ABSTRACT
Adenoid cystic carcinoma (ACC) is a head and neck cancer that frequently originates in salivary glands, but can also strike other exocrine glands such as the breast. A key molecular alteration found in the majority of ACC cases is MYB gene rearrangements, leading to activation of the oncogenic transcription factor MYB. In this study, we used immortalised breast epithelial cells and an inducible MYB transgene as a model of ACC. Molecular profiling confirmed that MYB-driven gene expression causes a transition into an ACC-like state. Using this new cell model, we identified BUB1 as a targetable kinase directly controlled by MYB, whose pharmacological inhibition caused MYB-dependent synthetic lethality, growth arrest and apoptosis of patient-derived cells and organoids.
Subject(s)
Carcinoma, Adenoid Cystic , Humans , Carcinoma, Adenoid Cystic/genetics , Carcinoma, Adenoid Cystic/metabolism , Carcinoma, Adenoid Cystic/pathology , M Phase Cell Cycle Checkpoints , Transcription Factors/genetics , Salivary Glands , Protein Serine-Threonine Kinases/geneticsABSTRACT
Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.
Subject(s)
Ovarian Neoplasms/diagnosis , Precision Medicine/methods , Prognosis , Spheroids, Cellular , Female , Humans , Middle Aged , Models, Biological , Ovarian Neoplasms/mortality , Ovarian Neoplasms/pathology , Progression-Free Survival , Treatment Outcome , Tumor Cells, CulturedABSTRACT
Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvß3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvß3 was expressed on 68% of microvessels in MYCN-amplified stage 3 neuroblastomas, but only on 34% (means) in MYCN-non-amplified tumors (p < 0.001; n = 54). PTEN, a tumor suppressor involved in αvß3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvß3 was expressed on 60% of tumor microvessels when PTEN was negative or focal, as compared to 32% of microvessels in tumors with diffuse PTEN expression (p < 0.001). In a MYCN transgenic mouse model, loss of one allele of PTEN promoted tumor growth, illustrating the potential role of PTEN in neuroblastoma pathogenesis. Interestingly, we report the novel dual PI-3K/BRD4 activity of SF1126 (originally developed as an RGD-conjugated pan PI3K inhibitor). SF1126 inhibits BRD4 bromodomain binding to acetylated lysine residues with histone H3 as well as PI3K activity in the MYCN amplified neuroblastoma cell line IMR-32. Moreover, SF1126 suppressed MYCN expression and MYCN associated transcriptional activity in IMR-32 and CHLA136, resulting in overall decrease in neuroblastoma cell viability. Finally, treatment of neuroblastoma tumors with SF1126 inhibited neuroblastoma growth in vivo. These data suggest integrin αvß3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvß3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma.
ABSTRACT
Nitrogen mustards, widely used as chemotherapeutics, have limited safety and efficacy. Mitochondria lack a functional nucleotide excision repair mechanism to repair DNA adducts and are sensitive to alkylating agents. Importantly, cancer cells have higher intrinsic mitochondrial membrane potential (Δψmt) than normal cells. Therefore, selectively targeting nitrogen mustards to cancer cell mitochondria based on Δψmt could overcome those limitations. Herein, we describe the design, synthesis, and evaluation of Mito-Chlor, a triphenylphosphonium derivative of the nitrogen mustard chlorambucil. We show that Mito-Chlor localizes to cancer cell mitochondria where it acts on mtDNA to arrest cell cycle and induce cell death, resulting in a 80-fold enhancement of cell kill in a panel of breast and pancreatic cancer cell lines that are insensitive to the parent drug. Significantly, Mito-Chlor delayed tumor progression in a mouse xenograft model of human pancreatic cancer. This is a first example of repurposing chlorambucil, a drug not used in breast and pancreatic cancer treatment, as a novel drug candidate for these diseases.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Chlorambucil/pharmacology , Mitochondria/drug effects , Pancreatic Neoplasms/pathology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Biological Transport , Cell Proliferation/drug effects , Chlorambucil/chemical synthesis , Chlorambucil/chemistry , Chlorambucil/metabolism , DNA Breaks, Double-Stranded/drug effects , DNA, Mitochondrial/genetics , Drug Resistance, Neoplasm/drug effects , Histones/metabolism , Humans , Mice , Mitochondria/genetics , Mitochondria/metabolism , Phosphorylation/drug effects , Reactive Oxygen Species/metabolism , S Phase Cell Cycle Checkpoints/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum, and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions.
