Inhibition Of Clonogenicity Research Articles

ET-56 * SENSITIZATION OF GLIOBLASTOMA CELLS TO IRRADIATION BY MODULATING THE GLUCOSE METABOLISM

Since adjuvant radiotherapy significantly reduces the mortality and increases median survival in patients with glioblastoma (GBM), the radioresistance of GBM cells and targets to modify their radiation tolerance are of significant interest. High glycolytic states of tumor cells are known to correlate strongly with radioresistance, thus the concept of metabolic targeting needs to be investigated in combination with radiotherapy. Dichloroacetate (DCA), currently being used to treat lactic acidosis, can modify tumor metabolism by activating mitochondrial activity to force glycolytic tumor cells into oxidative phosphorylation. DCA alone demonstrated modest anti-tumor effects on both in vitro and in vivo models of GBM. However, we herein describe a better efficacy when DCA is combined with radiotherapy. An enhanced inhibition of clonogenicity of a panel of GBM cells was observed when DCA was combined with irradiation. Further mechanistic investigation revealed that i) DCA sensitized GBM cells to irradiation by inducing the cell cycle arrest at G2/M phase, the most radiosensitive cell cycle phase; ii) DCA reduced mitochondrial reserve capacity, a measure of the ability of cells to resist oxidative stress; iii) DCA and irradiation synergistically boosted the oxidative stress and iv) induced DNA damage in GBM cells at a higher rate compared to each therapy alone. Metabolically, we observed that the glycolysis in GBM cells was induced post-irradiation by stabilizing the hypoxia inducible factor (HIF)-1α, which in turn activated its direct target pyruvate dehydrogenase kinase 1 (PDK1). This irradiation-induced glycolytic metabolism was blocked by the addition of DCA when the combination was administered. In conclusion, this study provides the proof of concept that DCA can effectively sensitize GBM cells to irradiation by modulating the metabolic state of tumor cells. These findings warrant further evaluation of the combination of DCA and radiotherapy in vivo using GBM xenografts.

Abstract 1600: Keeping glioblastoma (GBM)in check by targeting the CHK1-STAT3-CIP2A axis

Abstract Checkpoint kinase1 (CHK1) is a DNA damage kinase, which has been observed to be constitutively active in human cancers. Unfortunately a number of new generation CHK1 inhibitors, currently in clinical trials, have shown deleterious effects on normal cells, thus limiting their clinical utility. Therefore, identification and targeting of cancer specific effectors of CHK1 signaling has emerged as an attractive therapeutic alternative. Analysis of the REMBRANDT and TCGA datasets revealed a strong positive correlation for CHK1 and CIP2A expression in 422/522 and 356/454 glioma patients respectively. By contrast, there was negligible correlation observed in normal samples. Additionally, high mRNA expression of both CHK1 and CIP2A was associated with reduced overall survival in glioma patients and marked a more aggressive form of the disease. Notably, CIP2A amplification found in 14.72% cases in the Rembrandt study was associated with worse overall survival in GBM patients. Mechanistically, we identify STAT3 as a transcriptional mediator for CHK-dependent CIP2A expression in GBM cells. We demonstrate that CHK1 positively regulates CIP2A transcription by promoting phosphorylation of STAT3 at Tyrosine 705 both in vitro and in vivo. Further, depletion of STAT3 by siRNA or chemical inhibitors resulted in decreased CIP2A expression in GBM cells. Functionally, both CHK1 and CIP2A promoted viability, clonogenicity and anchorage-independent growth of GBM cells. Importantly, inhibition of cancer cell viability and clonogenicity by Chk1 inhibition can be partially rescued by exogenous overexpression of CIP2A. Higher CIP2A, p-S345-CHK1 and p-Ty705-STAT3 levels were observed in de novo and patient-derived GBM, and U251MG cells relative to levels in normal human astrocytes (NHA). Analogously, CHK1 and CIP2A mRNA expression is increased 10 to 15 fold in genetically engineered mouse(GEM) models of human GBM (Pdgf-Cre-driven Ptenf/f and Pdgf-Cre-driven Ptenf/f;/p53f/f double mutant mice compared with wild-type mice). Finally, using xenograft mouse models we show that both PF477736 (a small molecule inhibitor of CHK1) and CIP2A depletion inhibits tumor growth. These results highlight CHK1 and CIP2A expression as potential diagnostic and prognostic markers in human GBMs. Further, these findings identify CIP2A as a cancer specific therapeutic target downstream of constitutively active CHK1 and STAT3 signaling in GBM. Together, these data serves as a platform for using CIP2A expression in GBM, to either directly target CIP2A or to stratify patients for CHK1 and/or STAT3 inhibition. Note: This abstract was not presented at the meeting. Citation Format: Anchit Khanna, Brett Stringer, Bryan Day, Kathleen Ensbey, Han Shen, Andrew Boyd, Kerrie McDonald, John E. Pimanda. Keeping glioblastoma (GBM)in check by targeting the CHK1-STAT3-CIP2A axis. [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 1600. doi:10.1158/1538-7445.AM2014-1600

