Efficacy of focal adhesion kinase inhibition in non-small cell lung cancer with oncogenically activated MAPK pathways.

BACKGROUND: Focal adhesion kinase (FAK) is overexpressed in many types of tumours, including lung cancer. Y15, a small molecule which inhibits Y397 FAK autophosphorylation, decreases growth of human neuroblastoma, breast and pancreatic cancers. In this study, we investigated the in vitro and in vivo effects of Y15, and the underlying mechanism on non-small cell lung cancer cells. METHODS: The cytotoxic effects of Y15 targeting FAK signalling were evaluated. Gene-knockdown experiments were performed to determine the anti-cancer mechanism. Xenografts with RAS or EGFR mutations were selected for in vivo Y15 treatment. RESULTS: Y15 blocked autophosphorylation of FAK in a time- and dose-dependent manner. It caused dose-dependent decrease of lung cancer cell viability and clonogenicity. Y15 inhibited tumour growth of RAS-mutant (A549 with KRAS mutation and H1299 with NRAS mutation), as well as epidermal growth factor receptor (EGFR) mutant (H1650 and H1975) lung cancer xenografts. JNK activation is a mechanism underlying Y15-induced Bcl-2 and Mcl-1 downregulation. Moreover, knockdown of Bcl-2 or Bcl-xL potentiated the effects of Y15. The combination of various inhibitors of the Bcl-2 family of proteins with FAK inhibitors demonstrated synergy in multiple lung cancer cell lines in vitro. CONCLUSIONS: FAK inhibition demonstrated efficacy both in vitro and in vivo in lung cancers with either oncogenic RAS or EGFR mutations. In addition, FAK inhibition in combination with inhibitors of Bcl-2 family of anti-apoptotic proteins has synergistic activity in these MAPK-activated non-small cell lung cancer cell line models.

Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

In-depth LC-MS/MS analysis of the chicken ovarian cancer proteome reveals conserved and novel differentially regulated proteins in humans.

Ovarian cancer (OVC) remains the most lethal gynecological malignancy in the world due to the combined lack of early-stage diagnostics and effective therapeutic strategies. The development and application of advanced proteomics technology and new experimental models has created unique opportunities for translational studies. In this study, we investigated the ovarian cancer proteome of the chicken, an emerging experimental model of OVC that develops ovarian tumors spontaneously. Matched plasma, ovary, and oviduct tissue biospecimens derived from healthy, early-stage OVC, and late-stage OVC birds were quantitatively characterized by label-free proteomics. Over 2600 proteins were identified in this study, 348 of which were differentially expressed by more than twofold (p ≤ 0.05) in early- and late-stage ovarian tumor tissue specimens relative to healthy ovarian tissues. Several of the 348 proteins are known to be differentially regulated in human cancers including B2M, CLDN3, EPCAM, PIGR, S100A6, S100A9, S100A11, and TPD52. Of particular interest was ovostatin 2 (OVOS2), a novel 165-kDa protease inhibitor found to be strongly upregulated in chicken ovarian tumors (p = 0.0005) and matched plasma (p = 0.003). Indeed, RT-quantitative PCR and Western blot analysis demonstrated that OVOS2 mRNA and protein were also upregulated in multiple human OVC cell lines compared to normal ovarian epithelia (NOE) cells and immunohistochemical staining confirmed overexpression of OVOS2 in primary human ovarian cancers relative to non-cancerous tissues. Collectively, these data provide the first evidence for involvement of OVOS2 in the pathogenesis of both chicken and human ovarian cancer.

Identification of Piperazinyl-Difluoro-indene Derivatives Containing Pyridyl Groups as Potent FGFR Inhibitors against FGFR Mutant Tumor: Design, Synthesis, and Biological Evaluation.

The fibroblast growth factor receptor (FGFR) signaling pathway plays important roles in cellular processes such as proliferation, differentiation, and migration. In this study, we highlighted the potential of FGFR inhibitors bearing the (S)-3,3-difluoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-indene scaffold containing a crucial 3-pyridyl group for the treatment of FGFR mutant cancers. The representative compound (S)-23, which was identified through comprehensive evaluation, exhibited potent antiproliferative activity with GI50 in the range of 6.4-10.4 nM against FGFR1 fusion protein-carrying, FGFR2-amplified, and FGFR2 mutant cancer cell lines and good antiproliferative activity against FGFR3 translocation and mutant FGFR4 cancer cell lines, as well as potency assessment against FGFR1-4 kinases. Moreover, compound (S)-23 exhibited favorable pharmacokinetic properties, low potential for drug-drug interactions, and very potent antitumor activity in MFE-296 xenograft mouse models with a TGI of 99.1% at the dose of 10 mg/kg. These findings demonstrate that compound (S)-23 is a potential therapeutic agent for FGFR mutant tumors.

