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Non-small-cell lung cancer (NSCLC), a subtype of lung cancer, is one of the leading causes of cancer death in both men and women around the world. Circulating tumour DNA (ctDNA), tyrosine kinase inhibitors (TKIs), and immunotherapy have transformed our understanding of NSCLC and its treatment, from diagnosis to targeted NSCLC therapeutics. Quantifying ctDNA is convenient and precise, making clinical decisions easier. TKI-based targeted therapy and immunotherapy have also enhanced the quality of life of NSCLC patients. This article gives an update on ctDNA technologies and their implications for therapeutic options, including medications targeting the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) with TKIs such as osimertinib and lorlatinib, the emergence of various resistance mechanisms, the control of programmed cell death-1 (PD- 1), programmed cell death ligand-1 (PD-L1), and cytotoxic T-lymphocyte antigen-4 (CTLA-4) by immune checkpoint inhibitors (ICIs) in immunotherapy and the blood tumor mutational burden (bTMB) calculated by ctDNA assay as a novel biomarker for immunotherapy. NSCLC patients, on the other hand, nevertheless confront numerous hurdles. To produce more effective medications or therapies to treat NSCLC, additional research and trials are needed.

Gastrointestinal stromal tumours (GISTs) are the most prevalent kind of gastrointestinal mesenchymal tumour . Gain-of-function mutations in KIT or plateletderived growth factor receptor (PDGFRA) cause the carcinogenesis of GISTs, resulting in constitutive activation of the tyrosine kinase and its downstream signalling cascades. The KIT/PDGFRA inhibitor imatinib is the standard of therapy for patients with metastatic GISTs, and oncogenic KIT or PDGFRA mutations are attractive therapeutic targets for the treatment of GISTs. However, clinical resistance to imatinib and other tyrosine kinase inhibitors develops in the majority of GIST patients. Five mechanisms of resistance have been characterized: (1) acquisition of a secondary point mutation in KIT or PDGFRA; (2) genomic amplification of KIT; (3) activation of an alternative receptor tyrosine kinase; (4) loss of KIT oncoprotein expression; and (5) wild-type GIST. Sunitinib is being used as a second-line therapy for individuals who have failed imatinib, and regorafenib has been approved for patients who have failed both imatinib and sunitinib . Phase II/III trials are currently in progress to evaluate novel inhibitors and immunotherapies targeting KIT, its downstream effectors such as phosphatidylinositol 3-kinase, protein kinase B and mammalian target of rapamycin, heat shock protein 90, and histone deacetylase inhibitor. ETV1, AXL, insulin-like growth factor 1 receptor, KRAS, FAS receptor, protein kinase c theta, ANO1 (DOG1), CDC37, and aurora kinase A have all been identified as potential targets. These candidates should be tested in the clinic as potential new GIST treatment targets.

Non-small-cell lung cancer (NSCLC), a main subtype of lung cancer, is one of the most common causes of cancer death in men and women worldwide. Circulating tumor DNA (ctDNA), tyrosine kinase inhibitors (TKIs) and immunotherapy have revolutionized both our understanding of NSCLC, from its diagnosis to targeted NSCLC therapies, and its treatment. ctDNA quantification confers convenience and precision to clinical decision making. Furthermore, the implementation of TKI-based targeted therapy and immunotherapy has significantly improved NSCLC patient quality of life. This review provides an update on the methods of ctDNA detection and its impact on therapeutic strategies; therapies that target epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) using TKIs such as osimertinib and lorlatinib; the rise of various resistant mechanisms; and the control of programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), and cytotoxic T-lymphocyte antigen-4 (CTLA-4) by immune checkpoint inhibitors (ICIs) in immunotherapy; blood tumor mutational burden (bTMB) calculated by ctDNA assay as a novel biomarker for immunotherapy. However, NSCLC patients still face many challenges. Further studies and trials are needed to develop more effective drugs or therapies to treat NSCLC.

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of KIT secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or IR shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from IR amplification, IR mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107–17. ©2017 AACR.

This article has been withdrawn by the authors. Some of the SDHA enzyme activity data were flawed and were not performed and analyzed correctly. The withdrawing authors are in the process of correcting the data and re-evaluating them for resubmission.