Down-regulation of 5-HT1B and 5-HT1D receptors inhibits proliferation, clonogenicity and invasion of human pancreatic cancer cells.

Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa) patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT) contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA), induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT), concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D- mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification, and valuable new therapeutic targets for managing pancreatic cancer.

Abstract 3780: On the role of experimental microenvironmental conditions in targeted inhibition of the pH-regulating carbonic anhydrase IX in colorectal carcinoma cells

Abstract Tumor hypoxia, causing metabolic changes towards enhanced glycolysis and lactic acid production, is a common microenvironmental feature of solid tumors, and is recognized as a main mechanism both of therapeutic resistance and metastasis. The hypoxia-inducible factor-1α conducts transcription of a number of genes involved in adaption to the hypoxic condition. Among these is the tumor-specific carbonic anhydrase IX (CAIX), a membrane-bound protein that regulates extracellular (pHe) and intracellular (pHi) pH by catalyzing the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and protons, enabling tumor cell survival in the acidic microenvironment. To improve therapeutic outcome and reduce metastatic risk, targeted inhibition of CAIX is an attractive strategy. Here we report on effects of a CAIX inhibitor in a panel of colorectal carcinoma (CRC) cell lines, giving specific emphasis to the microenvironmental conditions. Five CRC cell lines were incubated for 24 hours under normoxic (21% O2) or hypoxic (0.2% O2) condition, either in the presence (22 mM HCO3-, 5% CO2) or absence (0 mM HCO3-, 0% CO2) of HCO3- in the culture medium. The cells were treated with 4-(3′-(3”,5”-dimethylphenyl)ureido)phenyl sulfamate (termed S4), considered to be a CAIX-specific inhibitor, and clonogenicity, pHe, and pHi (measured as cells labeled with a pH-sensitive dye) were assessed. In CO2/HCO3--buffered medium under normoxia, differential inhibition of clonogenicity following S4 treatment was observed among the five cell lines, correlating with individual CAIX levels: strong suppression in cell lines with high CAIX expression, weaker S4 effect in cell lines with low expression, and no effect in the CAIX-negative cell line. When incubating with this medium under hypoxia, despite significant induction of CAIX, no additional effect of S4 on clonogenicity was observed. Of note, when changing the cell culture medium to CO2/HCO3--free condition, S4 strongly inhibited clonogenicity of hypoxic HT29 cells (high CAIX expression) but not of the normoxic counterpart, while HCT116 cells (low CAIX expression) showed no clonogenic response to S4 with either oxygenation status. In all of the five cell lines, hypoxia caused significant acidification (decrease in pHe) of culture media. In parallel with the clonogenic response, S4 treatment significantly counteracted the hypoxia-induced pHe decline in HT29 cultures incubated in the CO2/HCO3--free condition. pHi was not altered with any of the tested conditions. This set of data demonstrated that the CO2/HCO3- buffering system determined CRC cell response to targeted CAIX inhibition. To enable reliable assessment of experimental S4 effects, strict control of the microenvironmental conditions was required. We believe this information may be of note for future evaluation of this specific therapeutic approach. Citation Format: Helga Helseth Hektoen, Kjersti Flatmark, Kirsti Solberg Landsverk, Marie Gron Saelen, Kathrine Roe, Anne Hansen Ree. On the role of experimental microenvironmental conditions in targeted inhibition of the pH-regulating carbonic anhydrase IX in colorectal carcinoma 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 3780. doi:10.1158/1538-7445.AM2014-3780

Selective release of a cyclopamine glucuronide prodrug toward stem-like cancer cell inhibition in glioblastoma.

