Oxoglutarate Carrier Inhibition Reduced Melanoma Growth and Invasion by Reducing ATP Production.
ABSTRACT: Recent findings indicate that (a) mitochondria in proliferating cancer cells are functional, (b) cancer cells use more oxygen than normal cells for oxidative phosphorylation, and (c) cancer cells critically rely on cytosolic NADH transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. In a spontaneous lung cancer model, tumor growth was reduced by 50% in heterozygous oxoglutarate carrier (OGC) knock-out mice compared with wild-type counterparts. To determine the mechanism through which OGC promotes tumor growth, the effects of the OGC inhibitor N-phenylmaleimide (NPM) on mitochondrial activity, oxygen consumption, and ATP production were evaluated in melanoma cell lines. NPM suppressed oxygen consumption and decreased ATP production in melanoma cells in a dose-dependent manner. NPM also reduced the proliferation of melanoma cells. To test the effects of NPM on tumor growth and metastasis in vivo, NPM was administered in a human melanoma xenograft model. NPM reduced tumor growth by approximately 50% and reduced melanoma invasion by 70% at a dose of 20 mg/kg. Therefore, blocking OGC activity may be a useful approach for cancer therapy.
Project description:Cancer cells exhibit mitochondrial cholesterol (mt-cholesterol) accumulation, which contributes to cell death resistance by antagonizing mitochondrial outer membrane (MOM) permeabilization. Hepatocellular mt-cholesterol loading, however, promotes steatohepatitis, an advanced stage of chronic liver disease that precedes hepatocellular carcinoma (HCC), by depleting mitochondrial GSH (mGSH) due to a cholesterol-mediated impairment in mGSH transport. Whether and how HCC cells overcome the restriction of mGSH transport imposed by mt-cholesterol loading to support mGSH uptake remains unknown. Although the transport of mGSH is not fully understood, SLC25A10 (dicarboxylate carrier, DIC) and SLC25A11 (2-oxoglutarate carrier, OGC) have been involved in mGSH transport, and therefore we examined their expression and role in HCC. Unexpectedly, HCC cells and liver explants from patients with HCC exhibit divergent expression of these mitochondrial carriers, with selective OGC upregulation, which contributes to mGSH maintenance. OGC but not DIC downregulation by siRNA depleted mGSH levels and sensitized HCC cells to hypoxia-induced ROS generation and cell death as well as impaired cell growth in three-dimensional multicellular HCC spheroids, effects that were reversible upon mGSH replenishment by GSH ethyl ester, a membrane permeable GSH precursor. We also show that OGC regulates mitochondrial respiration and glycolysis. Moreover, OGC silencing promoted hypoxia-induced cardiolipin peroxidation, which reversed the inhibition of cholesterol on the permeabilization of MOM-like liposomes induced by Bax or Bak. Genetic OGC knockdown reduced the ability of tumor-initiating stem-like cells to induce liver cancer. These findings underscore the selective overexpression of OGC as an adaptive mechanism of HCC to provide adequate mGSH levels in the face of mt-cholesterol loading and suggest that OGC may be a novel therapeutic target for HCC treatment.
Project description:Most cancer cells preferentially rely on glycolysis to produce the energy (adenosine triphosphate, ATP) for growth and proliferation. Emerging evidence demonstrates that the apoptosis in cancer cells could be closely associated with the inhibition of glycolysis. In this study, we have found that jolkinolide B (JB), a bioactive diterpenoid extracted from the root of Euphorbia fischeriana Steud, induced tumor cells apoptosis and decreased the production of ATP and lactic acid in mouse melanoma B16F10 cells. Furthermore, we found that JB downregulated the mRNA expression of glucose transporter genes (Glut1, Glut3 and Glut4) and glycolysis-related kinase genes (Hk2 and Ldha) in B16F10 cells. Moreover, treatment with JB upregulated the mRNA expression of pro-apoptosis genes (Bax), downregulated the mRNA expression of anti-apoptosis genes (Bcl-2, Caspase-3 and Caspase-9), decreased the potential of mitochondrial membrane and increased reactive oxygen species (ROS) levels in B16F10 cells. Finally, intragastric administration of JB suppressed tumor growth and induced tumor apoptosis in mouse xenograft model of murine melanoma B16F10 cells. Taken together, these results suggest that JB could induce apoptosis through the mitochondrial pathway and inhibit tumor growth. The inhibition of glycolysis could play a crucial role in the induction of apoptosis in JB-treated B16F10 cells.
