Potential Anticancer Drug Target Research Articles

Targeting autophagy to overcome drug resistance in cancer therapy.

Autophagy, a catabolic process, is activated by conditions of stress and nutrient deprivation, which occurs to maintain metabolic homeostasis by performing catabolic lysis of excessive or unnecessary proteins, and injured or aged organelles. Autophagy is regulated by various signaling pathways. Main regulators of autophagy are the PI3K-Akt-mTOR pathway, Beclin1, Bcl-2, Ras and p53. Autophagy plays dual role in cancer, shows both tumor suppressive and oncogenic activity. It is accepted that drug resistance in cancer cells can be overcome by inhibition of autophagy. Herein, we summarize autophagy as a potential target of anticancer drugs and targeting autophagy provides a promising therapeutic strategy to circumvent resistance and enhance the effect of anticancer therapies for cancer patients.

An engineered TIMP2-based and enediyne-integrated fusion protein for targeting MMP-14 shows potent antitumor efficacy.

Recent studies have shown that MMP-14 is highly expressed in a panel of human solid tumors and poses as a potential molecular target for anticancer drugs. Currently, major strategies for targeted therapeutics have mainly focused on the use of antibody or ligand-based agents. For seeking an alternative approach, it is of interest to employ endogenous proteins as drug delivery carriers. Considering the facts that TIMP2, the tissue inhibitor of metalloproteinase 2, shows specific interaction with MMP-14 and that Lidamycin (LDM), an extremely potent cytotoxic antitumor antibiotic, consists of an apoprotein (LDP) and a highly active enediyne (AE); we designed and prepared a TIMP2-based and enediyne-integrated fusion protein LDP(AE)-TIMP2 by DNA recombination and molecular reconstitution consecutively. Furthermore, the MMP-14 binding attributes of the active fusion protein were determined and its therapeutic efficacy against human esophageal carcinoma KYSE150 xenograft and human fibrosarcoma HT1080 xenograft models in nude mice was investigated. It is suggested that TIMP2, the endogenous and MMP-14 binding protein, might serve as a guided carrier for targeted therapeutics.

ShRNA Depletion of cIAP1 Sensitizes Human Ovarian Cancer Cells to Anticancer Agent-Induced Apoptosis.

Emerging evidence suggests a potential role of cellular inhibitor of apoptosis protein 1 (cIAP1) in the development of human ovarian cancer. However, its function in the progression of ovarian cancer has not been clearly determined. Our study aimed to investigate the effect of cIAP1 gene depletion on the chemosensitivity of ovarian cancer cells. We developed a novel short hairpin RNA (shRNA) plasmid specifically targeting cIAP1. Cell proliferation, invasion, and apoptosis of the shRNA-transfected cells were evaluated using MTT, Transwell chamber, and flow cytometric assays, respectively. The concentration of MMP-9 in the supernatant was detected by ELISA. Targeted depletion of cIAP1 by shRNA significantly reduced expression levels of cIAP1 mRNA and protein, leading to inhibition of cell proliferation and invasion capability in SKOV3 cells. At the same time, cIAP1 downregulation decreased the secretion of MMP-9. shRNA depletion of cIAP1 enhanced chemosensitivity of ovarian cancer cells to Taxol and carboplatin-induced apoptosis. cIAP1 is associated with tumor progression in human ovarian cancer. Therefore, cIAP1 might be a potential target for therapeutic anticancer drugs.

Molecular aspects on adriamycin interaction with hmga1 regulatory region and its inhibitory effect on HMGA1 expression in human cervical cancer