Carnitine palmitoyltransferase 1A promotes mitochondrial fission by enhancing MFF succinylation in ovarian cancer.

Mitochondria are dynamic organelles that are important for cell growth and proliferation. Dysregulated mitochondrial dynamics are highly associated with the initiation and progression of various cancers, including ovarian cancer. However, the regulatory mechanism underlying mitochondrial dynamics is still not fully understood. Previously, our study showed that carnitine palmitoyltransferase 1A (CPT1A) is highly expressed in ovarian cancer cells and promotes the development of ovarian cancer. Here, we find that CPT1A regulates mitochondrial dynamics and promotes mitochondrial fission in ovarian cancer cells. Our study futher shows that CPT1A regulates mitochondrial fission and function through mitochondrial fission factor (MFF) to promote the growth and proliferation of ovarian cancer cells. Mechanistically, we show that CPT1A promotes succinylation of MFF at lysine 302 (K302), which protects against Parkin-mediated ubiquitin-proteasomal degradation of MFF. Finally, the study shows that MFF is highly expressed in ovarian cancer cells and that high MFF expression is associated with poor prognosis in patients with ovarian cancer. MFF inhibition significantly inhibits the progression of ovarian cancer in vivo. Overall, CPT1A regulates mitochondrial dynamics through MFF succinylation to promote the development of ovarian cancer. Moreover, our findings suggest that MFF is a potential therapeutic target for ovarian cancer.

Dual targeting of mTORC1/C2 complexes enhances histone deacetylase inhibitor-mediated anti-tumor efficacy in primary HCC cancer in vitro and in vivo.

BACKGROUND & AIMS: The mammalian target of rapamycin (mTOR) plays a pivotal role in hepatocellular carcinoma (HCC). Previous studies indicated that inhibition of mTORC1 enhanced histone deacetylase inhibitors (HDACis)-mediated anti-tumor activity, accompanied with feedback activation of AKT. Therefore, dual targeting of mTORC1/C2 should be more efficient in suppressing AKT activity and in enhancing the anti-tumor activity of HDACi in HCC. METHODS: The interactions between mTOR kinase inhibitors (mTORKis) (i.e., Pp242, AZD8055, OSI027) and HDACis (i.e., SAHA, LBH589) were examined in vitro using HCC cell lines and in vivo using patient-derived primary HCC xenografts on SCID mice. RESULTS: mTORKis significantly enhanced HDACi-induced apoptosis in HCC cells. The inhibition of both mTORC1/2 not only efficiently blocked mTORC1 signaling, but also abrogated AKT-feedback activation caused by selective mTORC1 inhibition. The co-treatment of mTORKi and HDACi further inhibited AKT signaling and upregulated Bim. Dysfunction of mTORC2 by shRNA significantly lowered the threshold of HDACi-induced cytotoxicity by abrogating AKT activation. Knockdown of AKT1 sensitized Pp242/HDACi-induced apoptosis and ectopic expression of constitutively active AKT1 abrogated the combination-induced cytotoxicity, indicating AKT plays a vital role in the combination-induced effects. Knockdown of Bim prevented Pp242/HDACis-induced cytotoxicity in HCC. Lastly, in vivo studies indicated that the combination of AZD8055 and SAHA almost completely inhibited tumor-growth, without obvious adverse effects, by abrogating AKT and upregulating Bim; while either agent alone shows only 30% inhibition in primary HCC xenografts. CONCLUSIONS: Our findings suggest that a combining-regimen of mTORKi and HDACi may be an effective therapeutic strategy for HCC.

Hyper-SUMOylation of ERG Is Essential for the Progression of Acute Myeloid Leukemia.

