Phosphorylation of PDHA by AMPK Drives TCA Cycle to Promote Cancer Metastasis.

Cancer metastasis accounts for the major cause of cancer-related deaths. How disseminated cancer cells cope with hostile microenvironments in secondary site for full-blown metastasis is largely unknown. Here, we show that AMPK (AMP-activated protein kinase), activated in mouse metastasis models, drives pyruvate dehydrogenase complex (PDHc) activation to maintain TCA cycle (tricarboxylic acid cycle) and promotes cancer metastasis by adapting cancer cells to metabolic and oxidative stresses. This AMPK-PDHc axis is activated in advanced breast cancer and predicts poor metastasis-free survival. Mechanistically, AMPK localizes in the mitochondrial matrix and phosphorylates the catalytic alpha subunit of PDHc (PDHA) on two residues S295 and S314, which activates the enzymatic activity of PDHc and alleviates an inhibitory phosphorylation by PDHKs, respectively. Importantly, these phosphorylation events mediate PDHc function in cancer metastasis. Our study reveals that AMPK-mediated PDHA phosphorylation drives PDHc activation and TCA cycle to empower cancer cells adaptation to metastatic microenvironments for metastasis.

Synthesis, reactivity, and biological activity of gold(I) complexes modified with thiourea-functionalized tyrosine kinase inhibitors.

Thiourea-modified 3-chloro-4-fluoroanilino-quinazoline derivatives have been studied as potential receptor-targeted carrier ligands in linear gold(I) complexes. The molecules mimic the epidermal growth factor receptor (EGFR) tyrosine kinase-targeted inhibitor gefitinib. Thiourea groups were either directly attached to quinazoline-C6 (compounds 4, 5, and 7) or linked to this position via a flexible ethylamino chain (compound 9). Compound 7 acts as a thiourea-S/quinazoline-N1 mixed-donor ligand, giving the unexpected dinuclear complex [{Au(µ-7-S,N)}2]X2 (X = Cl(-), SCN(-)) (12a,b) (X-ray crystallography, electrospray mass spectrometry). Derivative 9 forms a stable linear complex, [Au(PEt3)(9-S)](NO3) (13). The biological activity of the carrier ligands and corresponding gold(I) complexes was studied in NCI-H460 and NCI-H1975 lung cancer cells. Compound 9 partially overcomes resistance to gefitinib in NCI-H1975, a lung cancer cell line characterized by a L858R/T790M mutation in EGFR (IC50 values of 1.7 and 30 µM, respectively). The corresponding gold complex (13) maintains activity in the low-micromolar concentration range similar to the metal-free carrier. Compound 9 and the corresponding [Au(PEt3)] complex, 13, inhibit EGFR kinase-mediated phosphorylation with sub-micromolar IC50 values similar to those observed for gefitinib under the same assay conditions. Potential mechanisms of action and reactions in biological media of this new type of hybrid agent, as well as shortcomings of the current design are discussed.

Investigating the cellular fate of a DNA-targeted platinum-based anticancer agent by orthogonal double-click chemistry.

Confocal fluorescence microscopy was used to study a platinum-based anticancer agent in intact NCI-H460 lung cancer cells. Orthogonal copper-catalyzed azide-alkyne cycloaddition (click) reactions were used to simultaneously determine the cell-cycle-specific localization of the azide-functionalized platinum-acridine agent 1 and monitor its effects on nucleic acid metabolism. Copper-catalyzed postlabeling showed advantages over copper-free click chemistry using a dibenzocyclooctyne (DIBO)-modified reporter dye, which produced high background levels in microscopic images and failed to efficiently label platinum adducts in chromatin. Compound 1 was successfully labeled with the fluorophore DIBO to yield 1* (characterized by in-line high-performance liquid chromatography/electrospray mass spectrometry). 1 and 1* show a high degree of colocalization in the confocal images, but the ability of 1* to target the (compacted) chromatin was markedly reduced, most likely owing to the steric bulk introduced by the DIBO tag. Nuclear platinum levels correlated inversely with the ability of the cells to synthesize DNA and cause cell cycle arrest, as confirmed by bivariate flow cytometry analysis. In addition, a decrease in the level of cellular transcription, shrinkage of the nucleolar regions, and redistribution of RNA into the cytosol were observed. Postlabeling in conjunction with colocalization experiments is a useful tool for studying the cell killing mechanism of this type of DNA-targeted agent.