High mobility group A1 (HMGA1), a non-histone chromosomal protein, is highly expressed in a wide range of human cancers including cervical, breast, and prostate cancers. Therefore, hmga1 gene is considered as an attractive potential target for anticancer drugs. We have chosen 27 bp DNA sequence from a regulatory region of hmga1 promoter and studied its interaction with adriamycin (ADM) and in vitro expression of HMGA1 in the presence of ADM in HeLa cell line. A variety of biophysical techniques were employed to understand the characteristics of [DNA–ADM] complex. Spectrophotometric titration data, DNA denaturation profiles, and quenching of fluorescence of ADM in the presence of DNA demonstrated a strong complexation between DNA and ADM with a high binding affinity (Ka) of 1.3 × 106 M−1 and a stoichiometry of 1:3 (drug:nucleotide). The energetics of binding obtained from isothermal titration calorimetry and differential scanning calorimetry suggest the binding to be exothermic and enthalpy (∆H, −6.7 ± 2.4 kcal M−1) and entropy (TΔS, 18.5 ± 6.4 kcal M−1) driven (20°C), which is typical of intercalative mode of binding. Further, results on decreased expression (by ~70%) of HMGA1 both at mRNA and protein levels in association with the observed cell death (by ~75%) in HeLa cell line, clearly confirm that ADM does target hmga1; however, the effect of ADM on genes other than hmga1 either directly or via hmga1-mediated pathways cannot be ruled out in the observed cytotoxicity. Therefore, hmga1 in general and particularly the regulatory region is a promising target for therapeutic strategy in combating cancer.

An Improved Nonradioactive Screening Method Identifies Genistein and Xanthohumol as Potent Inhibitors of Iodothyronine Deiodinases.

Deiodinases (DIO1, 2, and 3) are key enzymes in thyroid hormone (TH) activation and inactivation with impact on energy metabolism, development, cell differentiation, and a number of other physiological processes. The three DIO isoenzymes thus constitute sensitive rate-limiting components within the TH axis, prone to dysregulation by endocrine disruptive compounds or disease state. In animal models and cell culture experiments, they serve as readout for local TH status and disarrangement of the hormonal axis. Furthermore, some human diseases are characterized by apparent deiodinase dysregulation (e.g., the low triiodothyronine syndrome in critical illness). Consequently, these enzymes are targets of interest for the development of pharmacological compounds with modulatory activities. Until now, the portfolio of inhibitors for these enzymes is limited. In the clinics, the DIO1-specific inhibitor propylthiouracil is in use for treatment of severe hyperthyroidism. Other well-known inhibitors (e.g., iopanoic acid or aurothioglucose) are nonselective and block all three isoenzymes. Furthermore, DIO3 was shown to be a potential oncogenic gene, which is strongly expressed in some tumors and might, in consequence, protect tumor tissue form differentiation by TH. With respect to its role in tumorigenesis, specific inhibitors of DIO3 as a potential target for anticancer drugs would be highly desirable. To this end, a flexible and convenient assay for high-throughput screening is needed. We recently described a nonradioactive screening assay, utilizing the classic Sandell-Kolthoff reaction as readout for iodide release from the substrate molecules. While we used murine liver as enzyme source, the assay was limited to murine DIO1 activity testing. Here, we describe the use of recombinant proteins as enzyme sources within the assay, expanding its suitability from murine Dio1 to human DIO1, DIO2, and DIO3. As proof-of-concept, deiodination reactions catalyzed by these recombinant enzymes were monitored with various nonradioactive substrates and confirmed by liquid chromatography-tandem mass spectrometry. The contrast agent and known DIO inhibitor iopanoic acid was characterized as readily accepted substrate by DIO2 and Dio3. In a screening approach using established endocrine disrupting compounds, the natural food ingredient genistein was identified as a further DIO1-specific inhibitor, while xanthohumol turned out to potently block the activity of all three isoenzymes. A rapid nonradioactive screening method based on the Sandell-Kolthoff reaction is suitable for identification of environmental, nutritive and pharmacological compounds modulating activities of human deiodinase enzymes.

Functional insights from high resolution structures of mouse protein arginine methyltransferase 6.

PRMT6 is a protein arginine methyltransferase involved in transcriptional regulation, human immunodeficiency virus pathogenesis, DNA base excision repair, and cell cycle progression. Like other PRMTs, PRMT6 is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target. In the present study, we described six crystal structures of PRMT6 from Mus musculus, solved and refined at 1.34 Å for the highest resolution structure. The crystal structures revealed that the folding of the helix αX is required to stabilize a productive active site before methylation of the bound peptide can occur. In the absence of cofactor, metal cations can be found in the catalytic pocket at the expected position of the guanidinium moiety of the target arginine substrate. Using mass spectrometry under native conditions, we show that PRMT6 dimer binds two cofactor and a single H4 peptide molecules. Finally, we characterized a new site of in vitro automethylation of mouse PRMT6 at position 7.

