We have devised a chemocentric informatics methodology for drug discovery integrating independent approaches to mining biomolecular databases. As a proof of concept, we have searched for novel putative cognition enhancers. First, we generated Quantitative Structure- Activity Relationship (QSAR) models of compounds binding to 5-hydroxytryptamine-6 receptor (5HT6R), a known target for cognition enhancers, and employed these models for virtual screening to identify putative 5-HT6R actives. Second, we queried chemogenomics data from the Connectivity Map (http://www.broad.mit.edu/cmap/) with the gene expression profile signatures of Alzheimer’s disease patients to identify compounds putatively linked to the disease. Thirteen common hits were tested in 5-HT6R radioligand binding assays and ten were confirmed as actives. Four of them were known selective estrogen receptor modulators that were never reported as 5-HT6R ligands. Furthermore, nine of the confirmed actives were reported elsewhere to have memory-enhancing effects. The approaches discussed herein can be used broadly to identify novel drug-target-disease associations.
Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116). Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.
A new chemical series of antiproliferative compounds was identified via high-throughput screening on DU-145 human prostate carcinoma cell line (hit compound potency - 5.7 μM). Exploration of the two peripheral diversity vectors of the hit molecule in a hit-targeted library and testing of the resulting compounds led to SAR generalizations and identification of the 'best' pharmacophoric moieties. The latter were merged in a single compound that exhibited a 200-fold better potency than the original hit compound. Specific cancer cell cytotoxicity was confirmed for the most potent compounds.
The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid–based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)–based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent.
human peptide deformylase; high-throughput screening; fluorescence polarization; antiproliferative agents
Liver metastasis is a major cause of mortality in colorectal cancer (CRC). The current study was to investigate the ability of ulinastatin (UTI) and curcumin (CUR) to inhibit CRC liver metastases via modulating matrix metalloproteinase-9 (MMP-9) and E-cadherin expression. Human CRC HCT-116 cells were treated with compounds individually and in combination in order to understand the effect on cell migration and invasion. The HCT-116 cell line was established to stably express luciferase and green fluorescent protein (GFP) by lentiviral transduction (HCT-116-Luc-GFP). We identified an anti-metastasis effect of UTI and CUR on a CRC liver metastasis mouse model. Tumor development and therapeutic responses were dynamically tracked by bioluminescence imaging. Expression of MMP-9 and E-cadherin in metastatic tumors was detected by immunohistochemical assay. Results of wound healing and cell invasion assays suggest that treatment with UTI, CUR, and UTI plus CUR, respectively, significantly inhibit HCT-116 cell migration and invasion. Furthermore, results of CRC hepatic metastasis on a nude mouse model showed that treatment with UTI, CUR alone, and a combination notably inhibited hepatic metastases from CRC and prolonged survival of tumor-bearing mice, especially in the UTI plus CUR group. These results suggest that the combination of UTI and CUR together may offer greater inhibition against metastasis of CRC.
hepatic metastasis; bioluminescence imaging; therapy
The Mre11/Rad50/Nbs1 (MRN) complex is a regulator of cell cycle checkpoints and DNA repair. Defects in MRN can lead to defective S-phase arrest when cells are damaged. Such defects may elicit sensitivity to selected drugs providing a chemical synthetic lethal interaction that could be used to target therapy to tumors with these defects. The goal of this study was to identify these defects in the NCI60 panel of cell lines and identify compounds that might elicit selective cytotoxicity.
We screened the NCI60 panel in search of cell lines that express low levels of MRN proteins, or that fail to arrest in S-phase in response to the topisomerase I inhibitor SN38. The NCI COMPARE program was used to discover compounds that preferentially target cells with these phenotypes.
