IND-2, a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline derivative, circumvents multi-drug resistance and causes apoptosis in colon cancer cells.

Naturally occurring condensed quinolines have anticancer properties. In efforts to find active analogues, we designed and synthesized eight polycyclic heterocycles with a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline framework (IND series). The compounds were evaluated for activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231), ovarian (ov2008 and A2780), and hepatocellular (HepG2) cancer cells and against non-cancerous Madin Darby canine kidney (MDCK), mouse embryonic fibroblast (NIH/3T3), and human embryonic kidney cells (HEK293). IND-2, a 4-chloro-2-methyl pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline, exhibited more than ten-fold selectivity and potent cytotoxic activity against colon cancer cells relative to the other cancer and non-cancer cells. With five additional colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), IND-2 had similar cytotoxicity and selectivity, and sub-micromolar concentrations caused changes in the morphology of HCT-116 and HCT-15 cells. IND-2 did not activate the transactivating function of the pregnane X receptor (PXR), indicating that it does not induce PXR-regulated ABCB1 or ABCG2 transporters. Indeed, IND-2 was not a substrate of ABCB1 or ABCG2, and it induced cytotoxicity in HEK293 cells overexpressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. IND-2 was cytotoxic to resistant colon carcinoma S1-MI-80 cells, approximately three- and five-fold more than SN-38 and topotecan, respectively. In HCT-116 colon cancer cells, IND-2 produced concentration-dependent changes in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation. These findings suggest that, by increasing apoptosis, IND-2 has potential therapeutic efficacy for colorectal cancer.

Mutant tristetraprolin: a potent inhibitor of malignant glioma cell growth.

Malignant gliomas rely on the production of certain critical growth factors including VEGF, interleukin (IL)-6 and IL-8, to fuel rapid tumor growth, angiogenesis, and treatment resistance. Post-transcriptional regulation through adenine and uridine-rich elements of the 3' untranslated region is one mechanism for upregulating these and other growth factors. In glioma cells, we have shown that the post-transcriptional machinery is optimized for growth factor upregulation secondary to overexpression of the mRNA stabilizer, HuR. The negative regulator, tristetraprolin (TTP), on the other hand, may be suppressed because of extensive phosphorylation. Here we test that possibility by analyzing the phenotypic effects of a mutated form of TTP (mt-TTP) in which 8 phosphoserine residues were converted to alanines. We observed a significantly enhanced negative effect on growth factor expression in glioma cells at the post-transcriptional and transcriptional levels. The protein became stabilized and displayed significantly increased antiproliferative effects compared to wild-type TTP. Macroautophagy was induced with both forms of TTP, but inhibition of autophagy did not affect cell viability. We conclude that glioma cells suppress TTP function through phosphorylation of critical serine residues which in turn contributes to growth factor upregulation and tumor progression.

Amyotrophic lateral sclerosis-linked mutant SOD1 sequesters Hu antigen R (HuR) and TIA-1-related protein (TIAR): implications for impaired post-transcriptional regulation of vascular endothelial growth factor.

Down-regulation of vascular endothelial growth factor (VEGF) in the mouse leads to progressive and selective degeneration of motor neurons similar to amyotrophic lateral sclerosis (ALS). In mice expressing ALS-associated mutant superoxide dismutase 1 (SOD1), VEGF mRNA expression in the spinal cord declines significantly prior to the onset of clinical manifestations. In vitro models suggest that dysregulation of VEGF mRNA stability contributes to that decline. Here, we show that the major RNA stabilizer, Hu Antigen R (HuR), and TIA-1-related protein (TIAR) colocalize with mutant SOD1 in mouse spinal cord extracts and cultured glioma cells. The colocalization was markedly reduced or abolished by RNase treatment. Immunoanalysis of transfected cells indicated that colocalization occurred in insoluble aggregates and inclusions. RNA immunoprecipitation showed a significant loss of VEGF mRNA binding to HuR and TIAR in mutant SOD1 cells, and there was marked depletion of HuR from polysomes. Ectopic expression of HuR in mutant SOD1 cells more than doubled the mRNA half-life of VEGF and significantly increased expression to that of wild-type SOD1 control. Cellular effects produced by mutant SOD1, including impaired mitochondrial function and oxidative stress-induced apoptosis, were reversed by HuR in a gene dose-dependent pattern. In summary, our findings indicate that mutant SOD1 impairs post-transcriptional regulation by sequestering key regulatory RNA-binding proteins. The rescue effect of HuR suggests that this impairment, whether related to VEGF or other potential mRNA targets, contributes to cytotoxicity in ALS.