Project description:Nucleophosmin (NPM) is a multifunctional protein involved in a complex network of interactions. The role of NPM in oncogenesis is controversial. The NPM gene (NPM1) is mutated or rearranged in a number of hematological disorders, but such changes have not been detected in solid cancers. However, experiments with cultured NPM-null cells and with mice carrying a single inactivated NPM allele indicate a tumor suppressor function for NPM. To resolve the role of NPM in solid cancers, we examined its expression and localization in histologically normal breast tissue and a large array of human breast carcinoma samples (n = 1160), and also evaluated its association with clinicopathological variables and patient survival. The intensity and localization (nucleolar, nuclear, cytoplasmic) of NPM varied across clinical samples. No mutations explaining the differences were found, but the present findings indicate that expression levels of NPM affected its localization. Our study also revealed a novel granular staining pattern for NPM, which was an independent prognostic factor of poor prognosis. In addition, reduced levels of NPM protein were associated with poor prognosis. Furthermore, luminal epithelial cells of histologically normal breast displayed high levels of NPM and overexpression of NPM in the invasive MDA-MB-231 cells abrogated their growth in soft agar. These results support a tumor suppressive role for NPM in breast cancer.
Project description:Analysis of lung cancer response to chemotherapeutic agents showed the accumulation of a Taxol-induced protein that reacted with an anti-phospho-MEK1/2 antibody. Mass spectroscopy identified the protein as nucleophosmin/B23 (NPM), a multifunctional protein with diverse roles: ribosome biosynthesis, p53 regulation, nuclear-cytoplasmic shuttling, and centrosome duplication. Our work demonstrates that following cellular exposure to mitosis-arresting agents, NPM is phosphorylated and its chromatographic property is altered, suggesting changes in function during mitosis. To determine the functional relevance of NPM, its expression in tumor cells was reduced by siRNA. Cells with reduced NPM were treated with Taxol followed by microarray profiling accompanied by gene/protein pathway analyses. These studies demonstrate several expected and unexpected consequences of NPM depletion. The predominant downstream effectors of NPM are genes involved in cell proliferation, cancer, and the cell cycle. In congruence with its role in cancer, NPM is over-expressed in primary malignant lung cancer tissues. We also demonstrate a role for NPM in the expression of genes encoding SET (TAF1beta) and the histone methylase SET8. Additionally, we show that NPM is required for a previously unobserved G2/M upregulation of TAF1A, which encodes the rDNA transcription factor TAF(I)48. These results demonstrate multi-faceted functions of NPM that can affect cancer cells.
Project description:Tumor microenvironment favors tumor cells to promote their growth and metastasis such as migration, invasion, and angiogenesis. IL-1β, one of the inflammatory cytokines released from myeloid cells in tumor microenvironment, plays an important role in development and progress of tumor. The activation of inflammasome is a critical step to secrete mature IL-1β through stepwise reactions to activate capspase-1. Therefore, we investigated whether the inhibition of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in macrophages regulated the metastatic potential of tumor cells. NLRP3 inflammasome was activated by ATP in bone marrow-derived primary mouse macrophages. The metastatic potential of mouse melanoma cell line (B16F10) was determined by migration and invasion assays with transwell system. ATP-treated wild-type macrophages increased the migration and invasion of melanoma cells. However, NLRP3- or caspase-1-knockout macrophages exhibited greatly diminished ability to promote the migration and invasion of melanoma cells. In addition, treatment with celastrol, an inhibitor of NLRP3 inflammasome, reduced the potency of macrophages to stimulate migration and invasion of melanoma cells. The results demonstrate that inhibition of the NLRP3 inflammasome in macrophages by genetic deficiency or a pharmacological inhibitor is linked to suppression of the metastatic potential of tumor cells. The results would provide a novel anti-cancer strategy to modulate tumor microenvironment by suppressing NLRP3 inflammasome and consequently reducing IL-1β production.
