Human Colon Tumor Cell Research Articles

Abstract 701: A novel class of Ras selective inhibitors

Abstract Despite the fact that activating mutations in the various forms of Ras were identified as driving factors in oncogenesis over thirty years ago, there are still no effective therapeutics which act on this target. We recently synthesized and characterized a novel class of compounds that show striking potency and selectivity to inhibit the growth of tumor cells with mutant Ras. Through iterative structure-activity studies, we selected for and optimized Ras selectivity to achieve high potency with IC50 values in the low nanomolar range and selectivity indices of nearly 100-fold. For example, ADT-062 inhibited the growth of human HCT116 colon tumor cells that harbor mutant K-Ras with an IC50 value of 8.4 + 3.6 nM,. In contrast, human HT29 colon tumor cells that have wild type Ras displayed an IC50 value of 521 + 253 nM in response to ADT-062 treatment. When surveying a larger panel of colon tumor cell lines, we observed a direct correlation between potency and Ras activation status. Further, ADT-062 induced cell cycle arrest only in the Ras-mutant cell line. To study the mechanism by which this class of compounds selectively inhibits the growth of tumor cells with mutant K-Ras, we profiled lysates from treated HCT116 and HT29 colon tumor cells using high resolution LC-MS/MS. Over 100 ions were identified that were differentially affected by treatment with ADT-062 at its IC50 value in the respective cell line. Among the ions of interest, a component of the purine biosynthetic pathway, 5′-phosphoribosyl-N-formylglycinamide (FGAR), was found to be induced 200-fold (p = .0031) in K-Ras mutant cells compared with Ras wild type cells. Together, these findings suggest that this compound may cause a Ras selective disruption of purine metabolism and cell cycle progression which contributes to the selectivity by which this novel class of compounds inhibit the growth of tumor cells with mutant Ras. These findings may enable us to develop this compound class as a novel therapeutic which targets Ras driven tumors and tumors which may become resistant to other targeted therapeutics through secondary Ras mutations. Citation Format: Joshua C. Canzoneri, Xi Chen, Adam B. Keeton, Kevin Lee, Bernard Gary, Ethan B. Butler, William E. Grizzle, Landon Wilson, Stephen Barnes, Michael R. Boyd, Gary A. Piazza. A novel class of Ras selective inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 701. doi:10.1158/1538-7445.AM2015-701

Covering the Cover

Covering the Cover

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Maintenance of Clonogenic KIT+ Human Colon Tumor Cells Requires Secretion of Stem Cell Factor by Differentiated Tumor Cells

Colon tumors contain a fraction of undifferentiated stem cell-like cancer cells with high tumorigenic potential. Little is known about the signals that maintain these stem-like cells. We investigated whether differentiated tumor cells provide support. We established undifferentiated colonosphere cultures from human colon tumors and used them to generate stably differentiated cell lines. Antibody arrays were used to identify secreted factors. Expression of genes involved in stemness, differentiation, and the epithelial to mesenchymal transition was measured using reverse transcription quantitative polymerase chain reaction. Expression of KIT in human tumors was analyzed with gene expression arrays and by immunohistochemistry. Colonospheres were injected into the livers of CBy.Cg-Foxn1nu/J mice. After liver tumors had formed, hypoxia was induced by vascular clamping. Differentiated cells from various tumors, or medium conditioned by them, increased the clonogenic capacity of colonospheres. Stem cell factor (SCF) was secreted by differentiated tumor cells and supported the clonogenic capacity of KIT(+) colonosphere cells. Differentiated tumor cells induced the epithelial to mesenchymal transition in colonosperes; this was prevented by inhibition of KIT or SCF. SCF prevented loss of clonogenic potential under differentiation-inducing conditions. Suppression of SCF or KIT signaling greatly reduced the expression of genes that regulate stemness and the epithelial to mesenchymal transition and inhibited clonogenicity and tumor initiation. Bioinformatic and immunohistochemical analyses revealed a correlation between expression of KIT- and hypoxia-related genes in colon tumors, which was highest in relapse-prone mesenchymal-type tumors. Hypoxia induced expression of KIT in cultured cells and in human colon tumor xenografts and this contributed to the clonogenic capacity of the tumor cells. Paracrine signaling from SCF to KIT, between differentiated tumor cells and undifferentiated stem-like tumor cells, helps maintain the stem-like features of tumor cells, predominantly under conditions of hypoxia.