HCT116 cells were initially identified as defective in MRN and S phase arrest. Transfection with Mre11 also elevated Rad50 and Nbs1, and rescued the defective S-phase arrest. Cells of the NCI60 panel exhibited a large range of protein expression but a strong correlation existed between Mre11, Rad50 and Nbs1 consistent with complex formation determining protein stability. Mre11 mRNA correlated best with protein level suggesting it was the primary determinant of the overall level of the complex. Three other cell lines failed to arrest in response to SN38, two of which also had low MRN. However, other cell lines with low MRN still arrested suggesting low MRN does not predict an inability to arrest. Many compounds, including a family of benzothiazoles, correlated with the failure to arrest in S phase. The activity of benzothiazoles has been attributed to metabolic activation and DNA alkylation, but we note several cell lines in which sensitivity does not correlate with metabolism. We propose that the checkpoint defect imposes an additional mechanism of sensitivity on cells.
We have identified cells with possible defects in the MRN complex and S phase arrest, and a series of compounds that may preferentially target S phase-defective cells. We discuss limitations of the COMPARE program when attempting to identify compounds that selectively inhibit only a few cell lines.
Hereditary breast cancer is partly explained by germline mutations in BRCA1 and BRCA2. While patients carry heterozygous mutations, their tumors have typically lost the remaining wild-type allele. Selectively targeting BRCA-deficiency may therefore constitute an important therapeutic approach. Clinical trials applying this principle are underway, but it is unknown whether the compounds tested are optimal. It is therefore important to identify alternative compounds that specifically target BRCA-deficiency and to test new combination therapies to establish optimal treatment strategies.
We performed a high-throughput pharmaceutical screen on BRCA2-deficient mouse mammary tumor cells and isogenic controls with restored BRCA2 function. Subsequently, we validated positive hits in vitro and in vivo using mice carrying BRCA2-deficient mammary tumors.
Three alkylators – chlorambucil, melphalan and nimustine – displayed strong and specific toxicity against BRCA2-deficient cells. In vivo, these showed heterogeneous but generally strong BRCA2-deficient antitumor activity, with melphalan and nimustine outperforming cisplatin and the poly-(ADP-ribose)-polymerase (PARP) inhibitor olaparib (AZD2281) in this small study. In vitro drug combination experiments showed synergistic interactions between the alkylators and olaparib. Tumor intervention studies combining nimustine and olaparib resulted in recurrence-free survival exceeding 330 days in 3 out of 5 animals tested.
We generated and validated a platform for identification of compounds with specific activity against BRCA2-deficient cells that translates well to the preclinical setting. Our data call for the re-evaluation of alkylators – especially melphalan and nimustine – alone or in combination with PARP inhibitors for the treatment of breast cancers with a defective BRCA pathway.
KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers.
Computational approaches are becoming increasingly popular for the discovery of drug candidates against a target of interest. Proteins have historically been the primary targets of many virtual screening efforts. While in silico screens targeting proteins has proven successful, other classes of targets, in particular DNA, remain largely unexplored using virtual screening methods. With the realization of the functional importance of many non-cannonical DNA structures such as G-quadruplexes, increased efforts are underway to discover new small molecules that can bind selectively to DNA structures. Here, we describe efforts to build an integrated in silico and in vitro platform for discovering compounds that may bind to a chosen DNA target. Millions of compounds are initially screened in silico for selective binding to a particular structure and ranked to identify several hundred best hits. An important element of our strategy is the inclusion of an array of possible competing structures in the in silico screen. The best hundred or so hits are validated experimentally for binding to the actual target structure by a high-throughput 96-well thermal denaturation assay to yield the top ten candidates. Finally, these most promising candidates are thoroughly characterized for binding to their DNA target by rigorous biophysical methods, including isothermal titration calorimetry, differential scanning calorimetry, spectroscopy and competition dialysis.This platform was validated using quadruplex DNA as a target and a newly discovered quadruplex binding compound with possible anti-cancer activity was discovered. Some considerations when embarking on virtual screening and in silico experiments are also discussed.