Molecular interaction of a kinase inhibitor midostaurin with anticancer drug targets, S100A8 and EGFR: transcriptional profiling and molecular docking study for kidney cancer therapeutics.

The S100A8 and epidermal growth factor receptor (EGFR) proteins are proto-oncogenes that are strongly expressed in a number of cancer types. EGFR promotes cellular proliferation, differentiation, migration and survival by activating molecular pathways. Involvement of proinflammatory S100A8 in tumor cell differentiation and progression is largely unclear and not studied in kidney cancer (KC). S100A8 and EGFR are potential therapeutic biomarkers and anticancer drug targets for KC. In this study, we explored molecular mechanisms of interaction profiles of both molecules with potential anticancer drugs. We undertook transcriptional profiling in Saudi KCs using Affymetrix HuGene 1.0 ST arrays. We identified 1478 significantly expressed genes, including S100A8 and EGFR overexpression, using cut-off p value <0.05 and fold change ≥2. Additionally, we compared and confirmed our findings with expression data available at NCBI’s GEO database. A significant number of genes associated with cancer showed involvement in cell cycle progression, DNA repair, tumor morphology, tissue development, and cell survival. Atherosclerosis signaling, leukocyte extravasation signaling, notch signaling, and IL-12 signaling were the most significantly disrupted signaling pathways. The present study provides an initial transcriptional profiling of Saudi KC patients. Our analysis suggests distinct transcriptomic signatures and pathways underlying molecular mechanisms of KC progression. Molecular docking analysis revealed that the kinase inhibitor "midostaurin" has amongst the selected drug targets, the best ligand properties to S100A8 and EGFR, with the implication that its binding inhibits downstream signaling in KC. This is the first structure-based docking study for the selected protein targets and anticancer drug, and the results indicate S100A8 and EGFR as attractive anticancer targets and midostaurin with effective drug properties for therapeutic intervention in KC.

Spectroscopic, biological, and molecular modeling studies on the interactions of [Fe(III)-meloxicam] with G-quadruplex DNA and investigation of its release from bovine serum albumin (BSA) nanoparticles

The guanine-rich sequence, specifically in DNA, telomeric DNA, is a potential target of anticancer drugs. In this work, a mononuclear Fe(III) complex containing two meloxicam ligands was synthesized as a G-quadruplex stabilizer. The interaction between the Fe(III) complex and G-quadruplex with sequence of 5′-G3(T2AG3)3-3′ (HTG21) was investigated using spectroscopic methods, molecular modeling, and polymerase chain reaction (PCR) assays. The spectroscopic methods of UV–vis, fluorescence, and circular dichroism showed that the metal complex can effectively induce and stabilize G-quadruplex structure in the G-rich 21-mer sequence. Also, the binding constant between the Fe(III) complex and G-quadruplex was measured by these methods and it was found to be 4.53(±0.30) × 105 M−1). The PCR stop assay indicated that the Fe(III) complex inhibits DNA amplification. The cell viability assay showed that the complex has significant antitumor activities against Hela cells. According to the UV–vis results, the interaction of the Fe(III) complex with duplex DNA is an order of magnitude lower than G-quadruplex. Furthermore, the release of the complex incorporated in bovine serum albumin nanoparticles was also investigated in physiological conditions. The release of the complex followed a bi-phasic release pattern with high and low releasing rates at the first and second phases, respectively. Also, in order to obtain the binding mode of the Fe(III) complex with G-quadruplex, molecular modeling was performed. The molecular docking results showed that the Fe(III) complex was docked to the end-stacked of the G-quadruplex with a π–π interaction, created between the meloxicam ligand and the guanine bases of the G-quadruplex.

Intrinsic thermodynamics of trifluoromethanesulfonamide and ethoxzolamide binding to human carbonic anhydrase VII.

