Aberrant regulation of the MRP3 gene in non-small cell lung carcinoma.

INTRODUCTION: Multidrug-resistant protein-3 (MRP3), a membrane-bound transporter, facilitates efflux of toxic compounds, including certain chemotherapies, out of cells. Aberrant MRP3 expression has been linked to drug resistance in non-small cell lung carcinoma (NSCLC). We sought to determine if tumor MRP3 expression patterns correlate with the mutational status of upstream regulators, including nuclear factor erythroid-2-related factor 2 (Nrf2) and its functional repressor Keap1 in NSCLC cell lines and patient samples. METHODS: To identify putative Nrf2-binding sites in the MRP3 promoter and to evaluate Keap1, Nrf2, and p53 mutation status in four cell lines and 33 NSCLC surgically resected tumor specimens with regard to their impact on MRP3 levels. RESULTS: Chromatin immunoprecipitation analysis of the MRP3 promoter revealed an almost threefold increase in Nrf2 binding to the third putative Nrf2-binding sequence distal to the start site, demonstrating direct regulation of MRP3 by Nrf2. In NSCLC cell lines, elevated Nrf2 protein was observed in cell lines with increased MRP3 RNA expression. In patient tumor specimens, the presence of mutations in Keap1/Nrf2 correlated with MRP3 RNA levels (p < 0.05). p53 mutations were observed in 33% of cases, and all Keap1 mutant-positive tumors possessed a p53 mutation (n = 5; p = 0.0019). CONCLUSIONS: We demonstrate direct involvement between the transcription factor Nrf2 and the MRP3 promoter, which leads to the up-regulation of the MRP3 gene. In addition, we found a statistically significant correlation between the presence of Keap1/Nrf2 mutations and increased MRP3 messenger RNA levels in our NSCLC patient samples.

The HB22.7 Anti-CD22 monoclonal antibody enhances bortezomib-mediated lymphomacidal activity in a sequence dependent manner.

Most non-Hodgkin's lymphomas (NHL) initially respond to chemotherapy, but relapse is common and treatment is often limited by chemotherapy-related toxicity. Bortezomib, is a highly selective proteasome inhibitor with anti-NHL activity; it is currently FDA approved for second-line treatment of mantle cell lymphoma (MCL). Bortezomib exerts its activity in part through the generation of reactive oxygen species (ROS) and also by the induction of apoptosis.We previously validated CD22 as a potential target in treating NHL and have shown that the anti-CD22 ligand blocking antibody, HB22.7, has significant independent lymphomacidal properties in NHL xenograft models. We sought to determine whether or not these agents would work synergistically to enhance cytotoxicity. Our results indicate that treatment of NHL cell lines with HB22.7 six hours prior to bortezomib significantly diminished cell viability. These effects were not seen when the agents were administered alone or when bortezomib was administered prior to HB22.7. Additionally, HB22.7 treatment prior to bortezomib increased apoptosis in part through enhanced ROS generation. Finally, in a mouse xenograft model, administration of HB22.7 followed 24 hours later by bortezomib resulted in 23% smaller tumor volumes and 20% enhanced survival compared to treatment with the reverse sequence. Despite the increased efficacy of HB22.7 treatment followed by bortezomib, there was no corresponding decrease in peripheral blood cell counts, indicating no increase in toxicity. Our results suggest that pre-treatment with HB22.7 increases bortezomib cytotoxicity, in part through increased reactive oxygen species and apoptosis, and that this sequential treatment combination has robust efficacy in vivo.

Dual blockade of PKA and NF-κB inhibits H2 relaxin-mediated castrate-resistant growth of prostate cancer sublines and induces apoptosis.