drug discovery; in silico screening; SURFLEX-DOCK; DNA; G-quadruplex; high-throughput screening
In order to identify novel targets in pancreatic cancer cells, we utilized high-throughput RNAi (HT-RNAi) to identify genes that, when silenced, would decrease viability of pancreatic cancer cells. The HT-RNAi screen involved reverse transfecting the pancreatic cancer cell line BxPC3 with a siRNA library targeting 572 kinases. From replicate screens, approximately thirty-two kinases were designated as hits, of which twenty-two kinase targets were selected for confirmation and validation. One kinase identified as a hit from this screen was tyrosine kinase non-receptor 1 (TNK1), a kinase previously identified as having tumor suppressor-like properties in embryonic stem cells. Silencing of TNK1 with siRNA showed reduced proliferation in a panel of pancreatic cancer cell lines. Furthermore, we demonstrated that silencing of TNK1 led to increased apoptosis through a caspase-dependent pathway and that targeting TNK1 with siRNA can synergize with gemcitabine treatment. Despite previous reports that TNK1 affects Ras and NFκB signaling, we did not find similar correlations with these pathways in pancreatic cancer cells. Our results suggest that TNK1 in pancreatic cancer cells does not possess the same tumor suppressor properties seen in embryonic cells, but appears to be involved in growth and survival. The application of functional genomics using HT-RNAi screens has allowed us to identify TNK1 as a growth-associated kinase in pancreatic cancer cells.
HT-RNAi; TNK1; gemcitabine; pancreatic cancer
Loss of VHR phosphatase causes cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells. We recently reported that VHR is upregulated in several cervix cancer cell lines as well as in carcinomas of the uterine cervix. Here we report the development of multidentate small-molecule inhibitors of VHR that inhibit its enzymatic activity at nanomolar concentrations and exhibit antiproliferative effects on cervix cancer cells. Chemical library screening was used to identify hit compounds, which were further prioritized in profiling and kinetic experiments. SAR analysis was applied in the search for analogs with improved potency and selectivity, resulting in the discovery of novel inhibitors that are able to interact with both the phosphate-binding pocket and several distinct hydrophobic regions within VHR’s active site. This multidentate binding mode was confirmed by Xray crystallography. The inhibitors decreased the proliferation of cervix cancer cells, while growth of primary normal keratinocytes was not affected. These compounds may be a starting point to develop drugs for the treatment of cervical cancer.
Hepatocyte growth factor (HGF) is an important regulator of normal development and homeostasis, and dysregulated signaling through the HGF receptor, Met, contributes to tumorigenesis, tumor progression and metastasis in numerous human malignancies. The development of selective small-molecule inhibitors of oncogenic tyrosine kinases (TK) has led to well-tolerated, targeted therapies for a growing number of cancer types. To identify selective Met TK inhibitors, we used a high-throughput virtual screen of the 13.5 million compound ChemNavigator database to find compounds most likely to bind to the Met ATP binding site and to form several critical interactions with binding site residues predicted to stabilize the kinase domain in its inactive conformation. Subsequent biological screening of 70 in silico hit structures using cell-free and intact cell assays identified three active compounds with micromolar IC50 values. The predicted binding modes and target selectivity of these compounds are discussed and compared to other known Met TK inhibitors.
Cancer stem cells (CSCs) are a subpopulation generally thought to be responsible for cancer initiation and progression. Because CSCs are often rare in the total tumor cell population and differentiate rapidly when grown in culture, it has been challenging to uncover compounds that selectively target CSCs. We previously described CSC-emulating cells derived from breast cancer cell lines that maintained a stable undifferentiated state. We optimized a phenotypic assay with these cells and screened 1,280-bioactive compounds, identifying five that preferentially inhibited CSC-like cell proliferation. Using a compound-guided target identification approach, we found high topoisomerase I (Topo I) expression levels in breast CSC-like cells and primary breast CSCs. Structurally unrelated small molecules targeting Topo I preferentially inhibited CSC-like cells. These results illustrate the substantial power of this CSC phenotypic screening platform and promote Topo I as a potential molecular therapeutic target for therapies aimed at expunging CSCs.
cancer stem cell; Topoisomerase I; small molecule inhibitor; compound library screening
The KRAS oncogene is found in up to 30% of all human
2009, RNAi experiments revealed that lowering mRNA levels of a transcript
encoding the serine/threonine kinase STK33 was selectively toxic to
KRAS-dependent cancer cell lines, suggesting that small-molecule inhibitors
of STK33 might selectively target KRAS-dependent cancers. To test
this hypothesis, we initiated a high-throughput screen using compounds
in the Molecular Libraries Small Molecule Repository (MLSMR). Several
hits were identified, and one of these, a quinoxalinone derivative,
was optimized. Extensive SAR studies were performed and led to the
chemical probe ML281 that showed low nanomolar inhibition of purified
recombinant STK33 and a distinct selectivity profile as compared to
other STK33 inhibitors that were reported in the course of these studies.
