Elangovan Thavathiru

Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth, invasion and angiogenesis in vivo.

Aberrant activation of Wingless-type (Wnt)/β-catenin signaling is widespread in human cervical cancer. However, the underlying mechanisms of Wnt activation and the therapeutic potential of Wnt inhibition remain largely unknown. Here, we demonstrate that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, is downregulated in all human primary cervical tumors and cell lines analyzed. Our data reveal that WIF1 downregulation occurs due to promoter hypermethylation and is an early event in cervical oncogenesis. WIF1 re-expression upon 5-aza-2′-deoxycytidine treatment or WIF1 gene transfer induces significant apoptosis and G2/M arrest, and inhibits cervical cancer cell proliferation in vitro. Consistent with this, treatment of established mice tumor xenografts with peritumoral WIF1 gene transfer results in a significant inhibition of cancer growth and invasion. WIF1 treatment causes a significant decrease in intracellular WNT1 and TCF-4 proteins revealing novel Wnt-regulatory mechanisms. Thus, WIF1 causes a major cellular re-distribution of β-catenin and a significant inhibition of the Wnt/β-catenin pathway in tumor cells, as documented by a remarkable reversion in the expression of Wnt/β-catenin transcriptional target genes (E-cadherin, c-Myc, cyclin D1, CD44 and VEGF). Consequently, multiple critical events in tumor progression and metastasis such as cell proliferation, angiogenesis and invasion were inhibited by WIF1. In addition, WIF1 modulated the expression of specific anti-apoptotic and apoptotic proteins, thereby inducing significant apoptosis in vivo. Our findings demonstrate for the first time that WIF1 downregulation by epigenetic gene silencing is an important mechanism of Wnt activation in cervical oncogenesis. Of major clinical relevance, we show that peritumoral WIF1 gene transfer reduces not only cancer growth but also invasion in well-established tumors. Therefore, our data provide novel mechanistic insights into the role of WIF1 in cervical cancer progression, and the important preclinical validation of WIF1 as a potent drug target in cervical cancer treatment.

CAP37 activation of PKC promotes human corneal epithelial cell chemotaxis.

PURPOSE The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis. METHODS Immortalized HCECs were treated with pertussis toxin (10 and 1000 ng/mL), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro-31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 μM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr(505) phosphorylation, and PKCδ kinase activity was assessed in CAP37-treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay, respectively. RESULTS Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared with untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr(505). Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay. CONCLUSIONS These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ.

Insulin exerts direct effects on carcinogenic transformation of human endometrial organotypic cultures

Epidemiological studies suggest an association between elevated insulin levels and endometrial cancer. We studied the effects of insulin on normal endometrial cell proliferation with cytotoxicity assays. Organotypic cultures were used to determine the effects of insulin on the development of malignant histological features and anchorage independent growth. Western Blots were used to analyze the mitogen-activated protein kinases and AKT pathways. We found that insulin exerts direct effects on endometrial cells by increasing proliferation and promoting carcinogenesis. Our results suggest that this occurs through ERK 1/2 and glycogen synthase kinase-3β Ser9 phosphorylation.

Synthesis and evaluation of second generation Flex-Het scaffolds against the human ovarian cancer A2780 cell line.

Flexible Heteroarotinoids (Flex-Hets) are a class of substituted di-aryl compounds that exhibit potent anti-cancer activity without toxicity. They were derived from the more conformationally restricted, 2-atom linker Hets by substitution of the 2-atom linker with a 3-atom urea or thiourea linker, which conferred more potent inhibitory activity against cancer cell lines. The objectives of this structure activity relationship (SAR) study were to determine if a 4-atom acrylamide linker and various substitutions on the terminal aryl ring altered the anti-cancer activity of these second generation Flex-Het compounds compared to the parent Flex-Het compound, SHetA2, which has a thiourea linker and a nitro substituent. Biological activity was measured using a cytotoxicity assay of the human A2780 ovarian cancer cell line treated with a range of compound concentrations. Nitrogen-based substitutions on the terminal aryl group caused similar, but slightly reduced efficacies and potencies. Exceptions were systems that had a nitro group at the para position, the potencies of which were better than that of SHetA2 with efficacies that were only slightly reduced compared to SHetA2. Similarly, the potency of the system with a para dimethylamino group was greater than that of SHetA2. However, a 30% reduction in efficacy compared to SHetA2 was noted. While specific members with the 4-atom acrylamide linker did exhibit excellent potency, the efficacy was slightly below that of SHetA2. Thus, a gradient of activities was observed as the substituent on the aryl ring was altered.

