulative evidence has pointed out that pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene, PS) has excellent pharmacological benefits for the prevention and treatment for various types of cancer in their different stages of progression by evoking apoptotic or nonapoptotic anti-cancer activities. In this review article, we first update current knowledge regarding tumor progression toward accomplishment of metastasis. Subsequently, we review current literature regarding the anti-cancer activities of PS. Finally, we provide future perspectives to clinically utilize PS as novel cancer therapeutic remedies. We, therefore, conclude and propose that PS is one ideal alternative medicine to be administered in the diet as a nutritional supplement.

Ubiquitination of tumor suppressor PML regulates prometastatic and immunosuppressive tumor microenvironment.

The tumor microenvironment plays an important role in tumor growth and metastasis. However, the mechanism by which tumor cells regulate the cell and non-cell constituents of surrounding stroma remains incompletely understood. Promyelocytic leukemia (PML) is a pleiotropic tumor suppressor, but its role in tumor microenvironment regulation is poorly characterized. PML is frequently downregulated in many cancer types, including lung cancer. Here, we identify a PML ubiquitination pathway that is mediated by WD repeat 4-containing cullin-RING ubiquitin ligase 4 (CRL4WDR4). Clinically, this PML degradation pathway is hyperactivated in lung cancer and correlates with poor prognosis. The WDR4/PML axis induces a set of cell-surface or secreted factors, including CD73, urokinase-type plasminogen activator receptor (uPAR), and serum amyloid A2 (SAA2), which elicit paracrine effects to stimulate migration, invasion, and metastasis in multiple lung cancer models. In xenograft and genetically engineered mouse models, the WDR4/PML axis elevates intratumoral Tregs and M2-like macrophages and reduces CD8+ T cells to promote lung tumor growth. These immunosuppressive effects were all reversed by CD73 blockade. Our study identifies WDR4 as an oncoprotein that negatively regulates PML via ubiquitination to promote lung cancer progression by fostering an immunosuppressive and prometastatic tumor microenvironment, suggesting the potential of immune-modulatory approaches for treating lung cancer with aberrant PML degradation.

Pterostilbene prevents AKT-ERK axis-mediated polymerization of surface fibronectin on suspended lung cancer cells independently of apoptosis and suppresses metastasis.

BACKGROUND: Polymeric fibronectin (polyFN) assembled on suspended breast cancer cells is required for metastasis. Conceivably, drugs that target such polyFN may fight against cancer metastasis. While stilbene analogs trigger pro-apoptotic effect on attached cancer cells, whether they prevent polyFN assembly and metastasis of suspended cancer cells via an apoptosis-independent manner remains unexplored. METHODS: We depleted suspended Lewis lung carcinoma (LLC) cells of polyFN by silencing the endogenous FN expression or pterostilbene (PS) to examine whether metastasis of lung cancer cells could thus be suppressed. We investigated whether PS regulates AKT-ERK signaling axis to suppress polyFN assembly in suspended LLC cells independently of apoptosis. We tested the therapeutic effects of orally administered PS against cancer metastasis. RESULTS: Both FN-silencing and PS among the three stilbenoids indeed significantly reduced polyFN assembly and lung metastasis of suspended LLC cells in an apoptosis-independent manner. Mechanistically, PS-induced AKT phosphorylation (pAKT) and suppressed ERK phosphorylation (pERK) in suspended LLC cells, whereas pretreatment with a PI3K inhibitor, LY294002, effectively reduced pAKT, rescued pERK, and consequently reversed the PS-suppressed polyFN assembly on LLC cells; these pretreatment effects were then overturned by the ERK inhibitor U0126. Indeed, PS-suppressed lung metastasis was counteracted by LY294002, which was further overruled with U0126. Finally, we found that PS, when orally administered in experimental metastasis assays, both significantly prevented lung colonization and metastasis of LLC cells and reduced the already established tumor growth in the mouse lungs. CONCLUSIONS: PS suppressed AKT/ERK-regulated polyFN assembly on suspended LLC cells and pulmonary metastasis. PS possesses potency in both preventing and treating lung metastasis of lung cancer cells in apoptosis-independent and apoptosis-dependent manners, respectively.