Recent data suggest that inhibition of the Hedgehog pathway could be a therapeutic target for glioblastoma. Alkaloid cyclopamine inhibits Hedgehog signaling, depleting stem-like cancer cells derived from glioblastoma. However, this compound is toxic for somatic stem cells, preventing its use for clinical applications. In this study, we tested a derivatization product of cyclopamine in the form of cyclopamine glucuronide prodrug (CGP-2). This compound was used in vitro and in vivo toward glioblastoma-initiating cells (GIC). Results obtained in vitro indicate that CGP-2 is active only in the presence of β-glucuronidase, an enzyme detected in high levels in necrotic areas of glioblastomas. CGP-2 decreased proliferation and inhibited the self-renewal of all GIC lines tested. Hedgehog pathway blockade by 10 μmol/L of CGP-2 induced a 99% inhibition of clonogenicity on GICs, similar to cyclopamine treatment. Combination of CGP-2 with radiation decreased clonogenic survival in all GIC lines compared with CGP-2 alone. In a subcutaneous glioblastoma xenograft model, a two-week CGP-2 treatment prevented tumor growth with 75% inhibition at 8 weeks, and this inhibition was still significant after 14 weeks. Unlike cyclopamine, CGP-2 had no detectable toxic effects in intestinal crypts. Our study suggests that inhibition of the Hedgehog pathway with CGP-2 is more effective than conventional temozolomide adjuvant, with much lower concentrations, and seems to be an effective therapeutic strategy for targeting GICs.

Down-regulation of 5-HT1B and 5-HT1D receptors inhibits proliferation, clonogenicity and invasion of human pancreatic cancer cells.

Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa) patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT) contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA), induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT), concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B– and 5-HT1D–mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification, and valuable new therapeutic targets for managing pancreatic cancer.

A novel hydroxymethoxynaphthochalcone induces apoptosis through the p53-dependent caspase-mediated pathway in HCT116 human colon cancer cells

Flavonoids have always been studied in the context of therapies of human diseases. Among them, chalcone, an openchain flavonoid, has been used as a key precursor for synthetic lead compounds due to its diverse innate biological activity. Additionally, benzoflavone is known to induce xenobiotic-metabolizing enzyme activity, as well as have potent chemopreventive activity. Therefore, the combined structure of these two compounds should be useful for the discovery of new and/or increased biological activity. In this study, a chalcone derivative, 2′-hydroxy-2,4,6-trimethoxy-5′,6′-naphthochalcone (HMNC-74), was synthesized, and its anticancer activity was tested in the HCT116 human colon cancer cell line. An in silico docking study showed that HMNC-74 binds to tubulin. HMNC-74 exhibited the inhibition of clonogenicity of HCT116 cells and cell cycle arrest at the G2/M phase, followed by induction of apoptosis through, at least in part, p53-dependent caspase-7 activation. The results of this study show that HMNC-74 may be an effective chemotherapeutic agent.

Therapeutic effects of lentivirus-mediated shRNA targeting of cyclin D1 in human gastric cancer