Anti-CD73 and anti-OX40 immunotherapy coupled with a novel biocompatible enzyme prodrug system for the treatment of recurrent, metastatic ovarian cancer

Approximately 75% of ovarian cancer is diagnosed once metastasis to the peritoneal cavity has occurred. A large proportion of patients eventually develop platinum-resistive tumors, which are considered terminal. In order to provide an alternative a novel fusion protein, mCTH-ANXA5, has been developed for the treatment of recurrent, metastatic ovarian cancer. The fusion protein combines annexin V (ANXA5), an ovarian tumor and tumor vasculature targeting protein, with mutated cystathionine gamma-lyase (mCTH), an enzyme that converts selenomethionine (SeMet) into toxic methylselenol, which generates reactive oxygen species and eventual tumor cell death. In order to further enhance the therapeutic efficacy, anti-CD73 and anti-OX40 immunostimulants were combined with mCTH-ANXA5, resulting in an increase of survival by 100% from 12 to 24 days post-therapy and decrease tumor burden in mice with orthotopic metastatic ovarian cancer. Further evaluation of the combination therapy revealed a strong antibody-mediated immune response, and an increased infiltration of cytotoxic T-cells along with a decrease in tumor promoting immune cells. This study demonstrates the efficacy of a synergistic, multi-drug system by attacking the tumor as well as enlisting the bodys own defense system to treat the patient.

Bioanalytical method development and validation of HPLCUV assay for the quantification of SHetA2 in mouse and human plasma: Application to pharmacokinetics study

Background SHetA2 is an oral anticancer agent being investigated for cancer treatment and prevention. The aim of this study was to develop and validate a simple, cost-effective, and sensitive HPLC-UV method for the quantification of SHetA2 in biological samples and to apply the method to pharmacokinetic studies of the drug. Methods Sample preparation for mouse and human plasmas involved liquid-liquid precipitation and extraction using chilled acetonitrile with 2, 3-Diphenylquinoxaline as an internal standard. The separation of SHetA2 and internal standard was achieved via Waters XBridge™ BEH 130 C18 (3.5 μm, 2.1×150 mm) column coupled with a Waters XBridge™ C-18 (3.5 μm, 2.1×10 mm) guard column using 65% v/v acetonitrile: distilled water as a mobile phase in an isocratic mode with a flow rate of 0.18 ml/min. The analytes were eluted at a detection wavelength of 341 nm at a column temperature of 25°C. Results The method was validated across a range of 5-1000 ng/ml for SHetA2 in plasma, with a lower limit of quantification of 5 ng/ml. The method showed high recovery in human (79.9-81.8%) and mouse (95.4-109.2%) plasma with no matrix effect. The intra- and inter-day accuracy and precision studies demonstrated that the method was specific, sensitive, and reliable. Stability studies showed that SHetA2 is stable for 20 h postoperatively in the auto sampler, and for six weeks at -80°C in plasma. Repetitive freezing and thawing may be avoided by preparing the aliquots and storing them at -80°C. The developed method was successfully applied to study the plasma pharmacokinetics of SHetA2 in tumor-bearing nude mice after intravenous and oral administration. Conclusion A novel method for quantifying SHetA2 in mouse and human plasmas has been validated and is being applied for pharmacokinetic evaluation of SHetA2 in tumor-bearing mice. The developed method will be utilized for the quantification of SHetA2 in clinical studies.