Design of enzymatically cleavable prodrugs of a potent platinum-containing anticancer agent.

Using a versatile synthetic approach, a new class of potential ester prodrugs of highly potent, but systemically too toxic, platinum-acridine anticancer agents was generated. The new hybrids contain a hydroxyl group, which has been masked with a cleavable lipophilic acyl moiety. Both butanoic (butyric) and bulkier 2-propanepentanoic (valproic) esters were introduced. The goals of this design were to improve the drug-like properties (e.g., logD) and to reduce the systemic toxicity of the pharmacophore. Two distinct pathways by which the target compounds undergo effective ester hydrolysis, the proposed activating step, have been confirmed: platinum-assisted, self-immolative ester cleavage in a low-chloride environment (LC-ESMS, NMR spectroscopy) and enzymatic cleavage by human carboxylesterase-2 (hCES-2) (LC-ESMS). The valproic acid ester derivatives are the first example of a metal-containing agent cleavable by the prodrug-converting enzyme. They show excellent chemical stability and reduced systemic toxicity. Preliminary results from screening in lung adenocarcinoma cell lines (A549, NCI-H1435) suggest that the mechanism of the valproic esters may involve intracellular deesterification.

Redesigning the DNA-targeted chromophore in platinum-acridine anticancer agents: a structure-activity relationship study.

Platinum-acridine hybrid agents show low-nanomolar potency in chemoresistant non-small cell lung cancer (NSCLC), but high systemic toxicity in vivo. To reduce the promiscuous genotoxicity of these agents and improve their pharmacological properties, a modular build-click-screen approach was used to evaluate a small library of twenty hybrid agents containing truncated and extended chromophores of varying basicities. Selected derivatives were resynthesized and tested in five NSCLC cell lines representing large cell, squamous cell, and adenocarcinomas. 7-Aminobenz[c]acridine was identified as a promising scaffold in a hybrid agent (P1-B1) that maintained submicromolar activity in several of the DNA-repair proficient and p53-mutant cancer models, while showing improved tolerability in mice by 32-fold compared to the parent platinum-acridine (P1-A1). The distribution and DNA/RNA adduct levels produced by the acridine- and benz[c]acridine-based analogues in NCI-H460 cells (confocal microscopy, ICP-MS), and their ability to bind G-quadruplex forming DNA sequences (CD spectroscopy, HR-ESMS) were studied. P1-B1 emerges as a less genotoxic, more tolerable, and potentially more target-selective hybrid agent than P1-A1.

Modulation of oxidative phosphorylation and mitochondrial biogenesis by cigarette smoke influence the response to immune therapy in NSCLC patients.

The treatment regimen of non-small cell lung cancer (NSCLC) has drastically changed owing to the superior anti-cancer effects generated by the immune-checkpoint blockade (ICB). However, only a subset of patients experience benefit after receiving ICBs. Therefore, it is of paramount importance to increase the response rate by elucidating the underlying molecular mechanisms and identifying novel therapeutic targets to enhance the efficacy of IBCs in non-responders. We analyzed the progression-free survival (PFS) and overall survival (OS) of 295 NSCLC patients who received anti-PD-1 therapy by segregating them with multiple clinical factors including sex, age, race, smoking history, BMI, tumor grade and subtype. We also identified key signaling pathways and mutations that are enriched in patients with distinct responses to ICB by gene set enrichment analysis (GSEA) and mutational analyses. We found that former and current smokers have a higher response rate to anti-PD-1 treatment than non-smokers. GSEA results revealed that oxidative phosphorylation (OXPHOS) and mitochondrial related pathways are significantly enriched in both responders and smokers, suggesting a potential role of cellular metabolism in regulating immune response to ICB. We also demonstrated that all-trans retinoic acid (ATRA) which enhances mitochondrial function significantly enhanced the efficacy of anti-PD-1 treatment in vivo. Our clinical and bioinformatics based analyses revealed a connection between smoking induced metabolic switch and the response to immunotherapy, which can be the basis for developing novel combination therapies that are beneficial to never smoked NSCLC patients.

c-Met Mediated Cytokine Network Promotes Brain Metastasis of Breast Cancer by Remodeling Neutrophil Activities.