BackgroundGastric cancer is the second most common cause of cancer-related death in males and the fourth in females. Traditional treatment has poor prognosis because of recurrence and systemic side effects. Therefore, the development of new therapeutic strategies is an important issue. Lentivirus-mediated shRNA stably inhibits target genes and can efficiently transduce most cells. Since overexpressed cyclin D1 is closely related to human gastric cancer progression, inhibition of cyclin D1 using specific targeting could be an effective treatment method of human gastric cancer.MethodsThe therapeutic effect of lentivirus-mediated shRNA targeting of cyclin D1 (ShCCND1) was analyzed both in vitro and in vivo experiments.ResultsIn vitro, NCI-N87 cells with downregulation of cyclin D1 by ShCCND1 showed significant inhibition of cell proliferation, cell motility, and clonogenicity. Downregulation of cyclin D1 in NCI-N87 cells also resulted in significantly increased G1 arrest and apoptosis. In vivo, stable NCI-N87 cells expressing ShCCND1 were engrafted into nude mice. Then, the cancer-growth inhibition effect of lentivirus was confirmed. To assess lentivirus including ShCCND1 as a therapeutic agent, intratumoral injection was conducted. Tumor growth of the lentivirus-treated group was significantly inhibited compared to growth of the control group. These results are in accordance with the in vitro data and lend support to the mitotic figure count and apoptosis analysis of the tumor mass.ConclusionThe lentivirus-mediated ShCCND1 was constructed, which effectively inhibited growth of NCI-N87-derived cancer both in vitro and in vivo. The efficiency of shRNA knockdown and variation in the degree of inhibition is mediated by different shRNA sequences and cancer cell lines. These experimental results suggest the possibility of developing new gastric cancer therapies using lentivirus-mediated shRNA.

ERK2-dependent reactivation of Akt mediates the limited response of tumor cells with constitutive K-RAS activity to PI3K inhibition

K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells are resistant to EGFR targeting strategies. We investigated the impact of K-RAS activity irrespective of mutational status in the EGFR-independent increase in clonogenic cell survival. An analysis of the K-RAS activity status revealed a constitutively high K-RAS activity in K-RASmut NSCLC cells and also in head and neck squamous cell carcinoma (HNSCC) cells overexpressing wild-type K-RAS (K-RASwt). Similar to K-RAS-mutated cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Expression of mutated K-RAS stimulated Akt phosphorylation and increased plating efficiency. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in EGFR-dependent and EGFR-independent Akt activity. The short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in the repression of Akt activation, whereas long-term treatment (24 h) with inhibitors led to the reactivation of Akt and improved clonogenicity. The Akt re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, clonogenicity inhibition in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by targeting of EGFR downstream pathways, i.e., PI3K alone or the combination of PI3K and MAPK inhibitors.

Chk1 Targeting Reactivates PP2A Tumor Suppressor Activity in Cancer Cells

Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1-targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase protein phosphatase 2A (PP2A), which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of cancerous inhibitor of PP2A (CIP2A), a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma, where CIP2A was identified as an NMYC-independent prognostic factor. Because the Chk1-CIP2A-PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understanding how Chk1 inhibitors mediate single-agent anticancer efficacy. Furthermore, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy.

Efficacy of Chlorin e6-Mediated Sono-Photodynamic Therapy on 4T1 Cells

The present study aims to investigate the antitumor effect and possible mechanisms of chlorin e6 (Ce6)-mediated sono-photodynamic therapy (Ce6-SPDT) on murine 4T1 mammary cancer cells in vitro. Cellular uptake and intracellular distribution of Ce6 in 4T1 cells were detected by flow cytometry and confocal microscope. Cells after loading with 1 μg/mL Ce6 were exposed to ultrasound at 1.0 MHz for up to 1 minute with an intensity of 0.36 W/cm2 and laser light with total radiation dose of 1.2 J/cm2. Cell viability and clonogenicity were determined by MTT assay and colony formation assay. Apoptosis was analyzed by DAPI staining, Western blots were used to detect the activity of Caspase-3. DNA damage, mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) of 4T1 cells were also evaluated by flow cytometry. FD500 was employed to detect changes of membrane permeability after ultrasound. Ce6 rapidly entered 4T1 cells within 4 hours after it has been added and displayed a mitochondria-localization pattern. Compared with sonodynamic therapy (SDT) and photodynamic therapy (PDT) alone, the combined SPDT treatment further enhanced cell viability loss, DNA damage, and clonogenicity inhibition. DAPI staining and western blots analysis reflected that cells with apoptotic morphological characteristics and the activity of Caspase-3 were apparently increased in the combined group. Besides, SPDT caused obvious MMP loss and intracellular ROS generation at early 1 hour post treatment. Interestingly, the SPDT induced cell viability loss and cell apoptosis was greatly inhibited by pre-treatment with ROS scavenger N-acetylcysteine and Caspase inhibitor z-VAD-fmk. FD500 detection showed that ultrasound enhanced cell membrane permeability, implying much higher uptake of Ce6 might be involved in PDT therapy by pre-ultrasound treatment. The findings demonstrated that Ce6-mediated SPDT enhanced the antitumor efficacy on 4T1 cells compared with SDT and PDT alone, a Caspase-dependent apoptosis and loss of MMP, generation of ROS may be involved.