// Peipei Yang 1, * , Weicai Chen 1, * , Xuhui Li 2 , Grant Eilers 3 , Quan He 1 , Lili Liu 1 , Yeqing Wu 1 , Yuehong Wu 1 , Wei Yu 1 , Jonathan A. Fletcher 3 , Wen-Bin Ou 1, 2, 3 1 Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China 2 Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing, Zhejiang, China 3 Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA * These authors contributed equally to this work Correspondence to: Wen-Bin Ou, email: ouwenbin@tsinghua.org.cn Keywords: cyclin D1, MDM2, p53, upregulation, Nutlin-3 Received: January 10, 2016 Accepted: March 31, 2016 Published: April 26, 2016 ABSTRACT The MDM2-p53 pathway has a prominent oncogenic function in the pathogenesis of various cancers. Nutlin-3, a small-molecule antagonist of MDM2-p53 interaction, inhibits proliferation in cancer cells with wild-type p53. Herein, we evaluate the expression of MDM2, both the full length and a splicing variant MDM2-A, and the sensitivity of Nutlin-3 in different cancer cell lines. Included are seven cell lines with wild-type p53 (four mesothelioma, one breast cancer, one chondrosarcoma, and one leiomyosarcoma), two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one mesothelioma cell line harboring a p53 point mutation. Nutlin-3 treatment increased expression of cyclin D1, MDM2, and p53 in cell lines with wild-type p53. Additive effects were observed in cells containing wild-type p53 through coordinated attack on MDM2-p53 binding and cyclin D1 by lentivirual shRNA knockdown or small molecule inhibition, as demonstrated by immunoblots and cell viability analyses. Further results demonstrate that MDM2 binds to cyclin D1, and that an increase in cyclin D1 expression after Nutlin-3 treatment is correlated with expression and ubiquitin E3-ligase activity of MDM2. MDM2 and p53 knockdown experiments demonstrated inhibition of cyclin D1 by MDM2 but not p53. These results indicate that combination inhibition of cyclin D1 and MDM2-p53 binding warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53.

Receptor tyrosine kinase (RTK) anaplastic lymphoma kinase (ALK) serves a crucial role in brain development. ALK is located on the short arm of chromosome 2 (2p23) and exchange of chromosomal segments with other genes, including nucleophosmin (NPM), echinoderm microtubule-associated protein-like 4 (EML4) and Trk-fused gene (TFG), readily occurs. Such chromosomal translocation results in the formation of chimeric X-ALK fusion oncoproteins, which possess potential oncogenic functions due to constitutive activation of ALK kinase. These proteins contribute to the pathogenesis of various hematological malignancies and solid tumors, including lymphoma, lung cancer, inflammatory myofibroblastic tumors (IMTs), Spitz tumors, renal carcinoma, thyroid cancer, digestive tract cancer, breast cancer, leukemia and ovarian carcinoma. Targeting of ALK fusion oncoproteins exclusively, or in combination with ALK kinase inhibitors including crizotinib, is the most common therapeutic strategy. As is often the case for small-molecule tyrosine kinase inhibitors (TKIs), drug resistance eventually develops via an adaptive secondary mutation in the ALK fusion oncogene, or through engagement of alternative signaling mechanisms. The updated mechanisms of a variety of ALK fusions in tumorigenesis, proliferation and metastasis, in addition to targeted therapies are discussed below.

Oncogenic KIT or PDGFRA receptor tyrosine kinase (TK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is the standard of care for patients with metastatic GIST. However, approximately 10% of KIT-positive GIST metastases lose KIT expression at the time of clinical progression during imatinib therapy. In the present report, we performed TK-activation screens, using phosphotyrosine-TK double immunoaffinity purification and mass spectrometry, in GIST in vitro models lacking KIT expression. These studies demonstrated tyrosine-phosphorylated EGFR, AXL, and EPHA2 in four of six KIT-negative GIST lines (GIST62, GIST522, GIST54, GIST226, GIST48B, and GIST430B), and tyrosine-phosphorylated focal adhesion kinase (FAK) in each of the six KIT-negative lines. AXL expression was strong in KIT-negative or -weak clinical GIST samples that were obtained from progressing metastases during imatinib therapy. AXL knockdown inhibited viability in three KIT-negative GIST cell lines (GIST62, GIST54, and GIST522), but not in an AXL-negative, KIT-positive GIST control cell line (GIST430). AXL inhibition by R428, a specific AXL kinase inhibitor, reduced viability in AXL-activated GIST54. AXL knockdown in GIST62, GIST522, and GIST54 was accompanied by an increase in p21, p27, and p53 expression. By contrast, gefitinib-mediated EGFR inhibition, PF562271-mediated FAK inactivation, and shRNA-mediated knockdowns of EPHA2 and FAK had no effect on viability or colony formation of the KIT-negative GISTs. These findings highlight the potential relevance of AXL/p53 signaling as a therapeutic target in a subset of GISTs that have lost KIT oncoprotein expression.

