Gait speed is a biomarker of cancer-associated cachexia decline and recovery.

Background: Progressive functional decline is a key element of cancer-associated cachexia. No therapies have successfully translated to the clinic due to an inability to measure and improve physical function in cachectic patients. Major barriers to translating pre-clinical therapies to the clinic include lack of cancer models that accurately mimic functional decline and use of non-specific outcome measures of function, like grip strength. New approaches are needed to investigate cachexia-related function at both the basic and clinical science levels. Methods: Survival extension studies were performed by testing multiple cell lines, dilutions, and vehicle-types in orthotopic implantation of K-ras LSL.G12D/+ ; Trp53 R172H/+ ; Pdx-1-Cre (KPC) derived cells. 128 animals in this new model were then assessed for muscle wasting, inflammation, and functional decline using a battery of biochemical, physiologic, and behavioral techniques. In parallel, we analyzed a 156-subject cohort of cancer patients with a range of cachexia severity, and who required rehabilitation, to determine the relationship between gait speed via six-minute walk test (6MWT), grip strength (hGS), and functional independence measures (FIM). Cachectic patients were identified using the Weight Loss Grading Scale (WLGS), Fearon consensus criteria, and the Prognostic Nutritional Index (PNI). Results: Using a 100-cell dose of DT10022 KPC cells, we extended the survival of the KPC orthotopic model to 8-9 weeks post-implantation compared to higher doses used (p<0.001). In this Low-dose Orthotopic (LO) model, both progressive skeletal and cardiac muscle wasting were detected in parallel to systemic inflammation; skeletal muscle atrophy at the fiber level was detected as early as 3 weeks post-implantation compared to controls (p<0.001). Gait speed in LO animals declined as early 2 week post-implantation whereas grip strength change was a late event and related to end of life. Principle component analysis (PCA) revealed distinct cachectic and non-cachectic animal populations, which we leveraged to show that gait speed decline was specific to cachexia (p<0.01) while grip strength decline was not (p=0.19). These data paralleled our observations in cancer patients with cachexia who required rehabilitation. In cachectic patients (identified by WLGS, Fearon criteria, or PNI, change in 6MWT correlated with motor FIM score changes while hGS did not (r 2 =0.18, p<0.001). This relationship between 6MWT and FIM in cachectic patients was further confirmed through multivariate regression (r 2 =0.30, p<0.001) controlling for age and cancer burden. Conclusion: Outcome measures linked to gait are better associated with cachexia related function and preferred for future pre-clinical and clinical cachexia studies.

Synthesis of fluorinated triphenylphosphonium analogs that improve cancer cell selectivity and in vivo detection.

Triphenylphosphonium (TPP+) compounds like mito-metformin (MMe) target cancer cells by exploiting their hyperpolarized mitochondrial membrane potential. Here, we present a protocol for synthesizing TPP+ analogs with selectivity for mammalian cancer cells, reduced toxicity, and quantifiability using fluorine-19 nuclear magnetic resonance (19F-NMR). We describe steps for treating mammalian cells with mitochondria-targeted compounds, treating and preparing mouse tissue with these compounds, and 19F-NMR detection of MMe analogs in cells and tissue. TPP+-conjugated metformin analogs include para-methoxy (pMeO-MMe) and para-trifluoromethyl MMe (pCF3-MMe) and meta-trifluoromethyl MMe (mCF3-MMe).

Fluorinated triphenylphosphonium analogs improve cell selectivity and in vivo detection of mito-metformin.

Triphenylphosphonium (TPP+) conjugated compounds selectively target cancer cells by exploiting their hyperpolarized mitochondrial membrane potential. To date, studies have focused on modifying either the linker or the cargo of TPP+-conjugated compounds. Here, we investigated the biological effects of direct modification to TPP+ to improve the efficacy and detection of mito-metformin (MMe), a TPP+-conjugated probe we have shown to have promising preclinical efficacy against solid cancer cells. We designed, synthesized, and tested trifluoromethyl and methoxy MMe analogs (pCF3-MMe, mCF3-MMe, and pMeO-MMe) against multiple distinct human cancer cells. pCF3-MMe showed enhanced selectivity toward cancer cells compared to MMe, while retaining the same signaling mechanism. Importantly, pCF3-MMe allowed quantitative monitoring of cellular accumulation via 19F-NMR in vitro and in vivo. Furthermore, adding trifluoromethyl groups to TPP+ reduced toxicity in vivo while retaining anti-tumor efficacy, opening an avenue to de-risk these next-generation TPP+-conjugated compounds.

