Mitochondrial oxidative stress in the tumor microenvironment and cancer immunoescape: foe or friend?

The major concept of "oxidative stress" is an excess elevated level of reactive oxygen species (ROS) which are generated from vigorous metabolism and consumption of oxygen. The precise harmonization of oxidative stresses between mitochondria and other organelles in the cell is absolutely vital to cell survival. Under oxidative stress, ROS produced from mitochondria and are the major mediator for tumorigenesis in different aspects, such as proliferation, migration/invasion, angiogenesis, inflammation, and immunoescape to allow cancer cells to adapt to the rigorous environment. Accordingly, the dynamic balance of oxidative stresses not only orchestrate complex cell signaling events in cancer cells but also affect other components in the tumor microenvironment (TME). Immune cells, such as M2 macrophages, dendritic cells, and T cells are the major components of the immunosuppressive TME from the ROS-induced inflammation. Based on this notion, numerous strategies to mitigate oxidative stresses in tumors have been tested for cancer prevention or therapies; however, these manipulations are devised from different sources and mechanisms without established effectiveness. Herein, we integrate current progress regarding the impact of mitochondrial ROS in the TME, not only in cancer cells but also in immune cells, and discuss the combination of emerging ROS-modulating strategies with immunotherapies to achieve antitumor effects.

Lon upregulation contributes to cisplatin resistance by triggering NCLX-mediated mitochondrial Ca2+ release in cancer cells.

Mitochondria are the major organelles in sensing cellular stress and inducing the response for cell survival. Mitochondrial Lon has been identified as an important stress protein involved in regulating proliferation, metastasis, and apoptosis in cancer cells. However, the mechanism of retrograde signaling by Lon on mitochondrial DNA (mtDNA) damage remains to be elucidated. Here we report the role of Lon in the response to cisplatin-induced mtDNA damage and oxidative stress, which confers cancer cells on cisplatin resistance via modulating calcium levels in mitochondria and cytosol. First, we found that cisplatin treatment on oral cancer cells caused oxidative damage of mtDNA and induced Lon expression. Lon overexpression in cancer cells decreased while Lon knockdown sensitized the cytotoxicity towards cisplatin treatment. We further identified that cisplatin-induced Lon activates the PYK2-SRC-STAT3 pathway to stimulate Bcl-2 and IL-6 expression, leading to the cytotoxicity resistance to cisplatin. Intriguingly, we found that activation of this pathway is through an increase of intracellular calcium (Ca2+) via NCLX, a mitochondrial Na+/Ca2+ exchanger. We then verified that NCLX expression is dependent on Lon levels; Lon interacts with and activates NCLX activity. NCLX inhibition increased the level of mitochondrial calcium and sensitized the cytotoxicity to cisplatin in vitro and in vivo. In summary, mitochondrial Lon-induced cisplatin resistance is mediated by calcium release into cytosol through NCLX, which activates calcium-dependent PYK2-SRC-STAT3-IL-6 pathway. Thus, our work uncovers the novel retrograde signaling by mitochondrial Lon on resistance to cisplatin-induced mtDNA stress, indicating the potential use of Lon and NCLX inhibitors for better clinical outcomes in chemoresistant cancer patients.

Mitochondrial Lon-induced mtDNA leakage contributes to PD-L1-mediated immunoescape via STING-IFN signaling and extracellular vesicles.

