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1.
J Ovarian Res ; 15(1): 120, 2022 Nov 02.
Article in English | MEDLINE | ID: mdl-36324187

ABSTRACT

A leading theory for ovarian carcinogenesis proposes that inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, nonsteroidal anti-inflammatory drugs (NSAIDs) exert promising chemopreventive activity for ovarian cancer. Unfortunately, toxicity is associated with long-term use of NSAIDs due to their cyclooxygenase (COX) inhibitory activity. Previous studies suggest the antineoplastic activity of NSAIDs is COX independent, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. Here we evaluate PDE10A as a novel therapeutic target for ovarian cancer. Analysis of The Cancer Genome Atlas (TCGA) ovarian tumors revealed PDE10A overexpression was associated with significantly worse overall survival for patients. PDE10A expression also positively correlated with the upregulation of oncogenic and inflammatory signaling pathways. Using small molecule inhibitors, Pf-2545920 and a novel NSAID-derived PDE10A inhibitor, MCI-030, we show that PDE10A inhibition leads to decreased ovarian cancer cell growth and induces cell cycle arrest and apoptosis. We demonstrate these pro-apoptotic properties occur through PKA and PKG signaling by using specific inhibitors to block their activity. PDE10A genetic knockout in ovarian cancer cells through CRISP/Cas9 editing lead to decreased cell proliferation, colony formation, migration and invasion, and in vivo tumor growth. We also demonstrate that PDE10A inhibition leads to decreased Wnt-induced ß-catenin nuclear translocation, as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. These findings implicate PDE10A as novel target for ovarian cancer chemoprevention and treatment.


Subject(s)
Ovarian Neoplasms , beta Catenin , Female , Humans , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , beta Catenin/genetics , beta Catenin/metabolism , Carcinoma, Ovarian Epithelial/drug therapy , Carcinoma, Ovarian Epithelial/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Phosphoric Diester Hydrolases/genetics , Phosphoric Diester Hydrolases/metabolism , ras Proteins/metabolism
2.
Cancer Prev Res (Phila) ; 14(11): 995-1008, 2021 11.
Article in English | MEDLINE | ID: mdl-34584001

ABSTRACT

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic ß-catenin, inhibited Wnt-induced nuclear translocation of ß-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer. PREVENTION RELEVANCE: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/ß-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.


Subject(s)
Colonic Neoplasms , beta Catenin , Animals , Carcinogenesis , Colon/pathology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/prevention & control , Mice , Phosphodiesterase Inhibitors/pharmacology , Sulindac/pharmacology
3.
Mol Cancer Res ; 15(11): 1503-1516, 2017 11.
Article in English | MEDLINE | ID: mdl-28801308

ABSTRACT

EGFR signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non-small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild-type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells. Gene expression profiling in human bronchial epithelial cells (HBEC) revealed that MT-EGFR expression elevated transcripts related to cell cycle and replication in aerobic and hypoxic conditions and downregulated RAD50, a critical component of nonhomologous end joining and homologous recombination DNA repair pathways. NSCLCs and HBEC with MT-EGFR revealed elevated basal and hypoxia-induced γ-H2AX-associated DNA lesions that were coincident with replication protein A in the S-phase nuclei. DNA fiber analysis showed that, relative to WT-EGFR, MT-EGFR NSCLCs harbored significantly higher levels of stalled replication forks and decreased fork velocities in aerobic and hypoxic conditions. EGFR blockade by cetuximab significantly increased radiosensitivity in hypoxic cells, recapitulating MT-EGFR expression and closely resembling synthetic lethality of PARP inhibition.Implications: This study demonstrates that within an altered DNA damage response of hypoxic NSCLC cells, mutant EGFR expression, or EGFR blockade by cetuximab exerts a synthetic lethality effect and significantly compromises radiation resistance in hypoxic tumor cells. Mol Cancer Res; 15(11); 1503-16. ©2017 AACR.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , DNA Replication , DNA/metabolism , ErbB Receptors/genetics , Lung Neoplasms/genetics , A549 Cells , Acid Anhydride Hydrolases , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Hypoxia , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Cetuximab/pharmacology , DNA Damage , DNA Repair , DNA Repair Enzymes/genetics , DNA-Binding Proteins/genetics , Humans , Lung Neoplasms/drug therapy , Mutation , Radiation Tolerance/drug effects , Radiation-Sensitizing Agents/pharmacology
4.
Oncol Rep ; 32(2): 469-74, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24926795