Background Induced pluripotent stem cells (iPS) represent an innovative source for the standardized in vitro generation of macrophages (Mφ). Mφ show great promise in disease pathogenesis, particularly tuberculosis. However, there is no information about human iPS-derived (hiPS) macrophages (hiPS-Mφ) in response to tuberculosis infection. Methods In the present study, macrophages derived from hiPS were established via embryoid body (EB) formation by using feeder-free culture conditions, and the human monocyte cell line THP-1 (THP-1-Mφ) was used as control. iPS-Mφ were characterized by using morphology, Giemsa staining, nonspecific esterase staining (α-NAE), phagocytosis, and surface phenotype. Additionally, after treatment with Bacillus Calmette-Guérin (BCG) for 24 h, cell apoptosis was detected by using an Annexin V-FITC Apoptosis Detection assay. The production of nitric oxide (NO), expression of tumor necrosis factor alpha (TNF-α), activity of apoptosis-related protein cysteine-3 (Caspase-3) and expression of B-cell lymphoma-2 (Bcl-2) were analyzed. Results With respect to morphology, surface phenotype, and function, the iPS-Mφ closely resembled their counterparts generated in vitro from a human monocyte cell line. iPS-Mφ exhibited the typically morphological characteristics of macrophages, such as round, oval, fusiform and irregular characteristics. The cells were Giemsa-stained-positive, α-NAE-positive, and possessed phagocytic ability. iPS-Mφ express high levels of CD14, CD11b, CD40, CD68, and major histocompatibility complex II (MHC-II). Moreover, with regard to the apoptotic rate, the production of NO, expression of TNF-α, and activity of Caspase-3 and Bcl-2, iPS-Mφ closely resemble that of their counterparts generated in vitro from human monocyte cell line in response to BCG infection. The rate of apoptosis of BCG-treated iPS-Mφ was 37.77 ± 7.94% compared to that of the untreated group at 4.97 ± 1.60% (P < 0.01) by using Annexin V-FITC Apoptosis Detection. Additionally, the rate of apoptosis of BCG-treated THP-1-Mφ was 37.1 ± 2.84% compared to that of the untreated group at 6.19 ± 1.68% (P < 0.001). The expression of TNF-α and the production of NO were significantly increased (P < 0.001), and the activity of Caspase-3 was increased. However, the expression of Bcl-2 was inhibited (P < 0.001). Conclusions Our results demonstrate that Mφ derived from hiPS perform the immunological function in response to Bacillus Calmette-Guérin infection by undergoing apoptosis, increasing the production of NO and expression of TNF-α. Thus, our study may help to overcome the limitations of research into certain rare diseases due to the lack of adequate supply of disease-specific primary cells. Electronic supplementary material The online version of this article (10.1186/s13287-018-0800-x) contains supplementary material, which is available to authorized users.

The MDM2-p53 pathway plays a prominent role in well-differentiated liposarcoma (LPS) pathogenesis. Here, we explore the importance of MDM2 amplification and p53 mutation in LPS independently, to determine whether HDACi are therapeutically useful in LPS. We demonstrated that simultaneous knockdown of MDM2 and p53 in p53-mutant LPS lines resulted in increased apoptosis, anti-proliferative effects, and cell cycle arrest, as compared to either intervention alone. HDACi treatment resulted in the dephosphorylation and depletion of MDM2 and p53 without affecting CDK4 and JUN expression, irrespective of p53 mutational status in MDM2-amplified LPS. In control mesothelioma cell lines, HDACi treatment resulted in down-regulation of p53 in the p53 mutant cell line JMN1B, but resulted in no changes of MDM2 and p53 in two mesothelioma lines with normal MDM2 and wild-type p53. HDACi treatment substantially decreased LPS and mesothelioma proliferation and survival, and was associated with upregulation of PTEN and p21, and inactivation of AKT. Our findings indicate that wild-type p53 depletion by HDACi is MDM2 amplification-dependent. These findings underscore the importance of targeting both MDM2 and p53 in LPS and other cancers harboring p53 mutations. Moreover, the pro-apoptotic and anti-proliferative effect of HDACi warrants further evaluation as a therapeutic strategy in MDM2-amplified LPS.

Creatine kinase plays a key role in the energy homeostasis of vertebrate cells. Creatine kinase B (CKB), a cytosolic isoform of creatine kinase, shows upregulated expression in a variety of cancers. In this research, we confirmed that some ovarian cancer tissues had elevated CKB expression at the protein level. The functions of CKB in ovarian cancer progression were investigated in the ovarian cancer cell line Skov3, which has a high CKB expression. It was found that CKB knockdown inhibited Skov3 cell proliferation and induced apoptosis under hypoxia or hypoglycemia conditions. CKB depletion also sensitized Skov3 to chemotherapeutic agents. Furthermore, the CKB knockdown reduced glucose consumption and lactate production, and increased ROS production and oxygen consumption. This suggested that CKB knockdown decreased cytosolic glycolysis and resulted in a tumor suppressive metabolic state in Skov3 cells. Consequently, we found that the knockdown of CKB induced G2 arrest in cell cycle by elevating p21 expression and affected the PI3K/Akt and AMPK pathways. These findings provide new insights in the role of CKB in cancer cell survival and tumor progression. Our results also suggest that CKB depletion/inhibition in combination with chemotherapeutic agents might have synergistic effects in ovarian cancer therapy.

