MicroRNAs (miRNAs) are a class of small RNAs that have revealed a new level of gene regulation in the cell. After being processed by Drosha and Dicer RNase III endonucleases, mature miRNAs can inhibit the translation of mRNA by directing a RNA-induced silencing complex (RISC) to the target mRNA. miRNAs are making an impact in our understanding of cancer biology. Acting as either tumor suppressors or oncogenes, miRNAs regulate several genes known to play important roles in cancer. With the discovery of miRNAs comes the need for new techniques to study their activity. Bioinformatic tools can be used to predict mRNA targets of miRNA, but validation of miRNA regulation of predicted targets is imperative. miRNAs are differentially expressed in normal and tumor cells as well as between tumor subtypes. These differences may be useful as prognostic and predictive markers in cancer patients. The study of miRNAs holds much promise for improving diagnosis and treatment of cancer. [Mol Cancer Ther 2008;7(12):3655–60]

Livin is a member of the inhibitors of apoptosis protein gene family, which is highly expressed in a variety of human neoplasms. Several studies have shown that down-regulation of Livin expression increases the apoptotic rate, reduces tumor growth potential, and sensitizes tumor cells to chemotherapeutic drugs. Furthermore, emerging data reveal that Livin fragments cleavaged by caspases restored paradoxical proapoptotic activity during the apoptotic process, suggesting that Livin cleavage will become a highly potent proapoptotic agent in the future. In this article, we review the current understanding of the versatile roles of Livin in the apoptotic cascade and exploit the promising approach to interfere with Livin as a novel strategy for cancer therapy. [Mol Cancer Ther 2008;7(11):3661–9]

Angiogenesis is a hallmark of tumor development and metastasis and is now a validated target for cancer treatment. However, the survival benefits of antiangiogenic drugs have thus far been rather modest, stimulating interest in developing more effective ways to combine antiangiogenic drugs with established chemotherapies. This review discusses recent progress and emerging challenges in this field; interactions between antiangiogenic drugs and conventional chemotherapeutic agents are examined, and strategies for the optimization of combination therapies are discussed. Antiangiogenic drugs such as the anti-vascular endothelial growth factor antibody bevacizumab can induce a functional normalization of the tumor vasculature that is transient and can potentiate the activity of coadministered chemoradiotherapies. However, chronic angiogenesis inhibition typically reduces tumor uptake of coadministered chemotherapeutics, indicating a need to explore new approaches, including intermittent treatment schedules and provascular strategies to increase chemotherapeutic drug exposure. In cases where antiangiogenesis-induced tumor cell starvation augments the intrinsic cytotoxic effects of a conventional chemotherapeutic drug, combination therapy may increase antitumor activity despite a decrease in cytotoxic drug exposure. As new angiogenesis inhibitors enter the clinic, reliable surrogate markers are needed to monitor the progress of antiangiogenic therapies and to identify responsive patients. New targets for antiangiogenesis continue to be discovered, increasing the opportunities to interdict tumor angiogenesis and circumvent resistance mechanisms that may emerge with chronic use of these drugs. [Mol Cancer Ther 2008;07(12):3670–84]

This phase I study was designed to determine the maximum tolerated dose (MTD) and toxicity profile of the combination of gefitinib, capecitabine, and celecoxib in patients with advanced solid tumors. Patients were treated with escalating doses of gefitinib once daily, capecitabine twice daily (14 of 28 days), and celecoxib twice daily. Plasma samples for biomarkers were obtained at baseline and weekly for the first 2 cycles. Pharmacokinetic variables were correlated with toxicity and presence of biological effect. Tumor biopsies from 5 patients were analyzed for changes in tumor metabolic activity by nuclear magnetic resonance spectroscopy. [¹⁸F]fluorodeoxyglucose positron emission tomography was done as a correlate in 6 patients at the MTD. Thirty-nine patients received 168 cycles of therapy. The dose-limiting toxicities observed included nausea, dehydration and nausea, diarrhea, and stomatitis. The MTD was 250 mg/d gefitinib (days 1-14) and 2,000 mg/m²/d capecitabine divided twice daily (days 8-21) every 28 days. Celecoxib was eliminated due to concerns of increased risk for cardiovascular toxicity, although no patients in this study had cardiac events. One patient with cholangiocarcinoma had a confirmed partial response. Fourteen of 39 (36%) patients maintained prolonged stable disease for a median of 4 months (range, 3-24 months). [¹⁸F]fluorodeoxyglucose positron emission tomography scan and metabolomic analyses revealed differences in metabolic response to gefitinib versus capecitabine. The combination of gefitinib and capecitabine is well tolerated and appears to have activity against certain advanced solid tumors, providing a rationale for further evaluation in advanced solid malignancies. [Mol Cancer Ther 2008;7(12):3685–94]