First enantiospecific syntheses of marine merosesquiterpenes neopetrosiquinones a and B: evaluation of biological activity.

The first enantiospecific syntheses of neopetrosiquinones A (6) and B (7), two merosesquiterpenes isolated from the deep-water sponge Neopetrosia cf. proxima, from the labdane diterpene trans-communic acid (10) have been achieved. A key step of the synthetic sequence is the simultaneous aromatization of the C ring and the benzylic oxidation on C-7 of an advanced intermediate, mediated by the oxygen-DDQ system. The in vitro antiproliferative activities of neopetrosiquinone B (7) and of the synthetic intermediates 8 and 9 against human breast (MCF-7), lung (A-549), and colon (T-84) tumor cell lines have been assayed. The most potent was compound 9 (IC50 = 4.1 μM), which was twice as active as natural compound 7 (IC50 = 8.3 μM) against A-549 cells. In addition, the treatment with these compounds resulted in an induction of apoptosis. These findings indicate that the terpene benzoquinones reported here might be potentially useful as anticancer agents.

Abstract A59: A novel bispecific FAP-DR5 antibody inducing potent and tumor-specific death receptor 5 (DR5) activation by fibroblast activation protein (FAP) dependent crosslinking

Abstract Background: Activation of the extrinsic apoptosis pathway in tumor cells through agonistic death receptor 5 (DR5) antibodies has been evaluated in the clinic with limited success so far. In this context, several reports show that DR5 activation is strongly dependent on receptor hyperclustering on the cell surface. Therefore a therapeutic principle that induces DR5 hyperclustering specifically at the tumor site may provide superior efficacy, potency and safety compared to conventional DR5 agonistic antibodies. Fibroblast activation protein (FAP) is a marker for activated fibroblasts and abundantly expressed in cancer associated fibroblasts of various epithelial tumor indications and as a tumor antigen on tumors of mesenchymal origin. Due to its relative absence from normal tissues, FAP can be used as a tumor targeting antigen. Here, we are using the broad expression of FAP in tumor stroma for crosslinking of DR5 by a bispecific antibody. Aim: In order to achieve superior tumor targeting and tumor located DR5 hyperclustering we have generated a bispecific antibody, RG7386, comprised of an agonistic DR5 binder and a FAP targeting moiety. Results: RG7386 shows potent and selective binding to FAP and DR5 and can simultaneously bind to both targets. In in vitro co-culture assays, using human DLD1 colon tumor cells and FAP expressing fibroblasts, RG7386 induces potent, FAP dependent DR5 hyperclustering and apoptosis induction in DR5 positive tumor cells (IC50: 0.05 nM). In preclinical in vivo models with co-injection of DLD-1 tumor cells and fibroblasts as well as patient-derived colorectal cancer models, RG7386 shows FAP dependent efficacy and apoptosis induction superior to conventional DR5 antibodies. Furthermore the superior induction of apoptosis could be confirmed by in vivo and ex vivo analysis of cleaved Caspase-3 with imaging, Luminex and histopathology. Conclusion: RG7386 is a promising novel therapeutic entity for the treatment of solid tumors with FAP positive tumor stroma inducing DR5 activation by FAP dependent DR5 hypercrosslinking which results in potent anti-tumor activity. Citation Format: Katharina Wartha, Barbara Weiser, Thomas Friess, Meher Majety, Valeria Runza, Frank Herting, Thomas Weber, Werner Scheuer, Suzana Vega Harring, Hadassah Sade, Huifeng Niu. A novel bispecific FAP-DR5 antibody inducing potent and tumor-specific death receptor 5 (DR5) activation by fibroblast activation protein (FAP) dependent crosslinking. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A59. doi:10.1158/1538-7445.CHTME14-A59

Abstract A16: Overexpression and ubiquitination of kinase suppressor of Ras 1 (KSR1) in human colon tumor cells