Human carbonic anhydrase (CA) isozyme VII is a cytosolic protein that is highly expressed in the cortex, hippocampus, and thalamus regions within mammalian brain, and expression disorders can cause epilepsy and several cases of malignant brain tumors. Therefore, CA VII is a potential antiepileptic and anticancer drug target. There are numerous sulfonamides that target CAs nonspecifically. It is important to understand the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex in order to design specific inhibitors against CA VII. Isothermal titration calorimetry and fluorescent thermal shift assay were used to characterize the intrinsic thermodynamic parameters of trifluoromethanesulfonamide and ethoxzolamide binding to CA VII. Binding experiments were carried out at various pH in different buffers in order to dissect linked protonation of the water molecule bound to the CA VII active site, deprotonation of the sulfonamide group of the inhibitor, and protonation-deprotonation of buffer. Dissection of all those contributions yielded the intrinsic thermodynamic parameters of binding, such as Gibbs free energy, binding enthalpy, entropy, and protein pKa value. Thermal shift assay was also used to determine CA VII stability at various pH.

Recent Advances in Anticancer Drugs Development: G-Quadruplex as New Drug Target

Nucleic Acids are large biomolecules and indispensable for life. They include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids can adopt distinct non-canonical, highly compact secondary structure, G-quadruplex, in the dynamic region of chromosomal DNA and RNA transcripts, predominantly in telomeric sequences, and in promoter region of numerous genes including oncogenes such as Bcl-2 [1,2], VEGF [3,4], c-myc [5]. There are 376,000 putative quadruplex sequences (PQS) in the human genome that have been identified through genome-wide surveys based on a quadruplex folding rule [6], however, all of them may not exist in vivo. Recently, the existence of DNA G-quadruplex has been visualized on chromosomes in human cells [7]. These G-quadruplexes are an active target of drug discovery. The DNA G-quadruplexes formed in the promoter region of oncogene have been shown to be potential targets for anticancer drugs [8,9] and proteins. The formation of these quadruplexes in telomeres has been shown to regulate the activity of the enzyme telomerase, which maintains the length of telomeres and is involved in ~85% of all cancers. For example, Telomestatin [10,11], S2T1-6OTD (telomestatin synthetic Derivative) [12], SYUIQ-5 [13] interact with G-quadruplex formed in telomere and myc sequences and show their inhibitory activity in cancer cell growth. This has become a progressively large field of research. The G-quadruplexes are very condensed structures and formed by the ganosine (G)-rich DNA and RNA sequences and consists of several stacked G-tetrads. Each G-tetrad has four guanine arranged in a square planar arrangement and held together by hoogsteen hydrogen bonding. Besides this, each G-quadruplex structure is further stabilized by the presence of a monovalent cation, mainly potassium, which is localized in the center between each pair of tetrads. The G-quadruplexes could affect gene activity either by upregulation or down regulation, which can be achieved by inducing or stabilizing G-quadruplex formation through a G-quadruplex interacting molecule (small molecule drugs or protein) that can stabilize the G-quadruplex structure and thus perform their desired function [8]. The validation of drug-targeted G-quadruplex DNA and the modulation of cancer genes’ expression has been intensely increased in the recent past, thus opening new avenue for cancer research. Though a lot of research has been focused on DNA G-quadruplexes, there has lately been a rapid advancement in the area of RNA G-quadruplexes, chiefly in the 5 ′-UTRs (untranslated regions) of mRNAs. RNA G-quadruplexes in the 5’-UTRs of mRNAs impact posttranscriptional regulation of gene expression, which affects disruption of normal cell behavior in human diseases, particularly cancers. The recent in-vitro study on small molecule G-quadruplex binding compound that can selectively target RNA G-quadruplexes exposed a new and striking opportunity for RNA-directed drug design [14]. However, there is still a major challenge to understand the systematic effects and selectivity in in-vivo environment. It is distinct that the RNA G-quadruplex motif embodies a structurally attractive scaffold for small molecule targeting and therefore provides a productive area for future research [15].

Inhibition of C-terminal truncated PPM1D enhances the effect of doxorubicin on cell viability in human colorectal carcinoma cell line

PPM1D is a p53-inducible Ser/Thr phosphatase. One of the main functions of PPM1D in normal cells is to act as a negative regulator of the p53 tumor suppressor by dephosphorylating p53 and several kinases. PPM1D is considered an oncoprotein owing to both its functions and the fact that gene amplification and overexpression of PPM1D are reported in several tumors. Recently, PPM1D mutations resulting in C-terminal truncated alterations were found in brainstem gliomas and colorectal cancers, and these mutations enhanced the activity of PPM1D. Therefore, C-terminal truncated PPM1D should be also considered as a potential candidate target of anticancer drugs. Here we showed that combination treatment with PPM1D-specific inhibitor SPI-001 and doxorubicin suppressed cell viability of HCT-116 cells overexpressing C-terminal truncated PPM1D through p53 activation compared with doxorubicin alone. Our results suggest that combination treatment with PPM1D inhibitor and doxorubicin may be a potential anti-cancer treatment in PPM1D-mutated cancer cells.

