Wenqing Qi

Isoform-specific expression of 14-3-3 proteins in human lung cancer tissues.

14‐3‐3 Proteins play important roles in a wide range of vital regulatory processes, including signal transduction, apoptosis, cell cycle progression and DNA replication. In mammalian cells, 7 14‐3‐3 isoforms (β, γ, ϵ, η, σ, θ and ζ) have been identified and each of these seems to have distinct tissue localizations and isoform‐specific functions. Previous studies have shown that 14‐3‐3 protein levels are higher in human lung cancers as compared to normal tissues. It is unclear, however, which of the 14‐3‐3 isoform(s) are overexpressed in these cancers. In our study, the levels of all seven 14‐3‐3 isoforms were examined by RT‐PCR and Western blotting. We show that the message for only two isoforms, 14‐3‐3ϵ and ζ, could be detected in normal tissues. In lung cancer biopsies, however, four isoforms, 14‐3‐3β, γ, σ, and θ, in addition to 14‐3‐3ϵ and ζ, were present in abundance. The expression frequency of 14‐3‐3β, γ, σ and θ isoforms was 11, 10, 13 and 8 of the 14 biopsies examined, respectively. The data from immunohistochemical staining and Western blotting were consistent with the RT‐PCR results. Given the prevalence of elevated 14‐3‐3 expression in human lung cancers we propose that these proteins may be involved in lung cancer tumorigenesis and that specific 14‐3‐3 proteins may be useful as markers for lung cancer diagnosis and targets for therapy.

Reduction of 14-3-3 Proteins Correlates with Increased Sensitivity to Killing of Human Lung Cancer Cells by Ionizing Radiation

Abstract Qi, W. and Martinez J. D. Reduction of 14-3-3 Proteins Correlates with Increased Sensitivity to Killing of Human Lung Cancer Cells by Ionizing Radiation. Radiat. Res. 160, 217–223 (2003). The 14-3-3 proteins have a wide range of ligands and are involved in a variety of biological pathways. Importantly, 14-3-3 proteins are known to be overexpressed in some human lung cancers, suggesting that they may play a role in tumorigenesis. Here we examined 14-3-3 expression in several lung cancer-derived cell lines and found that four of the seven 14-3-3 isoforms, β, ϵ, θ and ζ, were highly expressed in both lung cancer cell lines and normal lung fibroblasts. Two isoforms, σ and γ, were present only at very low levels. Immunoprecipitation data showed 14-3-3ζ could bind to CDC25C in irradiated A549 cells, and suppression of 14-3-3ζ in A549 cells with antisense resulted in a decrease in CDC25C localization in cytoplasm and CDC2 phosphorylation on Tyr15. As a consequence, CDC2 activity remained elevated which resulted in release from radiation-induced G2/M-phase arrest. Moreover, 16% 14-3-3ζ antisense-transfected cells underwent apoptosis when exposed to 10 Gy ionizing radiation. These data indicate that 14-3-3ζ is involved in G2 checkpoint activation and that inhibition of 14-3-3 may be a useful approach to sensitize human lung cancers to ionizing radiation.

Phase I pharmacokinetic and pharmacodynamic study of the pan-PI3K/mTORC vascular targeted pro-drug SF1126 in patients with advanced solid tumours and B-cell malignancies

BACKGROUND SF1126 is a peptidic pro-drug inhibitor of pan-PI3K/mTORC. A first-in-human study evaluated safety, dose limiting toxicities (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of SF1126, in patients with advanced solid and B-cell malignancies. PATIENTS AND METHODS SF1126 was administered IV days 1 and 4, weekly in 28day-cycles. Dose escalation utilised modified Fibonacci 3+3. Samples to monitor PK and PD were obtained. RESULTS Forty four patients were treated at 9 dose levels (90-1110 mg/m(2)/day). Most toxicity was grade 1 and 2 with a single DLT at180 mg/m(2) (diarrhoea). Exposure measured by peak concentration (C(max)) and area under the time-concentration curve (AUC(0-)(t)) was dose proportional. Stable disease (SD) was the best response in 19 of 33 (58%) evaluable patients. MTD was not reached but the maximum administered dose (MAD) was 1110 mg/m(2). The protocol was amended to enrol patients with CD20+ B-cell malignancies at 1110 mg/m(2). A CLL patient who progressed on rituximab [R] achieved SD after 2 months on SF1126 alone but in combination with R achieved a 55% decrease in absolute lymphocyte count and a lymph node response. PD studies of CLL cells demonstrated SF1126 reduced p-AKT and increased apoptosis indicating inhibition of activated PI3K signalling. CONCLUSION SF1126 is well tolerated with SD as the best response in patients with advanced malignancies.

Aurora inhibitor MLN8237 in combination with docetaxel enhances apoptosis and anti-tumor activity in mantle cell lymphoma.