Although breast cancer molecular subtypes have been extensively defined by means of gene expression profiling over the past decade, little is known, at the proteomic level, as to how signaling pathways are differentially activated and serve to control proliferation in different breast cancer subtypes. We used reverse-phase protein arrays to examine phosphorylation status of 100 proteins in a panel of 30 breast cancer cell lines and showed distinct pathway activation differences between different subtypes that are not obvious from previous gene expression studies. We also show that basal levels of phosphorylation of key signaling nodes may have diagnostic utility in predicting response to selective inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. Finally, we show that reverse-phase protein arrays allow the parallel analysis of multiple pharmacodynamic biomarkers of response to targeted kinase inhibitors and that inhibitors of epidermal growth factor receptor and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase result in compensatory up-regulation of the phosphatidylinositol 3-kinase/Akt signaling pathway. [Mol Cancer Ther 2008;7(12):3695–706]

The microsomal antiestrogen-binding site (AEBS) is a high-affinity membranous binding site for the antitumor drug tamoxifen that selectively binds diphenylmethane derivatives of tamoxifen such as PBPE and mediates their antiproliferative properties. The AEBS is a hetero-oligomeric complex consisting of 3β-hydroxysterol-⁸-⁷-isomerase and 3β-hydroxysterol-⁷-reductase. High-affinity AEBS ligands inhibit these enzymes leading to the massive intracellular accumulation of zymostenol or 7-dehydrocholesterol (DHC), thus linking AEBS binding to the modulation of cholesterol metabolism and growth control. The aim of the present study was to gain more insight into the control of breast cancer cell growth by AEBS ligands. We report that PBPE and tamoxifen treatment induced differentiation in human breast adenocarcinoma cells MCF-7 as indicated by the arrest of cells in the G₀-G₁ phase of the cell cycle, the increase in the cell volume, the accumulation and secretion of lipids, and a milk fat globule protein found in milk. These effects were observed with other AEBS ligands and with zymostenol and DHC. Vitamin E abrogates the induction of differentiation and reverses the control of cell growth produced by AEBS ligands, zymostenol, and DHC, showing the importance of the oxidative processes in this effect. AEBS ligands induced differentiation in estrogen receptor-negative mammary tumor cell lines SKBr-3 and MDA-MB-468 but with a lower efficiency than observed with MCF-7. Together, these data show that AEBS ligands exert an antiproliferative effect on mammary cancer cells by inducing cell differentiation and growth arrest and highlight the importance of cholesterol metabolism in these effects. [Mol Cancer Ther 2008;7(12):3707–18]

p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (PI3K), specifically interacts with retinoblastoma protein (Rb) through the unique NH₂ terminus of p55PIK, N24. This interaction is critical for cell proliferation and cell cycle progression. To examine p55PIK as a potential target for cancer therapy, we generated an adenovirus expressing N24 (Ad-N24-GFP) and studied its effects on the proliferation of cultured cancer cells, including human colon (HT29) and thyroid (FTC236) cancer cells. Ad-N24-GFP blocked cell proliferation and induced cell cycle arrest in all cancer cell lines tested. N24 induced cell cycle arrest at G₀-G₁ phase in cell lines that expressed Rb. Interestingly, N24 inhibited cell proliferation by blocking cell cycle transition at both S and G₂-M phases in FTC236 cells, which did not express Rb. When Rb was knocked down by short hairpin RNA in HT29 cells, N24 also inhibited cell cycle progression at S and G₂-M phases, suggesting that p55PIK regulates cell cycle progression by Rb-dependent and Rb-independent mechanisms. Finally, Ad-N24-GFP markedly decreased the growth of xenograft tumors derived from HT29 and FTC236 cancer cells in athymic nude mice. Our data strongly suggest that N24 peptide is an effective anticancer therapy, which specifically inhibits PI3K signaling pathways mediated by p55PIK. Moreover, they show that the regulatory subunit of an enzyme, in addition to its catalytic subunit, can be an important target for drug development. [Mol Cancer Ther 2008;07(12):3719–28]