Abstract A293: Novel allosteric small molecule FAK inhibitors effectively inhibit cancer cell viability and clonogenicity.

Abstract Introduction: Focal Adhesion Kinase is overexpressed and activated in many types of tumors and plays an important role in survival. Recently, we developed FAK inhibitor, 1,2,4,5-benzenetetraamine tetrahydrochloride, called Y15, which inhibited FAK autophosphorylation and blocked breast, neuroblastoma, pancreatic, brain and colon cancer tumor growth. Experimental procedures: In this study, eight novel chemical derivatives of Y15, called Y15-I1-I8 were synthesized and tested in a panel of different cancer cell lines. To test the effect on FAK autophosphorylation we performed in vitro ADP-Glo kinase assay with recombinant FAK protein and Western blotting with Y397-FAK antibody. To test the in vivo effect of FAK inhibitors we performed MTT assay, AnnexinV apoptosis and clonogenicity assays in different cancer cell lines. As a control, we used Novartis NVP-TAE-226 FAK inhibitor. Results: By in vitro kinase assay Y15-I1-8 derivatives effectively inhibited FAK autophosphorylation with recombinant purified full length FAK protein. Western blotting demonstrated decreased Y397-FAK and Y418-Src phosphorylation in many cancer cell lines. In addition, these novel FAK inhibitors demonstrated decreased viability, increased apoptosis and inhibition of clonogenicity in lung, melanoma, glioblastoma, pancreatic, breast, and colon cancer cell lines. Some of the novel derivatives were better than parental Y15 inhibitor. Conclusion: Targeting FAK autophosphorylation with novel allosteric derivatives of Y15 is an effective therapy approach. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A293. Citation Format: Vita M. Golubovskaya, Iryna Lebedyeva, Baotran Ho, Michael Yemma, Neha Singh, Makayla Arcara, David Ostrov, Alan Katritzky, William Cance. Novel allosteric small molecule FAK inhibitors effectively inhibit cancer cell viability and clonogenicity. [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 A293.

Focal adhesion kinase inhibition as a therapeutic strategy in differentiated thyroid cancer.

e13556 Background: Thyroid cancer, themost common endocrine malignancy, can be curable with resection of low stage tumors, but new treatments are needed for advanced differentiated cancers with radioiodine resistance. Focal Adhesion Kinase (FAK), a tyrosine kinase involved in protein-protein interactions regulating crucial cellular functions, is upregulated in thyroid cancer, and is a drug target along with its autophosphorylation site Y397. Clinical trials are ongoing for FAK inhibitors PF04554878 and GSK2256098. 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15), a small molecule FAK scaffolding inhibitor targeting the Y397 autophosphorylation site, and TAE226, a FAK/Insulin-like Growth Factor-1 Receptor inhibitor are in preclinical studies . In this report, we compared the effects of these FAK inhibitors on thyroid cancer cell lines. Methods: Four human thyroid cancer cell lines were cultured in vitro: K1, BCPAP, TPC1 derived from papillary thyroid carcinoma, and TT derived from medullary thyroid carcinoma. Western blots detected expression of FAK and pY397 FAK. Clonogenicity and MTS assays measured effects of the inhibitors on cell viability and colony formation. Results: The most significant dose-dependent decrease of FAK Y397 autophosphorylation and p125FAK expression was seen with Y15, with significant decrease at 10μM and near complete inhibition at 50μM. PF04554878, GSK2256098 and TAE226 at 50μM showed lowered pY397 expression, but less effect than Y15 at 10μM concentrations. TAE226 had the most effect on clonogenicity, with complete colony inhibition at doses 1μM and higher. Least effect was with GSK2256098. Y15 and PF04554878 had moderate inhibition of clonogenicity. Y15 had greatest inhibition of cell viability, with IC50s less than 15μM, two to over five-fold more potent than TAE226, GSK2256098, and PF04554878. Conclusions: All FAK inhibitors caused dose-dependent effects on thyroid cancer cell viability, clonogenicity and expression of pY397 FAK. Y15 had the most potent effects on cell viability and expression of FAK and p397 FAK. Our data suggests targeting the FAK scaffold, as Y15 does, is more effective than targeting the kinase function as a potential therapeutic agent for thyroid cancer.