ABSTRACT

Cellular resistance to platinum anticancer compounds is governed by no less than two molecular processes; DNA repair and cellular accumulation of drug. Gli1 is an upstream regulator of nucleotide excision repair, effecting this process through c-jun. We, therefore, investigated whether Gli1 plays a role in cellular accumulation of cisplatin. Using a Gli1-specific shRNA, we explored the role of Gli1 in the cellular accumulation and efflux of cisplatin, in cisplatin-resistant A2780-CP70 human ovarian cancer cells. When Gli1 is inhibited, cellular uptake of cisplatin was approximately 33% of the level of uptake under control conditions. When Gli1 is inhibited, cellular efflux of cisplatin was completely abrogated, over a 12-h period of observation. We assayed nuclear lysates from these cells, for the ability to bind the DNA sequence that is the Gli-binding site (GBS) in the 5'UTR for each of five known cisplatin transmembrane transporters. Four of these transporters are active in cisplatin uptake; and, one is active in cisplatin efflux. In each case, nuclear lysate from A2780-CP70 cells binds the GBS of the respective cisplatin transport gene. We conclude that Gli1 plays a strong role in total cellular accumulation of cisplatin in these cells; and, that the combined effects on cellular accumulation of drug and on DNA repair may indicate a role for Gli1 in protecting cellular DNA from lethal types of DNA damage.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Cisplatin/pharmacokinetics , Drug Resistance, Neoplasm , Ovarian Neoplasms/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Binding Sites , Cation Transport Proteins/metabolism , Cell Line, Tumor , Copper Transporter 1 , DNA Damage , Female , Humans , Organic Cation Transport Proteins/metabolism , Zinc Finger Protein GLI1
5.
Int J Oncol ; 44(3): 655-61, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24366538

ABSTRACT

The Hedgehog pathway is molecularly linked to increased resistance to cisplatin and increased repair of platinum-DNA damage, through C-JUN. GLI1, which has five known isoforms, is a positive transcriptional regulator in Hedgehog. Southwestern blot assay, EMSA and ChIP assays indicate that only one of five isoforms of GLI1 may be responsible for the Hedgehog link with C-JUN and thus, increased platinum-DNA adduct repair. Cancer tissues express this 130-kDa isoform at levels 6-fold higher than non-malignant tissues; and this isoform exists in abundance in six of seven ovarian cancer cell lines examined.


Subject(s)
Drug Resistance, Neoplasm/genetics , JNK Mitogen-Activated Protein Kinases/genetics , Ovarian Neoplasms/genetics , Transcription Factors/genetics , Cisplatin/therapeutic use , DNA Damage/drug effects , Female , Hedgehog Proteins/metabolism , Humans , Ovarian Neoplasms/drug therapy , Protein Isoforms/genetics , Protein Isoforms/metabolism , Transcription Factors/metabolism , Tumor Cells, Cultured , Zinc Finger Protein GLI1
6.
Clin Cancer Res ; 14(24): 8080-6, 2008 Dec 15.
Article in English | MEDLINE | ID: mdl-19088023

ABSTRACT

PURPOSE: The purpose of this study is to investigate the molecular mechanism of miR-192 in colon cancer. EXPERIMENTAL DESIGN: Human colon cancer cell lines with different p53 status were used as our model system to study the effect of miR-192 on cell proliferation, cell cycle control, and mechanism of regulation. RESULTS: Our results show that one of the key miR-192 target genes is dihydrofolate reductase (DHFR). miR-192 affects cellular proliferation through the p53-miRNA circuit. Western immunoblot analyses indicated that the expression of DHFR was significantly decreased by miR-192. Further investigation revealed that such suppression was due to translational arrest rather than mRNA degradation. More profound inhibition of cellular proliferation was observed by ectopic expression of miR-192 in colon cancer cell lines containing wild-type p53 than cells containing mutant p53. Thus, the effect of miR-192 on cellular proliferation is mainly p53 dependent. Overexpression of miR-192 triggered both G1 and G2 arrest in HCT-116 (wt-p53) cells but not in HCT-116 (null-p53) cells. The cell cycle checkpoint control genes p53 and p21 were highly overexpressed in cells that overexpressed miR-192. Endogenous miR-192 expression was increased in HCT-116 (wt-p53) and RKO (wt-p53) cells treated with methotrexate, which caused an induction of p53 expression. Chromatin immunoprecipitation-quantitative reverse transcription-PCR analysis revealed that the p53 protein interacted with the miR-192 promoter sequence. CONCLUSION: These results indicate that miR-192 may be another miRNA candidate that is involved in the p53 tumor suppressor network with significant effect on cell cycle control and cell proliferation.