Hypoxia-inducible factor (HIF)-1 plays a key role in tumor promotion by inducing ~60 genes required for tumor adaptation to hypoxia; thus, it is viewed as a target for cancer therapy. For this reason, YC-1, which down-regulates HIF-1 and HIF-2 at the post-translational level, is being developed as a novel anticancer drug. We here found that YC-1 acts in a novel manner to inhibit HIF-1. In the Gal4 reporter system, which is not degraded by YC-1, YC-1 was found to significantly inactivate the COOH-terminal transactivation domain (CAD) of HIF-1, whereas it failed to inactivate CAD(N803A) mutant. In coimmunoprecipitation assays, YC-1 stimulated factor inhibiting HIF (FIH) binding to CAD even in hypoxia, whereas it failed to increase the cellular levels of hydroxylated Asn⁸⁰³ of CAD. It was also found that YC-1 prevented p300 recruitment by CAD in mammalian two-hybrid and coimmunoprecipitation assays. The involvement of FIH in YC-1-induced CAD inactivation was confirmed in EPO-enhancer and Gal4 reporter systems using FIH small interfering RNA and dimethyloxalylglycine FIH inhibitor. Indeed, FIH inhibition rescued HIF target gene expressions repressed by YC-1. In cancer cell lines other than Hep3B, YC-1 inhibits HIF-1 via the FIH-dependent CAD inactivation as well as via the protein down-regulation. Given these results, we suggest that the functional inactivation of HIF- contributes to the YC-1-induced deregulation of hypoxia-induced genes. [Mol Cancer Ther 2008;7(12):3729–38]

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to prevent colorectal tumorigenesis. Although antitumor effects of NSAIDs are mainly due to inhibition of cyclooxygenase activity, there is increasing evidence that cyclooxygenase-independent mechanisms may also play an important role. The early growth response-1 (EGR-1) gene is a member of the immediate-early gene family and has been identified as a tumor suppressor gene. Tolfenamic acid is a NSAID that exhibits anticancer activity in a pancreatic cancer model. In the present study, we investigated the anticancer activity of tolfenamic acid in human colorectal cancer cells. Tolfenamic acid treatment inhibited cell growth and induced apoptosis as measured by caspase activity and bioelectric impedance. Tolfenamic acid induced EGR-1 expression at the transcription level, and analysis of the EGR-1 promoter showed that a putative ETS-binding site, located at –400 and –394 bp, was required for activation by tolfenamic acid. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed that this sequence specifically bound to the ETS family protein epithelial-specific ETS-1 (ESE-1) transcription factor. Tolfenamic acid also facilitated translocation of endogenous and exogenous ESE-1 to the nucleus in colorectal cancer cells, and gene silencing using ESE-1 small interfering RNA attenuated tolfenamic acid-induced EGR-1 expression and apoptosis. Overexpression of EGR-1 increased apoptosis and decreased bioelectrical impedance, and silencing of endogenous EGR-1 prevented tolfenamic acid-induced apoptosis. These results show that activation of ESE-1 via enhanced nuclear translocation mediates tolfenamic acid-induced EGR-1 expression, which plays a critical role in the activation of apoptosis. [Mol Cancer Ther 2008;7(12):3739–50]

It is well recognized that nitric oxide (NO) is involved in tumor progression, including melanoma. Measurement of proliferative and metastatic capacity by MTS and Matrigel invasion assays, respectively, was done and showed that NO-treated melanoma cells exhibited a higher capacity compared with control, especially metastatic Lu1205 cells. Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1) is a multifunctional protein and its role in tumor biology has attracted considerable attention. To determine whether APE/Ref-1 plays a role in mediating NO stimulation of melanoma progression, we investigated the effect of DETA/NO on levels of APE/Ref-1 and related downstream targets [activator protein-1 (AP-1)/JunD, matrix metalloproteinase-1 (MMP-1), Bcl-2, and inducible nitric oxide synthase (iNOS)] by Western blot and reverse transcription-PCR analysis. Following DETA/NO treatment, APE/Ref-1 and other downstream molecules were induced. Knockdown of APE/Ref-1 or AP-1/JunD by specific small interfering RNA markedly reversed the induction by NO stress of target proteins. These results present evidence for the existence of a functional feedback loop contributing to progression and metastasis of melanoma cells. Resveratrol has been shown to be an APE/Ref-1 inhibitor and significant decreases in AP-1/JunD, MMP-1, Bcl-2, and iNOS protein levels occurred after exposure to resveratrol. This phenolic antioxidant may be an appropriate choice for combining with other compounds that develop resistance by up-regulation of these molecules. [Mol Cancer Ther 2008;7(12):3751–60]