Abstract Kinase Suppressor of Ras 1 (KSR1) is required for oncogenic Ras-induced transformation in mouse embryonic fibroblasts (MEFs) and human colon tumor cells, but is dispensable for normal cellular development. It is also overexpressed in a variety of human tumors, including human colon tumor cells. Seven human colon tumor cell lines with mutated and activated Ras (GEO, CBS, FET, SW480, HCT15, DLD1, and HCT116), one with wild type Ras (CaCo2), and non-transformed human colonic epithelial cells (HCECs) were assessed for KSR1 by western blot. Compared to HCECs, all eight tumor cell lines showed a marked decrease in electrophoretic motility. Seven of eight cell lines had a clear increase in KSR1 expression regardless of Ras mutation status. The purpose of the study is to examine the mechanism by which KSR1 is up-regulated in cancer cells. Previous studies indicate that KSR proteins interact with multiple E3 ubiquitin ligase complexes including members homologous to the E6AP carboxyl terminus (HECT) domain family and the really interesting new gene (RING) family, including Cullin (Cul) 1, Cul4A, Cul4B, Cul7, damage-specific DNA binding protein 1 (DDB1), and DDB1-Cullin associating factor 1 (DCAF1), and ubiquitin protein ligase E3 component N-recognin 5 (UBR5/EDD1). Using tagged expression vectors and immunoprecipitation techniques, we investigated whether KSR1 is a substrate or a component of E3 ligase complexes. Both FLAG-tagged and endogenous KSR1 are ubiquitinated in the human colon tumor cell line, HCT-116. In HEK 293T cells, truncated KSR1 constructs were used to determine that KSR1 ubiquitination is dependent on the presence of the putative kinase domain in the C-terminal region. Furthermore, the kinase domain is sufficient for KSR1 ubiquitination by Cul4A/4B RING ligases (CRLs); however, silencing of Cul4A/4B by RNAi does not inhibit KSR1 ubiquitination. These data indicate that multiple Cullin family members may function to ubiquitinate KSR1. Additionally, KSR1 does not exclusively bind to Cullins, but also interacts with other components of the multi-subunit CRLs, including DDB1 and DCAF1. Therefore, KSR1 may not only be a direct substrate but also a member of the large complex helping to modulate activity of the CRL. Overall, these data suggest that Cul4A/4B-dependent ubiquitination of the KSR1 putative kinase may function to modulate KSR1-dependent signaling. Citation Format: Jamie L. McCall, Ken Kono, Kurt W. Fisher, Manabu Furukawa, Robert E. Lewis. Overexpression and ubiquitination of kinase suppressor of Ras 1 (KSR1) in human colon tumor cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A16. doi: 10.1158/1557-3125.RASONC14-A16

Abstract A07: Novel effectors of K-Ras-mediated and KSR1 dependent colon tumorigenesis