Even at the highest concentration tested (10 μM), ML281 had
no effect on the viability of KRAS-dependent cancer cells. These results
are consistent with other recent reports using small-molecule STK33
inhibitors. Small molecules having different chemical structures and
kinase-selectivity profiles are needed to fully understand the role
of STK33 in KRAS-dependent cancers. In this regard, ML281 is a valuable
addition to small-molecule probes of STK33.
STK33 inhibitor; KRAS synthetic lethality; MLPCN probe
Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor kinase that plays a major role in cancer cell survival and metastasis.
We performed computer modeling of the p53 peptide containing the site of interaction with FAK, predicted the peptide structure and docked it into the three-dimensional structure of the N-terminal domain of FAK involved in the complex with p53. We screened small molecule compounds that targeted the site of the FAK-p53 interaction and identified compounds (called Roslins, or R compounds) docked in silico to this site.
By different assays in isogenic HCT116p53+/+ and HCT116 p53-/- cells we identified a small molecule compound called Roslin 2 (R2) that bound FAK, disrupted the binding of FAK and p53 and decreased cancer cell viability and clonogenicity in a p53-dependent manner. In addition, dual-luciferase assays demonstrated that the R2 compound increased p53 transcriptional activity that was inhibited by FAK using p21, Mdm-2, and Bax-promoter targets. R2 also caused increased expression of p53 targets: p21, Mdm-2 and Bax proteins. Furthermore, R2 significantly decreased tumor growth, disrupted the complex of FAK and p53, and up-regulated p21 in HCT116 p53+/+ but not in HCT116 p53-/- xenografts in vivo. In addition, R2 sensitized HCT116p53+/+ cells to doxorubicin and 5-fluorouracil.
Thus, disruption of the FAK and p53 interaction with a novel small molecule reactivated p53 in cancer cells in vitro and in vivo and can be effectively used for development of FAK-p53 targeted cancer therapy approaches.
Focal adhesion kinase; p53Cancer; Small molecule; p21; Tumor; Apoptosis
Identifying effective drug combinations that significantly improve over single agents is a challenging problem. Pairwise combinations already represent a huge screening effort. Beyond two drug combinations the task seems unfeasible.
In this work we introduce a method to uncover drug combinations with a putative effective response when presented to a heterogeneous population of malignant agents (strains), such as cancer cell lines or viruses. Using data quantifying the effect of single drugs over several individual strains, we search for minimal drug combinations that successfully target all strains. We show that the latter problem can be mapped to a minimal hitting set problem in mathematics. We illustrate this approach using data for the NCI60 panel of tumor derived cell lines, uncovering 14 anticancer drug combinations.
The drug-response graph and the associated minimal hitting set method can be used to uncover effective drug combinations in anticancer drug screens and drug development programs targeting heterogeneous populations of infectious agents such as HIV.
RNA interference (RNAi) has the potential to be more selective than small molecule inhibitors and can be used to target proteins, such as RAS, that are currently undruggable. The purpose of our study was to determine the optimal co-targeting strategy of the commonly mutated PI3K/AKT/mTOR and RAS pathways by a selective RNAi approach in colorectal cancer (CRC) cell lines possessing co-existent PIK3CA and KRAS mutations.
Human CRC cell lines HCT116 and DLD-1 were treated with a panel of siRNAs directed against the PI3K/AKT/mTOR and RAS pathways; proliferation, apoptosis, and protein expression were assessed. Combined treatment with siRNA and 5-fluorouracil (5-FU) was then evaluated.