Oncostatin M Receptor-Targeted Antibodies Suppress STAT3 Signaling and Inhibit Ovarian Cancer Growth.

Although patients with advanced ovarian cancer may respond initially to treatment, disease relapse is common, and nearly 50% of patients do not survive beyond five years, indicating an urgent need for improved therapies. To identify new therapeutic targets, we performed single-cell and nuclear RNA-seq data set analyses on 17 human ovarian cancer specimens, revealing the oncostatin M receptor (OSMR) as highly expressed in ovarian cancer cells. Conversely, oncostatin M (OSM), the ligand of OSMR, was highly expressed by tumor-associated macrophages and promoted proliferation and metastasis in cancer cells. Ovarian cancer cell lines and additional patient samples also exhibited elevated levels of OSMR when compared with other cell types in the tumor microenvironment or to normal ovarian tissue samples. OSMR was found to be important for ovarian cancer cell proliferation and migration. Binding of OSM to OSMR caused OSMR-IL6ST dimerization, which is required to produce oncogenic signaling cues for prolonged STAT3 activation. Human monoclonal antibody clones B14 and B21 directed to the extracellular domain of OSMR abrogated OSM-induced OSMR-IL6ST heterodimerization, promoted the internalization and degradation of OSMR, and effectively blocked OSMR-mediated signaling in vitro. Importantly, these antibody clones inhibited the growth of ovarian cancer cells in vitro and in vivo by suppressing oncogenic signaling through OSMR and STAT3 activation. Collectively, this study provides a proof of principle that anti-OSMR antibody can mediate disruption of OSM-induced OSMR-IL6ST dimerization and oncogenic signaling, thus documenting the preclinical therapeutic efficacy of human OSMR antagonist antibodies for immunotherapy in ovarian cancer. SIGNIFICANCE: This study uncovers a role for OSMR in promoting ovarian cancer cell proliferation and metastasis by activating STAT3 signaling and demonstrates the preclinical efficacy of antibody-based OSMR targeting for ovarian cancer treatment.

Synchronous effects of targeted mitochondrial complex I inhibitors on tumor and immune cells abrogate melanoma progression.

Metabolic heterogeneity within the tumor microenvironment promotes cancer cell growth and immune suppression. We determined the impact of mitochondria-targeted complex I inhibitors (Mito-CI) in melanoma. Mito-CI decreased mitochondria complex I oxygen consumption, Akt-FOXO signaling, blocked cell cycle progression, melanoma cell proliferation and tumor progression in an immune competent model system. Immune depletion revealed roles for T cells in the antitumor effects of Mito-CI. While Mito-CI preferentially accumulated within and halted tumor cell proliferation, it also elevated infiltration of activated effector T cells and decreased myeloid-derived suppressor cells (MDSC) as well as tumor-associated macrophages (TAM) in melanoma tumors in vivo. Anti-proliferative doses of Mito-CI inhibited differentiation, viability, and the suppressive function of bone marrow-derived MDSC and increased proliferation-independent activation of T cells. These data indicate that targeted inhibition of complex I has synchronous effects that cumulatively inhibits melanoma growth and promotes immune remodeling.

Targeted biologic inhibition of both tumor cell-intrinsic and intercellular CLPTM1L/CRR9-mediated chemotherapeutic drug resistance.