Abstract Purpose of the study: The purpose of the study is to identify new effectors of K-Ras mediated and KSR1 dependent colon tumorigenesis. Background: Gene targeting and RNA interference identified Kinase suppressor of Ras1 (KSR1) as an effector of oncogenic Ras-induced tumor formation in mouse embryonic fibroblast (MEF) and human colon tumor cells but dispensable for normal development. Therefore, KSR1 and its downstream signaling molecules are potential targets for therapy in colon tumorigenesis. Previous analyses identified nuclear co-activator Peroxisome proliferator-activated receptor gamma co-activator 1(PGC1) and transcription factor Estrogen Related Receptor α (ERRα) as effectors of Ras-induced and KSR1-dependent cell transformation in MEFs. To identify molecules that are essential for Ras-driven and KSR1-dependent colon tumorigenesis, we performed high-throughput screens with the human colon tumor cell line HCT116, using genome-wide siRNA and natural product libraries. The siRNA screen identified AMP-activated protein kinase subunit γ1 (AMPKγ1) as an effector of KSR1-dependent signaling. Results: Compared to non-targeting siRNA, RNAi of AMPKγ1 in HCT116 cells decreased anchorage-independent growth in polyHEMA-coated plates by 6-fold in 24 h (p<10-3) and by 11-fold in 48 hours (p<10-3). Moreover, RNAi of AMPKγ1 differentially induced apoptosis of HCT116 cells (21%) compared to immortalized normal human colon epithelial cells (HCEC) (0.7%; p<10-4). Similarly, RNAi of AMPK catalytic α subunits (AMPKα1/AMPKα2) induced the death of HCT116 cells (21.5%) compared to non-targeting control siRNA (1.3%). RNAi of K-Ras, KSR1 and AMPKγ1 decreased protein expression of PGC1β and ERRα in HCT116 cells. Protein and mRNA expression of PGC1β and ERRα were significantly up-regulated in colon cancer cell lines compared to HCEC, indicating these are important effectors of K-Ras mediated KSR1 and AMPK-driven cell transformation. Furthermore, stable knockdown of PGC1β and ERRα decreased colony formation by 7-fold (p<10-3) and 3.5-fold (p<10-3) in soft agar and delayed tumor formation in nude mice (p<10-4). In-situ hybridization in human tumor samples revealed PGC1β mRNA to be significantly up-regulated in primary tumor stage I, stage II, and stage IV metastatic tumor samples (p<0.05) compared to normal colonic epithelium. ERRα mRNA was up-regulated in primary tumor stage I, II, III and stage IV metastatic tumors (p<0.05). Assay of natural product library for molecules that phenocopy RNAi of KSR1 and AMPK inhibitor Compound C identified 4-OH-Staurosporine. 4-OH-Staurosporine is a competitive inhibitor ATP binding to recombinant AMPK (α1β1γ1) (Ki=23.5 nM). 4-OH-Staurosporine treatment decreased phosphorylation of AMPK substrates acetyl-CoA carboxylase (ACC) at Ser89 and Raptor at Ser792 and expression of PGC1β and ERRα in a panel of human colon tumor cell lines (HCT116, HCT15, SW480, CACO2, GEO). Treatment with 3.75μM 4-OH-Staurosporine also decreased viability of colon tumor cells (HCT116, 52%; HCT15, 15%; SW480, 12%, CACO2, 58%; DLD1, 16%; GEO, 15%) compared with HCEC (p<10-3). Conclusion: These results demonstrate the importance of AMPK and its downstream signaling effectors PGC1β and ERRα in maintaining colon tumor cell survival driven by oncogenic Ras and demonstrate the value of using KSR1 as a reference standard to detect genes essential to colon tumor survival. Citation Format: Binita Das, Kurt Fisher, Hyunseok Kim, Deanna J. Volle, Deandra R. Smith, Robert S. Livergood, John MacMillan, Michael White, Robert Lewis. Novel effectors of K-Ras-mediated and KSR1 dependent colon tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A07. doi: 10.1158/1557-3125.RASONC14-A07

Abstract B36: Targeting oncogenic RAS with small molecule PKC-delta inhibitors

Abstract Somatic point mutations of RAS genes at codons 12, 13, and 61 are the most common dominant oncogenic lesions in human cancer, making aberrant RAS signaling an important therapeutic target. Unfortunately, the oncogenic RAS/GAP switch is an exceedingly difficult target for rational drug discovery. We have developed a novel, alternative strategy to circumvent this limitation of targeting RAS directly. Mutated, constitutively-activated RAS is lethal to the tumor cell unless a survival pathway, mediated by a novel PKC isozyme, PKC-delta, is also active. This ”non-oncogene” dependency of RAS-mutant cells on PKC-delta activity can be exploited therapeutically. We have demonstrated the selective susceptibility of human pancreatic, lung, colon, ovarian and neuroendocrine tumor cells with mutant K- or HRAS alleles to PKC-delta inhibition. Importantly, unlike the classical PKC isozymes, PKC-delta is not required for the survival of normal cells, and its inhibition or down-regulation in normal cells and organisms has no adverse effects. Furthermore, we demonstrate that cancer stem-like cells derived from RAS-mutant tumor specimens and cell lines are equally or more susceptible to PKC-delta inhibition as the non-stem-like population, both in culture and in xenograft studies. There is currently no treatment option that targets melanoma with NRAS mutations, which is the second most frequently mutated gene in melanoma. We report here that PKC-delta inhibition is cytotoxic in melanomas with primary NRAS mutations. Novel small-molecule inhibitors of PKC-delta were designed as chimeric hybrids of two naturally-occurring PKC-delta inhibitors, staurosporine and rottlerin. The specific hypothesis we have interrogated and validated is the concept that combining two domains of two naturally-occurring PKC-delta inhibitors into a chimeric or hybrid structure retains biochemical and biological activity, and improves selectivity for the specific PKC-delta isozyme. We have devised a potentially general synthetic protocol to make these chimeric species using Molander trifluorborate coupling chemistry. Inhibition of PKC-delta, by siRNA or small molecule inhibitors, suppressed the growth of multiple melanoma cell lines carrying NRAS mutations, mediated via caspase-dependent apoptosis. Following PKC-delta inhibition, the stress-responsive JNK pathway was activated, leading to the activation of H2AX. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKC-delta inhibition mitigated the induction of caspase-dependent apoptosis. Furthermore, PKC-delta inhibition effectively induced cytotoxicity in BRAF-mutant melanoma cell lines that had evolved resistance to a BRAF inhibitor, suggesting the potential clinical application of targeting PKC-delta in patients who have relapsed following treatment with BRAF inhibitors. Collectively, these findings demonstrate that inhibition of PKC-delta by novel small molecule inhibitors represents a novel and targeted therapeutic approach to tumors bearing mutant RAS alleles. Citation Format: Asami Takashima, Zhihong Chen, Brandon English, Robert M. Williams, Douglas V. Faller. Targeting oncogenic RAS with small molecule PKC-delta inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B36. doi: 10.1158/1557-3125.RASONC14-B36