PIK3CA and KRAS siRNAs were most effective as single treatments; combined treatments with PIK3CA + KRAS siRNA resulted in a more pronounced inhibition of CRC proliferation. Either KRAS siRNA alone or combined PIK3CA + KRAS siRNA treatments increased apoptosis in HCT116 cells but not in the DLD-1 cell line. Inhibition of 4E-BP1 phosphorylation correlated with increased apoptosis. Additionally, siRNA treatment combined with 5-FU further inhibited CRC cell proliferation.
Combined PIK3CA + KRAS siRNA treatments offer an effective therapy against CRCs with co-existing mutations in both pathways. Decreased 4E-BP1 phosphorylation correlates with increased apoptosis and may provide a biomarker indicative of treatment success. Furthermore, siRNA directed to PIK3CA and KRAS may be used to enhance the effects of current chemotherapy.
small interfering RNA (siRNA); RNA interference (RNAi); colorectal cancer; PI3K; KRAS; chemotherapy
The combination of gemcitabine plus erlotinib has shown a small but statistically significant survival advantage when compared to gemcitabine alone in patients with advanced pancreatic cancer. However, the overall survival rate with the erlotinib and gemcitabine combination is still low. In this study we sought to identify gene targets that, when inhibited, would enhance the activity of EGFR-targeted therapies in pancreatic cancer cells.
A high-throughput RNAi screen was carried out to identify candidate genes. Selected gene hits were further confirmed and mechanisms of action were further investigated using various assays.
Six gene hits from siRNA screening were confirmed to significantly sensitize BxPC-3 pancreatic cancer cells to erlotinib. One of the hits, MAPK1, was selected for further mechanistic studies. Combination treatments of erlotinib plus two MAP kinase kinase (MEK) inhibitors, RDEA119 and AZD6244, showed significant synergistic effect for both combinations (RDEA119-erlotinib and AZD6244-erlotinib) compared to the corresponding single drug treatments in pancreatic cancer cell lines with wild-type KRAS (BxPC-3 and Hs 700T) but not in cell lines with mutant KRAS (MIA PaCa-2 and PANC-1). The enhanced antitumor activity of the combination treatment was further verified in the BxPC-3 and MIA PaCa-2 mouse xenograft model. Examination of the MAPK signaling pathway by Western blotting indicated effective inhibition of the EGFR signaling by the drug combination in KRAS wildtype cells but not in KRAS mutant cells.
Overall, our results suggest that combination therapy of an EGFR and MEK inhibitors may have enhanced efficacy in patients with pancreatic cancer.
pancreatic cancer; EGFR; MEK; KRAS; combination chemotherapy
Identifying effective small molecules that specifically target the p53 pathway in cancer has been an exciting, though challenging, approach for the development of anti-cancer therapy. We recently identified Inauhzin (INZ) as a novel p53 activator, selectively and efficiently suppressing tumor growth without displaying genotoxicity and with little toxicity to normal cells. In order to reveal the structural features essential for anti-cancer activity of this small molecule, we have synthesized a panel of INZ analogs and evaluated their ability to induce cellular p53 and to inhibit cell growth in cell-based assays. This study as described here leads to the discovery of INZ analog 37 that displays much better potency than INZ in both of p53 activation and cell growth inhibition in several human cancer cell lines including H460 and HCT116+/+ cells. This INZ analog exhibited much less effect on p53-null H1299 cells and HCT116−/− cells, and importantly no toxicity on normal human p53-containing WI-38 cells. Hence, our results not only unveil key chemical features for INZ activity, but also identify the newly synthesized INZ analog 37 as a better small molecule for further development of anti-cancer therapy.