Abstract 1798: Mortalin precursor as potential marker for chemoprevention with SHetA2

Introduction: The flexible heteroarotinoid (Flex-Het), SHetA2, is a novel anticancer drug that induces both intrinsic and extrinsic apoptosis and autophagy in cancer cells, but not in normal cells. Protein isolation / mass spectrometry analysis using SHetA2-coupled polystyrene magnetic beads yielded three SHetA2-binding proteins all belonging to HSPA family namely, HSPA9/mortalin, HSPA8/Hsc70 and HSPA5/BiP. Mortalin, in addition to its vital chaperoning roles in other organelles of the cell, is essential for import and folding of nuclear-encoded mitochondrial proteins. The precursor form of mortalin has a 46-amino acid N-terminal region that functions as a mitochondrial localization sequence (MLS) and is cleaved by proteases after import into the mitochondrial matrix. Hypothesis: SHetA2 binding to mortalin causes alterations that can be measured to study mechanism and monitor drug effects in animal models and clinical trials. Methods: Western blots of whole cell or subcellular protein extracts made from cultures of normal human fallopian tube secretory or mammary epithelial cells, rat mammary tumors or human ovarian cancer cell lines treated with SHetA2 or solvent were probed with an antibody that recognizes total mortalin, a custom-made rabbit polyclonal antibody specific for the mortalin MLS (PA8238) or antibodies to loading control proteins. Immunocytochemistry using these antibodies was performed with an automated (Leica Bond III) IHC work station on cells treated with SHetA2 or solvent. Results: Using a commercial antibody against total mortalin, we observed a lower-mobility band closely moving above the specific (mature) mortalin band in Western blots. Our mortalin MLS-specific antibody recognized only the lower-mobility band confirming it as the precursor form of mortalin in the SHetA2-treated cell extracts. Subcellular fractionation of the drug-treated cells revealed that the precursor protein accumulated only in the cytoplasm and not in the mitochondria. Staining of whole cells with an antibody to total mortalin showed no effect of SHetA2 treatment on the punctate pattern of expression consistent with mitochondrial localization. In contrast, staining with the mortalin MLS-specific antibody demonstrated that SHetA2 increased the intensity of the diffuse cytoplasmic staining consistent with localization of mortalin throughout the cytoplasm. These results were consistently observed among the various cell types. Conclusions: SHetA2 is the only known drug that blocks the translocation of the precursor mortalin to mitochondria and its processing to mature protein. Such cytoplasmic accumulation of the precursor form of mortalin can potentially serve as pharmacodynamic endpoint to study SHetA2’s effect in laboratory experimental tumor models as well as in human clinical trials. Funding: NCI PREVENT Task Order HHSN26100002 Citation Format: Elangovan Thavathiru, Vishal Chandra, Rajani Rai, Doris Benbrook. Mortalin precursor as potential marker for chemoprevention with SHetA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1798. doi:10.1158/1538-7445.AM2017-1798