Subject(s)
Antineoplastic Agents/therapeutic use , Cyclotides/therapeutic use , Signal Transduction/drug effects , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Cell Cycle Proteins , Cell Line, Tumor , Cyclotides/chemistry , Cyclotides/genetics , Female , Humans , Mice, Nude , Models, Molecular , Molecular Sequence Data , Neoplasms/drug therapy , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Protein Engineering , Protein Interaction Maps/drug effects , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/chemistryABSTRACT
BACKGROUND: Pancreatic cancer is one of the deadliest cancers with a 5-year survival rate of 6%. Therapeutic options are very limited and there is an unmet medical need for safe and efficacious treatments. Cancer cell metabolism and mitochondria provide unexplored targets for this disease. We recently identified a novel class of triphenylphosphonium salts, TP compounds, with broad- spectrum anticancer properties. We examined the ability of our prototypical compound TP421- chosen for its fluorescent properties - to inhibit the growth of pancreatic cancer cells and further investigated the molecular mechanisms by which it exerts its anticancer effects. METHODOLOGY/PRINCIPAL FINDINGS: TP421 exhibited sub-micromolar IC(50) values in all the pancreatic cancer cell lines tested using MTT and colony formation assays. TP421 localized predominantly to mitochondria and induced G(0)/G(1) arrest, ROS accumulation, and activation of several stress-regulated kinases. Caspase and PARP-1 cleavage were observed indicating an apoptotic response while LC3B-II and p62 were accumulated indicating inhibition of autophagy. Furthermore, TP421 induced de-phosphorylation of key signaling molecules involved in FAK mediated adhesion that correlated with inhibition of cell migration. CONCLUSIONS/SIGNIFICANCE: TP421 is a representative compound of a new promising class of mitochondrial-targeted agents useful for pancreatic cancer treatment. Because of their unique mechanism of action and efficacy further development is warranted.
Subject(s)
Apoptosis/drug effects , Coumarins/administration & dosage , Drug Discovery , Mitochondria , Organophosphorus Compounds/administration & dosage , Pancreatic Neoplasms/drug therapy , Autophagy/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Fluorescence , Humans , Inhibitory Concentration 50 , Mitochondria/drug effects , Mitochondria/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Phosphorylation , Signal Transduction/drug effectsABSTRACT
Integrins are heterodimeric, transmembrane receptors that function as mechanosensors, adhesion molecules and signal transduction platforms in a multitude of biological processes. As such, integrins are central to the etiology and pathology of many disease states. Therefore, pharmacological inhibition of integrins is of great interest for the treatment and prevention of disease. In the last two decades several integrin-targeted drugs have made their way into clinical use, many others are in clinical trials and still more are showing promise as they advance through preclinical development. Herein, this review examines and evaluates the various drugs and compounds targeting integrins and the disease states in which they are implicated.
ABSTRACT
Herein, we discovered a series of propynoic acid carbamoyl methyl-amides (PACMAs) with potent cytotoxicity against a panel of cancer cell lines. These compounds interrupted cell cycle progression at low micromolar concentrations and induced early and late stage apoptosis. A representative compound suppressed tumor growth without apparent toxicity in an MDA-MB-435 mouse xenograft model. We used a Kinexus 628-antibody microarray and the Ingenuity Pathway Analysis (IPA) bioinformatics tools to better understand their mechanisms. The IPA analysis revealed the initiation of Nrf2-mediated oxidative stress through modulating the expression of SOD1 and STIP1 by compound 1. The involvement of the oxidative stress pathway was further validated by measuring the levels of the PACMA-induced mitochondrial superoxide species. To our knowledge, this is the first report on the discovery and biological evaluations of PACMAs as anticancer agents. Their broad-spectrum in vitro cytotoxicity, possibly through an oxidative stress-mediated pathway, and in vivo efficacy warrant further preclinical investigations.