Project description:Nucleophosmin (NPM) is a nucleolar phosphoprotein that is involved in many cellular processes and has both oncogenic and growth suppressing activities. NPM is localized primarily in nucleoli but shuttles between the nucleus and the cytoplasm, and sustained cytoplasmic distribution contributes to its tumor promoting activities. Plakoglobin (PG, ?-catenin) is a homolog of ?-catenin with dual adhesive and signaling functions. These proteins interact with cadherins and mediate adhesion, while their signaling activities are regulated by association with various intracellular partners. Despite these similarities, ?-catenin has a well-defined oncogenic activity, whereas PG acts as a tumor/metastasis suppressor through unknown mechanisms. Comparison of the proteomic profiles of carcinoma cell lines with low- or no PG expression with their PG-expressing transfectants has identified NPM as being upregulated upon PG expression. Here, we examined NPM subcellular distribution and in vitro tumorigenesis/metastasis in the highly invasive and very low PG expressing MDA-MB-231 (MDA-231) breast cancer cells and their transfectants expressing increased PG (MDA-231-PG) or NPM shRNA (MDA-231-NPM-KD) or both (MDA-231-NPM-KD+PG). Increased PG expression increased the levels of nucleolar NPM and coimmunoprecipitation studies showed that NPM interacts with PG. PG expression or NPM knockdown decreased the growth rate of MDA-231 cells substantially and this reduction was decreased further in MDA-231-NPM-KD+PG cells. In in vitro tumorigenesis/metastasis assays, MDA-231-PG cells showed substantially lower and MDA-231-NPM-KD cells substantially higher invasiveness relative to the MDA-231 parental cells, and the co-expression of PG and NPM shRNA led to even further reduction of the invasiveness of MDA-231-PG cells. Furthermore, examination of the levels and localization of PG and NPM in primary biopsies of metastatic infiltrating ductal carcinomas revealed coordinated expression of PG and NPM. Together, the data suggest that PG may regulate NPM subcellular distribution, which may potentially change the function of the NPM protein from oncogenic to tumor suppression.
Project description:Endometrial cancer incidence rates are growing, especially in countries with rapid socioeconomic transitions. Despite recent advances in chemotherapy, hormone therapy, and targeted therapy, advanced/recurrent disease remains a clinical challenge. Palbociclib-a selective inhibitor of cyclin-dependent kinases (CDK) 4/6-has therapeutic potential against estrogen receptor (ER)-positive and HER2-negative breast cancer. However, the question as to whether it can be clinically useful in endometrial cancer remains open. Here, we show that combined treatment with palbociclib and megesterol acetate exerts synergistic antiproliferative effects against endometrial cancer cells. Treatment of cancer cells with palbociclib suppressed NPM/B23 phosphorylation at threonine 199 (Thr199). We further demonstrated that CDK6 acts as a NPM/B23 kinase. Palbociclib-induced NPM/B23 dephosphorylation sensitized endometrial cancer cells to megesterol acetate through the upregulation of ER? expression. Immunohistochemistry revealed an overexpression of phospho-NPM/B23 (Thr199) in human endometrial cancer, and phospho-NPM/B23 (Thr199) expression levels were inversely associated with Er? in clinical specimen. In a xenograft tumor model, the combination of palbociclib and megesterol acetate successfully inhibited tumor growth. Taken together, our data indicate that palbociclib promoted NPM/B23 dephosphorylation at Thr199-an effect mediated by disruption of CDK6 kinase activity. We conclude that palbociclib holds promise for the treatment of endometrial cancer when used in combination with megesterol acetate.