Abstract 4679: A novel assay to predict susceptibility to tobacco-induced disease.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Tobacco misuse is the leading preventable cause of morbidity and mortality in the world. Tobacco-induced DNA damage is one of the main mechanisms contributing to the pathogenesis of cancer, stroke, heart and pulmonary diseases. Tobacco-induced DNA damage is modulated by genetic and epigenetic factors, as well as life-style choices, and is expected to be a major determinant of the individual susceptibility to tobacco-induced diseases. Technical limitations allow only a few types of DNA damage to be quantified in human samples. Recently, we filled a major methodological gap in the field of DNA damage by developing a novel and highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo levels of DNA damage. Aims: (1) To standardize PADDA for the detection of oxidative DNA damage, one of the main types of tobacco-induced damage. (2) To define the levels of persistent DNA damage in the oral mucosa of smokers. (3) To determine if persistent nucleotide damage at p53 co-localizes with tobacco-induced cancer mutational hotspots. Methods: To standardize the assay, PADDA was used on a high-throughput setting to quantify DNA damage in oral cell lines exposed to very low doses of hydrogen peroxide. DNA damage was mapped and quantified on the p53 gene by PADDA in oral epithelial cells collected from smokers and non-smokers by oral scrapings. The location of p53 nucleotide damage was compared with reported tobacco-induced p53 cancer mutational hotspots. Saliva cotinine levels were used to confirm smoking status. Data were analyzed by chi-square goodness of fit and exact non-parametric tests. Results: Our data documented PADDAs ability to detect dose-dependent increase in DNA damage in human oral cells. DNA damage was significantly higher in smokers than in non-smokers. Moreover, we documented for the first time that the persistent DNA damage in the oral mucosa of smokers has significantly higher mutagenic potential than that present in non-smokers and persists mainly at p53 nucleotides that are hotspots for mutation in tobacco-induced cancers. Conclusion: PADDA detects dose-dependent DNA damage response, a crucial test of its accuracy and a prerequisite for its use in biomonitoring. PADDA documents the extent of tobacco-induced DNA damage in vivo, and reinforces the importance of smoking cessation. Of major clinical importance, we show, for the first time, that tobacco-induced DNA damage persists preferentially in p53 nucleotides that are hotspots for mutation. Application of this assay to large series of smokers and former smokers has a major potential to establish biomarkers of susceptibility to tobacco-induced disease, which can guide preventive and diagnostic strategies. Funding: This work was supported by the Oklahoma Tobacco Research Center and the Oklahoma Center for the Advancement of Science & Technology. Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Vengatesh Ganapathy, Wilbur K. Mills, Elangovan Thavathiru, Ilangovan Ramachandran, Leslie Chandler, Antonio Reis, Lurdes VF Queimado. A novel assay to predict susceptibility to tobacco-induced disease. [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 4679. doi:10.1158/1538-7445.AM2013-4679

Abstract 3703: SHetA2 decreases mitoses and growth, and alters pathology of MNU-induced rat mammary tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: SHetA2 (NSC721689) is a non-toxic chemoprevention drug that has completed pre-clinical testing by the NCI-RAID and RAPID programs and will be evaluated in a Phase 0 clinical trial under development. Previous studies found that SHetA2 reversed the cancerous phenotype and prevented carcinogenesis in organotypic cultures of human ovarian cancer and endometrial cells. The objective of this project was to evaluate the effects of oral SHetA2 on histologic features of tumors induced by the nitrosomethylurea (MNU) carcinogen in Sprague-Dawley rats. Methods: SHetA2 was administered to 3 treatment groups of 15 animals each in a Teklad diet formulated to contain doses of 0 (control), 25 (low dose) and 50 (high dose) mg/kg/day. Five days after initiation of treatment, each rat was injected with 11.25 mg of MNU into the peritoneum and the animals were continued on their respective diets. The first palpable tumors were noted 48 days after MNU injection. Tumors volumes were measured weekly. Four animals in the control group were euthanized early on days 58, 63, 78 and 82, one animal in the low dose group was euthanized on day 78 and one animal in the high dose group was euthanized on day 74 due to the excessive sizes of their tumors. The remaining animals were euthanized on day 85 and the tumors were collected for histologic evaluation. H&E stained slides of the tumors were reviewed for histologic features in a blinded manner. A linear mixed model was used to compare the growth rate of tumor volumes. The fixed effects included treatment group and days. An unstructured covariance matrix was used in the model. The numbers of mitoses in the tumors of the different treatment groups were compared using a linear mixed model. Results: Tumor growth rate was significantly lower in the high dose group in comparison to the control group (p=.004) and the low dose (p=.003). No significant difference (p=.716) in growth rate was observed between low dose and control groups. The number of mitoses counted in the tumors was significantly smaller in the high dose than in the control group (p=.004), but no significant difference was observed between low dose and control groups (p=.945). The histology of the tumors included fibrocystic disease, intraductal papillomatosis, adenoid cystic carcinoma, papillary carcinoma, papillary and adenoid cystic carcinoma and carcinoma. When compared per animal, there were no significant differences in the histologies between the treatment groups, however when each tumor was considered an individual data point, there was a statistical trend that both doses of SHetA2 decreased the percentage of papillary carcinoma and increased the percentages of adenoid cystic carcinoma, (high dose vs control: p=.058; low dose vs control: p=.058). Conclusion: SHetA2 administered in the diet reduced cell proliferation, growth and aggressiveness of the pathologic diagnosis of mammary tumors in MNU-treated rats. Citation Format: Stan Lightfoot, Daniel Zhao, Elangovan Thavathiru, Doris Mangiaracina Benbrook. SHetA2 decreases mitoses and growth, and alters pathology of MNU-induced rat mammary tumors. [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 3703. doi:10.1158/1538-7445.AM2013-3703