Recurrence of therapy-resistant tumors is a principal problem in solid tumor oncology, particularly in ovarian cancer. Despite common complete responses to first line, platinum-based therapies, most women with ovarian cancer recur, and eventually, nearly all with recurrent disease develop platinum resistance. Likewise, both intrinsic and acquired resistance contribute to the dismal prognosis of pancreatic cancer. Our previous work and that of others has established CLPTM1L (cleft lip and palate transmembrane protein 1-like)/CRR9 (cisplatin resistance related protein 9) as a cytoprotective oncofetal protein that is present on the tumor cell surface. We show that CLPTM1L is broadly overexpressed and accumulated on the plasma membrane of ovarian tumor cells, while weakly or not expressed in normal tissues. High expression of CLPTM1L is associated with poor outcome in ovarian serous adenocarcinoma. Robust re-sensitization of resistant ovarian cancer cells to platinum-based therapy was achieved using human monoclonal biologics inhibiting CLPTM1L in both orthotopic isografts and patient-derived cisplatin resistant xenograft models. Furthermore, we demonstrate that in addition to cell-autonomous cytoprotection by CLPTM1L, extracellular CLPTM1L confers resistance to chemotherapeutic killing in an ectodomain-dependent fashion, and that this intercellular resistance mechanism is inhibited by anti-CLPTM1L biologics. Specifically, exosomal CLPTM1L from cisplatin-resistant ovarian carcinoma cell lines conferred resistance to cisplatin in drug-sensitive parental cell lines. CLPTM1L is present in extracellular vesicle fractions of tumor culture supernatants and in patients' serum with increasing abundance upon chemotherapy treatment. These findings have encouraging implications for the use of anti-CLPTM1L targeted biologics in the treatment of therapy-resistant tumors.

STING Activated Tumor-Intrinsic Type I Interferon Signaling Promotes CXCR3 Dependent Antitumor Immunity in Pancreatic Cancer.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is a lethal chemoresistant cancer that exhibits early metastatic spread. The highly immunosuppressive PDA tumor microenvironment renders patients resistant to emerging immune-targeted therapies. Building from our prior work, we evaluated stimulator of interferon genes (STING) agonist activation of PDA cell interferon-α/ß-receptor (IFNAR) signaling in systemic antitumor immune responses. METHODS: PDA cells were implanted subcutaneously to wild-type, IFNAR-, or CXCR3-knockout mice. Tumor growth was monitored, and immune responses were comprehensively profiled. RESULTS: Human and mouse STING agonist ADU-S100 reduced local and distal tumor burden and activated systemic antitumor immune responses in PDA-bearing mice. Effector T-cell infiltration and inflammatory cytokine and chemokine production, including IFN-dependent CXCR3-agonist chemokines, were elevated, whereas suppressive immune populations were decreased in treated tumors. Intratumoral STING agonist treatment also generated inflammation in distal noninjected tumors and peripheral immune tissues. STING agonist treatment of type I IFN-responsive PDA tumors engrafted to IFNAR-/- recipient mice was sufficient to contract tumors and stimulate local and systemic T-cell activation. Tumor regression and CD8+ T-cell infiltration were abolished in PDA engrafted to CXCR3-/- mice treated with STING agonist. CONCLUSIONS: These data indicate that STING agonists promote T-cell infiltration and counteract immune suppression in locally treated and distant tumors. Tumor-intrinsic type I IFN signaling initiated systemic STING-mediated antitumor inflammation and required CXCR3 expression. STING-mediated induction of systemic immune responses provides an approach to harness the immune system to treat primary and disseminated pancreatic cancers.

Mitochondria-targeted magnolol inhibits OXPHOS, proliferation, and tumor growth via modulation of energetics and autophagy in melanoma cells.

INTRODUCTION: Melanoma is an aggressive form of skin cancer for which there are no effective drugs for prolonged treatment. The existing kinase inhibitor antiglycolytic drugs (B-Raf serine/threonine kinase or BRAF inhibitors) are effective for a short time followed by a rapid onset of drug resistance. PRESENTATION OF CASE: Here, we show that a mitochondria-targeted analog of magnolol, Mito-magnolol (Mito-MGN), inhibits oxidative phosphorylation (OXPHOS) and proliferation of melanoma cells more potently than untargeted magnolol. Mito-MGN also inhibited tumor growth in murine melanoma xenografts. Mito-MGN decreased mitochondrial membrane potential and modulated energetic and mitophagy signaling proteins. DISCUSSION: Results indicate that Mito-MGN is significantly more potent than the FDA-approved OXPHOS inhibitor in inhibiting proliferation of melanoma cells. CONCLUSION: These findings have implications in the treatment of melanomas with enhanced OXPHOS status due to metabolic reprogramming or drug resistance.

Increased formation of reactive oxygen species during tumor growth: Ex vivo low-temperature EPR and in vivo bioluminescence analyses.