Mutant copper-zinc superoxide dismutase associated with amyotrophic lateral sclerosis binds to adenine/uridine-rich stability elements in the vascular endothelial growth factor 3'-untranslated region.

Vascular endothelial growth factor (VEGF) is a neurotrophic factor essential for maintenance of motor neurons. Loss of this factor produces a phenotype similar to amyotrophic lateral sclerosis (ALS). We recently showed that ALS-producing mutations of Cu/Zn-superoxide dismutase (SOD1) disrupt post-transcriptional regulation of VEGF mRNA, leading to significant loss of expression [Lu et al., J. Neurosci.27 (2007), 7929]. Mutant SOD1 was present in the ribonucleoprotein complex associated with adenine/uridine-rich elements (ARE) of the VEGF 3'-untranslated region (UTR). Here, we show by electrophoretic mobility shift assay that mutant SOD1 bound directly to the VEGF 3'-UTR with a predilection for AREs similar to the RNA stabilizer HuR. SOD1 mutants A4V and G37R showed higher affinity for the ARE than L38V or G93A. Wild-type SOD1 bound very weakly with an apparent K(d) 11- to 72-fold higher than mutant forms. Mutant SOD1 showed an additional lower shift with VEGF ARE that was accentuated in the metal-free state. A similar pattern of binding was observed with AREs of tumor necrosis factor-alpha and interleukin-8, except only a single shift predominated. Using an ELISA-based assay, we demonstrated that mutant SOD1 competes with HuR and neuronal HuC for VEGF 3'-UTR binding. To define potential RNA-binding domains, we truncated G37R, G93A and wild-type SOD1 and found that peptides from the N-terminal portion of the protein that included amino acids 32-49 could recapitulate the binding pattern of full-length protein. Thus, the strong RNA-binding affinity conferred by ALS-associated mutations of SOD1 may contribute to the post-transcriptional dysregulation of VEGF mRNA.

Novel DNA-binding properties of the RNA-binding protein TIAR.

TIA-1 related protein binds avidly to uridine-rich elements in mRNA and pre-mRNAs of a wide range of genes, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). The protein has diverse regulatory roles, which in part depend on the locus of binding within the transcript, including translational control, splicing and apoptosis. Here, we observed selective and potent inhibition of TIAR-RNP complex formation with IL-8 and VEGF 3'-untranslated regions (3'-UTRs) using thymidine-rich deoxyoligonucleotide (ODN) sequences derived from the VEFG 3'-UTR. We show by ultraviolet crosslinking and electrophoretic mobility shift assays that TIAR can bind directly to single-stranded, thymidine-rich ODNs but not to double-stranded ODNs containing the same sequence. TIAR had a nearly 6-fold greater affinity for DNA than RNA (K(d)app = 1.6x10(-9) M versus 9.4 x 10(-9) M). Truncation of TIAR indicated that the high affinity DNA-binding site overlaps with the RNA-binding site involving RNA recognition motif 2 (RRM2). However, RRM1 alone could also bind to DNA. Finally, we show that TIAR can be displaced from single-stranded DNA by active transcription through the binding site. These results provide a potential mechanism by which TIAR can shuttle between RNA and DNA ligands.

Molecular Biomarkers of Colorectal Cancer and Cancer Disparities: Current Status and Perspective.