Identification of antipsychotic drug fluspirilene as a potential p53-MDM2 inhibitor: a combined computational and experimental study.

The inhibition of tumor suppressor p53 protein due to its direct interaction with oncogenic murine double minute 2 (MDM2) protein, plays a central role in almost 50% of all human tumor cells. Therefore, pharmacological inhibition of the p53-binding pocket on MDM2, leading to p53 activation, presents an important therapeutic target against these cancers expressing wild-type p53. In this context, the present study utilized an integrated virtual and experimental screening approach to screen a database of approved drugs for potential p53-MDM2 interaction inhibitors. Specifically, using an ensemble rigid-receptor docking approach with four MDM2 protein crystal structures, six drug molecules were identified as possible p53-MDM2 inhibitors. These drug molecules were then subjected to further molecular modeling investigation through flexible-receptor docking followed by Prime/MM-GBSA binding energy analysis. These studies identified fluspirilene, an approved antipsychotic drug, as a top hit with MDM2 binding mode and energy similar to that of a native MDM2 crystal ligand. The molecular dynamics simulations suggested stable binding of fluspirilene to the p53-binding pocket on MDM2 protein. The experimental testing of fluspirilene showed significant growth inhibition of human colon tumor cells in a p53-dependent manner. Fluspirilene also inhibited growth of several other human tumor cell lines in the NCI60 cell line panel. Taken together, these computational and experimental data suggest a potentially novel role of fluspirilene in inhibiting the p53-MDM2 interaction. It is noteworthy here that fluspirilene has a long history of safe human use, thus presenting immediate clinical potential as a cancer therapeutic. Furthermore, fluspirilene could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53-MDM2 inhibitors against several types of cancer. Importantly, the combined computational and experimental screening protocol presented in this study may also prove useful for screening other commercially-available compound databases for identification of novel, small molecule p53-MDM2 inhibitors.

Abstract 2322: β-catenin-dependent TCF/LEF transcriptional regulation of phosphodiesterase expression in colon cancer cells