Previous studies have shown that reactive oxygen species (ROS) such as superoxide or hydrogen peroxide generated at low levels can exert a tumor-promoting role via a redox-signaling mechanism. Reports also suggest that both tumorigenesis and tumor growth are associated with enhanced ROS formation. However, whether ROS levels or ROS-derived oxidative marker levels increase during tumor growth remains unknown. In this study, in vivo bioluminescence imaging with a boronate-based pro-luciferin probe was used to assess ROS formation. Additionally, probe-free cryogenic electron paramagnetic resonance was used to quantify a characteristic aconitase [3Fe4S]+ center that arises in the tumor tissue of mouse xenografts from the reaction of the native [4Fe4S]2+ cluster with superoxide. Results indicated that tumor growth is accompanied by increased ROS formation, and revealed differences in oxidant formation in the inner and outer sections of tumor tissue, respectively, demonstrating redox heterogeneity. Studies using luciferin and pro-luciferin probes enabled the assessment of tumor size, ROS formation, and bioenergetic status (e.g., ATP) in luciferase-transfected mice tumor xenografts. Probe-free ex vivo low-temperature electron paramagnetic resonance can also be translated to clinical studies.

STING agonist inflames the pancreatic cancer immune microenvironment and reduces tumor burden in mouse models.

Pancreatic cancer is characterized by an immune suppressive stromal reaction that creates a barrier to therapy. A murine transgenic pancreatic cancer cell line that recapitulates human disease was used to test whether a STimulator of Interferon Genes (STING) agonist could reignite immunologically inert pancreatic tumors. STING agonist treatment potently changed the tumor architecture, altered the immune profile, and increased the survival of tumor-bearing mice. Notably, STING agonist increased numbers and activity of cytotoxic T cells within tumors and decreased levels of suppressive regulatory T cells. Further, STING agonist treatment upregulated costimulatory molecule expression on cross-presenting dendritic cells and reprogrammed immune-suppressive macrophages into immune-activating subtypes. STING agonist promoted the coordinated and differential cytokine production by dendritic cells, macrophages, and pancreatic cancer cells. Cumulatively, these data demonstrate that pancreatic cancer progression is potently inhibited by STING agonist, which reignited immunologically cold pancreatic tumors to promote trafficking and activation of tumor-killing T cells.

Diacylglycerol kinase ζ (DGKζ) and Casitas b-lineage proto-oncogene b-deficient mice have similar functional outcomes in T cells but DGKζ-deficient mice have increased T cell activation and tumor clearance.

Targeting negative regulators downstream of the T cell receptor (TCR) represents a novel strategy to improve cancer immunotherapy. Two proteins that serve as critical inhibitory regulators downstream of the TCR are diacylglycerol kinase ζ (DGKζ), a regulator of Ras and PKC-θ signaling, and Casitas b-lineage proto-oncogene b (Cbl-b), an E3 ubiquitin ligase that predominantly regulates PI(3)K signaling. We sought to compare the signaling and functional effects that result from deletion of DGKζ, Cbl-b, or both (double knockout, DKO) in T cells, and to evaluate tumor responses generated in a clinically relevant orthotopic pancreatic tumor model. We found that whereas deletion of Cbl-b primarily served to enhance NF-κB signaling, deletion of DGKζ enhanced TCR-mediated signal transduction downstream of Ras/Erk and NF-κB. Deletion of DGKζ or Cbl-b comparably enhanced CD8+ T cell functional responses, such as proliferation, production of IFNγ, and generation of granzyme B when compared with WT T cells. DKO T cells demonstrated enhanced function above that observed with single knockout T cells after weak, but not strong, stimulation. Deletion of DGKζ, but not Cbl-b, however, resulted in significant increases in numbers of activated (CD44hi) CD8+ T cells in both non-treated and tumor-bearing mice. DGKζ-deficient mice also had enhanced control of pancreatic tumor cell growth compared to Cbl-b-deficient mice. This represents the first direct comparison between mice of these genotypes and suggests that T cell immunotherapies may be better improved by targeting TCR signaling molecules that are regulated by DGKζ as opposed to molecules regulated by Cbl-b.