Background: Improved mesothelioma patient survival will require development of novel and more effective pharmacological interventions. TP53 genomic mutations are uncommon in mesothelioma, and recent data indicate that p53 remains functional, and therefore is a potential therapeutic target in these cancers. In addition, the tumour suppressor NF2 is inactivated by genomic mechanisms in more than 80% of mesothelioma, causing upregulation of FAK activity. Because FAK is a negative regulator of p53, NF2 regulation of FAK–p53–MDM2 signalling loops were evaluated. Methods: Interactions of FAK–p53 or NF2–FAK were evaluated by phosphotyrosine-p53 immunoaffinity purification and tandem mass spectrometry, and p53, FAK, and NF2 immunoprecipitations. Activation and/or expression of FAK, p53, and NF2 were also evaluated in mesotheliomas. Effects of combination MDM2 and FAK inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, expression of cell cycle checkpoints, and cell cycle alterations. Results: We observed constitutive activation of FAK, a known negative regulator of p53, in each of 10 mesothelioma cell lines and each of nine mesothelioma surgical specimens, and FAK was associated with p53 in five of five mesothelioma cell lines. In four mesotheliomas with wild-type p53, FAK silencing by RNAi induced expression and phosphorylation of p53. However, FAK regulation of mesothelioma proliferation was not restricted to p53-dependent pathways, as demonstrated by immunoblots after FAK knockdown in JMN1B mesothelioma cells, which have mutant/inactivated p53, compared with four mesothelioma cell lines with nonmutant p53. Additive effects were obtained through a coordinated reactivation of p53, by FAK knockdown/inhibition and MDM2 inhibition, as demonstrated by immunoblots, cell viability, and cell-cycle analyses, showing increased p53 expression, apoptosis, anti-proliferative effects, and cell-cycle arrest, as compared with either intervention alone. Our results also indicate that NF2 regulates the interaction of FAK–p53 and MDM2–p53. Conclusions: These findings highlight novel therapeutic opportunities in mesothelioma.

The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR) and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3) in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90). Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17demethoxygeldanamycin (17-AAG) had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of down-stream signaling intermediates (AKT, mitogen-activated protein kinase, and S6); upregulated the p53, p21, and p27 cell cycle checkpoints; induced G2 phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G1 apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.

Mesothelioma is an asbestos-associated and notoriously chemotherapy-resistant neoplasm. Activation of the receptor tyrosine kinases (RTKs), epidermal growth factor receptor and MET, has been described in subsets of mesothelioma, suggesting that TKs might represent therapeutic targets in this highly lethal disease. We employed proteomic screening by phosphotyrosine immunoaffinity purification and tandem mass spectrometry to characterize RTK activation in mesothelioma cell lines. These assays demonstrated expression and activation of the AXL protein, which is an RTK with known oncogenic properties in non-mesothelial cancer types. AXL was expressed and activated strongly in 8 of 9 mesothelioma cell lines and 6 of 12 mesothelioma biopsies, including each of 12 mesotheliomas with spindle-cell histology. Somatic AXL mutations were not found, but all mesotheliomas expressed an alternatively spliced AXL transcript with in-frame deletion of exon 10, and six of seven mesothelioma cell lines expressed the AXL ligand, growth arrest-specific 6 (GAS6). GAS6 expression appeared to be functionally relevant, as indicated by modulation of AXL tyrosine phosphorylation by knockdown of endogeneous GAS6, and by administration of exogenous GAS6. AXL silencing by lentivirus-mediated short hairpin RNA suppressed mesothelioma migration and cellular proliferation due to G1 arrest. The AXL inhibitor DP-3975 inhibited cell migration and proliferation in mesotheliomas with strong AXL activation. DP-3975 response in these tumors was characterized by inhibition of PI3-K/AKT/mTOR and RAF/MAPK signaling. AXL inhibition suppressed mesothelioma anchorage-independent growth, with reduction in colony numbers and size. These studies suggest that AXL inhibitors warrant clinical evaluation in mesothelioma.

Gastrointestinal stromal tumors are clinically distinct mesenchymal tumors, which generally result from expression of mutant KIT or PDGFRA receptor tyrosine kinase oncogenes. Most gastrointestinal stromal tumors feature strong expression of KIT that serves as a crucial diagnostic adjunct. However, a subset of tumors lacks KIT expression and otherwise may also be difficult to distinguish from other sarcomas, including leiomyosarcoma. Because various carbonic anhydrase isozymes have been identified as potential treatment targets against different cancers, we evaluated carbonic anhydrase II expression in 175 gastrointestinal stromal tumors. Western blotting experiments indicated that carbonic anhydrase II is highly expressed in gastrointestinal stromal tumor cell lines. Immunohistochemically, 95% of gastrointestinal stromal tumors showed positive signal. The carbonic anhydrase II expression in gastrointestinal stromal tumors did not correlate with particular KIT or PDGFRA mutation types. Carbonic anhydrase II immunoreactivity was absent or low in other mesenchymal tumor categories analyzed. High carbonic anhydrase II expression was associated with a better disease-specific survival rate than low or no expression (Mantel-Cox test, p