Evaluation of antiproliferative and antioxidant activities of the organic extract and its polar fractions from the Mediterranean gorgonian Eunicella singularis

The present study was conducted to evaluate the antiproliferative and antioxidant activities of organic extract and its polar fractions from Eunicella singularis (Esper 1794). Organic extract and two fractions of E. singularis (F2 and F3) were screened for the presence of phenolic compounds, terpenoids and glycosides. The antiproliferative activity of E. singularis organic extract and its polar fractions was evaluated on human cancer cell lines (A549, lung cell carcinoma; HCT15, colon cell carcinoma and MCF7, breast adenocarcinoma), using the MTT colorimetric method and clonogenic assay, as well as the antioxidant activity, using the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the FRAP assays. The fractions F2 and F3 showed significant total phenolic content (40 and 35.72mg gallic-acid equivalent/g dried sample), respectively, and important antiproliferative properties against the cancer cell lines. The IC50 values, ranged from 36 to 274μg/ml for A549; 93 to 426μg/ml for HCT15; and 52 to 225μg/ml for MCF7 and in the clonogenic inhibition assay from 18 to 134μg/ml for A549; 43 to 357μg/ml for HCT15; and 17 to 160μg/ml for MCF7. Using the DPPH method, the fraction F2 exhibited the strongest radical scavenging activity, with IC50 0.08mg/ml, which approaches the activity of the powerful antioxidant standard, ascorbic acid (IC50=0.064mg/ml). The reducing power of the samples was in the following order: F2>organic extract>F3. These results suggest that E. singularis fractions might be used as a potential source of natural antioxidant and antitumor agents. The purification and determination of the chemical structures of compounds in these active fractions are under investigation. The results could provide a compound(s) with a promising role in future medicines.

Cell cycle inhibition therapy that targets stathmin in in vitro and in vivo models of breast cancer

Stathmin is the founding member of a family of microtubule-destabilizing proteins that have a critical role in the regulation of mitosis. Stathmin is expressed at high levels in breast cancer and its overexpression is linked to disease progression. Although there is a large body of evidence to support a role for stathmin in breast cancer progression, the validity of stathmin as a viable therapeutic target for breast cancer has not been investigated. Here, we used a bicistronic adenoviral vector that co-expresses green fluorescent protein and a ribozyme that targets stathmin messenger RNA in preclinical breast cancer models with different estrogen receptor (ER) status. We examined the effects of anti-stathmin ribozyme on the malignant phenotype of breast cancer cells in vitro and in xenograft models in vivo both as a single agent and in combination with chemotherapeutic agents. Adenovirus-mediated gene transfer of anti-stathmin ribozyme resulted in a dose-dependent inhibition of proliferation and clonogenicity associated with a G2/M arrest and increase in apoptosis in both ER-positive and ER-negative breast cancer cell lines. This inhibition was markedly enhanced when stathmin-inhibited breast cancer cells were exposed to low concentrations of taxol, which resulted in virtually complete loss of the malignant phenotype. Interestingly, breast cancer xenografts treated with low doses of anti-stathmin therapy and taxol showed regression in a majority of tumors, while some tumors stopped growing completely. In contrast, combination of anti-stathmin ribozyme and adriamycin resulted in only a modest inhibition of growth in vitro and in breast cancer xenografts in vivo. Although inhibition of tumor growth was observed in both the combination treatment groups compared with groups treated with single agent alone, combination of anti-stathmin therapy and taxol had a more profound inhibition of tumorigenicity, as both agents target the microtubule pathway. Clinically, these findings are highly relevant because taxol is one of the most active chemotherapeutic agents in breast cancer. These studies provide the proof-of-principle that stathmin provides an attractive molecular target, which could serve as a primary focus of novel approaches to breast cancer.