BACKGROUND: Mitochondrial Lon is a chaperone and DNA-binding protein that functions in protein quality control and stress response pathways. The level of Lon regulates mitochondrial DNA (mtDNA) metabolism and the production of mitochondrial reactive oxygen species (ROS). However, there is little information in detail on how mitochondrial Lon regulates ROS-dependent cancer immunoescape through mtDNA metabolism in the tumor microenvironment (TME). METHODS: We explored the understanding of the intricate interplay between mitochondria and the innate immune response in the inflammatory TME. RESULTS: We found that oxidized mtDNA is released into the cytosol when Lon is overexpressed and then it induces interferon (IFN) signaling via cGAS-STING-TBK1, which upregulates PD-L1 and IDO-1 expression to inhibit T-cell activation. Unexpectedly, upregulation of Lon also induces the secretion of extracellular vehicles (EVs), which carry mtDNA and PD-L1. Lon-induced EVs further induce the production of IFN and IL-6 from macrophages, which attenuates T-cell immunity in the TME. CONCLUSIONS: The levels of mtDNA and PD-L1 in EVs in patients with oral cancer function as a potential diagnostic biomarker for anti-PD-L1 immunotherapy. Our studies provide an insight into the immunosuppression on mitochondrial stress and suggest a therapeutic synergy between anti-inflammation therapy and immunotherapy in cancer.

Identification of Novel Cdc7 Kinase Inhibitors as Anti-Cancer Agents that Target the Interaction with Dbf4 by the Fragment Complementation and Drug Repositioning Approach.

BACKGROUND: Cdc7-Dbf4 is a conserved serine/threonine kinase that plays an important role in initiation of DNA replication and DNA damage tolerance in eukaryotic cells. Cdc7 has been found overexpressed in human cancer cell lines and tumor tissues, and the knockdown of Cdc7 expression causes an p53-independent apoptosis, suggesting that Cdc7 is a target for cancer therapy. Only a handful Cdc7 kinase inhibitors have been reported. All Cdc7 kinase inhibitors, including PHA-767491, were identified and characterized as ATP-competitive inhibitors. Unfortunately, these ATP-competitive Cdc7 inhibitors have no good effect on clinical trial. METHODS: Here, we have developed a novel drug-screening platform to interrupt the interaction between Cdc7 and Dbf4 based on Renilla reniformis luciferase (Rluc)-linked protein-fragment complementation assay (Rluc-PCA). Using drug repositioning approach, we found several promising Cdc7 inhibitors for cancer therapy from a FDA-approved drug library. FINDINGS: Our data showed that dequalinium chloride and clofoctol we screened inhibit S phase progression, accumulation in G2/M phase, and Cdc7 kinase activity. In addition, in vivo mice animal study suggests that dequalinium chloride has a promising anti-tumor activity in oral cancer. Interestingly, we also found that dequalinium chloride and clofoctol sensitize the effect of platinum compounds and radiation due to synergistic effect. In conclusion, we identified non-ATP-competitive Cdc7 kinase inhibitors that not only blocks DNA synthesis at the beginning but also sensitizes cancer cells to DNA damage agents. INTERPRETATION: The inhibitors will be a promising anti-cancer agent and enhance the therapeutic effect of chemotherapy and radiation for current cancer therapy. FUND: This work was supported by grants from the Ministry of Science and Technology, Ministry of Health and Welfare, and National Health Research Institutes, Taiwan.

Mitochondrial Lon sequesters and stabilizes p53 in the matrix to restrain apoptosis under oxidative stress via its chaperone activity.

Mitochondrial Lon is a multi-function matrix protease with chaperone activity. However, little literature has been undertaken into detailed investigations on how Lon regulates apoptosis through its chaperone activity. Accumulating evidences indicate that various stresses induce transportation of p53 to mitochondria and activate apoptosis in a transcription-independent manner. Here we found that increased Lon interacts with p53 in mitochondrial matrix and restrains the apoptosis induced by p53 under oxidative stress by rescuing the loss of mitochondrial membrane potential (Δψm) and the release of cytochrome C and SMAC/Diablo. Increased chaperone Lon hampers the transcription-dependent apoptotic function of p53 by reducing the mRNA expression of p53 target genes. The ATPase mutant (K529R) of chaperone Lon decreases the interaction with p53 and fails to inhibit apoptosis. Furthermore, the chaperone activity of Lon is important for mitochondrial p53 accumulation in an mtHsp70-dependent manner, which is also important to prevent the cytosolic distribution of p53 from proteasome-dependent degradation. These results indicate that the chaperone activity of Lon is important to bind with mitochondrial p53 by which increased Lon suppresses the apoptotic function of p53 under oxidative stress. Furthermore, mitochondrial Lon-mtHsp70 increases the stability/level of p53 through trafficking and retaining p53 in mitochondrial matrix and preventing the pool of cytosolic p53 from proteasome-dependent degradation in vitro and in clinic.