Subject(s)
Gene Expression Regulation, Enzymologic , MicroRNAs/physiology , Tetrahydrofolate Dehydrogenase/genetics , Tumor Suppressor Protein p53/physiology , Cell Cycle , Cell Proliferation , HCT116 Cells , HT29 Cells , Humans , Methotrexate/pharmacology , Promoter Regions, Genetic
7.
PLoS One ; 3(12): e4054, 2008.
Article in English | MEDLINE | ID: mdl-19112505

ABSTRACT

Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU) treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53) and HCT116 (null-p53) colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt) treatment. Decreased Orc6 expression in HCT-116 (wt-p53) cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53) cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53) cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53) cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45beta and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.


Subject(s)
Cisplatin/pharmacology , Colonic Neoplasms/drug therapy , Fluorouracil/pharmacology , Gene Expression Regulation, Neoplastic , Genes, p53 , Origin Recognition Complex/biosynthesis , Origin Recognition Complex/physiology , Cell Line, Tumor , Cell Nucleus/metabolism , Colonic Neoplasms/metabolism , Cyclin-Dependent Kinase Inhibitor p27/biosynthesis , G1 Phase , Gene Expression Profiling , Humans , Mitogen-Activated Protein Kinase 8/metabolism , Phosphorylation , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics
8.
Cancer Genomics Proteomics ; 5(1): 1-35, 2008.
Article in English | MEDLINE | ID: mdl-18359977

ABSTRACT

Various in vitro and in vivo experimental models have been used for the discovery of genes and pathways involved in melanoma and other types of cancer. However, in many cases, the results from various tumor models failed to be validated successfully in clinical studies. Limited information is available on how closely these models reflect the in vivo physiological conditions. In this study, a comprehensive genomics approach was used to systematically compare the expression patterns of snap frozen samples obtained from patients with primary melanoma, lymph node metastasis, and distant metastases, and compare these patterns to those of their corresponding cell lines and tumor xenografts in nude mice. The GE Healthcare 20k human genome array was used and the expression data was normalized and analyzed using GeneSpring 7.2 software. Based on the expression analysis, the correlation rate between the snap frozen primary patient samples vs. derived cell lines was 66%, with 1687 differentially expressed genes. The correlation rate between the snap frozen primary patient samples and the tumor xenografts was 75%, with 1,374 differentially expressed genes, and the correlation rate comparing tumor xenografts to derived cell lines ranged between 58% and 84%. These results demonstrated significant gene expression differences between tumor materials with different in vitro and in vivo growth microenvironments. Such studies can help us to distinguish between genes up- or down-regulated as a result of the microenvironment and those stably expressed independently of the tumor milieu. With the extensive use of cell lines and xenografts in cancer research, the information obtained using our approach may help to better interpret results generated from different tumor models by understanding common differences, as well as similarities at the gene expression level, information that may have important practical and biological implications.


Subject(s)
Gene Expression Regulation, Neoplastic , Melanoma/genetics , Animals , Cell Line, Tumor , Cluster Analysis , Gene Expression Profiling , Genome, Human , Humans , Melanoma/metabolism , Melanoma/pathology , Mice , Mice, Nude , Neoplasm Metastasis , Oligonucleotide Array Sequence Analysis , Transplantation, Heterologous
9.
RNA ; 13(10): 1668-74, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17698639

ABSTRACT

microRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the translational level by mainly interacting with 3' UTRs of their target mRNAs. Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery. Currently there is a lack of systematic analysis on the stability of miRNAs and optimized conditions for expression analysis using FFPE samples. In this study, the expression of miRNAs from FFPE samples was analyzed using high-throughput locked nucleic acid-based miRNA arrays. The effect of formalin fixation on the stability of miRNAs was also investigated using miRNA real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The stability of miRNAs of archived colorectal cancer FFPE specimens was characterized with samples dating back up to 10 yr. Our results showed that the expression profiles of miRNAs were in good correlation between 1 mug of fresh frozen and 1-5 mug of FFPE samples (correlation coefficient R (2) = 0.86-0.89). Different formalin fixation times did not change the stability of miRNAs based on real-time qRT-PCR analysis. There are no significant differences of representative miRNA expression among 40 colorectal cancer FFPE specimens. This study provides a foundation for miRNA investigation using FFPE samples in cancer and other types of diseases.


Subject(s)
MicroRNAs/analysis , Tissue Fixation/methods , Animals , Formaldehyde/metabolism , Frozen Sections/methods , Humans , Paraffin Embedding/methods
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