The human immunodeficiency virus protease inhibitor ritonavir inhibits lung cancer cells, in part, by inhibition of survivin.

INTRODUCTION: Ritonavir is a potential therapeutic agent in lung cancer, but its targets in lung adenocarcinoma are unknown, as are candidate biomarkers for its activity. METHODS: RNAi was used to identify genes whose expression affects ritonavir sensitivity. Synergy between ritonavir, gemcitabine, and cisplatin was tested by isobologram analysis. RESULTS: Ritonavir inhibits growth of K-ras mutant lung adenocarcinoma lines A549, H522, H23, and K-ras wild-type line H838. Ritonavir causes G0/G1 arrest and apoptosis. Associated with G0/G1 arrest, ritonavir down-regulates cyclin-dependent kinases, cyclin D1, and retinoblastoma protein phosphorylation. Associated with induction of apoptosis, ritonavir reduces survivin messenger RNA and protein levels more than twofold. Ritonavir inhibits phosphorylation of c-Src and signal transducer and activator of transcription protein 3, which are important events for survivin gene expression and cell growth, and induces cleavage of PARP1. Although knock down of survivin, c-Src, or signal transducer and activator of transcription protein 3 inhibits cell growth, only survivin knock down enhances ritonavir inhibition of growth and survivin overexpression promotes ritonavir resistance. Ritonavir was tested in combination with gemcitabine or cisplatin, exhibiting synergistic and additive effects, respectively. The combination of ritonavir/gemcitabine/cisplatin is synergistic in the A549 line and additive in the H522 line, at clinically feasible ritonavir concentrations (<10 µM). CONCLUSIONS: Ritonavir is of interest for lung adenocarcinoma therapeutics, and survivin is an important target and potential biomarker for its sensitivity. Ritonavir cooperation with gemcitabine/cisplatin might be explained by involvement of PARP1 in repair of cisplatin-mediated DNA damage and survivin in repair of gemcitabine-mediated double-stranded DNA breaks.

Prediction of postoperative recurrence-free survival in non-small cell lung cancer by using an internationally validated gene expression model.

PURPOSE: This study was performed to discover prognostic genomic markers associated with postoperative outcome of stage I to III non-small cell lung cancer (NSCLC) that are reproducible between geographically distant and demographically distinct patient populations. EXPERIMENTAL DESIGN: American patients (n = 27) were stratified on the basis of recurrence and microarray profiling of their tumors was performed to derive a training set of 44 genes. A larger Korean patient validation cohort (n = 138) was also stratified by recurrence and screened for these genes. Four reproducible genes were identified and used to construct genomic and clinicogenomic Cox models for both cohorts. RESULTS: Four genomic markers, DBN1 (drebrin 1), CACNB3 (calcium channel beta 3), FLAD1 (PP591; flavin adenine dinucleotide synthetase), and CCND2 (cyclin D2), exhibited highly significant differential expression in recurrent tumors in the training set (P < 0.001). In the validation set, DBN1, FLAD1 (PP591), and CACNB3 were significant by Cox univariate analysis (P ≤ 0.035), whereas only DBN1 was significant by multivariate analysis. Genomic and clinicogenomic models for recurrence-free survival (RFS) were equally effective for risk stratification of stage I to II or I to III patients (all models P < 0.0001). For stage I to II or I to III patients, 5-year RFS of the low- and high-risk patients was approximately 70% versus 30% for both models. The genomic model for overall survival of stage I to III patients was improved by addition of pT and pN stage (P < 0.0013 vs. 0.010). CONCLUSION: A 4-gene prognostic model incorporating the multivariate marker DBN1 exhibits potential clinical utility for risk stratification of stage I to III NSCLC patients.

Temporal quantitative proteomics by iTRAQ 2D-LC-MS/MS and corresponding mRNA expression analysis identify post-transcriptional modulation of actin-cytoskeleton regulators during TGF-beta-Induced epithelial-mesenchymal transition.

To gain insights into how TGF-beta regulates epithelial-mesenchymal transition (EMT), we assessed the time course of proteins and mRNAs during EMT by multiplex iTRAQ labeling and 2D-LC-MS/MS, and by hybridization, respectively. Temporal iTRAQ analysis identified 66 proteins as differentially expressed during EMT, including newly associated proteins calpain, fascin and macrophage-migration inhibitory factor (MIF). Comparing protein and mRNA expression overtime showed that all the 14 up-regulated proteins involved in the actin-cytoskeleton remodeling were accompanied by increases in corresponding mRNA expression. Interestingly, siRNA mediated knockdown of cofilin1 potentiated TGF-beta-induced EMT. Further analysis of cofilin1 and beta-actin revealed an increase in their mRNA stability in response to TGF-beta, contributing to the observed increase in mRNA and protein expression. These results are the first demonstration of post-transcriptional regulation of cytoskeletal remodelling and a key role for cofilin1 during TGF-beta-induced EMT.

Chemotherapeutic drugs induce PPAR-gamma expression and show sequence-specific synergy with PPAR-gamma ligands in inhibition of non-small cell lung cancer.