Cdc7-Dbf4-mediated phosphorylation of HSP90-S164 stabilizes HSP90-HCLK2-MRN complex to enhance ATR/ATM signaling that overcomes replication stress in cancer.

Cdc7-Dbf4 kinase plays a key role in the initiation of DNA replication and contributes to the replication stress in cancer. The activity of human Cdc7-Dbf4 kinase remains active and acts as an effector of checkpoint under replication stress. However, the downstream targets of Cdc7-Dbf4 contributed to checkpoint regulation and replication stress-support function in cancer are not fully identified. In this work, we showed that aberrant Cdc7-Dbf4 induces DNA lesions that activate ATM/ATR-mediated checkpoint and homologous recombination (HR) DNA repair. Using a phosphoproteome approach, we identified HSP90-S164 as a target of Cdc7-Dbf4 in vitro and in vivo. The phosphorylation of HSP90-S164 by Cdc7-Dbf4 is required for the stability of HSP90-HCLK2-MRN complex and the function of ATM/ATR signaling cascade and HR DNA repair. In clinically, the phosphorylation of HSP90-S164 indeed is increased in oral cancer patients. Our results indicate that aberrant Cdc7-Dbf4 enhances replication stress tolerance by rewiring ATR/ATM mediated HR repair through HSP90-S164 phosphorylation and by promoting recovery from replication stress. We provide a new solution to a subtyping of cancer patients with dominant ATR/HSP90 expression by combining inhibitors of ATR-Chk1, HSP90, or Cdc7 in cancer combination therapy.

Loss of GDF10/BMP3b as a prognostic marker collaborates with TGFBR3 to enhance chemotherapy resistance and epithelial-mesenchymal transition in oral squamous cell carcinoma.

Growth differentiation factor-10 (GDF10), commonly referred as BMP3b, is a member of the transforming growth factor-ß (TGF-ß) superfamily. GDF10/BMP3b has been considered as a tumor suppressor, however, little is known about the molecular mechanism of its roles in tumor suppression in oral cancer. Clinical significance of GDF10 downregulation in oral squamous cell carcinoma (OSCC) was evaluated using three independent cohorts of OSCC patients. The molecular mechanisms of GDF10 in the suppression of cell survival, cell migration/invasion and epithelial-mesenchymal transition (EMT) were investigated by using oral cancer cell lines. The present study shows that GDF10 is downregulated during oral carcinogenesis, and GDF10 expression is also an independent risk factor for overall survival of OSCC patients. Overexpression of GDF10 attenuates cell proliferation, transformation, migration/invasion, and EMT. GDF10-inhibited EMT is mediated by ERK signaling but not by typical TGF-ß signaling. In addition, overexpression of GDF10 promotes DNA damage-induced apoptosis and sensitizes the response to all-trans retinoic acid (ATRA) and camptothecin (CPT). Intriguingly, the expression of GDF10 is induced by type III TGF-ß receptor (TGFBR3) through TGF-ß-SMAD2/3 signaling. Our findings suggest that TGFBR3 is an upstream activator of GDF10 expression and they share the same signaling to inhibit EMT and migration/invasion. These results support that GDF10 acts as a hinge to collaborate with TGFBR3 in the transition of EMT-MET program. Taken together, we illustrated the clinical significance and the molecular mechanisms of tumor-suppressive GDF10 in OSCC.

Cdc6 cooperates with c-Myc to promote genome instability and epithelial to mesenchymal transition EMT in zebrafish.