Abstract 4298: microRNA-200c mediates the tumor suppressive effects of Wnt inhibitory factor 1 in human malignant salivary gland cells.

Background: The important role of Wingless-type (Wnt)/β-catenin pathway is well established in various human tumors. Previously, we have demonstrated that Wnt inhibitory factor 1 (WIF1) is significantly downregulated in human salivary gland tumors. The tumor suppressor microRNA, miR-200c, is downregulated in many cancers and is known to repress migration and invasion of cancer cells. Aims: To determine the tumor suppressive effects of WIF1 and delineate the mechanisms by which WIF1 regulates microRNAs in human malignant salivary gland cells. Methods: Human malignant salivary gland cells (Carcinoma ExPleomorphic adenoma, CaExPA79) were transiently transfected with either empty vector or pCI blast-WIF1 and cell proliferation was determined by MTT assay at various time points. CaExPA79 cells transfected with WIF1 for 48 h were used for total RNA isolation and gene expression studies. To determine the effect of WIF1 restoration on salivary gland cancer cells, CaExPA79 cells were treated with 50 μM of 5-aza-2′-deoxycytidine (DAC), a demethylating agent, for 4 days. After DAC treatment, the cells were either used for cell cycle analysis or total RNA isolation and real-time RT-PCR analysis. To determine the effect of WIF1 on miR-200c, CaExPA79 cells were transfected with pCI blast-WIF1 along with miR200c-Luc reporter vector and Renilla luciferase expressing plasmid pRL-TK for 24 h. Luciferase activity was determined using Dual-Luciferase Reporter Assay System. Results: WIF1 significantly inhibited the proliferation of CaExPA79 at 24, 48 and 72 h. Treatment with DAC increased the WIF1 mRNA expression and resulted in a significant accumulation of cells in S phase. DAC treatment also resulted in a significant increase in miR-200c expression. WIF1 over-expression resulted in a significant increase in miR-200c expression and led to a significant reduction in the expression of miR-200c targeted genes as documented by luciferase reporter gene assays. Conclusions: WIF1 inhibits salivary gland cancer cell proliferation and induces cell cycle arrest. Most importantly, our findings suggest that WIF1 is a positive transcriptional regulator of the tumor suppressor microRNA miR-200c and leads to a corresponding downregulation of its downstream targets. Taken together, this study reveals a novel mechanism by which WIF1 elicits it9s tumor suppressive effects on salivary gland cancer cells. Grant support: This work was supported by the Oklahoma Center for the Advancement of Science & Technology (LQ) (HR08-018). LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Ilangovan Ramachandran, David Obeso, Sripathi M. Sureban, Elangovan Thavathiru, Antonio Reis, Lurdes Queimado. microRNA-200c mediates the tumor suppressive effects of Wnt inhibitory factor 1 in human malignant salivary gland cells. [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 4298. doi:10.1158/1538-7445.AM2013-4298