Despite recent advances in targeted therapies, patients with pancreatic adenocarcinoma continue to have poor survival highlighting the urgency to identify novel therapeutic targets. Our previous investigations have implicated chemokine receptor CXCR4 and its selective ligand CXCL12 in the pathogenesis and progression of pancreatic intraepithelial neoplasia and invasive pancreatic cancer; hence, CXCR4 is a promising target for suppression of pancreatic cancer growth. Here, we combined in silico structural modeling of CXCR4 to screen for candidate anti-CXCR4 compounds with in vitro cell line assays and identified NSC56612 from the National Cancer Institute's (NCI) Open Chemical Repository Collection as an inhibitor of activated CXCR4. Next, we identified that NSC56612 is structurally similar to the established anti-malarial drugs chloroquine and hydroxychloroquine. We evaluated these compounds in pancreatic cancer cells in vitro and observed specific antagonism of CXCR4-mediated signaling and cell proliferation. Recent in vivo therapeutic applications of chloroquine in pancreatic cancer mouse models have demonstrated decreased tumor growth and improved survival. Our results thus provide a molecular target and basis for further evaluation of chloroquine and hydroxychloroquine in pancreatic cancer. Historically safe in humans, chloroquine and hydroxychloroquine appear to be promising agents to safely and effectively target CXCR4 in patients with pancreatic cancer.
Epithelial to Mesenchymal Transition (EMT) is a key step towards metastasis. MCF7 breast cancer cells conditionally expressing the EMT master regulator SNAI1 were used to identify 8 early expressed miRNAs and their targets which may contribute to the EMT process. Potential targets of miRNAs were identified by matching lists of in silico predicted targets and of inversely expressed mRNAs. MiRNAs were ranked based on the number of predicted hits, highlighting miR-661, a miRNA with so far no reported role in EMT. MiR-661 was found required for efficient invasion of breast cancer cells by destabilizing two of its predicted mRNA targets, the cell-cell adhesion protein Nectin-1 and the lipid transferase StarD10, resulting, in turn, in the down-regulation of epithelial markers. Re-expression of Nectin-1 or StarD10 lacking the 3′-untranslated region counteracted SNAI1-induced invasion. Importantly, analysis of public transcriptomic data from a cohort of 295 well-characterized breast tumor specimen revealed that expression of StarD10 is highly associated with markers 8 of luminal subtypes while its loss negatively correlated with the EMT-related, basal-like subtype. Collectively, our non-a priori approach revealed a non-predicted link between SNAI1-triggered EMT and the down-regulation of Nectin-1 and StarD10 via the up-regulation of miR-661 which may contribute to the invasion of breast cancer cells and poor disease outcome.
Breast Neoplasms; genetics; metabolism; pathology; Cell Adhesion Molecules; genetics; metabolism; Cell Dedifferentiation; drug effects; genetics; physiology; Epithelial Cells; metabolism; physiology; Female; Gene Expression; physiology; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; drug effects; physiology; Humans; Mesenchymal Stem Cells; metabolism; physiology; MicroRNAs; genetics; metabolism; physiology; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Phosphoproteins; genetics; metabolism; RNA, Messenger; genetics; metabolism; RNA, Small Interfering; pharmacology; Transcription Factors; genetics; metabolism; physiology; Tumor Cells, Cultured; Validation Studies as Topic; EMT; miR-661; SNAI1; StarD10; Nectin-1; breast cancer cell invasion
Genetic and genomic studies highlight the substantial complexity and heterogeneity of human cancers and emphasize the general lack of therapeutics that can match this complexity. With the goal of expanding opportunities for drug discovery, we describe an approach that makes use of a phenotype-based screen combined with the use of multiple cancer cell lines. In particular, we have used the NCI-60 cancer cell line panel that includes drug sensitivity measures for over 40,000 compounds assayed on 59 independent cells lines. Targets are cancer-relevant phenotypes represented as gene expression signatures that are used to identify cells within the NCI-60 panel reflecting the signature phenotype and then connect to compounds that are selectively active against those cells. As a proof-of-concept, we show that this strategy effectively identifies compounds with selectivity to the RAS or PI3K pathways. We have then extended this strategy to identify compounds that have activity towards cells exhibiting the basal phenotype of breast cancer, a clinically-important breast cancer characterized as ER-, PR-, and Her2- that lacks viable therapeutic options. One of these compounds, Simvastatin, has previously been shown to inhibit breast cancer cell growth in vitro and importantly, has been associated with a reduction in ER-, PR- breast cancer in a clinical study. We suggest that this approach provides a novel strategy towards identification of therapeutic agents based on clinically relevant phenotypes that can augment the conventional strategies of target-based screens.