Subject(s)
Alkynes/pharmacology , Amides/chemistry , Antineoplastic Agents/pharmacology , Drug Discovery , Propionates/pharmacology , Alkynes/chemistry , Alkynes/pharmacokinetics , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Apoptosis/drug effects , Caspase 9/drug effects , Cell Division/drug effects , Cell Line, Tumor , Drug Evaluation, Preclinical , Humans , Magnetic Resonance Spectroscopy , Mice , Oxidative Stress , Propionates/chemistry , Propionates/pharmacokinetics , Tumor Suppressor Protein p53/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Increasing knowledge of the relationship between p53 and MDM2 has led to development of potential small molecule inhibitors useful for clinical studies. Herein, we discuss the patented (2006-2010) inhibitors of p53-MDM2 interaction. The anticancer agents discussed in this review belong to several different chemical classes including benzodiazepinediones, cis-imidazolines, oxindoles, spiro-oxindoles, and numerous miscellaneous groups. This review also provides comprehensive information on inhibitors of p53-MDM2 interaction that are currently being tested in clinical trials. It is important to note that many of the disclosed inhibitors need further validation to be considered as bona fide inhibitors of p53-MDM2 interaction and some will not be further considered for future studies. On the other hand, JNJ-26854165, a novel tryptamine derivative and RG7112, a cis-imidazoline representative have shown promising results in early phases of trials in cancer patients. AT-219, a spiroindolinone in late stage preclinical studies is a likely candidate to proceed into clinical trials. It remains to be seen how these inhibitors will perform in future clinical studies as single agents and in combination with the currently approved chemotherapeutic agents.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Design , Genes, p53 , Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Antineoplastic Agents/chemistry , Clinical Trials as Topic , Humans , Neoplasms/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/genetics , Small Molecule Libraries , Tumor Suppressor Protein p53/agonists , Tumor Suppressor Protein p53/geneticsABSTRACT
BACKGROUND: Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers. METHODOLOGY/PRINCIPAL FINDINGS: A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 as having IC50 concentrations in the submicromolar range. TP treatment induced cell cycle arrest independent of p53 status, as determined by analysis of DNA content in propidium iodide stained cells. In a mouse model of human breast cancer, TP-treated mice showed significantly decreased tumor growth compared to vehicle or paclitaxel treated mice. No toxicities or organ damage were observed following TP treatment. Immunohistochemical staining of tissue sections from TP187-treated tumors demonstrated a decrease in cellular proliferation and increased caspase-3 cleavage. The fluorescent properties of analog TP421 were exploited to assess subcellular uptake of TP compounds, demonstrating mitochondrial localization. Following mitochondrial uptake cells exhibited decreased oxygen consumption and concomittant increase in mitochondrial superoxide production. Proteomics analysis of results from a 600 target antibody microarray demonstrated that TP compounds significantly affected signaling pathways relevant to growth and proliferation. CONCLUSIONS/SIGNIFICANCE: Through our continued interest in designing compounds targeting cancer-cell metabolism, the Warburg effect, and mitochondria we recently discovered a series of novel, small-molecule compounds containing a triphenylphosphine moiety that show remarkable activity in a panel of cancer cell lines as well as in a mouse model of human breast cancer. The mechanism of action includes mitochondrial localization causing decreased oxygen consumption, increased superoxide production and attenuated growth factor signaling.