The brain is one of the most common metastatic sites among breast cancer patients, especially in those who have Her2-positive or triple-negative tumors. The brain microenvironment has been considered immune privileged, and the exact mechanisms of how immune cells in the brain microenvironment contribute to brain metastasis remain elusive. In this study, we found that neutrophils are recruited and influenced by c-Met high brain metastatic cells in the metastatic sites, and depletion of neutrophils significantly suppressed brain metastasis in animal models. Overexpression of c-Met in tumor cells enhances the secretion of a group of cytokines, including CXCL1/2, G-CSF, and GM-CSF, which play critical roles in neutrophil attraction, granulopoiesis, and homeostasis. Meanwhile, our transcriptomic analysis demonstrated that conditioned media from c-Met high cells significantly induced the secretion of lipocalin 2 (LCN2) from neutrophils, which in turn promotes the self-renewal of cancer stem cells. Our study unveiled the molecular and pathogenic mechanisms of how crosstalk between innate immune cells and tumor cells facilitates tumor progression in the brain, which provides novel therapeutic targets for treating brain metastasis.

Delivery of an ectonucleotidase inhibitor with ROS-responsive nanoparticles overcomes adenosine-mediated cancer immunosuppression.

Tumor evasion of immune destruction is associated with the production of immunosuppressive adenosine in the tumor microenvironment (TME). Anticancer therapies can trigger adenosine triphosphate (ATP) release from tumor cells, causing rapid formation of adenosine by the ectonucleotidases CD39 and CD73, thereafter exacerbating immunosuppression in the TME. The goal of this study was to develop an approach to facilitate cancer therapy-induced immunogenic cell death including ATP release and to limit ATP degradation into adenosine, in order to achieve durable antitumor immune response. Our approach was to construct reactive oxygen species (ROS)-producing nanoparticles that carry an ectonucleotidase inhibitor ARL67156 by electronic interaction and phenylboronic ester. Upon near-infrared irradiation, nanoparticle-produced ROS induced ATP release from MOC1 cancer cells in vitro and triggered the cleavage of phenylboronic ester, facilitating the release of ARL67156 from the nanoparticles. ARL67156 prevented conversion of ATP to adenosine and enhanced anticancer immunity in an MOC1-based coculture model. We tested this approach in mouse tumor models. Nanoparticle-based ROS-responsive drug delivery reprogramed the immunogenic landscape in tumors, eliciting tumor-specific T cell responses and tumor regression, conferring long-term survival in mouse models. We demonstrated that TME reprograming sets the stage for response to anti-programmed cell death protein 1 (PD1) immunotherapy, and the combination resulted in tumor regression in a 4T1 breast cancer mouse model that was resistant to PD1 blockade. Furthermore, our approach also induced immunological effects in patient-derived organotypic tumor spheroid model, suggesting potential translation of our nanoparticle approach for treating human cancers.

Intrapleural nano-immunotherapy promotes innate and adaptive immune responses to enhance anti-PD-L1 therapy for malignant pleural effusion.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

Diet Alters Entero-Mammary Signaling to Regulate the Breast Microbiome and Tumorigenesis.

Obesity and poor diet often go hand-in-hand, altering metabolic signaling and thereby impacting breast cancer risk and outcomes. We have recently demonstrated that dietary patterns modulate mammary microbiota populations. An important and largely open question is whether the microbiome of the gut and mammary gland mediates the dietary effects on breast cancer. To address this, we performed fecal transplants between mice on control or high-fat diets (HFD) and recorded mammary tumor outcomes in a chemical carcinogenesis model. HFD induced protumorigenic effects, which could be mimicked in animals fed a control diet by transplanting HFD-derived microbiota. Fecal transplants altered both the gut and mammary tumor microbiota populations, suggesting a link between the gut and breast microbiomes. HFD increased serum levels of bacterial lipopolysaccharide (LPS), and control diet-derived fecal transplant reduced LPS bioavailability in HFD-fed animals. In vitro models of the normal breast epithelium showed that LPS disrupts tight junctions (TJ) and compromises epithelial permeability. In mice, HFD or fecal transplant from animals on HFD reduced expression of TJ-associated genes in the gut and mammary gland. Furthermore, infecting breast cancer cells with an HFD-derived microbiome increased proliferation, implicating tumor-associated bacteria in cancer signaling. In a double-blind placebo-controlled clinical trial of patients with breast cancer administered fish oil supplements before primary tumor resection, dietary intervention modulated the microbiota in tumors and normal breast tissue. This study demonstrates a link between the gut and breast that mediates the effect of diet on cancer. SIGNIFICANCE: This study demonstrates that diet shifts the microbiome in the gut and the breast tumor microenvironment to affect tumorigenesis, and oral dietary interventions can modulate the tumor microbiota in patients with breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3890/F1.large.jpg.