Targeted therapies have been used to combat many tumor types; however, few have effectively improved the overall survival in women with epithelial ovarian cancer, begging for a better understanding of this deadly disease and identification of essential drivers of tumorigenesis that can be targeted effectively. Therefore, we used a loss-of-function screening approach to help identify molecular vulnerabilities that may represent key points of therapeutic intervention. We employed an unbiased high-throughput lethality screen using a 24,088 siRNA library targeting over 6,000 druggable genes and studied their effects on growth and/or survival of epithelial ovarian cancer (EOC) cell lines. The top 300 “hits” affecting the viability of A1847 cells were rescreened across additional EOC cell lines and non-tumorigenic, human immortalized ovarian epithelial cell lines. Fifty-three gene candidates were found to exhibit effects in all tumorigenic cell lines tested. Extensive validation of these hits refined the list to four high quality candidates (HSPA5, NDC80, NUF2, and PTN). Mechanistic studies show that silencing of three genes leads to increased apoptosis, while HSPA5 silencing appears to alter cell growth through G1 cell cycle arrest. Furthermore, two independent gene expression studies show that NDC80, NUF2 and PTN were significantly aberrantly overexpressed in serous adenocarcinomas. Overall, our functional genomics results integrated with the genomics data provide an important unbiased avenue towards the identification of prospective therapeutic targets for drug discovery, which is an urgent and unmet clinical need for ovarian cancer.
Isoform selective inhibitors of the sirtuins (NAD+-dependent histone deacetylases) should enable an in depth study of the molecular biology underpinning these targets and how they are deregulated in diseases such as cancer and neurodegeneration. Herein, we present the discovery of structurally novel SIRT2 inhibitors. Hit molecule 8 was discovered through the chemical synthesis and biological characterization of a small-molecule compound library based around the 10,11-dihydro-5H-dibenz[b,f]azepine scaffold. In vitro screening assays revealed compound 8 to have an IC50 of 18 μM against SIRT2 and to exhibit more than 30-fold selectivity compared to SIRT1. Cellular assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed hit 8 to have antiproliferative activity at a concentration of 30 μM. Computational studies were performed to predict the SIRT2 binding mode and to rationalise the observed selectivity.
AIM: To investigate the expression pattern of γ-synuclein in colorectal cancer (CRC) tissues, and to study the effects of γ-synuclein on CRC cell line HCT116 biological features in vitro.
METHODS: The expression pattern of γ-synuclein was determined in 54 CRC tissues and 30 tumor-matched nonneoplastic adjacent tissues (NNAT) 5 cm away from the tumor via real-time quantitative reverse transcription PCR (RT-PCR) and immunohistochemistry. The relationship between γ-synuclein protein expression and clinicopathological factors of CRC tissues was analyzed. Three small interfering RNA (siRNA) targeting γ-synuclein mRNA plasmids were constructed and transfected into the CRC cell line HCT116. The stable cell lines were selected with G-418 for 28 d, and the biological features of these cells were examined by cell growth curve, soft agar assay, and cell migration and invasion assays in vitro.
RESULTS: The expression of γ-synuclein mRNA and protein was much higher in CRC tissue samples than in NNAT samples (P = 0.02, P = 0.036). There was a significant correlation between the γ-synuclein protein expression and clinical stage and lymph node involvement of CRC (P = 0.02, P = 0.033). In functional analysis we found that down-regulation of γ-synuclein expression in HCT116 cells could inhibit the growth, colony formation rate, and migration and invasion ability of HCT116 cells.
CONCLUSION: Increased expression of γ-synuclein in CRC tissues and the biological effects of reduced γ-synuclein expression on HCT116 cells suggest that γ-synuclein may play a positive role in the progression of CRC.
γ-synuclein; Colorectal cancer; Expression; Cell proliferation; Colony formation; Migration; Invasion