Abstract 1295: Inhibition of clonogenicity and xenograft tumor growth by activation and/or over-expression of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ).

Abstract The peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) regulates a variety of biological processes but the function of PPARβ/δ in carcinogenesis remains controversial. Stable human cancer cell lines (A431, MDA-MB-231 and MCF7) that constitutively over-express PPARβ/δ were produced to effectively examine the role of this receptor in human cancer models. Ligand activation of PPARβ/δ both in MDA-MB-231 (ER-negative) and MCF7 (ER-positive) cells over-expressing PPARβ/δ cells caused significantly decreased clonogenicity compared to controls. Over-expression of PPARβ/δ reduced tumor volumes and tumor weights of ectopic xenografts from both MDA-MB-231 and MCF7cells compared to controls. Ligand activation of PPARβ/δ caused an even greater reduction of tumor volumes and tumor weights compared to controls. Interestingly, similar results were observed in the human epidermoid carcinoma cell line A431 and other human cancer cell lines. Ligand activation of PPARβ/δ strongly inhibited clonogenicity in A431 cells over-expressing PPARβ/δ. Xenografts from A431 control cells rapidly developed tumors while tumors were barely detectable in A431 cells over-expressing PPARβ/δ. These observations demonstrate the inhibitory effect of activating or over-expressing PPARβ/δ on tumorigenesis in human cancer cell lines. This suggests that PPARβ/δ suppression of tumorigenicity may be mediated through common mechanisms. Citation Format: Pei-Li Yao, Michael G. Borland, Prasad Krishnan, Bokai Zhu, Frank J. Gonzalez, Jeffrey M. Peters. Inhibition of clonogenicity and xenograft tumor growth by activation and/or over-expression of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1295. doi:10.1158/1538-7445.AM2013-1295

Antiproliferative activity and phenolics of the Mediterranean seaweed Laurencia obusta

Cancer remains one of the most dreaded diseases causing an astonishingly high death rate, second only to cardiac arrest. The fact that conventional and newly emerging treatment procedures like chemotherapy, catalytic therapy, photodynamic therapy and radiotherapy have not succeeded in reverting the outcome of the disease to any drastic extent, has made researchers investigate alternative treatment options. In this paper we have tested the in vitro antiproliferative effects of methanol extract and its fractions from the Mediterranean seaweed Laurencia obusta using the MTT colorimetric assay and clonogenic inhibition. Among the series, the fraction F3 inhibited the growth and colony formation of the three cell lines studied in a concentration-related manner and these results were correlated with its total phenolic and flavonoid contents.

Novel FGFR inhibitor ponatinib suppresses the growth of non-small cell lung cancer cells overexpressing FGFR1

Lung cancer is still the leading cause of cancer-related deaths worldwide. Identifying new oncogenic drivers and developing efficient inhibitors through molecular targeting approaches are crucial for improving therapies. The aim of this study was to investigate whether targeting fibroblast growth factor receptor 1 (FGFR1) with ponatinib inhibits the cell growth in both established and primary lung cancer cells overexpressing FGFR1. Eighty-eight non-small cell lung cancer (NSCLC) and paired normal tissue specimens were analyzed by real-time RT-PCR for FGFR1 gene expression. We identified four cell lines and two newly established primary lung cancer cultures that showed high FGFR1 expression levels, and evaluated the effect of the novel FGFR1 inhibitor ponatinib on cell growth. Approximately 50% (30 out of 59) NSCLC specimens expressed FGFR1>2-fold compared with their adjacent normal counterparts using quantitative RT-PCR. Ponatinib treatment of established NSCLC cell lines expressing higher levels of FGFR1 resulted in marked cell growth inhibition and suppression of clonogenicity. This growth inhibition was associated with inactivation of FGFR1 and its downstream targets. FGFR1 knockdown by shRNA achieved similar results when compared to treatment with ponatinib. Furthermore, ponatinib was able to significantly inhibit the growth of primary lung cancer cultures in vitro. Our data indicate that pharmacological inhibition of FGFR1 kinase activity with ponatinib may be effective for the treatment of lung cancer patients whose tumors overexpress FGFR1.