Mitochondria-Targeted Analogues of Metformin Exhibit Enhanced Antiproliferative and Radiosensitizing Effects in Pancreatic Cancer Cells.

Metformin (Met) is an approved antidiabetic drug currently being explored for repurposing in cancer treatment based on recent evidence of its apparent chemopreventive properties. Met is weakly cationic and targets the mitochondria to induce cytotoxic effects in tumor cells, albeit not very effectively. We hypothesized that increasing its mitochondria-targeting potential by attaching a positively charged lipophilic substituent would enhance the antitumor activity of Met. In pursuit of this question, we synthesized a set of mitochondria-targeted Met analogues (Mito-Mets) with varying alkyl chain lengths containing a triphenylphosphonium cation (TPP(+)). In particular, the analogue Mito-Met10, synthesized by attaching TPP(+) to Met via a 10-carbon aliphatic side chain, was nearly 1,000 times more efficacious than Met at inhibiting cell proliferation in pancreatic ductal adenocarcinoma (PDAC). Notably, in PDAC cells, Mito-Met10 potently inhibited mitochondrial complex I, stimulating superoxide and AMPK activation, but had no effect in nontransformed control cells. Moreover, Mito-Met10 potently triggered G1 cell-cycle phase arrest in PDAC cells, enhanced their radiosensitivity, and more potently abrogated PDAC growth in preclinical mouse models, compared with Met. Collectively, our findings show how improving the mitochondrial targeting of Met enhances its anticancer activities, including aggressive cancers like PDAC in great need of more effective therapeutic options. Cancer Res; 76(13); 3904-15. ©2016 AACR.

Expression of nuclear Methyl-CpG binding protein 2 (Mecp2) is dependent on neuronal stimulation and application of Insulin-like growth factor 1.

Methyl-CpG binding protein 2 (MECP2) is a chromosome-binding protein that regulates the development and maintenance of brain circuits. Altered function of the protein product of MECP2 plays an important role in the etiology of many neurodevelopmental disorders. Mutations involving a loss of function are implicated in the etiology of Rett syndrome, intellectual disability, psychosis and severe encephalopathy. Conversely, MECP2 duplications have been identified in autism and intellectual disability. MECP2 action is dependent on neuronal function, as the DNA binding is modulated by activity, and it is phosphorylated in response to stimulation. Although MECP2 is considered a major risk factor for neurodevelopmental disorders, and it is a mediator of activity-dependent mechanisms, the expression levels in response to neuronal activity have never been measured. We studied the expression of Mecp2 protein and RNA in mice neuronal cultures in response to different stimulation conditions and in the presence of insulin-like growth factor1 (IGF1): a growth factor involved in brain development and plasticity. The stimulation protocols were selected according to their ability to induce different forms of synaptic plasticity: rapid depolarization, feed-forward plasticity (LTP, LTD) and feedback forms of plasticity (TTX, KCl). We find a significant reduction of Mecp2 protein nuclear expression in neurons in response to stimuli that induce a potentiation of neuronal response, suggesting that Mecp2 protein expression is modulated by neuronal activation. Application of IGF1 to the cultures induces an increase in the expression of Mecp2 transcript and nuclear Mecp2 protein in neurons. These results show that Mecp2 is responsive to neuronal stimulation and IGF1, and different stimuli have different effects on Mecp2 expression; this differential response may have downstream effects on functional mechanisms regulating brain development and plasticity.

Pancreatic Cancer Cell Migration and Metastasis Is Regulated by Chemokine-Biased Agonism and Bioenergetic Signaling.