Leukemia is a malignant disease of hematopoietic tissue characterized by the differentiation arrest and malignant proliferation of immature hematopoietic precursor cells in bone marrow. ERG (ETS-related gene) is an important member of the E26 transformation-specific (ETS) transcription factor family that plays a crucial role in physiological and pathological processes. However, the role of ERG and its modification in leukemia remains underexplored. In the present study, we stably knocked down or overexpressed ERG in leukemia cells and observed that ERG significantly promotes the proliferation and inhibits the differentiation of AML (acute myeloid leukemia) cells. Further experiments showed that ERG was primarily modified by SUMO2, which was deconjugated by SENP2. PML promotes the SUMOylation of ERG, enhancing its stability. Arsenic trioxide decreased the expression level of ERG, further promoting cell differentiation. Furthermore, the mutation of SUMO sites in ERG inhibited its ability to promote the proliferation and inhibit the differentiation of leukemia cells. Our results demonstrated the crucial role of ERG SUMOylation in the development of AML, providing powerful targeted therapeutic strategies for the clinical treatment of AML.

Discovery of a Candidate Containing an (S)-3,3-Difluoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-inden Scaffold as a Highly Potent Pan-Inhibitor of the BCR-ABL Kinase Including the T315I-Resistant Mutant for the Treatment of Chronic Myeloid Leukemia.

BCR-ABL kinase inhibition is an effective strategy for the treatment of chronic myeloid leukemia (CML). Herein, we report compound 3a-P1, bearing a difluoro-indene scaffold, as a novel potent pan-inhibitor against BCR-ABL mutants, including the most refractory T315I mutant. As the privileged (S)-isomer compared to its (R)-isomer 3a-P2, 3a-P1 exhibited potent antiproliferative activities against K562 and Ku812 CML cells and BCR-ABL and BCR-ABLT315I BaF3 cells, with IC50 values of 0.4, 0.1, 2.1, and 4.7 nM, respectively. 3a-P1 displayed a good safety profile in a battery of assays, including single-dose toxicity, hERG K+, and genotoxicity. It also showed favorable mice pharmacokinetic properties with a good oral bioavailability (32%), a reasonable half-life (4.61 h), and a high exposure (1386 h·ng/mL). Importantly, 3a-P1 demonstrated a higher potency than ponatinib in a mice xenograft model of BaF3 harboring BCR-ABLT315I. Overall, the results indicate that 3a-P1 is a promising drug candidate for the treatment of CML to overcome the imatinib-resistant T315I BCR-ABL mutation.

AIM2 promotes the development of non-small cell lung cancer by modulating mitochondrial dynamics.

Mitochondrial fusion and fission dynamics fine-tune cellular calcium homeostasis, ATP production capacity and ROS production and play important roles in cell proliferation and migration. Dysregulated mitochondrial dynamics is closely related to tumor development, but the mechanism of mitochondrial dynamics dysregulation and its role in the development of lung cancer remains unclear. Here, we demonstrate that the DNA sensor protein absent in melanoma 2 (AIM2) is highly expressed in non-small cell lung cancer (NSCLC) cells and that high AIM2 expression is associated with poor prognosis in patients with NSCLC. High expression of AIM2 contributes to tumor cell growth and proliferation independent of inflammasome activation in vitro and in vivo. Further studies have shown that AIM2 colocalizes with mitochondria in NSCLC cells and that AIM2 knockdown leads to enhanced mitochondrial fusion and decreased cell proliferation. Mechanistic studies have shown that AIM2 downregulation promotes MFN2 upregulation, thereby enhancing mitochondrial fusion. Moreover, we found that mitochondrial fusion driven by AIM2 knockdown leads to a decrease of cellular reactive oxygen species (ROS) production, which further causes inactivation of the MAPK/ERK signaling pathway. Together, we discovered a novel function of AIM2 in promoting NSCLC development by regulating mitochondrial dynamics and revealed its underlying mechanism. Our work provides new intervention targets for the treatment of NSCLC.

BH3 mimetic ABT-263 enhances the anticancer effects of apigenin in tumor cells with activating EGFR mutation.