The PEA-15/PED protein regulates cellular survival and invasiveness in colorectal carcinomas

The PEA-15/PED (phosphoprotein enriched in astrocytes 15kD/phosphoprotein enriched in diabetes) protein is a multifunctional phosphoprotein involved in various signaling pathways which determine survival, proliferation, and migration of cancer cells. Here, we investigated the expression and cellular functions of PEA-15 in colorectal carcinoma (CRC). PEA-15 is expressed in the majority of human CRC, predominantly in well differentiated tumor areas. A tissue microarray analysis of 1262 human CRC specimens from the DACHS study showed that PEA-15 expression is significantly associated with a low pT stadium as defined by limited invasion into the bowel wall. Moreover, patients with PEA-15-positive CRC exhibited a significantly longer tumor-specific survival time. To investigate the functional relevance of PEA-15 expression on a cellular level, we over-expressed PEA-15 in several CRC cell lines. Increased expression of PEA-15 resulted in a strong inhibition of clonogenicity, proliferation, and invasiveness of CRC cells. These effects were associated with a PEA-15-dependent down-regulation of integrin αvβ5 as well as with elevated levels of the phosphorylated MAP kinase ERK1/2. Moreover, expression of PEA-15 resulted in significant protection from cell death induced by cytotoxic drugs (5-FU, cisplatin), by the death ligand TRAIL, or by serum withdrawal. In conclusion, the PEA-15 protein regulates invasiveness, proliferation, and apoptosis resistance in CRC cells. PEA-15 might play an important role in chemoresistance, progression and metastasis in CRC.

Gene specific overwriting of epigenetic signatures to modulate the expression of selected tumor-promoting genes in cancer

Epidermal Growth Factor Receptor-2 (HER-2) and Estrogen receptor-alpha (ER-a) have been found to be dysregulated in several types of cancer. Their dysregulation is associated with aberrant epigenetic modifications. Additionally, expression of ER-a and HER-2 is inversely correlated; their crosstalk has been shown to be involved in endocrine therapy resistance mechanisms [1]. Therapies targeting either receptor result in cell proliferation inhibition for some cancer types; the anti-cancer effect might be further optimized to be more efficient and applicable for more cancer types by co-targeting the genes at the DNA level. As epigenetic modifications provide a way to modify expression of genes in a sustained manner we aim to downregulate HER-2 and ER-a by inducing epigenetic silencing marks specifically onto their promoters (Epigenetic Editing [2]). We also aim to discover the possible crosstalk of ER-a with HER-2 and other important genes in cancer. Methods and materials Towards downregulation of HER-2 and ER-a, expression and epigenetic modification status of promoter of these genes were assessed in a panel of cancer cell lines (bisulfite sequencing and ChIP). Designed zinc finger proteins (ZFPs) targeting genes promoters were fused to a transcription repressor domain [SKD, histone methyltransferases (G9a, SUV39H1), or a DNA methyltransferase domain (C141S [3])] and expressed in cancer cells through viral transduction. Results HER2-ZFP fused to G9a or SUV39H1 induced the intended silencing histone methylation marks (H3K9me2, H3K9me3) on the HER-2 promoter. Up to 4-fold induced H3K9me2 mark was associated with up to 54 ± 2.9% downregulation of HER-2 expression (P<0.0001). Of note, downregulation of HER-2 by induced H3K9 methylation mark was associated with inhibition of cell proliferation and clonogenicity. Additionally, up to 50% downregulation of ER-a was obtained by a ZFP specif c to ER-a fused to SKD, G9a, and C141S. Conclusions These results show that targeted induction of epigenetic marks is instructive in downregulation of HER-2 and ERa expression. We conclude that Epigenetic Editing might provide a novel and synergistic approach to modulate (onco)genes. In addition, investigation of ER-a crosstalk with HER-2 and other genes might be promising to better interfere with pathways involved in cancer.