Probing platinum-adenine-n3 adduct formation with DNA minor-groove binding agents.

Me-lex(py/py), an adenine-N3-selective alkylating agent, and the reversible minor-groove binder netropsin were used to probe the formation of unusual minor-groove adducts by the cytotoxic hybrid agent PT-ACRAMTU ([PtCl(en)(ACRAMTU)](NO(3))(2); en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea). PT-ACRAMTU was found by chemical footprinting to inhibit specific Me-lex-mediated DNA cleavage at several adenine sites but not at nonspecific guanine, which is consistent with the platination of adenine-N3. In a cell proliferation assay, a significant decrease in cytotoxicity was observed for PT-ACRAMTU, when cancer cells were pretreated with netropsin, suggesting that minor-groove adducts in cellular DNA contribute to the biological activity of the hybrid agent.

The Novel Phospholipid Mimetic KPC34 Is Highly Active Against Acute Myeloid Leukemia with Activated Protein Kinase C.

Acute myeloid leukemia (AML) is an aggressive malignancy with poor outcomes. Nucleoside analogs are subject to resistance mechanisms including downregulation of equilibrative nucleoside transporter (ENT1) and deoxycytidine kinase (dCK). KPC34 is a novel phospholipid mimetic that when cleaved by phospholipase C (PLC) liberates gemcitabine monophosphate and a diacylglycerol mimetic that inhibits the classical isoforms of protein kinase C (PKC). KPC34 acts independently of ENT1 and dCK. KPC34 was active against all AML cell lines tested with IC50s in the nanomolar range. Enforced expression of PLC increased response to KPC34 in vivo. In an orthotopic, xenograft model, KPC34 treatment resulted in a significant increase in survival compared to control animals and those treated with high-dose cytarabine. In a PDX model with activated PKC, there was a significant survival benefit with KPC34, and at progression, there was attenuation of PKC activation in the resistant cells. In contrast, KPC34 was ineffective against a syngeneic, orthotopic AML model without activated PKC. However, when cells from that model were forced to express PKC, there were significantly increased sensitivity in vitro and survival benefit in vivo. These data suggest that KPC34 is active against AML and that the presence of activated PKC can be a predictive biomarker.

Synthesis and biological evaluation of platinum-acridine hybrid agents modified with bipyridine non-leaving groups.

The use of 2,2'-bipyridines (4,4'-R(2)-2,2'-bpy; R=H, Me, OMe, CF(3)) as non-leaving groups (L-L) in platinum-acridinylthiourea conjugates, [PtCl(L-L)(ACRAMTU)](NO(3))(2), has been investigated. All bpy-substituted complexes (2-5) show micromolar activity in HL-60 (leukemia) and H460 (lung) cancer cell lines but proved to be significantly less potent than the prototypical compound (1) containing aliphatic ethane-1,2-diamine. NMR and mass spectrometry data indicate that bpy accelerates the reaction of platinum with DNA nitrogen, but the resulting adducts are more labile than those formed by the prototype.

Optimization of Tissue Microarrays from Banked Human Formalin-Fixed Paraffin Embedded Tissues in the Cancer Research Setting.

The tissue microarray (TMA) is a powerful tool for cancer biomarker discovery and validation. Tens to hundreds of samples can be evaluated simultaneously for molecular marker status at the protein or nucleic acid level. Although fully automated or semiautomated technologies for TMA creation provide excellent precision with respect to core transfer, they do not obviate the need for technical expertise to successfully generate high-quality TMA blocks and derivative sections. We have leveraged our expanding bank of formalin-fixed paraffin embedded paired tumor and normal tissues in our academic cancer center to provide a rich source of input material for cancer research TMAs. In this study, we report a stepwise optimization of TMA generation parameters, including paraffin wax selection, tempering protocol, and sectioning conditions, to achieve the best ribbon sectioning.

Tuning the DNA conformational perturbations induced by cytotoxic platinum-acridine bisintercalators: effect of metal cis/trans isomerism and DNA threading groups.

Four highly charged, water soluble platinum-acridine bisintercalating agents have been synthesized. Depending on the cis/trans isomerism of the metal and the nature of the acridine side chains, bisintercalation induces/stabilizes the classical Watson-Crick B-form or a non-B-form. Circular dichroism spectra and chemical footprinting experiments suggest that 4, the most active derivative in HL-60 cells, produces a structurally severely perturbed DNA with features of a Hoogsteen base-paired biopolymer.