Notch 1 signaling pathway is the potential target of novel anticancer drugs for the treatment of human nasopharyngeal cancer

Activation of Notch signaling pathway in cancer stem cells plays a crucial role in the regulation of self–renewal and maintenance of side population cells. In the present study, we have identified cancer stem like 2.7% side population cells from nasopharyngeal carcinoma samples whose prevalence was signifi-cantly reduced to 0.3% upon verapamil treatment. The protein level of Notch1 and Hes-1 are highly up-regulated in fluorescence-activated cell sorting purified side population cells and thus leads to the elevated expression of stem cell surface proteins (Oct-4, Sox2 and Nanog), which are essential for side population cells self-renewal. In addition, these nasopharyngeal carcinoma side population cells are CD133 and CD44 positive and they possess enhanced cell proliferation rate, highly tumorgenic and invasive. Our findings suggest that Notch1 signaling is a potential target of novel anticancer drugs, which could efficiently target and eradicate the cancer stem cells. DOI: http://dx.doi.org/10.3329/bjp.v9i4.20342

Identification of prohibitin 1 as a potential prognostic biomarker in human pancreatic carcinoma using modified aqueous two-phase partition system combined with 2D-MALDI-TOF-TOF-MS/MS.

Membrane proteins are an important source of potential targets for anticancer drugs or biomarkers for early diagnosis. In this study, we used a modified aqueous two-phase partition system combined with two-dimensional (2D) matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS, 2D-MALDI-TOF-TOF-MS/MS) analysis to isolate and identify membrane proteins in PANC-1 pancreatic cancer cells. Using this method, we identified 55 proteins, of which 31 (56.4%) were membrane proteins, which, according to gene ontology annotation, are associated with various cellular processes including cell signal transduction, differentiation, and apoptosis. Immunohistochemical analysis showed that the expression level of one of the identified mitochondria membrane proteins, prohibitin 1 (PHB1), is correlated with pancreatic carcinoma differentiation; PHB1 is expressed at a higher level in normal pancreatic tissue than in well-differentiated carcinoma tissue. Further studies showed that PHB1 plays a proapoptotic role in human pancreatic cancer cells, which suggests that PHB1 has antitumorigenic properties. In conclusion, we have provided a modified method for isolating and identifying membrane proteins and demonstrated that PHB1 may be a promising biomarker for early diagnosis and therapy of pancreatic (and potentially other) cancers.

AKR1C3 Inhibition Therapy in Castration-Resistant Prostate Cancer and Breast Cancer: Lessons from Responses to SN33638.