Oncogenic KIT or PDGFRA receptor tyrosine kinase mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is standard of care for patients with metastatic GIST. However, most of these patients eventually develop clinical resistance to imatinib and other KIT/PDGFRA kinase inhibitors and there is an urgent need to identify novel therapeutic strategies. We reported previously that protein kinase C-theta (PKCtheta) is activated in GIST, irrespective of KIT or PDGFRA mutational status, and is expressed at levels unprecedented in other mesenchymal tumors, therefore serving as a diagnostic marker of GIST. Herein, we characterize biological functions of PKCtheta in imatinib-sensitive and imatinib-resistant GISTs, showing that lentivirus-mediated PKCtheta knockdown is accompanied by inhibition of KIT expression in three KIT+/PKCtheta+ GIST cell lines, but not in a comparator KIT+/PKCtheta- Ewing's sarcoma cell line. PKCtheta knockdown in the KIT+ GISTs was associated with inhibition of the phosphatidylinositol-3-kinase/AKT signaling pathway, upregulation of the cyclin-dependent kinase inhibitors p21 and p27, antiproliferative effects due to G(1) arrest and induction of apoptosis, comparable to the effects seen after direct knockdown of KIT expression by KIT short-hairpin RNA. These novel findings highlight that PKCtheta warrants clinical evaluation as a potential therapeutic target in GISTs, including those cases containing mutations that confer resistance to KIT/PDGFRA kinase inhibitors.

Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 μm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 μm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (

Chaperone GroEL was treated with different concentrations of hydrogen peroxide. The conformational states of GroEL were monitored by protein intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate fluorescence, and far-UV CD measurements. The results show that GroEL has unusual ability to resist oxidative stress. GroEL kept its quaternary structure and activity even when treated with 10 mM hydrogen peroxide. Two fragments were formed when GroEL was treated with high concentrations of hydrogen peroxide (more than 20 mM). It is suggested that GroEL, as a molecular chaperone, is related to oxidative process in vivo.

The effects of osmolytes, including dimethysulfoxide, sucrose, glycine and proline, on the unfolding and inactivation of guanidine-denatured creatine kinase were studied by observing the fluorescence emission spectra, the CD spectra and the inactivation of enzymatic activity. The results showed that low concentrations of dimethysulfoxide (

Background Ovarian cancer has the highest mortality rate of all gynecologic malignancy. The receptor tyrosine kinases (RTKs), including EGFR, ERBB2, PDGFR, VEGFR and MET, are activated in subsets of ovarian cancer, suggesting that these kinases might represent novel therapeutic targets. However, clinical trials have not or just partially shown benefit to ovarian cancers treated with EGFR, ERBB2, or PDGFR inhibitors. Despite multiple RTK activation in ovarian cancer pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. We hypothesized that a coordinated network of multi-RTK activation is important for the tumorigenesis of ovarian cancers. Results Herein, we demonstrate co-activation of multiple RTKs (EGFR, ERBB2, ERBB4, MET and/or AXL) in individual ovarian cancer cell lines and primary tumors. We also show that coordinate inhibition of this multi-kinase signaling has substantially greater effect on ovarian cancer proliferation and survival, compared to inhibition of individual activated kinases. The inhibition of this multi-RTK signaling by HSP90 suppression results in profound pro-apoptotic and anti-proliferative effects, and is associated with the inactivation of RTK downstream PI3-K/AKT/mTOR and RAF/MAPK signaling. Conclusion These studies suggest that anti-multiple RTK strategy could be useful in the treatment of ovarian cancer.

Renal Medullary Carcinoma (RMC) is an aggressive malignancy that affects young black individuals with sickle cell trait. No effective treatment is available, resulting in an ominous clinical course, with overall survival averaging less than four months. We report rearrangement of the ALK receptor tyrosine kinase in a pediatric case of RMC harboring a t(2;10)(p23;q22) translocation. Mass spectrometry-based proteomic evaluation identified a novel ALK oncoprotein in which the cytoskeletal protein vinculin (VCL) was fused to the ALK kinase domain. The resulting VCL-ALK fusion does not contain known self-association domains, but includes the talin binding domains of vinculin. We demonstrate coprecipitation of strongly tyrosine phosphorylated talins with the VCL-ALK oncoprotein, suggesting that ALK oncogenic crossphosphorylation is mediated by interactions between neighboring VCL-ALK proteins on a talin scaffold. This report widens the spectrum of ALK-related tumors and ALK fusion partners, and provides a rationale for treating RMC with targeted ALK inhibitors.