Auroras (A and B) are oncogenic serine/threonine kinases that play key roles in the mitotic phase of the eukaryotic cell cycle. Analysis of the leukemia lymphoma molecular profiling project (LLMPP) database indicates Aurora over-expression correlates with poor prognosis. A tissue microarray (TMA) composed of 20 paired mantle cell lymphoma (MCL) patients demonstrated >75% of patients had high levels Aurora expression. Aurora A and B were also found elevated in 13 aggressive B-NHL cell lines. MLN8237, an Aurora inhibitor induced G2/M arrest with polyploidy and abrogated Aurora A and histone-H3 phosphorylation. MLN8237 inhibited aggressive B-NHL cell proliferation at an IC(50) of 10-50 nM and induced apoptosis in a dose- and time-dependent manner. Low dose combinations of MLN8237+docetaxel enhanced apoptosis by ~3-4-fold in cell culture compared to single agents respectively. A mouse xenograft model of MCL demonstrated that MLN8237 (10 or 30 mg/kg) or docetaxel (10mg/kg) alone had modest anti-tumor activity. However, MLN8237 plus docetaxel demonstrated a statistically significant tumor growth inhibition and enhanced survival compared to single agent therapy. Together, our results suggest that MLN8237 plus docetaxel may represent a novel therapeutic strategy that could be evaluated in early phase trials in relapsed/refractory aggressive B-cell NHL.

NSC348884, a nucleophosmin inhibitor disrupts oligomer formation and induces apoptosis in human cancer cells

Nucleophosmin (NPM), a multifunctional nucleolar phosphoprotein is dysregulated in human malignancies leading to anti-apoptosis and inhibition of differentiation. We evaluated the precise three-dimensional structure of NPM based on the highly conserved structure of Xenopus NO38 and its requirement to form dimers and pentamers via its N-terminal domain (residues, 1–107). We hypothesized that a small molecular inhibitor (SMI) that could disrupt the formation of dimers would inhibit aberrant NPM function(s) in cancer cells. Molecular modeling, pharmacophore design, in silico screening and interactive docking identified NSC348884 as a putative NPM SMI that disrupts a defined hydrophobic pocket required for oligomerization. NSC348884 inhibited cell proliferation at an IC50 of 1.7–4.0 μM in distinct cancer cell lines and disrupted NPM oligomer formation by native polyacrylamide gel electrophoresis assay. Treatment of several different cancer cell types with NSC348884 upregulated p53 (increased Ser15 phosphorylation) and induced apoptosis in a dose-dependent manner that correlated with apoptotic markers: H2AX phosphorylation, poly(ADP-ribose) polymerase cleavage and Annexin V labeling. Further, NSC348884 synergized doxorubicin cytotoxicity on cancer cell viability. The data together show that NSC348884 is an SMI of NPM oligomer formation, upregulates p53, induces apoptosis and synergizes with chemotherapy. Hence, an SMI to NPM may be a useful approach to anticancer therapy.

MP470, a novel receptor tyrosine kinase inhibitor, in combination with Erlotinib inhibits the HER family/PI3K/Akt pathway and tumor growth in prostate cancer

BackgroundProstate cancer is a common disease in men and at present there is no effective therapy available due to its recurrence despite androgen deprivation therapy. The epidermal growth factor receptor family (EGFR/HER1, HER2/neu and HER3)/PI3K/Akt signaling axis has been implicated in prostate cancer development and progression. However, Erlotinib, an EGFR tyrosine kinase inhibitor, has less effect on proliferation and apoptosis in prostate cancer cell lines. In this study, we evaluate whether MP470, a novel receptor tyrosine kinase inhibitor alone or in combination with Erlotinib has inhibitory effect on prostate cancer in vitro and in vivo.MethodsThe efficacy of MP470 or MP470 plus Erlotinib was evaluated in vitro using three prostate cancer cell lines by MTS and apoptosis assays. The molecular mechanism study was carried out by phosphorylation antibody array, immunoblotting and immunohistochemistry. A LNCaP mouse xenograft model was also used to determine the tumor growth inhibition by MP470, Erlotinib or the combination treatments.ResultsMP470 exhibits low μM IC50 in prostate cancer cell lines. Additive effects on both cytotoxicity and induction of apoptosis were observed when LNCaP were treated with MP470 in combination with Erlotinib. This combination treatment completely inhibited phosphorylation of the HER family members (HER1, 2, 3), binding of PI3K regulatory unit p85 to HER3 and downstream Akt activity even after androgen depletion. Furthermore, in a LNCaP mouse xenograft model, the MP470-Erlotinib combination produced 30–65% dose-dependent tumor growth inhibition (TGI).ConclusionWe propose that MP470-Erlotinib targets the HER family/PI3K/Akt pathway and may represent a novel therapeutic strategy for prostate cancer.