This review provides updates on the efforts for the development of prognostic and predictive markers in colorectal cancer based on the race/ethnicity of patients. Since the clinical consequences of genetic and molecular alterations differ with patient race and ethnicity, the usefulness of these molecular alterations as biomarkers needs to be evaluated in different racial/ethnic groups. To accomplish personalized patient care, a combined analysis of multiple molecular alterations in DNA, RNA, microRNAs (miRNAs), metabolites, and proteins in a single test is required to assess disease status in a precise way. Therefore, a special emphasis is placed on issues related to utility of recently identified genetic and molecular alterations in genes, miRNAs, and various "-omes" (e.g., proteomes, kinomes, metabolomes, exomes, methylomes) as candidate molecular markers to determine cancer progression (disease recurrence/relapse and metastasis) and to assess the efficacy of therapy in colorectal cancer in relation to patient race and ethnicity. This review will be useful for oncologists, pathologists, and basic and translational researchers.

Association between Hepatitis C Virus Infection, p53 Phenotypes, and Gene Variants of Adenomatous Polyposis Coli in Hepatocellular Carcinomas.

OBJECTIVE: To investigate the clinical value of p53 codon 72 single nucleotide polymorphisms (SNPs) and variants of adenomatous polyposis coli (APC) in hepatocellular carcinomas (HCCs). METHODS: DNA and RNA from 51 HCCs and their matching, uninvolved liver tissues were analyzed for p53 mutations, and the methylation and expression of APC variants were determined. Proliferation of each HCC was assessed by Ki67 immunohistochemistry. The results were correlated with the demographic and clinicopathologic features and patient survival. RESULTS: Of 51 HCCs, 12% exhibited missense p53 mutations. SNP analysis of p53 codon 72 demonstrated the highest prevalence of the Arg/Arg (56%) phenotype, followed by Arg/Pro (33%) and Pro/Pro (11%). Four of five cases with the Pro/Pro phenotype were African Americans (AAs). All five cases with the Pro/Pro phenotype had hepatitis C virus (HCV) infections, a high Ki67 index, and lower median survival (15.5 months) compared to those with Arg/Arg or Arg/Pro phenotypes (32 months). The overall frequency of APC methylation was 31%, which was found predominantly in Caucasians. There was lower mRNA expression of APC variants-2 and -3 in both HCCs and corresponding adjacent, uninvolved liver tissues as compared to APC variant-1. The expression of APC variant-3, but not variants-1 and -2, was lower in HCCs relative to uninvolved tissues. Expression of all APC variants was lower in HCCs with APC methylation relative to HCCs without APC methylation, and low expression of APC variant-2 was associated with the Pro/Pro phenotype. CONCLUSIONS: These findings suggest that, for AA patients with HCCs, the p53 Pro/Pro phenotype and low expression of APC variant-2 are associated with aggressive tumor behavior, HCV infection, and poor clinical outcome.

IL-1beta induces stabilization of IL-8 mRNA in malignant breast cancer cells via the 3' untranslated region: Involvement of divergent RNA-binding factors HuR, KSRP and TIAR.

IL-8 plays an integral role in promoting the malignant phenotype in breast cancer, and its production is directly influenced by inflammatory cytokines in the tumor microenvironment. Here, we show that activation of IL-1beta receptors on malignant HS578t and MDA-MB-231 breast cancer cells strongly induces IL-8 expression and that RNA stabilization is persistently activated at least 12-24 hr after stimulation. SB 203580 and rapamycin reversed the RNA stabilization effect of IL-1beta in a dose-dependent manner, suggesting involvement of the p38/MAP kinase and mTOR pathways. A luciferase reporter assay indicated that the stabilization effect was dependent on cis elements in the 3'-untranslated region (UTR) of the IL-8 transcript. By UV cross-linking, we identified multiple cellular factors that interact with the IL-8 3'UTR, ranging 34-76 kDa. Immunoprecipitation analysis indicated that HuR, KSRP and TIAR bound to one or more loci in the 3'UTR. While the cross-linking patterns were similar, quantitative immunoprecipitation of native IL-8 RNA from IL-1beta-stimulated cytoplasmic extract revealed a 20-fold greater association of transcript with the stabilizing factor HuR vs. the destabilizing factor KSRP. In conclusion, IL-1beta is a potent cytokine stimulus for IL-8 RNA stabilization in breast cancer cells, possibly by enhanced binding of cytoplasmic HuR to the 3'UTR.