In the current issue of Frontiers in Oncology – Cancer Endocrinology Section, Yin et al. report that the aldo-keto reductase family 1 member C3 (AKR1C3) inhibitor SN33638 was able to reduce testosterone production, PSA expression, and cell proliferation only in some castration-resistant prostate cancer (CRPC) cells, overexpressing the enzyme, but not the majority of a panel of prostate and breast cell lines investigated, and revealing a novel caveat in developing AKR1C3 inhibitors for cancer therapy. Prostate and breast cancers are the most common cancer and the second-leading cause of cancer-related deaths in males and females, respectively (1, 2). The mainstay treatment for advanced or metastatic prostate or ER-positive breast carcinoma is androgen-deprivation therapy or anti-estrogen therapy. Although initially these treatments are beneficial and reduce tumor burden, tumors ultimately become resistant to these therapies. There are few treatment options for this stage of cancers (termed CRPC and tamoxifen-resistant breast cancer). Thus, new approaches to treat or prevent these hormone-insensitive cancers or their progression are in great need. The AKR1C3, also known as 17β-hydroxysteroid dehydrogenase type 5, is capable to produce intratumorally testosterone and 17β-estradiol by reducing the androgen precursors and estrone, respectively. The local conversion of less potent hormones to more potent ones will lead to nuclear receptor activation and tumor progression. Therefore, AKR1C3 has recently been regarded as a potential anti-cancer drug target in both CRPC and ER-positive breast cancer (3, 4). The AKR1C3 inhibitor SN33638 is a lead compound newly synthesized by Dr. Jamieson’s group (5). It had low nanomolar potency against AKR1C3 and >300-fold selectivity for AKR1C3 over the other AKR1C isoforms. Equipped by this powerful compound, Yin et al. (6) treated a panel of CRPC and ER-positive breast cancer cell lines, in the presence of hormone or prostaglandin precursors, prior to evaluation of cell proliferation and levels of 11β-prostaglandin F2α (11β-PGF2α), testosterone production, PSA expression, and cell proliferation (6). They observed that AKR1C3 mRNA was upregulated in CRPC in a meta-analysis of patient samples. 11β-PGF2α and testosterone levels in the cell line panel correlated with AKR1C3 protein expression. SN33638 prevented 11β-PGF2α formation in cell lines that expressed AKR1C3, but only partially inhibited testosterone formation and subsequent cell proliferation and/or PSA expression only in high (LAPC4 AKR1C3) or moderate (22RV1) AKR1C3-expressing cell lines. This may be explained by that there is AKR1C3-independent steroid hormone production in most cells, and the data suggest that AKR1C3 is not an effective drug target in most CRPC cancer patients. For ER-positive breast cancers, their finding on AKR1C3 inhibition is even less promising. They observed AKR1C3 mRNA was downregulated in patient samples, a poor correlation between AKR1C3 protein expression and 17β-estradiol production, while SN33638 was largely ineffective at preventing estrone reduction or cell proliferation. These results call into question of the possibility of using AKR1C3 inhibitors in treating breast cancer patients. In summary, Yin et al. (6) revealed a novel finding that AKR1C3 inhibitor therapy is unlikely to be beneficial for the treatment of most CRPC and ER-positive breast cancers. Nevertheless, given the role of AKR1C3 in intratumoral steroidogenesis, we still hold enthusiastically the hope that a subpopulation of CRPC patients with high AKR1C3 might benefit from AKR1C3 inhibitor therapy, whose tumors are “addicted” from AKR1C3 to produce testosterone. The study by Yin et al. (6) provides a valuable framework for future preclinical or clinical studies aiming at verifying this hypothesis that AKR1C3 inhibition suppresses tumor formation in a selected population of AKR1C3-high CRPC patients.

Multimodal Effects of Small Molecule ROCK and LIMK Inhibitors on Mitosis, and Their Implication as Anti-Leukemia Agents

Accurate chromosome segregation is vital for cell viability. Many cancer cells show chromosome instability (CIN) due to aberrant expression of the genes involved in chromosome segregation. The induction of massive chromosome segregation errors in such cancer cells by small molecule inhibitors is an emerging strategy to kill these cells selectively. Here we screened and characterized small molecule inhibitors which cause mitotic chromosome segregation errors to target cancer cell growth. We screened about 300 chemicals with known targets, and found that Rho-associated coiled-coil kinase (ROCK) inhibitors bypassed the spindle assembly checkpoint (SAC), which delays anaphase onset until proper kinetochore-microtubule interactions are established. We investigated how ROCK inhibitors affect chromosome segregation, and found that they induced microtubule-dependent centrosome fragmentation. Knockdown of ROCK1 and ROCK2 revealed their additive roles in centrosome integrity. Pharmacological inhibition of LIMK also induced centrosome fragmentation similar to that by ROCK inhibitors. Inhibition of ROCK or LIMK hyper-stabilized mitotic spindles and impaired Aurora-A activation. These results suggested that ROCK and LIMK are directly or indirectly involved in microtubule dynamics and activation of Aurora-A. Furthermore, inhibition of ROCK or LIMK suppressed T cell leukemia growth in vitro, but not peripheral blood mononuclear cells. They induced centrosome fragmentation and apoptosis in T cell leukemia cells. These results suggested that ROCK and LIMK can be a potential target for anti-cancer drugs.