Background: Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11- cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear. Principal Findings: We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 49-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108–135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees. Significance: Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III., National Institutes of Health (U.S.) (grant GM28289), National Eye Institute (Grant Grant EY11716), National Science Foundation (U.S.) (grant EIA-0225609)

The MDM2-p53 pathway plays a prominent role in various cancer pathogenesis. Nutlin-3, a small-molecule antagonist of MDM2, inhibits proliferation in cancer cells with wild-type p53 by blocking the interaction of MDM2 and p53. However, different cancer cells differ greatly sensitivity of Nutlin-3 because of MDM2 amplification and/or p53 mutation status. Herein, we evaluate the sensitivity of Nutlin-3 in different cancer cell lines including four mesothelioma cell lines, one breast cancer line, and one leiomyosarcoma cell line with nonmutant p53 expression, two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one control mesothelioma cell line JMN1B harboring p53 point mutation. Unexpectedly, Nutlin-3 treatment induced cyclin D1 expression accompanied with inhibition of MDM2-p53 interaction in nonmutant cancer cell lines. Additive effects were obtained through a coordinated attack on MDM2-p53 interaction and cyclin D1, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that lentiviral mediated cyclin D1 knockdown or drug inhibition, and block of MDM2-p53 interaction, in cancer cell lines containing wild-type p53, induced greater anti-proliferative effects, compared to either intervention alone. Further results demonstrated that upregulation of cyclin D1 after Nutlin-3 treatment to some extent was dependent on expression and ubiquitin E3-ligase activity of MDM2. These compelling anti-proliferative responses indicate that combination inhibition of cyclin D1 and MDM2-p53 interaction warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53. Citation Format: Wenbin Ou, Weicai Chen, Jiaqing Zhu, Grant Eilers, Xuhui Li, Peipei Yang, Hailong Li, Fanguo Meng, Jonathan Fletcher. Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2707. doi:10.1158/1538-7445.AM2015-2707

Fatty acid synthase (FASN), a key enzyme for de novo lipogenesis, is overexpressed in many malignant tumours and is associated with aggressive biological behaviour. FASN expression and its possible relationship with more aggressive behaviour in gastrointestinal stromal tumours (GISTs) have not been addressed to date. Here, FASN expression was assessed by immunohistochemistry in 60 primary GISTs (28 low/intermediate risk and 32 high risk) and seven metastatic GISTs. Sixteen smooth muscle gastrointestinal tumours were used as controls. FASN was overexpressed in 36 of 60 GISTs (60%): in 12 of 28 (42%) low/intermediate-risk GISTs and in 24 of 32 (75%) high-risk GISTs (p

The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H 2 O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK.

The lactic acid induced unfolding and the salt-induced folding of creatine kinase (CK) were studied by enzyme activity, fluorescence emission spectra, circular dichroism spectra, and native polyacrylamide gel electrophoresis. The results showed that the kinetics of CK inactivation was a monophase process. Lactic acid caused inactivation and unfolding of CK with no aggregation during CK denaturation. The unfolding of the whole molecule and the inactivation of CK in solutions of different concentration of lactic acid were compared. Much lower lactic acid concentration values were required to bring about inactivation than were required to produce significant conformational changes of the enzyme molecule. At higher concentrations of lactic acid (more than 0.2 mM) the CK dimers were partially dissociated, as proved by native polyacrylamide gel electrophoresis. NaCl induced the molten globule state with a compact structure after CK was denatured with 0.8 mM lactic acid, and the increasing of anions led to a tight side-chain. The above results suggest that the effect of lactic acid differed from that of other denaturants such as guanidine hydrochloride, HCl, or urea during CK folding, and the molten globule state indicates that intermediates exist during CK folding.Key words: lactic acid, creatine kinase, salt-induced, unfolding, molten globule state.

The aspartate (Asp)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, circular dichroism (CD) spectra, native polyacrylamide gel electrophoresis and ultraviolet difference spectra. The results showed that Asp caused inactivation and unfolding of CK, with no aggregation during CK denaturation. The kinetics of CK unfolding followed a one phase process. At higher concentrations of Asp (>2.5 mM), the CK dimers were partially dissociated. Inactivation occurred before noticeable conformational change during CK denaturation. Asp denatured CK was mostly reactivated and refolded by dilution. KCl induced the molten globule state with compact structure after CK was denatured with 10 mM Asp. These results suggest that the effect of Asp differed from that of other denaturants such as guanidine, HCl or urea during CK unfolding. Asp is a reversible protein denaturant and the molten globule state indicates that intermediates exist during CK folding.

The influence of osmolytes, including dimethysulfoxide, glycine, proline and sucrose, on the refolding and reactivation courses of guanidine-denatured creatine kinase was studied by fluorescence emission spectra, circular dichroism spectra, recovery of enzymatic activity and aggregation. The results showed that low concentrations of dimethysulfoxide (