BACKGROUND: Mutated epidermal growth factor receptor (EGFR) is one of the most successful targets in cancer targeted therapy. While this treatment has benefited many patients with an activating EGFR mutation (EGFRm), almost all those who initially benefited will eventually develop acquired drug resistance (ADR) after a certain period of time. New therapeutic strategies need to be explored to treat EGFRm tumors and overcome or minimize this recurring ADR. RESULTS: Our data showed that apigenin alone has only mild inhibitory effects on EGFRm tumor cells. By drug screening, we found that ABT-263 can significantly enhance the antitumor activities of apigenin in tumor cells harbouring an activating EGFR mutation and AZD9291-resistant H1975 cells. Mechanistically, apigenin upregulated the expression of Noxa in EGFRm tumor cells by targeting the AKT-FoxO3a pathway, thereby synergizing with ABT-263 to suppress tumor cell growth and proliferation in vitro and in vivo. CONCLUSIONS: Our study provides a rationale for the clinical application of the combination treatment of apigenin and BH3 mimetics in the treatment of EGFRm tumors.

Aberrant alternative splicing of human zinc finger gene ZNF268 in human hematological malignancy.

The human ZNF268 gene was initially described as a gene associated with early human embryogenesis and was later implicated in human leukemia due to the identification of an alternatively splice form in leukemia patients. To systematically evaluate the correlation of ZNF268 with human hematological malignancy, expression of different alternatively spliced forms of ZNF268 mRNA in peripheral blood of 45 patients with hematological malignancies and 17 healthy donors were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR. We demonstrated that presence of ZNF268a, ZNF268c, ZNF268f and ZNF268g were significantly different between the patients and healthy donors (P<0.05). Our study thus suggests that aberrant alternative splicing of ZNF268 is a potential prognostic factor of and may contribute to human hematological malignancies.

Identification of the DNA binding element of the human ZNF300 protein.

The human ZNF300 gene is a member of the KRAB/C(2)H(2) zinc finger gene family, the members of which are known to be involved in various developmental and pathological processes. Here, we show that the ZNF300 gene encodes a 68-kDa nuclear protein that binds DNA in a sequence-specific manner. The ZNF300 DNA binding site, C(t/a)GGGGG(c/g)G, was defined via a random oligonucleotide selection assay, and the DNA binding site was further confirmed by electrophoretic mobility shift assays. A potential ZNF300 binding site was found in the promoter region of the human IL-2Rbeta gene. The results of electrophoretic mobility shift assays indicated that ZNF300 bound to the ZNF300 binding site in the IL-2Rbeta promoter in vitro. Transient co-transfection assays showed that ZNF300 could activate the IL-2Rbeta promoter, and that the activation was abrogated by the mutation of residues in the ZNF300 binding site. Identifying the DNA binding site and characterizing the transcriptional regulation property of ZNF300 would provide critical insights into its potential as a transcriptional regulator.

Hyper-SUMOylation of K+ Channels in Sudden Unexplained Death in Epilepsy: Isolation and Primary Culture of Dissociated Hippocampal Neurons from Newborn Mice for Subcellular Localization.

The physiological characteristics of rat and murine hippocampal neurons are widely studied, especially because of the involvement of the hippocampus in learning, memory, and neurological functions. Primary cultures of hippocampal neurons are commonly used to discover cellular and molecular mechanisms in neurobiology. By isolating and culturing individual hippocampal neurons, neuroscientists are able to investigate the activity of neurons at the individual cell and single synapse level, and to analyze properties related to cellular structure, cellular trafficking, and individual protein subcellular localization or protein-protein interaction using a variety of biochemical techniques. Conclusions addressed from such research are critical for testing theories related to memory, learning, and neurological functions. Here, we will describe how to isolate and culture primary hippocampal cells from newborn mice. The hippocampus may be isolated from newborn mice in as short as 2 min, and the cell cultures can be maintained for up to 2 weeks, and then ready for investigation of subcellular localization of K+ channel proteins and interaction with SUMO-specific protease 2 (SENP2). The protocol provides a fast and efficient technique for the culture of neuronal cells from mouce hippocampal tissue, and will ensure the immunocytochemistry detection of subcellular localization or protein-protein interactions in neurological research.

Dihydroartemisinin suppresses STAT3 signaling and Mcl-1 and Survivin expression to potentiate ABT-263-induced apoptosis in Non-small Cell Lung Cancer cells harboring EGFR or RAS mutation.