Preclinical studies have shown that peroxisome proliferator-activated receptor gamma (PPAR-gamma) ligands can exert antitumor effects against non-small cell lung cancer (NSCLC) and a variety of other cancers. In this study, we investigate the potential use of a PPAR-gamma ligand, troglitazone (Tro), in combination with either of two chemotherapeutic agents, cisplatin (Cis) or paclitaxel (Pac), for the treatment of NSCLC. In vitro, treatment of NSCLC cell lines with Tro potentiated Cis- or Pac-induced growth inhibition. The potentiation of growth inhibition was observed only when Cis or Pac treatment was followed by Tro and not vice versa, demonstrating a sequence-specific effect. Median effect analysis revealed a synergistic interaction between Tro and Cis in the inhibition of NSCLC cell growth and confirmed the sequence-specific effect. We also found that Cis or Pac up-regulated the expression of PPAR-gamma protein, accounting for the observed sequence-specific synergy. Similarly, experiments performed using a NSCLC xenograft model demonstrated enhanced effectiveness of combined treatment with Cis and PPAR-gamma ligands, Tro or pioglitazone. Tumors from Cis-treated mice also demonstrated enhanced PPAR-gamma expression. Together, our data demonstrate a novel sequence-specific synergy between PPAR-gamma ligands and chemotherapeutic agents for lung cancer treatment.

Effects of HIV protease inhibitor ritonavir on Akt-regulated cell proliferation in breast cancer.

PURPOSE: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. EXPERIMENTAL DESIGN: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)-positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. RESULTS: ER-positive estradiol-dependent lines (IC50, 12-24 micromol/L) and ER-negative (IC50, 45 micromol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-alpha levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 +/- 8 micromol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (K(D), 7.8 micromol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90alpha short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. CONCLUSIONS: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.

HPV16 E5 protein disrupts the c-Cbl-EGFR interaction and EGFR ubiquitination in human foreskin keratinocytes.

The E5 protein of human papillomavirus type 16 (HPV16) is a small hydrophobic protein, which localizes to the cell membrane, Golgi apparatus and endosomes. HPV16 E5 enhances the activation of the epidermal growth factor (EGFR). The activated EGFR is downregulated through the endocytic pathway, where E5 has been shown to inhibit endosomal acidification and trafficking. Ubiquitination of the activated EGFR plays a role in this downregulation. c-Cbl is a ubiquitin ligase that associates with the activated EGFR and targets it for degradation. Since E5 has been shown to form a complex with the EGFR, we tested the hypothesis that E5 affects the interaction of c-Cbl with the EGFR. We found a significant decrease of c-Cbl bound to the EGFR and of ubiquitinated EGFR in the presence of E5. E5 did not affect c-Cbl steady-state level, phosphorylation or translocation to the membrane. This novel result suggests that HPV16 E5 may, at least in part, upregulate EGFR-mediated signal transduction by inhibiting the interaction of c-Cbl with the EGFR, thereby decreasing c-Cbl-mediated degradation of the EGFR.

Expression of the unconventional myosin Myo1c alters sodium transport in M1 collecting duct cells.

Epithelial cells rely on proper targeting of cellular components to perform their physiological function. This dynamic process utilizes the cytoskeleton and involves movement of vesicles to and from the plasma membrane, thus traversing the actin cortical cytoskeleton. Studies support both direct interaction of actin with channels and an indirect mechanism whereby actin may serve as a track in the final delivery of the channel to the plasma membrane. Actin-dependent processes are often mediated via a member of the myosin family of proteins. Myosin I family members have been implicated in multiple cellular events occurring at the plasma membrane. In these studies, we investigated the function of the unconventional myosin I Myo1c in the M1 mouse collecting duct cell line. Myo1c was observed to be concentrated at or near the plasma membrane, often in discrete membrane domains. To address the possible role of Myo1c in channel regulation, we expressed a truncated Myo1c, lacking ATP and actin domains, in M1 cells and compared electrophysiological responses to control M1 cells, M1 cells expressing the empty vector, and M1 cells expressing the full-length Myo1c construct. Interestingly, cells expressing the Myo1c constructs had modulated antidiuretic hormone (ADH)-stimulated short-circuit current and showed little inhibition of short-circuit current with amiloride addition. Evaluation of enhanced green fluorescent protein-Myo1c constructs supports the importance of the IQ region in targeting the Myo1c to its respective cellular domain. These data are consistent with Myo1c participating in the regulation of the Na+ channel after ADH stimulation.

Calpain regulates enterocyte brush border actin assembly and pathogenic Escherichia coli-mediated effacement.

This study identifies calpain as being instrumental for brush border (BB) microvillus assembly during differentiation and effacement during bacterial pathogenesis. Calpain activity is decreased by 25-80% in Caco 2 lines stably overexpressing calpastatin, the physiological inhibitor of calpain, and the effect is proportional to the calpastatin/calpain ratio. These lines exhibit a 2.5-fold reduction in the rate of microvillus extension. Apical microvillus assembly is reduced by up to 50%, as measured by quantitative fluorometric microscopy (QFM) of ezrin, indicating that calpain recruits ezrin to BB microvilli. Calpain inhibitors ZLLYCHN2, MDL 28170, and PD 150606 block BB assembly and ezrin recruitment to the BB. The HIV protease inhibitor ritonavir, which inhibits calpain at clinically relevant concentrations, also blocks BB assembly, whereas cathepsin and proteasome inhibitors do not. Microvillus effacement is inhibited after exposure of calpastatin-overexpressing cells to enteropathogenic Escherichia coli. These results suggest that calpain regulates BB assembly as well as pathological effacement, and indicate that it is an important regulator involved in HIV protease inhibitor toxicity and host-microbial pathogen interactions.