Patients with pancreatic ductal adenocarcinoma (PDAC) invariably succumb to metastatic disease, but the underlying mechanisms that regulate PDAC cell movement and metastasis remain little understood. In this study, we investigated the effects of the chemokine gene CXCL12, which is silenced in PDAC tumors, yet is sufficient to suppress growth and metastasis when re-expressed. Chemokines like CXCL12 regulate cell movement in a biphasic pattern, with peak migration typically in the low nanomolar concentration range. Herein, we tested the hypothesis that the biphasic cell migration pattern induced by CXCL12 reflected a biased agonist bioenergetic signaling that might be exploited to interfere with PDAC metastasis. In human and murine PDAC cell models, we observed that nonmigratory doses of CXCL12 were sufficient to decrease oxidative phosphorylation and glycolytic capacity and to increase levels of phosphorylated forms of the master metabolic kinase AMPK. Those same doses of CXCL12 locked myosin light chain into a phosphorylated state, thereby decreasing F-actin polymerization and preventing cell migration in a manner dependent upon AMPK and the calcium-dependent kinase CAMKII. Notably, at elevated concentrations of CXCL12 that were insufficient to trigger chemotaxis of PDAC cells, AMPK blockade resulted in increased cell movement. In two preclinical mouse models of PDAC, administration of CXCL12 decreased tumor dissemination, supporting our hypothesis that chemokine-biased agonist signaling may offer a useful therapeutic strategy. Our results offer a mechanistic rationale for further investigation of CXCL12 as a potential therapy to prevent or treat PDAC metastasis.

Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: Role of mitochondrial bioenergetics and energy-sensing mechanism.

One of the proposed mechanisms for tumor proliferation involves redox signaling mediated by reactive oxygen species such as superoxide and hydrogen peroxide generated at moderate levels. Thus, the antiproliferative and anti-tumor effects of certain antioxidants were attributed to their ability to mitigate intracellular reactive oxygen species (ROS). Recent reports support a role for mitochondrial ROS in stimulating tumor cell proliferation. In this study, we compared the antiproliferative effects and the effects on mitochondrial bioenergetic functions of a mitochondria-targeted cationic carboxyproxyl nitroxide (Mito-CP), exhibiting superoxide dismutase (SOD)-like activity and a synthetic cationic acetamide analog (Mito-CP-Ac) lacking the nitroxide moiety responsible for the SOD activity. Results indicate that both Mito-CP and Mito-CP-Ac potently inhibited tumor cell proliferation. Both compounds altered mitochondrial and glycolytic functions, and intracellular citrate levels. Both Mito-CP and Mito-CP-Ac synergized with 2-deoxy-glucose (2-DG) to deplete intracellular ATP, inhibit cell proliferation and induce apoptosis in pancreatic cancer cells. We conclude that mitochondria-targeted cationic agents inhibit tumor proliferation via modification of mitochondrial bioenergetics pathways rather than by dismutating and detoxifying mitochondrial superoxide.

A novel mitochondrially-targeted apocynin derivative prevents hyposmia and loss of motor function in the leucine-rich repeat kinase 2 (LRRK2(R1441G)) transgenic mouse model of Parkinson's disease.

Recently, we demonstrated that dimeric apocynin prevented loss of motor function in the leucine-rich repeat kinase 2 (LRRK2(R1441G)) transgenic (tg) mouse (treated with 200mg/kg, three times per week) [B.P. Dranka et al., Neurosci. Lett. 549 (2013) 57-62]. Here we extend those studies by treating LRRK2(R1441G) mice with an orally-available, mitochondrially-targeted apocynin derivative. We hypothesized that the increased mitochondrial permeability of Mito-apocynin, due to the triphenylphosphonium moiety, would allow improvement of Parkinson's disease (PD) symptoms at lower doses than those required for diapocynin. Tests of motor coordination (pole test, Rotor-Rod) revealed a significant deficit in coordinated motor function in LRRK2(R1441G) mice by 15 months of age. Decreased performance on the pole test and Rotor-Rod in the LRRK2(R1441G) mice was prevented with Mito-apocynin treatment (3mg/kg, three times per week). Decreased olfactory function is an early indication of PD in human patients. LRRK2(R1441G) tg mice displayed deficits in sense of smell in both the hidden treat test, and a radial arm maze test. Interestingly, treatment with Mito-apocynin prevented this hyposmia, and animals retained normal ability to identify either a scented treat or a food pellet as well as wild type littermates. Together, these data demonstrate that the mitochondria-targeted apocynin analog is effective in preventing early PD-like symptoms in the LRRK2(R1441G) mouse model.

Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death.

BACKGROUND: Recent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells. METHODS: In this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed. RESULTS: Both Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer. CONCLUSIONS: We conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect.

An adenosine-mediated signaling pathway suppresses prenylation of the GTPase Rap1B and promotes cell scattering.