Abstract Cyclic nucleotide phosphodiesterases (PDE) regulate cyclic nucleotides, cAMP and cGMP, blocking signaling by hydrolyzing the 3′,5′-phosphodiester bond resulting in their degradation to inactive forms. We have previously reported that certain PDE isozymes (e.g. PDE5) are elevated in several types of cancer, which can disrupt cyclic nucleotide signaling to suppress apoptosis. The inhibition of PDE5 by agents such as sulindac sulfide (SS) results in increased cGMP levels and reduced β-catenin/TCF/LEF mediated transcription leading to the selective inhibition of colon cancer cell proliferation and induction of apoptosis. Currently, little is known regarding the transcriptional regulation of PDE5 in cancer cells. We therefore analyzed the promoter regions of PDE5, as well as PDE10, for transcription factor binding sites using the Matinspector Genomatix Software. Matinspector analysis predicted that both PDE5 and PDE10 promoters contain binding sites for TCF/LEF-1, an important transcriptional factor that is regulated by Wnt/β-catenin signaling, which plays an important role in colorectal cancer. To determine if Wnt/β-catenin signaling can regulate PDE5 and PDE10 expression, human HCT116 colon tumor cells and normal colonic epithelial cell line NCM-460 were treated with the GSK-3β inhibitor, lithium chloride (LiCl) to inhibit β-catenin degradation and stimulate TCF/LEF transcriptional activity. TCF/LEF transcriptional activity was measured by mRNA levels of genes (e.g. survivin, cyclin D1 and c-myc) that are known to be regulated by Wnt/β-catenin signaling. Survivin, cyclin D1 and c-myc mRNA were confirmed to be significantly increased after LiCl treatment in comparison to control cells treated with NaCl. Translocation of β-catenin to the nucleus was also evident after treatment with LiCl. Levels of PDE5 and 10 mRNA were found to be significantly elevated after LiCl treatment in comparison to NaCl treated cells. Inhibitors such as SS, the novel derivative SS-D1 and a tadalafil (a selective PDE5 inhibitor) derivative TA-D1 demonstrated selective inhibition of colon cancer cell growth in comparison to normal colonocytes. The potential mechanism of these selective inhibitors may be through the inhibition of Wnt/β-catenin signaling and thus the inhibition of β-catenin/TCF/LEF mediated transcription. This study demonstrates a novel regulatory mechanism of PDE5 and PDE10 expression by the β-catenin-dependent TCF/LEF transcription factor in colon cancer cells and identifies a potential mechanism for novel chemopreventive agents. Defining the role of this regulatory mechanism in cancer progression may provide valuable insight into targeting PDE5 and/or PDE10 by small molecules for cancer chemoprevention. Citation Format: Alexandra M. Fajardo, Bernard D. Gary, Kevin J. Lee, Adam B. Keeton, Bing Zhu, Xi Chen, Ashraf H. Abadi, Gary A. Piazza. β-catenin-dependent TCF/LEF transcriptional regulation of phosphodiesterase expression in colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2322. doi:10.1158/1538-7445.AM2014-2322

Abstract 1773: Phosphodiesterase 10A: A novel target for selective inhibition of colon tumor cell growth and Wnt/β-catenin signaling

Abstract The cyclic nucleotide phosphodiesterase10A (PDE10) has been studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we report that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes. Similarly, PDE10 is elevated in colon tumors from human clinical specimens as well as intestinal tumors from the ApcMin/+ mouse model compared with non-involved tissue or normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 was evident by the ability of specific inhibitors and siRNA knockdown to suppress colon tumor cell growth without significantly affecting the growth of normal colonocytes. Stable knockdown of PDE10 by shRNA also inhibits colony formation and increases doubling time of colon tumor cells. Inhibition of PDE10 results in the selective activation of cGMP/PKG signaling in colon tumor cells to suppress β-catenin levels and T-cell factor (TCF) transcriptional activity. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations demonstrate that PDE10 is essential for colon tumor cell growth and suggest that inhibitors may have therapeutic potential for the treatment or prevention of colorectal cancer. Citation Format: Nan Li, Kevin Lee, Yaguang Xi, Bing Zhu, Bernary D. Gary, Verónica Ramírez-Alcántara, Evrim Gurpinar, Joshua C. Canzoneri, Alexandra Fajardo, Sara Sigler, John T. Piazza, Xi Chen, Joel Andrews, Wenyan Lu, Yonghe Li, Suzanne Russo, Larry Yet, Adam B. Keeton, William E. Grizzle, Gary A. Piazza. Phosphodiesterase 10A: A novel target for selective inhibition of colon tumor cell growth and Wnt/β-catenin signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1773. doi:10.1158/1538-7445.AM2014-1773

Synthesis and antitumor activity of tetrahydrocarbazole hybridized with dithioate derivatives

The present study reported the synthesis of tetrahydrocarbazoles hybridized with dithioate derivatives. Three series were synthesized namely alkyl dithiocarbonates (4a–d), heterocyclic dithiocarbamates (6a–g) and dialkyl dithiocarbamate (7). The synthesized compounds were tested in vitro on human breast adenocarcinoma cell line (MCF7) and the human colon tumor cell line (HCT116). Most of the synthesized compounds exploited potent antitumor activity, especially compound 6f [4-chlorophenylpiperazine derivative], which showed cytotoxic activity against MCF7 superior to doxorubicin with IC50 value of 7.24 nM/mL.

Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and β-catenin-dependent TCF transcriptional activity

The cyclic nucleotide phosphodiesterase10A (PDE10) has been mostly studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we show that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes, as well as in colon tumors from human clinical specimens and intestinal tumors from ApcMin/+ mice compared with normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 was evident by the ability of small molecule inhibitors and siRNA knockdown to suppress colon tumor cell growth with reduced sensitivity of normal colonocytes. Stable knockdown of PDE10 by shRNA also inhibits colony formation and increases doubling time of colon tumor cells. PDE10 inhibition selectively activates cGMP/PKG signaling to suppress β-catenin levels and T-cell factor (TCF) transcriptional activity in colon tumor cells. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations suggest a novel role of PDE10 in colon tumorigenesis and that inhibitors may be useful for the treatment or prevention of colorectal cancer.

Novel merosesquiterpene exerts a potent antitumor activity against breast cancer cells in vitro and in vivo

This article describes the antitumor properties of a new family of merosesquiterpenes, which were synthesized by Diels–Alder cycloaddition of the labdane diene trans-communic acid, highly abundant in Cupressus sempervirens, or its methyl ester, with the appropriate dienophile. These compounds demonstrated potent cytotoxic activity in vitro against human breast, colon, and lung tumor cells. We highlight the elevated activity (IC50: 0.35 ± 0.10 μM) and specificity (TI: 9) of compound 13 against the MCF-7 line, which corresponds to the most prevalent breast cancer cell subtype, luminal A. It was found that compound 13 exerts its anti-tumor action by inducing oxidative stress, arresting the cell cycle in stages G0–G1, and activating apoptosis, which are all associated with low cyclin D1 regulation, pRb hypophosphorylation, increased expression of p27 and p53, and poly (ADP-ribose) polymerase (PARP) fractioning. Epithelial–mesenchymal transition, a phenomenon associated with metastasis promotion and a worsened prognosis also appeared to be inhibited by compound 13. In addition, it markedly reduced tumor development in immunocompetent C57BL/6 mice with allografts of E0771 mouse breast tumor cells (luminal A subtype). According to these findings, this new family of compounds, especially compound 13, may be highly useful in the treatment of human breast cancer.

Novel non-cyclooxygenase inhibitory derivatives of naproxen for colorectal cancer chemoprevention.

A structure-based medicinal chemistry strategy was applied to design new naproxen derivatives that show growth inhibitory activity against human colon tumor cells through a cyclooxygenase (COX)-independent mechanism. In vitro testing of the synthesized compounds against the human HT-29 colon tumor cell line revealed enhanced growth inhibitory activity compared to the parent naproxen with 3a showing IC50 of 11.4 μM (two orders of magnitude more potent than naproxen). Selectivity of 3a was investigated against a panel of three tumor and one normal colon cell lines and showed up to six times less toxicity against normal colonocytes. Compound 3a was shown to induce dose-dependent apoptosis of HT116 colon tumor cells as evidenced by measuring the activity of caspases-3 and 7. None of the synthesized compounds showed activity against COX-1 or COX-2 isozymes, confirming a COX-independent mechanism of action. Compound 3k was found to have no ulcerogenic effect in rats as indicated by electron microscope scanning of the stomach after oral administration. A pharmacophore model was developed for elucidating structure-activity relationships and subsequent chemical optimization for this series of compounds as colorectal cancer chemopreventive drugs.

Prospective virtual screening for novel p53–MDM2 inhibitors using ultrafast shape recognition

The p53 protein, known as the guardian of genome, is mutated or deleted in approximately 50% of human tumors. In the rest of the cancers, p53 is expressed in its wild-type form, but its function is inhibited by direct binding with the murine double minute 2 (MDM2) protein. Therefore, inhibition of the p53-MDM2 interaction, leading to the activation of tumor suppressor p53 protein presents a fundamentally novel therapeutic strategy against several types of cancers. The present study utilized ultrafast shape recognition (USR), a virtual screening technique based on ligand-receptor 3D shape complementarity, to screen DrugBank database for novel p53-MDM2 inhibitors. Specifically, using 3D shape of one of the most potent crystal ligands of MDM2, MI-63, as the query molecule, six compounds were identified as potential p53-MDM2 inhibitors. These six USR hits were then subjected to molecular modeling investigations through flexible receptor docking followed by comparative binding energy analysis. These studies suggested a potential role of the USR-selected molecules as p53-MDM2 inhibitors. This was further supported by experimental tests showing that the treatment of human colon tumor cells with the top USR hit, telmisartan, led to a dose-dependent cell growth inhibition in a p53-dependent manner. It is noteworthy that telmisartan has a long history of safe human use as an approved anti-hypertension drug and thus may present an immediate clinical potential as a cancer therapeutic. Furthermore, it could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53-MDM2 inhibitors against variety of cancers. Importantly, the present study demonstrates that the adopted USR-based virtual screening protocol is a useful tool for hit identification in the domain of small molecule p53-MDM2 inhibitors.