Subject(s)
Antineoplastic Agents/pharmacology , Organophosphorus Compounds/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Caspase 3/metabolism , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Drug Screening Assays, Antitumor , Female , Humans , Immunohistochemistry , Mice , Mitochondria/metabolism , Organophosphorus Compounds/pharmacokinetics , Proteomics , Superoxides/metabolism , Transplantation, HeterologousABSTRACT
Inhibition of integrins alphavbeta3 and alphavbeta5 in human brain microvascular endothelial cells (HBMECs) by the function-blocking peptide RGDfV induces loss of spreading on vitronectin, cell detachment, and apoptosis. We demonstrate that cell detachment is not required for apoptosis because plating on bovine serum albumin-blocked poly-L-lysine (allows attachment, but not integrin ligation and cell spreading) also induced apoptosis. Latrunculin B (LatB), which inhibits F-actin polymerization, induced transient loss of HBMEC spreading on vitronectin, but not their detachment, and induced apoptosis despite recovery of cell spreading. However, LatB did not cause apoptosis in 5 tumor cell lines. In HBMECs, both LatB and RGDfV induced transient Y412 and Y245 phosphorylation of endogenous c-Abl, a nonreceptor tyrosine kinase that reciprocally regulates F-actin. LatB also induced nuclear translocation of c-Abl in HBMECs. STI-571 (imatinib), a targeted therapy for BCR-ABL1(+) leukemias and inhibitor of c-Abl, platelet-derived growth factor receptor, and c-Kit, decreased endothelial apoptosis. LatB-induced HBMEC apoptosis, and its inhibition by STI-571 also occurred in a 3-dimensional collagen model, supporting physiologic relevance. Last, siRNA to c-Abl (but not nonspecific siRNA) also inhibited RGDfV- and LatB-induced apoptosis. Thus, endogenous c-Abl mediates endothelial apoptosis induced by inhibition of integrins alphavbeta3/alphavbeta5 or by LatB-induced disruption of F-actin.
Subject(s)
Actins/antagonists & inhibitors , Apoptosis/physiology , Endothelial Cells/cytology , Integrin alphaVbeta3/antagonists & inhibitors , Proto-Oncogene Proteins c-abl/physiology , Receptors, Vitronectin/antagonists & inhibitors , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/ultrastructure , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cattle , Cell Adhesion , Cell Line, Tumor , Cell Shape/drug effects , Culture Media/pharmacology , Culture Media, Serum-Free , Endothelial Cells/drug effects , Fetal Blood , Genes, abl , Glioblastoma/pathology , Humans , Medulloblastoma/pathology , Membrane Potential, Mitochondrial , Neuroblastoma/pathology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , RNA Interference , RNA, Small Interfering/genetics , Thiazolidines/pharmacologyABSTRACT
Cancer cells are characterized by self-sufficiency in the absence of growth signals, their ability to evade apoptosis, resistance to anti-growth signals, sustained angiogenesis, uncontrolled proliferation, and invasion and metastasis. Alterations in cellular bioenergetics are an emerging hallmark of cancer. The mitochondrion is the major organelle implicated in the cellular bioenergetic and biosynthetic changes accompanying cancer. These bioenergetic modifications contribute to the invasive, metastatic and adaptive properties typical in most tumors. Moreover, mitochondrial DNA mutations complement the bioenergetic changes in cancer. Several cancer management therapies have been proposed that target tumor cell metabolism and mitochondria. Glycolytic inhibitors serve as a classical example of cancer metabolism targeting agents. Several TCA cycle and OXPHOS inhibitors are being tested for their anticancer potential. Moreover, agents targeting the PDC/PDK (pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase) interaction are being studied for reversal of Warburg effect. Targeting of the apoptotic regulatory machinery of mitochondria is another potential anticancer field in need of exploration. Additionally, oxidative phosphorylation uncouplers, potassium channel modulators, and mitochondrial redox are under investigation for their anticancer potential. To this end there is an increased demand for agents that specifically hit their target. Delocalized lipophilic cations have shown tremendous potential in delivering anticancer agents selectively to tumor cells. This review provides an overview of the potential anticancer agents that act by targeting cancer cell metabolism and mitochondria, and also brings us face to face with the emerging opportunities in cancer therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Delivery Systems/methods , Drug Discovery/methods , Mitochondria/enzymology , Neoplasms/metabolism , Apoptosis Regulatory Proteins/drug effects , Citric Acid Cycle/drug effects , Energy Metabolism/drug effects , Glycolysis/drug effects , Humans , Mitochondria/drug effects , Models, Biological , Neoplasms/drug therapy , Oxidation-Reduction/drug effectsABSTRACT