Aberration in DNA replication is a major cause to genome instability that is a hallmark of cancer cells. Cell division cycle 6 (Cdc6) and c-Myc have a critical role in the initiation of DNA replication. However, whether their interaction induces epithelial-mesenchymal transition (EMT) and promotes tumorigenesis in in vivo animal model remains unclear. Since using zebrafish as a cancer model has been restricted by the late onset of tumorigenesis and extreme difficulty in transformation on skin, we tried to establish a novel non-melanoma skin model in zebrafish to study their role in tumorigenesis. A stable transgenic zebrafish was created by using tol2 transposon, in which cdc6 and c-myc were co-overexpressed in epidermis driven by a skin-specific krt4 promoter. Intriguingly, co-overexpression of cdc6 and c-myc in transgenic zebrafish skin triggered tumor-like transformation, apoptosis attenuation, genomic instability, and EMT, hallmarks of malignant tumorigenesis. Our findings and other characteristics of zebrafish, including optical clarity and small molecule treatment, provide the future utility of this model for easy and non-invasive detection and for identification of new anti-cancer drug.

Increased Cdc7 expression is a marker of oral squamous cell carcinoma and overexpression of Cdc7 contributes to the resistance to DNA-damaging agents.

Cdc7-Dbf4 kinase (Dbf4-dependent kinase, DDK) is an essential factor of DNA replication and DNA damage response (DDR), which is associated with tumorigenesis. However, Cdc7 expression has never been associated to the outcome of oral squamous cell carcinoma (OSCC) patients, and the mechanism underlying cancer cell survival mediated by Cdc7 remains unclear. The Cdc7 protein expression of 105 OSCC tumor and 30 benign tissues was examined by immunohistochemistry assay. Overall survival rates of 80 OSCC patients were measured using Kaplan-Meier estimates and the log-rank tests. Cdc7 overexpression by adenovirus system was used to scrutinize the underlying mechanism contributed to cancer cell survival upon DDR. In silico analysis showed that increased Cdc7 is a common feature of cancer. Cdc7 overexpression was found in 96 of 105 (91.4%) studied cases of OSCC patients. Patients with higher Cdc7 expression, either categorized into two groups: Cdc7 high expression (2+ to 3+) versus Cdc7 low expression (0 to 1+) [hazard ratios (HR)=2.6; 95% confidence interval (CI)=1.28-5.43; P=0.0087] or four groups (0 to 3+) [HR=1.71; 95% CI=1.20-2.44; P=0.0032], exhibited a poorer outcome. Multivariate analysis showed that Cdc7 is an independent marker for survival prediction. Overexpressed Cdc7 inhibits genotoxin-induced apoptosis to increase the survival of cancer cells. In summary, Cdc7 expression, which is universally upregulated in cancer, is an independent prognostic marker of OSCC. Cdc7 inhibits genotoxin-induced apoptosis and increases survival in cancer cells upon DDR, suggesting that high expression of Cdc7 enhances the resistance to chemotherapy.

Dopaminergic and serotoninergic deficiencies in young adult rats prenatally exposed to the bacterial lipopolysaccharide.