Phosphodiesterase 10, a novel target in colon cancer.

503 Background: Elevation of intracellular cGMP is known to inhibit tumor proliferation and induce apoptosis, although the phosphodiesterase (PDE) isozymes that regulate cGMP levels in tumor cells have not been well studied. We report first evidence that PDE10 is elevated in colon tumors compared with normal colon and suggest that PDE10 inhibitors can be used for the treatment or prevention of colon cancer. Methods: PDE10 protein and mRNA levels were measured in human colon tumor cells (HT29, HCT116, SW480, Caco2), normal colonocytes (NCM460), human clinical samples, and ApcMin/+ mouse model. Two chemically distinct PDE10 selective inhibitors, PQ-10 and Pf-2545920, were tested against the cell lines. The NCI-60 panel of human tumor cell lines was also screened against Pf-2545920 to identify potential differences in sensitivity among histologically diverse tumor types. We also performed siRNA knockdown studies in colonocytes and tumor cell lines. To determine the effect of the PDE10 siRNA knockdown on cyclic nucleotide hydrolysis, whole cell lysates from transfected cells were assayed for PDE activity using cGMP or cAMP as substrates. Results: PDE10 levels were low in normal colonocytes (NCM460) and elevated in tumor cell lines. Similarly, PDE10 was elevated human clinical specimens and the ApcMin+/ mouse model compared with normal mucosa. PDE10 inhibitors and siRNA selectively inhibited colonic tumor growth while stable knockdown inhibited colony formation and increased doubling time. Pf-2545920 also supressed growth of all cell lines within the NCI-60 panel. In comparison with lysates from vector control cells, transfection with PDE10 siRNA reduced cGMP hydrolysis by ~35% in both HCT116 and HT29 cell lines, but did not affect cGMP hydrolysis in colonocytes; siRNA did not significantly affect cAMP degradation in all 3 cell lines. Conclusions: PDE10 plays a role in colon tumorgenesis whereby inhibitors can selectively suppress tumor cell growth. The mechanism by which PDE10 inhibition affects growth appears to involve activation of cGMP/PKG signalling. PDE10 represents a novel anticancer target for the treament and prevention of colon cancer.

6,11-Bisthia Steroidal Analogues: Synthesis, Characterization, and Evaluation of Antimicrobial-, Antituberculosis-, and Antitumor Activity

Aims: Synthesis, characterization, antimicrobial-, antituberculosis-, and antitumor activity of a few 6,11-bisthiasteroidal analogues have been carried out. Methodology: Subsequent reaction of the mixture 3 with phenylhydrazine hydrochloride/hydrazine hydrochloride under reflux in ethanol afforded the pyrazole derivatives 4/5. The isoxazole moiety in compounds 6 was built up from 3 with hydroxylamine hydrochloride. The pyrimidine derivatives 7 and 8 were prepared from the diketoester mixture 3 with urea and thiourea respectively. The benzodiazepine derivatives 9 and 10 were prepared from the condensation of 3 with o-phenylenediamine and 1,2diphenylethane-1,2-diamine, respectively. Results: Compound 10 showed higher bioactivity against Klebsiella pneumoniae and Escherichia coli than the standard, chloroamphenicol, and equipotent to clotrimazole in inhibiting the growth of Candida albicans. These compounds were screened for their cytotoxic activity against human colon tumor cell line (HCT116) and human cervical cancer cell line (HeLa). Compound 10 displayed the highest activity among the tested compounds with IC50 equal to 12 μM for HeLa cell, and 11 μM for HCT116 cell, respectively. Again, Research Article American Chemical Science Journal, 4(1): 58-73, 2014 59 compound 10 was found to be the most promising being active against M. tuberculosis (H37Rv) with MIC 7.5 μM. Conclusion: Compound 10 displayed better antimicrobial-, antituberculosis-, and antitumor activity than the other derivatives.