High-level copy number gains of established and potential drug target genes in gastric cancer as a lead for treatment development and selection

The overall survival rate of patients with advanced gastric cancer is poor. Therefore, there is an urgent need for new treatment options for these patients. The identification of drug target genes located on DNA regions exhibiting high-level copy number gains (CNG) may be an effective approach, as has e.g. previously been shown for HER2. The aim of the present study was to identify putative drug targets in patients with gastric cancer by applying this strategy. Genome-wide array comparative genomic hybridization (array CGH) data available from 183 primary gastric cancer samples were analyzed through Ingenuity Pathway Analysis (IPA) to assess whether any established or potential anticancer drug target genes showed high-level CNG, including focal amplifications. A total of 147 high-level gained regions were identified in the gastric cancer samples, harboring 167 genes that had previously been annotated as drug target genes. Thirty (18 %) of these genes showed high-level gains in at least 2 % of the tumors. The identified drug target genes included those for drugs known to be active in advanced (gastric) cancer, targets for targeted therapies in clinical development, as well as targets for drugs currently used for other indications but of potential interest for anticancer treatment. In addition, 12 potential drug target genes were identified, including genes involved in growth factor signaling and cell cycle regulation. The majority of gastric cancers carried one or more high-level CNGs or focal amplifications encompassing putative drug target genes. A number of the associated drugs are currently not being considered for treatment of gastric cancer. Based on these results we hypothesize that DNA copy number profiling may be a useful tool to identify new drug targets and to guide individualized treatment strategies in patients with gastric cancer.

Crystal Structures of the Human G3BP1 NTF2-Like Domain Visualize FxFG Nup Repeat Specificity

Ras GTPase Activating Protein SH3 Domain Binding Protein (G3BP) is a potential anti-cancer drug target implicated in several cellular functions. We have used protein crystallography to solve crystal structures of the human G3BP1 NTF2-like domain both alone and in complex with an FxFG Nup repeat peptide. Despite high structural similarity, the FxFG binding site is located between two alpha helices in the G3BP1 NTF2-like domain and not at the dimer interface as observed for nuclear transport factor 2. ITC studies showed specificity towards the FxFG motif but not FG and GLFG motifs. The unliganded form of the G3BP1 NTF2-like domain was solved in two crystal forms to resolutions of 1.6 and 3.3 Å in space groups P212121 and P6322 based on two different constructs, residues 1–139 and 11–139, respectively. Crystal packing of the N-terminal residues against a symmetry related molecule in the P212121 crystal form might indicate a novel ligand binding site that, however, remains to be validated. The crystal structures give insight into the nuclear transportation mechanisms of G3BP and provide a basis for future structure based drug design.

Abstract B299: The role of Necl-5 in hepatocarcinogenesis.

Abstract Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy of adults and the third leading cause of cancer mortality in the world. Hepatocarcinogenesis is a multistep process, usually caused by disturbing cell-cell adhesion and associated with a change of surface molecules. Recently, it is noticed that Necl-5 (Nectin-like 5) could regulate cell adhesion and migration, moreover, overexpressed in many cancers, such as colon cancer and lung cancer. However, the expression of Necl-5 in HCC is still unknown. Hence, we examined the chemically induced hepatocarcinogenesis using Necl-5 knock out mice and the expression of Necl-5 in human HCC. It was significantly showed that HCC was easily generated in Necl-5 knock out mice compared with wild type mice. In human cancerous tissues, the expression of Necl-5 was significantly higher in well differentiated HCC than moderately and poorly differentiated HCC. The expression of CD226, the ligand of Necl-5, was significantly lower in cancerous tissues compared with non-cancerous tissue. It was suggested that there was a negative regulation between Necl-5 and CD226. It was known that NK cells were activated by CD226 and played a role in early stage of tumorigenesis and participated in immune surveillance. Lacked of the Necl-5 gene indirectly impair immune surveillance to induce mice was easier generated cancer. Those findings suggested that Necl-5 might be a potential target of anti-cancer drugs in the future. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B299. Citation Format: Xue Chen, Yoshihiko Yano, Yoshitake Hayashi. The role of Necl-5 in hepatocarcinogenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B299.