Project description:Mutations leading to aberrant cytoplasmic localization of nucleophosmin (NPM) are the most frequent genetic alteration in acute myelogenous leukemia (AML). NPM binds the Arf tumor suppressor and protects it from degradation. The AML-associated NPM mutant (NPMmut) also binds p19Arf but is unable to protect it from degradation, which suggests that inactivation of p19Arf contributes to leukemogenesis in AMLs. We report here that NPM regulates turnover of the c-Myc oncoprotein by acting on the F-box protein Fbw7gamma, a component of the E3 ligase complex involved in the ubiquitination and proteasome degradation of c-Myc. NPM was required for nucleolar localization and stabilization of Fbw7gamma. As a consequence, c-Myc was stabilized in cells lacking NPM. Expression of NPMmut also led to c-Myc stabilization because of its ability to interact with Fbw7gamma and delocalize it to the cytoplasm, where it is degraded. Because Fbw7 induces degradation of other growth-promoting proteins, the NPM-Fbw7 interaction emerges as a central tumor suppressor mechanism in human cancer.
Project description:NPM-ALK? T-cell anaplastic large-cell lymphoma (ALCL) is an aggressive type of cancer. Standard treatment of NPM-ALK? ALCL is CHOP polychemotherapy. Although patients initially respond favorably to CHOP, resistance, relapse, and death frequently occur. Recently, selective targeting of ALK has emerged as an alternative therapeutic strategy. ASP3026 is a second-generation ALK inhibitor that can overcome crizotinib resistance in non-small cell lung cancer, and is currently being evaluated in clinical trials of patients with ALK? solid tumors. However, NPM-ALK? ALCL patients are not included in these trials. We studied the effects of ASP3026 on NPM-ALK? ALCL cell lines in vitro and on systemic lymphoma growth in vivo. ASP3026 decreased the viability, proliferation, and colony formation, as well as induced apoptotic cell death of NPM-ALK? ALCL cells. In addition, ASP3026 significantly reduced the proliferation of 293T cells transfected with NPM-ALK mutants that are resistant to crizotinib and downregulated tyrosine phosphorylation of these mutants. Moreover, ASP3026 abrogated systemic NPM-ALK? ALCL growth in mice. Importantly, the survival of ASP3026-treated mice was superior to that of control and CHOP-treated mice. Our data suggest that ASP3026 is an effective treatment for NPM-ALK? ALCL, and support the enrollment of patients with this lymphoma in the ongoing clinical trials.
Project description:Reliance on aerobic glycolysis is one of the hallmarks of cancer. Although pyruvate kinase M2 (PKM2) is a key mediator of glycolysis in cancer cells, lack of selective agents that target PKM2 remains a challenge in exploiting metabolic pathways for cancer therapy. We report that unlike its structural analog shikonin, a known inhibitor of PKM2, lapachol failed to induce non-apoptotic cell death ferroxitosis in hypoxia. However, melanoma cells treated with lapachol showed a dose-dependent inhibition of glycolysis and a corresponding increase in oxygen consumption. Accordingly, in silico studies revealed a high affinity-binding pocket for lapachol on PKM2 structure. Lapachol inhibited PKM2 activity of purified enzyme as well as in melanoma cell extracts. Blockade of glycolysis by lapachol in melanoma cells led to decreased ATP levels and inhibition of cell proliferation. Furthermore, perturbation of glycolysis in melanoma cells with lapachol sensitized cells to mitochondrial protonophore and promoted apoptosis. These results present lapachol as an inhibitor of PKM2 to interrogate metabolic plasticity in tumor cells.