Non-small cell lung cancer (NSCLC) is the most common malignancy worldwide. A significant fraction of NSCLC carries activating mutations in epidermal growth factor receptor (EGFR) or RAS oncogene. Dihydroartemisinin (DHA) is a semisynthetic derivative of the herbal antimalarial drug artemisinin that has been recently reported to exhibit anti-cancer activity. To develop new therapeutic strategies for NSCLC, we investigated the interactions between DHA and ABT-263 in NSCLC cells harboring EGFR or RAS mutation. Our data indicated that DHA synergized with ABT-263 to trigger Bax-dependent apoptosis in NSCLC cells in culture. DHA treatment antagonized ABT-263-induced Mcl-1 upregulation and sensitized NSCLC cells to ABT-263-triggered apoptosis. Additionally, DHA treatment caused downregulation of Survivin and upregulation of Bim, which also contribute to cotreatment-induced cytotoxicity. Moreover, DHA effectively suppressed STAT3 phosphorylation, and STAT3 inactivation resulted in the downregulation of Mcl-1 and Survivin, functioning to enhance ABT-263-induced cytotoxicity. Finally, cotreatment of DHA and ABT-263 significantly inhibited xenograft growth in nude mice. Together, DHA effectively inhibits STAT3 activity and modulates expression of Mcl-1, Survivin and Bim, thereby synergizing with ABT-263 to trigger apoptosis in NSCLC cells harboring EGFR or RAS mutation. Our data provide a novel therapeutic strategy for EGFR or RAS mutant NSCLC treatment.

Apigenin in cancer therapy: anti-cancer effects and mechanisms of action.

Apigenin is a common dietary flavonoid that is abundantly present in many fruits, vegetables and Chinese medicinal herbs and serves multiple physiological functions, such as strong anti-inflammatory, antioxidant, antibacterial and antiviral activities and blood pressure reduction. Therefore, apigenin has been used as a traditional medicine for centuries. Recently, apigenin has been widely investigated for its anti-cancer activities and low toxicity. Apigenin was reported to suppress various human cancers in vitro and in vivo by multiple biological effects, such as triggering cell apoptosis and autophagy, inducing cell cycle arrest, suppressing cell migration and invasion, and stimulating an immune response. In this review, we focus on the most recent advances in the anti-cancer effects of apigenin and their underlying mechanisms, and we summarize the signaling pathways modulated by apigenin, including the PI3K/AKT, MAPK/ERK, JAK/STAT, NF-κB and Wnt/ß-catenin pathways. We also discuss combinatorial strategies to enhance the anti-cancer effect of apigenin on various cancers and its use as an adjuvant chemotherapeutic agent to overcome cancer drug resistance or to alleviate other adverse effects of chemotherapy. The functions of apigenin against cancer stem cells are also summarized and discussed. These data demonstrate that apigenin is a promising reagent for cancer therapy. Apigenin appears to have the potential to be developed either as a dietary supplement or as an adjuvant chemotherapeutic agent for cancer therapy.

KRAB-containing zinc finger gene ZNF268 encodes multiple alternatively spliced isoforms that contain transcription regulatory domains.

The ZNF268 gene was originally isolated from an early human embryo cDNA library. Several different transcripts have been isolated for the ZNF268 gene and developmental expression studies suggest that ZNF268 plays a role in the development of human fetal liver and the differentiation of blood cells. In our effort to study the functions of ZNF268 in different organs during development and in pathogenesis, we have now identified 3 novel splicing isoforms, ZNF268e, ZNF268f and ZNF268g, in human fetal tissues and human tumor derived cell lines. The 8 alternatively spliced mRNAs discovered so for are predicted to encode 3 protein isoforms. Expression analysis showed that different mRNA isoforms have different expression profiles. In particular, ZNF268c mRNA was detected only in tumor cells, and ZNF268f appeared to be tissue-specific. By Western blot analysis, all 3 ZNF268 protein isoforms, ZNF268a, ZNF268b1 and ZNF268b2, were expressed in tumor cell lines, while only two protein products, ZNF268b1 and ZNF268b2, were detected in human fetal tissues. Subcellular localization analysis showed that ZNF268a and ZNF268b2 distributed diffusely throughout the cell, while ZNF268b1 mainly localized in the cytoplasm. Moreover, using a CAT reporter system fused to the Gal4 DNA binding domain of the ZNF268 gene, the ZNF268a and b2 activated the CAT reporter gene expression, while the KRAB domain, corresponding to the ZNF268b1 repressed the reporter gene expression. Taken together, our results showed that multiple ZNF268 splicing products encode multiple ZNF268 protein isoforms with different subcellular localization, and that the ZNF268 gene may function as a transcriptional activator in the growth and differentiation of cells in development and/or pathogenesis.