Abstract C62: Phase I trial of ABC294640, a first-in-class sphingosine kinase-2 inhibitor.

Abstract Sphingosine kinases (SK1 and SK2) are new potential targets for anticancer drugs because SK inhibitors elevate ceramide levels thereby promoting apoptosis of tumor cells, and concomitantly reduce sphingosine 1-phosphate (S1P) levels thereby attenuating tumor cell proliferation and migration, as well as reducing host angiogenesis and inflammation. ABC294640 is a first-in-class orally-available inhibitor of SK2 that has activity both as a single-agent and in drug combinations in models of liver, kidney, pancreas, breast, ovary, colon and lung cancer, as well as leukemia and multiple myeloma. ABC294640 is currently in Phase I testing in patients with advanced solid tumors. The primary objectives are to identify the MTD, to determine the dose limiting toxicities (DLTs) and to evaluate the safety of ABC294640. Secondary objectives are to determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties of ABC294640, and to assess its antitumor activity. The study employs a standard 3+3 dose-escalation design. To date, this Phase I trial has enrolled 16 patients at the following dose levels: 250 mg po qd (6 patients), 250 mg po bid (4 patients), 500 mg po bid (4 patients) and 750 mg po bid (2 patients). In general, the drug has been well tolerated, with the most common side effects being low-grade fatigue and nausea. One pancreatic cancer patient at 250 mg po qd developed grade 4 DLT of hyperglycemia as disease progressed, which was possibly related to drug. Additionally, a patient at 750 mg po bid with a malignant neoplasm of the ovary experienced a grade 3 DLT of nausea and vomiting that was possibly drug-related. After 2 cycles of treatment (8 weeks), tumor imaging demonstrated that 6 of 10 patients had stable disease. Notably 2 patients have had prolonged stabilization of disease: 1 patient with metastatic cholangiocarcinoma (16 months), and 1 patient with metastatic/recurrent bladder cancer (11 months). The MTD has not yet been reached, and dose escalation is continuing. The t1/2 for ABC294640 was approximately 4 hours, and through at least the first 3 cohorts, PK parameters (Cmax and AUC) were proportional to the dose of ABC294640 received. Importantly, the average Cmax for patients receiving 500 mg ABC294640 (16.4 μM) is sufficient for anticancer efficacy in mouse models. This study includes the first-ever analysis of plasma S1P levels as a potential PD biomarker for activity of a sphingolipid-targeted drug, and will establish the degree of intra-and inter-patient variability needed for assessment of this PD marker in future clinical trials. Overall, this first-in-human study provides key information about the safety, toxicities, PK and PD of ABC294640 that are needed to support future Phase II and III clinical trials. (Supported by: P30 CA138313 from the National Cancer Institute to MUSC; R01 FD004102 from the Food and Drug Administration to Apogee; and SAP#4100061668 from the Pennsylvania Department of Health to Apogee.) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C62. Citation Format: Melanie B. Thomas, Carolyn D. Britten, Elizabeth Garrett-Mayer, Steve H. Chin, Tricia A. Bentz, Alan Brisendine, Terri L. Matson, Susan Cusack, Lynn W. Maines, Yan Zhuang, Charles D. Smith. Phase I trial of ABC294640, a first-in-class sphingosine kinase-2 inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C62.

The Chemokine CXCL8 in Carcinogenesis and Drug Response

Although the functions of chemokines in the regulation of immune processes have been studied in some detail, the role of these biomolecules in cancer is not fully understood. Chemokines mediate migration of immune cells and other functions related to immunity. They are also involved in oncogenesis and in tumor progression, invasion, and metastasis through mechanisms similar to their roles in immune functions. Various chemokines also promote cell proliferation and resistance to apoptosis of stressed cells. Consequently, chemokines and their receptors present potential therapeutic targets for anticancer drugs. The chemokine CXCL8, also known as interleukin-8 (IL8), is a proinflammatory molecule that has functions within the tumor microenvironment. Due to its potent angiogenic effects and the activity of the chemokine and its receptors in the promotion of invasion and metastasis, CXCL8 and its receptors are now considered as attractive targets for cancer therapy. This review relates the current understanding of the regulation, signaling, and functions of CXCL8 that contribute to tumor growth and metastasis, and of its role in drug response.