We have reported previously that prenatal bacterial lipopolysaccharide (LPS) exposure at the gestation window of vulnerability could consistently lead to dopamine (DA) neuron loss in the substantia nigra (SN). Thus, we suggested that prenatal LPS exposure might represent as a risk factor for the development of Parkinson's disease (PD). Here, we report that the same exposure could lead to tryptophan hydroxylase (TPH, a serotonin neuron marker) immunoreactive cell loss in the dorsal raphe nucleus (DRN). Twenty two pups born to saline or LPS-injected gravid female rats at E10.5 were used in the current study. Twelve male pups at age of 4 months (6 from each of two prenatal groups) were used for the tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) immunochemistry studies. The other 10 (5 from each of two prenatal groups) males were used in the biochemistry studies. A 29% THir neuron loss in the substantia nigra (F(1,11)=17.573, P=0.002) and a 31% TPHir neuron loss (F(1,11)=44.005, P<0.001) in the DRN were seen. Significant DA and 5-hydroxytryptamine (5-HT) reductions (P<0.05) were found in the frontal cortex, nucleus accumbens, striatum, amygdala, hippocampus, and hypothalamus. The losses of DA and 5-HT were accompanied by the significant increases in homovanillic acid over DA and 5-hydroxyindoleacetic acid over 5-HT ratios in the most areas tested. These data further validate prenatal LPS exposure as a model of PD since DA and 5-HT changes similar to those seen in PD patients. They also suggest that prenatal LPS might be a risk factor for other diseases including mood disorders.

Enhanced oral bioavailability of paclitaxel by D-alpha-tocopheryl polyethylene glycol 400 succinate in mice.

Paclitaxel is widely used to treat several types of solid tumors. The commercially available paclitaxel formulation contains Cremophor/ethanol as solubilizers. This study evaluated the effects of D-alpha-tocopheryl polyethylene glycol 400 succinate (TPGS 400) on the oral absorption of paclitaxel in mice. Mice were given an intravenous (18mg/kg) or oral (100mg/kg) dose of paclitaxel solubilized in Cremophor/ethanol or in TPGS 400/ethanol formulations. Paclitaxel plasma concentrations and pharmacokinetic parameters were determined. The maximal plasma concentrations of paclitaxel after an oral dose were 1.77+/-0.17 and 3.39+/-0.49microg/ml for Cremophor/ethanol and TPGS 400/ethanol formulations, respectively, with a similar time at 40-47min to reach the maximal plasma concentrations. The oral bioavailability of paclitaxel in TPGS 400/ethanol (7.8%) was 3-fold higher than that in Cremophor/ethanol (2.5%). On the other hand, the plasma pharmacokinetic profiles of intravenous paclitaxel demonstrated a superimposition for the two formulations. Furthermore, TPGS 400 concentration-dependently increased the intracellular retention of Rhodamine 123 in Caco-2 cells and enhanced paclitaxel permeability in monolayer Caco-2 cultures. TPGS 400 at concentrations up to 1mM did not inhibit testosterone 6beta-hydroxylase, a cytochrome P450 isozyme 3A in liver microsomes metabolizing paclitaxel. Our results indicated that TPGS 400 enhances the oral bioavailability of paclitaxel in mice and the enhancement may result from an increase in intestinal absorption of paclitaxel.

Substituted pyrrolidine-2,4-dicarboxylic acid amides as potent dipeptidyl peptidase IV inhibitors.

A series of substituted pyrrolidine-2,4-dicarboxylic acid amides were synthesized as potential antidiabetic agents, and many of them showed good in vitro DPP-IV inhibition (IC50 = 2-250 nM) with selectivity over DPP-II, DPP8, and FAP enzymes. Selected compounds 8c and 11a showed in vivo plasma DPP-IV inhibition after oral administration in Wistar rats.

Synthesis and structure-activity relationship of 2-aminobenzophenone derivatives as antimitotic agents.

A new type of inhibitor of tubulin polymerization was discovered on the basis of the combretastatin molecular skeleton. The lead compounds in this series, compounds 6 and 7, strongly inhibited tubulin polymerization in vitro and significantly arrested cells at the G(2)/M phase. Compounds 6 and 7 yielded 50- to 100-fold lower IC(50) values than did combretastatin A-4 against Colo 205, NUGC3, and HA22T human cancer cell lines as well as similar or greater growth inhibitory activities than did combretastain A-4 against DLD-1, HR, MCF-7, DU145, HONE-1, and MES-SA/DX5 human cancer cell lines. Structure-activity relationship information revealed that introduction of an amino group at the ortho position of the benzophenone ring plays an integral role for increased growth inhibition.