Liposarcoma (LPS) is the most common sarcoma of humans. There are no systemic therapeutic regimens known to improve survival when complete surgical resection is not feasible, underscoring the need for an improved molecular understanding of LPS to stimulate the development of effective targeted therapies. The MDM2-p53 pathway plays a prominent role in WDLPS pathogenesis, with the vast majority of human tumors harboring either MDM2 amplifications or p53 mutations. Nutlin-3, a small-molecular antagonist of MDM2, inhibits cell proliferation via blocking the interaction of MDM2-p53 in LPS with wild-type p53. We wonder whether MDM2 overexpression due to amplification or/and mutant p53 still plays a crucial oncogenic role in LPS containing p53 mutation. Herein, we developed isogenic liposarcoma lines in which the parental forms were MDM2 amplification and p53 wild-type, whereas sublines had mutation p53 expression, showed Nutlin-3 resistance. mRNA sequencing and immunoblotting showed that MDM2 and CDK4 were overexpression in the parental lines and sublines, whereas MDM2 and CDK4 expression was low in mesothelioma and GIST cell lines. HDAC inhibitor (HDACi, SAHA and LBH589) treatment resulted in the dephosphorylation and degradation of MDM2 and p53, but little affected CDK4 and JUN expression, irrespective of p53 mutational status in LPS with MDM2 amplification, and in a mesothelioma cell line JMN1B with p53 mutation, but not two mesothelioma lines containing normal MDM2 level and wild-type p53. Our findings indicate that regulation of wild-type 53 degradation by HDACi is MDM2 amplification-dependent. HDAC inhibition by SAHA and LBH589 had a substantially effect on LPS and mesothelioma proliferation and survival associated with upregulation of the PTEN and p21, inhibition of cell proliferation marker cyclin A and PCNA expression, induction of G1 or G2 phase arrest; induction of apoptosis showing an increase of caspase 3/7 activity and expression, PARP cleavage, and the sub-G1 apoptotic population. Moreover, we characterize biological functions of MDM2-p53 axis in nutlin-3-sensitive and nutlin-3-resistant LPS, showing MDM2 knockdown in the four LPS lines and p53 knockdown in the three mutant-p53 LPS lines resulted in anti-proliferative effects. Additive effects were obtained through a coordinated attack on MDM2 and p53, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that MDM2 and p53 knockdown, in LPS cell lines containing the p53 mutation, induced greater cell apoptosis and anti-proliferative effects, compared to either intervention alone, which is comparable to the effects seen after HDAC inhibition by SAHA and LBH589. These compelling pro-apoptotic and anti-proliferative responses indicate that HDAC inhibition warrants clinical evaluation as a novel therapeutic strategy in LPS, including nutlin-3 resistant sublines with the p53 mutation. Note: This abstract was not presented at the meeting. Citation Format: Wen-bin Ou, Hailong Li, Li Liu, Shengmei Zhou, Ye Kuang, Ziqin Yan, Zhe Jiang, Si Shi, Fanguo Meng, Qing Sheng, Haimeng Zhou, Jonathan A. Fletcher. HDACi inhibits liposarcoma via targeting of the MDM2-p53 signaling axis and PTEN, irrespective of p53 mutational status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5136. doi:10.1158/1538-7445.AM2014-5136

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.

Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with this asbestos-associated disease. The receptor tyrosine kinases EGFR and MET are activated in subsets of mesothelioma, suggesting these kinases might represent novel therapeutic targets. However, clinical trials have not shown activity for EGFR inhibitors in mesothelioma. We had previously demonstrated activation of multiple receptor tyrosine kinases, including EGFR, MET, and AXL, in individual mesothelioma cell lines, targeting HSP90 suppressed the multiple activated RTKs in mesothelioma cell lines, resulting in apo-apoptotic and anti-proliferative effects. Thus, we hypothesized that a coordinated network of multi-RTK activation contributes collectively to activate PI3-K/AKT/mTOR and/or RAF/MAPK signalings for the tumorigenesis of mesothelioma. Herein, we demonstrate abnormal activation of the PI3-K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, as compared with normal mesothelial cells. Multiple receptor tyrosine kinases, including EGFR, MET, and AXL, in individual mesothelioma cell lines, collectively activated survival signaling intermediate AKT, but not MAPK. The anti-proliferative responses of the coordinate inhibition of these multi-kinase signaling were comparable with suppression of PI3-K/AKT/mTOR pathway. Dual targeting AKT/mTOR signaling has substantially greater effect on mesothelioma proliferation and survival, compared to inhibition of individual activated kinases and downstream signaling alone, suggesting that no one activated RTK is the predominant oncogenic control mechanism in mesothelioma and PI3-K/AKT/mTOR is a crucial survival pathway. The AKT/mTOR signaling inhibition by BEZ235 resulting in pro-apoptotic and anti-proliferative effects in mesothelioma, was associated with the blockage of MDM2-p53 interaction. Citation Format: Shengmei Zhou, Li Liu, Weijiang Hu, Hailong Li, Fanguo Meng, Haimeng Zhou, Jonathan A. Fletcher, Wenbin Ou. Dual targeting of the AKT/mTOR signaling pathwayinhibits mesothelioma: Targeted therapies against multiple activated receptor tyrosine kinases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2168. doi:10.1158/1538-7445.AM2013-2168