Matrix ligation of integrins alphavbeta3/alphavbeta5 is critical for endothelial survival and angiogenesis. We have previously shown that ceramide, a proapoptotic lipid second messenger, increases during endothelial anoikis (detachment-induced apoptosis). We now show that RGDfV, an integrin alphavbeta3/alphavbeta5 cyclic function-blocking peptide, increased ceramide and decreased sphingomyelin in human brain microvascular endothelial cells (HBMECs) plated on vitronectin, suggesting that sphingomyelin hydrolysis contributes to RGDfV-induced ceramide increase. Desipramine and imipramine, inhibitors of acid sphingomyelinase (ASMase), suppressed RGDfV-induced ceramide increase. Importantly, desipramine, imipramine, and a third ASMase inhibitor, SR33557, but not inhibitors of neutral sphingomyelinase, suppressed RGDfV-induced apoptosis, suggesting that ASMase was required for integrin-mediated apoptosis. Myriocin, an inhibitor of de novo ceramide synthesis, had no effect on RGDfV-induced HBMEC apoptosis. Interestingly, ASMase inhibitors also suppressed the RGDfV-induced loss of spreading on vitronectin. RGDfV induced a similar increase in ceramide and apoptosis in HBMECs on poly-l-lysine or vitronectin, although cells detached only from vitronectin, indicating that cell detachment was not required for RGDfV-induced apoptosis. Our results suggest involvement of ASMase and ceramide in endothelial apoptosis induced by inhibition of integrins alphavbeta3/alphavbeta5, and propose a novel molecular mechanism for the antiangiogenic effect of RGDfV.
Subject(s)
Apoptosis , Ceramides/metabolism , Endothelium, Vascular/cytology , Integrin alphaVbeta3/antagonists & inhibitors , Integrins/antagonists & inhibitors , Receptors, Vitronectin/antagonists & inhibitors , Angiogenesis Inhibitors/pharmacology , Brain/blood supply , Brain/cytology , Humans , Integrin alphaVbeta3/physiology , Integrins/physiology , Oligopeptides/pharmacology , Receptors, Vitronectin/physiology , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelin Phosphodiesterase/physiology , Sphingomyelins/metabolismABSTRACT
Stress stimuli can mediate apoptosis by generation of the lipid second messenger, ceramide. Herein we investigate the molecular mechanism of ceramide signaling in endothelial apoptosis induced by fenretinide (N-(4-hydroxyphenyl)retinamide (4-HPR)). 4-HPR, a synthetic derivative of retinoic acid that induces ceramide in tumor cell lines, has been shown to have antiangiogenic effects, but the molecular mechanism of these is largely unknown. We report that 4-HPR was cytotoxic to endothelial cells (50% cytotoxicity at 2.4 microm, 90% at 5.36 microm) and induced a caspase-dependent endothelial apoptosis. 4-HPR (5 microm) increased ceramide levels in endothelial cells 5.3-fold, and the increase in ceramide was required to achieve the apoptotic effect of 4-HPR. The 4-HPR-induced increase in ceramide was suppressed by inhibitors of ceramide synthesis, fumonisin B(1), myriocin, and l-cycloserine, and 4-HPR transiently activated serine palmitoyltransferase, demonstrating that 4-HPR induced de novo ceramide synthesis. Sphingomyelin levels were not altered by 4-HPR, and desipramine had no effect on ceramide level, suggesting that sphingomyelinase did not contribute to the 4-HPR-induced ceramide increase. Finally, the pancaspase inhibitor, t-butyloxycarbonyl-aspartyl[O-methyl]-fluoromethyl ketone, suppressed 4-HPR-mediated apoptosis but not ceramide accumulation, suggesting that ceramide is upstream of caspases. Our results provide the first evidence that increased ceramide biosynthesis is required for 4-HPR-induced endothelial apoptosis and present a molecular mechanism for its antiangiogenic effects.