Effect of linkage geometry on biological activity in thiourea- and guanidine-substituted acridines and platinum-acridines.

Novel thiourea- and guanidine-modified acridine-4-carboxamides (4, 7) and a corresponding platinum-intercalator conjugate (4') have been synthesized and evaluated as cytotoxic agents in human promyelocytic leukemia, HL-60, and a non-small cell lung cancer, NCI-H460. Modification of thiourea sulfur in derivative 4 with a DNA platinating moiety, giving 4', resulted in a pronounced cytotoxic enhancement, and the conjugate proved to be the most active of the newly synthesized compounds in NCI-H460 cells. Conjugate 4' represents a new chemotype with potential applications in the treatment of chemoresistant tumors.

Effect of the diamine nonleaving group in platinum-acridinylthiourea conjugates on DNA damage and cytotoxicity.

The following complexes of type [PtCl(R)(ACRAMTU)](NO3)2 (ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea)), derived from prototype 1 (with R = ethane-1,2-diamine), were synthesized: 2 (with R = (1R,2R)-1,2-diaminocyclohexane), 3 (with R = propane-1,3-diamine), 4 (with R = N1,N1,N2,N2-tetramethylethane-1,2-diamine), and 5 (with R = 2,2'-bipyridine). The DNA sequence specificity of the conjugates and their antiproliferative potential in HL-60 and H460 cells were investigated. Conjugate 3 showed the strongest non-cisplatin-type DNA damage in polymerase stop assays and superior cell kill efficacy in H460 lung cancer (IC50 = 70 nM).

The novel phospholipid mimetic KPC34 is highly active against preclinical models of Philadelphia chromosome positive acute lymphoblastic leukemia.

Philadelphia chromosome positive B cell acute lymphoblastic leukemia (Ph+ ALL) is an aggressive cancer of the bone marrow. The addition of tyrosine kinase inhibitors (TKIs) has improved outcomes but many patients still suffer relapse and novel therapeutic agents are needed. KPC34 is an orally available, novel phospholipid conjugate of gemcitabine, rationally designed to overcome multiple mechanisms of resistance, inhibit the classical and novel isoforms of protein kinase C, is able to cross the blood brain barrier and is orally bioavailable. KPC34 had an IC50 in the nanomolar range against multiple ALL cell lines tested but was lowest for Ph+ lines. In mice bearing either naïve or resistant Ph+ ALL, KPC34 treatment resulted in significantly improved survival compared to cytarabine and gemcitabine. Treatment with KPC34 and doxorubicin was more effective than doxorubicin and cytarabine. Mice with recurrence of their ALL after initial treatment with cytarabine and doxorubicin saw dramatic improvements in hind limb paralysis after treatment with KPC34 demonstrating activity against established CNS disease. Consistent with this KPC34 was better than gemcitabine at reducing CNS leukemic burden. These promising pre-clinical results justify the continued development of KPC34 for the treatment of Ph+ALL.

Synthesis, biological activity, and DNA-damage profile of platinum-threading intercalator conjugates designed to target adenine.

PT-ACRAMTU {[PtCl(en)(ACRAMTU)](NO3)2, 2; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, 1, en = ethane-1,2-diamine} is the prototype of a series of DNA-targeted adenine-affinic dual intercalating/platinating agents. Several novel 4,9-disubstituted acridines and the corresponding platinum-acridine conjugates were synthesized. The newly introduced 4-carboxamide side chains contain H-bond donor/acceptor functions designed to promote groove- and sequence-specific platinum binding. In HL-60 (leukemia) and H460 (lung) cancer cells, IC50 values in the micromolar to millimolar range were observed. Several of the intercalators show enhanced cytotoxicity compared to prototype 1, but conjugate 2 appears to be the most potent hybrid agent. Enzymatic digestion assays in conjunction with liquid chromatography-electrospray mass spectrometry analysis indicate that the new conjugates produce PT-ACRAMTU-type DNA damage. Platinum-modified 2'-deoxyguanosine, dG, and several dinucleotide fragments, d(NpN)*, were detected. One of the conjugates showed significantly higher levels of binding to A-containing sites than conjugate 2 (35 +/- 3% vs 24 +/- 3%). Possible structure-activity relationships are discussed.