During metastasis, cancer cells acquire the ability to dissociate from each other and migrate, which is recapitulated in vitro as cell scattering. The small guanosine triphosphatase (GTPase) Rap1 opposes cell scattering by promoting cell-cell adhesion, a function that requires its prenylation, or posttranslational modification with a carboxyl-terminal isoprenoid moiety, to enable its localization at cell membranes. Thus, signaling cascades that regulate the prenylation of Rap1 offer a mechanism to control the membrane localization of Rap1. We identified a signaling cascade initiated by adenosine A2B receptors that suppressed the prenylation of Rap1B through phosphorylation of Rap1B, which decreased its interaction with the chaperone protein SmgGDS (small GTPase guanosine diphosphate dissociation stimulator). These events promoted the cytosolic and nuclear accumulation of nonprenylated Rap1B and diminished cell-cell adhesion, resulting in cell scattering. We found that nonprenylated Rap1 was more abundant in mammary tumors than in normal mammary tissue in rats and that activation of adenosine receptors delayed Rap1B prenylation in breast, lung, and pancreatic cancer cell lines. Our findings support a model in which high concentrations of extracellular adenosine, such as those that arise in the tumor microenvironment, can chronically activate A2B receptors to suppress Rap1B prenylation and signaling at the cell membrane, resulting in reduced cell-cell contact and promoting cell scattering. Inhibiting A2B receptors may be an effective method to prevent metastasis.

Mitochondria-targeted drugs synergize with 2-deoxyglucose to trigger breast cancer cell death.

Cancer cells are long known to exhibit increased aerobic glycolysis, but glycolytic inhibition has not offered a viable chemotherapeutic strategy in part because of the systemic toxicity of antiglycolytic agents. However, recent studies suggest that a combined inhibition of glycolysis and mitochondrial function may help overcome this issue. In this study, we investigated the chemotherapeutic efficacies of mitochondria-targeted drugs (MTD) in combination with 2-deoxy-d-glucose (2-DG), a compound that inhibits glycolysis. Using the MTDs, termed Mito-CP and Mito-Q, we evaluated relative cytotoxic effects and mitochondrial bioenergetic changes in vitro. Interestingly, both Mito-CP and Mito-Q synergized with 2-DG to decrease ATP levels in two cell lines. However, with time, the cellular bioenergetic function and clonogenic survival were largely restored in some cells. In a xenograft model of human breast cancer, combined treatment of Mito-CP and 2-DG led to significant tumor regression in the absence of significant morphologic changes in kidney, liver, or heart. Collectively, our findings suggest that dual targeting of mitochondrial bioenergetic metabolism with MTDs and glycolytic inhibitors such as 2-DG may offer a promising chemotherapeutic strategy.

Diapocynin and apocynin administration fails to significantly extend survival in G93A SOD1 ALS mice.

NADPH oxidase has recently been identified as a promising new therapeutic target in ALS. Genetic deletion of NADPH oxidase (Nox2) in the transgenic SOD1(G93A) mutant mouse model of ALS was reported to increase survival remarkably by 97 days. Furthermore, apocynin, a widely used inhibitor of NADPH oxidase, was observed to dramatically extend the survival of the SOD1(G93A) ALS mice even longer to 113 days (Harraz et al. J Clin Invest 118: 474, 2008). Diapocynin, the covalent dimer of apocynin, has been reported to be a more potent inhibitor of NADPH oxidase. We compared the protection of diapocynin to apocynin in primary cultures of SOD1(G93A)-expressing motor neurons against nitric oxide-mediated death. Diapocynin, 10 µM, provided significantly greater protection compared to apocynin, 200 µM, at the lowest statistically significant concentrations. However, administration of diapocynin starting at 21 days of age in the SOD1(G93A)-ALS mouse model did not extend lifespan. Repeated parallel experiments with apocynin failed to yield protection greater than a 5-day life extension in multiple trials conducted at two separate institutions. The maximum protection observed was an 8-day extension in survival when diapocynin was administered at 100 days of age at disease onset. HPLC with selective ion monitoring by mass spectrometry revealed that both apocynin and diapocynin accumulated in the brain and spinal cord tissue to low micromolar concentrations. Diapocynin was also detected in the CNS of apocynin-treated mice. The failure to achieve significant protection with either apocynin or diapocynin raises questions about the utility for treating ALS patients.