Aurora A inhibitor (MLN8237) plus Vincristine plus Rituximab is synthetic lethal and a potential curative therapy in aggressive B-cell non-Hodgkin lymphoma

Purpose: Aurora A and B are oncogenic serine/threonine kinases that regulate mitosis. Overexpression of Auroras promotes resistance to microtubule-targeted agents. We investigated mechanistic synergy by inhibiting the mitotic spindle apparatus in the presence of MLN8237 [M], an Aurora A inhibitor with either vincristine [MV] or docetaxel [MD] in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The addition of rituximab [R] to MV or MD was evaluated for synthetic lethality. Experimental Design: Aggressive B-NHL cell subtypes were evaluated in vitro and in vivo for target modulation and anti-NHL activity with single agents, doublets, and triplets by analyzing cell proliferation, apoptosis, tumor growth, survival, and mechanisms of response/relapse by gene expression profiling with protein validation. Results: MV is synergistic whereas MD is additive for cell proliferation inhibition in B-NHL cell culture models. Addition of rituximab to MV is superior to MD, but both significantly induce apoptosis compared with doublet therapy. Mouse xenograft models of mantle cell lymphoma showed modest single-agent activity for MLN8237, rituximab, docetaxel, and vincristine with tumor growth inhibition (TGI) of approximately 10% to 15%. Of the doublets, MV caused tumor regression, whereas TGI was observed with MD (approximately 55%–60%) and MR (approximately 25%–50%), respectively. Although MV caused tumor regression, mice relapsed 20 days after stopping therapy. In contrast, MVR was curative, whereas MDR led to TGI of approximately 85%. Proliferation cell nuclear antigen, Aurora B, cyclin B1, cyclin D1, and Bcl-2 proteins of harvested tumors confirmed response and resistance to therapy. Conclusions: Addition of rituximab to MV is a novel therapeutic strategy for aggressive B-NHL and warrants clinical trial evaluation. Clin Cancer Res; 18(8); 2210–9. ©2012 AACR.

Alisertib (MLN8237) an investigational agent suppresses Aurora A and B activity, inhibits proliferation, promotes endo-reduplication and induces apoptosis in T-NHL cell lines supporting its importance in PTCL treatment

Peripheral T-cell lymphomas (PTCL) are a diverse group of rare non-Hodgkin lymphomas (NHL) that carry a poor prognosis and are in need of effective therapies. Alisertib (MLN8237) an investigational agent that inhibits Aurora A Ser/Thr kinase has shown activity in PTCL patients. Here we demonstrate that aurora A and B are highly expressed in T-cell lymphoma cell lines. In PTCL patient samples aurora A was positive in 3 of 24 samples and co-expressed with aurora B. Aurora B was positive in tumor cells in 22 of 32 samples. Of the subtypes of PTCL, aurora B was over-expressed in PTCL (NOS) [73%], T-NHL [100%], ALCL (Alk-Neg) [100%] and AITL [100%]. Treatment with MLN8237 inhibited PTCL cell proliferation in CRL-2396 and TIB-48 cells with an IC50 of 80-100nM. MLN8237 induced endo-reduplication in a dose and time dependent manner in PTCL cell lines leading to apoptosis demonstrated by flow cytometry and PARP-cleavage at concentrations achieved in early phase clinical trials. Moreover, inhibition of HisH3 and aurora A phosphorylation was dose dependent and strongly correlated with endo-reduplication. The data provide a sound rationale for aurora inhibition in PTCL as a therapeutic modality and warrants clinical trial evaluation.

Overexpression of 14-3-3γ causes polyploidization in H322 lung cancer cells

The 14‐3‐3 proteins are a family of highly conserved proteins that participate in a wide variety of cellular processes. Mounting evidence suggests that 14‐3‐3 proteins have a role in human cancers, however their role in tumorigenesis is unclear. Here we report that over‐expression of 14‐3‐3 γ protein in human lung cancer cell line H322 results in abnormal DNA replication and polyploidization. Cells that overexpress 14‐3‐3 γ are resistant to microtubule inhibitors and can reenter the cell cycle in the absence of mitosis suggesting that elevated levels of 14‐3‐3 γ may enable cells to bypass the mitotic checkpoint. Taken together, our data indicate that 14‐3‐3γ may contribute to tumorigenesis by promoting genomic instability.

Caffeine Induces TP53-Independent G1-Phase Arrest and Apoptosis in Human Lung Tumor Cells in a Dose-Dependent Manner

Abstract Qi, W., Qiao, D. H. and Martinez, J. D. Caffeine Induces TP53-Independent G1-Phase Arrest and Apoptosis in Human Lung Tumor Cells in a Dose-Dependent Manner. Radiat. Res. 157, 166–174 (2002). Caffeine is a model radiosensitizing agent that is thought to work by abrogating the radiation-induced G2-phase checkpoint. In this study, we examined the effect that various concentrations of caffeine had on cell cycle checkpoints and apoptosis in cells of a human lung carcinoma cell line and found that a concentration of 0.5 mM caffeine could abrogate the G2-phase arrest normally seen after exposure to ionizing radiation. Surprisingly, at a concentration of 5 mM, caffeine not only induced apoptosis by itself and acted synergistically to enhance radiation-induced apoptosis, but also induced a TP53-independent G1-phase arrest. Examination of the molecular mechanisms by which caffeine produced these effects revealed that caffeine had opposing effects on different cyclin-dependent kinases. CDK2 activity was suppressed by caffeine, whereas activity of CDC2 was enhanced by suppressing phosphorylation on Tyr15 and by interfering with 14-3-3 binding to CDC25C. These data indicate that the effect of caffeine on cell cycle checkpoints and apoptosis is dependent on dose and that caffeine acts through differential regulation of cyclin-dependent kinase activity.