Ovarian cancer is a leading cause of cancer death among women in Western Europe and the United States, which has the highest mortality rate of all gynecologic malignancy. Although more than 70% patients have increased 5-year survival rates after surgery followed by chemotherapy and second-line therapies, the low overall cure rates and the intolerable side effects of systemic chemotherapy asks for the development of novel and more effective pharmacological interventions. The increasing evidences suggest that receptor tyrosine kinase (RTK) activation participates in the oncogenic progression from nonneoplastic mesothelial lining of the ovaries or the fallopian tube epithelium to epithelial ovarian cancer. The RTKs, including EGFR, ERBB2, PDGFR, VEGFR and MET, are activated in subsets of ovarian cancer, suggesting that these kinases might represent novel therapeutic targets. However, clinical trials have not or just partially shown benefit to ovarian cancers treated with EGFR, ERBB2, or PDGFR inhibitors. Despite multiple RTK activation in ovarian cancer pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. We hypothesized that a coordinated network of multi-RTK activation is important for the tumorigenesis of ovarian cancers. Herein, we demonstrate co-activation of multiple RTKs (EGFR, ERBB2, ERBB4, MET and/or AXL) in individual ovarian cancer cell lines and primary tumors. We also show that coordinate inhibition of this multi-kinase signaling has substantially greater effect on ovarian cancer proliferation and survival, compared to inhibition of individual activated kinases. The inhibition of this multi-RTK signaling by HSP90 suppression results in profound pro-apoptotic and anti-proliferative effects, and is associated with the inactivation of RTK downstream PI3-K/AKT/mTOR and RAF/MAPK signaling. These studies suggest that anti-multiple RTK strategy could be useful in the treatment of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1092. doi:1538-7445.AM2012-1092

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is standard of care for patients with metastatic GIST. However, most patients eventually develop clinical resistance to imatinib and other KIT/PDGFRA kinase inhibitors due to acquisition of secondary mutations in the KIT kinase domain, genomic amplification of KIT, or activation of alternate oncoproteins. Approximately 10% of KIT-dependent GIST metastases lose KIT expression at time of clinical progression during imatinib therapy. We performed whole transcriptome sequencing in KIT-expressing GIST vs. KIT-negative GIST, and thereby identified strong cyclin D1 expression only in KIT-negative GISTs. KIT-negative GISTs showed complete loss of the GIST biomarker, PKCθ, and restoration of PKCθ expression in these cells resulted in loss of Cyclin D1 expression. Lentivirus-mediated CCND1 (Cyclin D1) short hairpin RNA (shRNA) knockdown resulted in profound anti-proliferative and pro-apoptotic effects in KIT-negative GIST cell lines, and was associated with activation (dephosphorylation) of the tumor suppressor Rb and upregulation of the p27 CDK inhibitor. c-Jun expression levels paralleled the Cyclin D1 expression in the GIST lines, and c-Jun shRNA knockdown resulted in downregulation of cyclin D1 in the KIT-negative GIST lines, and was accompanied by profound anti-proliferative effects, including p27 upregulation, and pro-apoptotic effects as evidenced by PARP cleavage. By contrast, cyclin D1 and c-Jun silencing showed little anti-proliferative and pro-apoptotic effects in KIT-positive, KIT-dependent GIST lines. These findings show that cyclin D1 and c-Jun overexpression serve a crucial oncogenic role in KIT-independent GISTs, including those enabling clinical resistance to imatinib or sunitinib targeted kinase inhibitor therapies. Our findings also show that PKCθ downregulation and c-Jun upregulation coordinately enable cyclin D1 overexpression in KIT-independent GISTs. These novel findings highlight the relevance of c-Jun/cyclin D1 as novel therapeutic targets in GISTs that have lost KIT oncoprotein expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 952. doi:10.1158/1538-7445.AM2011-952

Mesothelioma is a chemotherapy-resistant neoplasm, as is evident in the dismal survival for patients with this asbestos-associated disease. Conventional chemotherapies and radiation therapy have only limited efficacy, and improved survival will require development of novel and more effective pharmacological interventions. Although p53 genomic mutations are uncommon in mesothelioma, we hypothesized that functional p53 inactivation (resulting from NF2-dysregulated FAK activation) might be a frequent event. Using proteomic methods, with tandem mass spectrometry analyses of mesotheliomas after double immuno-affinity purifications for p53 and phosphotyrosine, we find that FAK is consitutively activated in 10 of 10 mesothelioma cell lines and 9 of 9 mesothelioma surgical specimens, and associated with p53 in 6 of 6 mesothelioma cell lines. In four mesotheliomas with non-mutant p53, FAK silencing by lentiviral short hairpin RNA (shRNA) induced expression and phosphorylation of p53 at Ser15. However, FAK regulation of mesothelioma proliferation was not restricted to p53-dependent pathways, as demonstrated by immunoblots after FAK knockdown in JMN1B mesothelioma cells, which have mutant/inactivated p53, compared to MESO924, MESO257, MESO296, and MESO428 cells, which have nonmutant p53. Additive effects were obtained through a coordinated attack on p53, as demonstrated by immunoblots, cell viability and cell cycle analyses, showing that FAK knockdown and MDM2 inhibition (nutlin-3) induced greater p53 expression, cell apoptosis, anti-proliferative effects and cell cycle arrest, compared to either intervention alone. These findings highlight novel therapeutic opportunities in mesothelioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5031.