We previously demonstrated that H2 relaxin (RLN2) facilitates castrate-resistant (CR) growth of prostate cancer (CaP) cells through PI3K/Akt/ß-catenin-mediated activation of the androgen receptor (AR) pathway. As inhibition of this pathway caused only ~50% reduction in CR growth, the goal of the current study was to identify additional RLN2-activated pathways that contribute to CR growth. Next-generation sequencing-based transcriptome and gene ontology analyses comparing LNCaP stably transfected with RLN2 versus LNCaP-vector identified differential expression of genes associated with cell proliferation (12.7% of differentially expressed genes), including genes associated with the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and nuclear factor-kappaB (NF-κB) pathways. Subsequent molecular analyses confirmed that the cAMP/PKA and NF-κB pathways play a role in facilitating H2 relaxin-mediated CR growth of CaP cells. Inhibition of PKA-attenuated RLN2-mediated AR activity inhibited proliferation and caused a small but significant increase in apoptosis. Combined inhibition of the PKA and NF-κB signaling pathways via inhibition of PKA and Akt induced significant apoptosis and dramatically reduced clonogenic potential, outperforming docetaxel, the standard of care treatment for CR CaP. Immunohistochemical analysis of tissue microarrays in combination with multispectral quantitative imaging comparing RLN2 levels in patients with benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia, and CaP determined that RLN2 is significantly upregulated in CaP vs BPH (p = 0.002). The combined data indicate RLN2 overexpression is frequent in CaP patients and provides a growth advantage to CaP cells. A near-complete inhibition of RLN2-induced CR growth can be achieved by simultaneous blockade of both pathways.

Multidrug-resistant protein-3 gene regulation by the transcription factor Nrf2 in human bronchial epithelial and non-small-cell lung carcinoma.

Multidrug-resistant proteins (MRPs) are members of the ATP-binding cassette superfamily that facilitate detoxification by transporting toxic compounds, including chemotherapeutic drugs, out of cells. Chemotherapy, radiation, and other xenobiotic stresses have been shown to increase levels of select MRPs, although the underlying mechanism remains largely unknown. Additionally, MRP3 is suspected of playing a role in the drug resistance of non-small-cell lung carcinoma (NSCLC). Analysis of the MRP3 promoter revealed the presence of multiple putative electrophile-responsive elements (EpREs), sequences that suggest possible regulation of this gene by Nrf2, the key transcription factor that binds to EpRE. The goal of this investigation was to determine whether MRP3 induction was dependent upon the transcription factor Nrf2. Keap1, a key regulator of Nrf2, sequesters Nrf2 in the cytoplasm, preventing entry into the nucleus. The electrophilic lipid peroxidation product 4-hydroxy-2-nonenal (HNE) has been shown to modify Keap1, allowing Nrf2 to enter the nucleus. We found that HNE up-regulated MRP3 mRNA and protein levels in cell lines with wild-type Keap1 (the human bronchial epithelial cell line HBE1 and the NSCLC cell line H358), but not in the Keap1-mutant NSCLC cell lines (A549 and H460). Cell lines with mutant Keap1 had constitutively higher MRP3 that was not increased by HNE treatment. In HBE1 cells, silencing of Nrf2 with siRNA inhibited induction of MRP3 by HNE. Finally, we found that silencing Nrf2 also increased the toxicity of cisplatin in H358 cells. The combined results therefore support the hypothesis that MRP3 induction by HNE involves Nrf2 activation.

Schedule-dependent apoptosis in K-ras mutant non-small-cell lung cancer cell lines treated with docetaxel and erlotinib: rationale for pharmacodynamic separation.

BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) given concurrently with chemotherapy in 4 large randomized clinical trials did not improve patient outcomes compared with chemotherapy alone in advanced non-small-cell lung cancer (NSCLC). We hypothesized that the lack of benefit resulted from a negative interaction between chemotherapy and EGFR TKIs. MATERIALS AND METHODS: Herein, we report the cell cycle and apoptotic effects of treatment with erlotinib and docetaxel in the NSCLC cell lines A549 and Calu-1, both of which are mutant for K-ras and wild-type for EGFR. RESULTS: Treatment with erlotinib resulted in accumulation of cells in G(1) phase in A549 cells, with no evidence of apoptosis. Docetaxel treatment led to apoptosis as assessed by increased sub-G1 DNA content and cleavage of caspase 3 and poly (ADP-ribose) polymerase. The sequence of docetaxel followed by erlotinib resulted in significantly enhanced apoptosis compared with single-agent docetaxel in both cell lines. However, in the reverse sequence of erlotinib followed by docetaxel, a reduction of apoptosis was observed. We hypothesize that cell cycle arrest induced by erlotinib accounts for these findings in the presence of wild-type EGFR and that pharmacodynamic separation of the 2 drug classes will ameliorate these effects. CONCLUSION: These studies provide a rationale for intermittent dosing of EGFR TKIs with chemotherapy in order to enhance cytotoxicity.