Cooperation between human DAF and CD59 in protecting cells from human complement-mediated lysis.

The complement (C) regulatory proteins decay accelerating factor (DAF, CD55) and CD59 could protect host cells using different mechanisms from C-mediated damage at two distinct levels within the C pathway. Co-expression of DAF and CD59 would be an effective strategy to help overcome host C-induced xenograft hyperacute rejection. In this study, we made a construct of recombinant expression vector containing DAF and CD59 cDNA and the stable cell lines were obtained by G418 selection. Extraneous genes integration and co-expression were identified by PCR, RT-PCR and Western blot analysis. Human c-mediated cytolysis assays showed that NIH/3T3 cells transfected stably with pcDNA3-CD59, pcDNA3-DAF, and pcDNA3-CD59DAF-DP were protected from Cmediated damage and that synchronously expressed human CD59 and DAF provided the most excellent protection for host cells as compared with either human CD59 or DAF expressed alone. Therefore, the construct represents an effective and efficacy strategy to overcome C-mediated damage in cells and, ultimately, in animals.

[Cloning and characterization of two novel alternatively spliced transcripts of ZNF268].

ZNF268 gene encodes a typical KRAB-containing zinc finger protein, which has an amino-terminal KRAB domain and 24 carboxyl-terminal C2H2 zinc finger motifs. There is evidence that ZNF268 is expressed in human early embryos, and that it played a role in the development of human fetal liver. In this study, we use immunohistochemistry to show that ZNF268 was also expressed in hematopoietic stem cells in 3-5 week-old human embryos. ZNF268 expression was also detected in the blood cells from 7 healthy adult donors by RT-PCR. Furthermore, 4 alternative transcripts were detected in the 7 samples. By cloning and sequencing, two novel transcripts, ZNF268c and ZNF268d were identified. We predict that there is association between ZNF268 and development of hematopoietic cells.

Carnitine palmitoyltransferase 1A functions to repress FoxO transcription factors to allow cell cycle progression in ovarian cancer.

Cancer cells rely on hyperactive de novo lipid synthesis for maintaining malignancy. Recent studies suggest involvement in cancer of fatty acid oxidation, a process functionally opposite to lipogenesis. A mechanistic link from lipid catabolism to oncogenic processes is yet to be established. Carnitine palmitoyltransferase 1 (CPT1) is a rate-limiting enzyme of fatty acid ß-oxidation (FAO) that catalyzes the transfer of long-chain acyl group of the acyl-CoA ester to carnitine, thereby shuttling fatty acids into the mitochondrial matrix for ß-oxidation. In the present study, we demonstrated that CPT1A was highly expressed in most ovarian cancer cell lines and primary ovarian serous carcinomas. Overexpression of CPT1A correlated with a poor overall survival of ovarian cancer patients. Inactivation of CPT1A decreased cellular ATP levels and induced cell cycle arrest at G0/G1, suggesting that ovarian cancer cells depend on or are addicted to CPT1A-mediated FAO for cell cycle progression. CPT1A deficiency also suppressed anchorage-independent growth and formation of xenografts from ovarian cancer cell lines. The cyclin-dependent kinase inhibitor p21WAF1 (p21) was identified as most consistently and robustly induced cell cycle regulator upon inactivation of CPT1A. Furthermore, p21 was transcriptionally upregulated by the FoxO transcription factors, which were in turn phosphorylated and activated by AMP-activated protein kinase and the mitogen-activated protein kinases JNK and p38. Our results established the oncogenic relevance of CPT1A and a mechanistic link from lipid catabolism to cell cycle regulation, suggesting that CPT1A could be a prognostic biomarker and rational target for therapeutic intervention of cancer.

Co-expression of the complement regulatory proteins human DAF and CD59 with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects.

The human complement regulatory proteins (hCRPs) decay accelerating factor (DAF/CD55) and protectin CD59 transfected into non-human cells could confer protection against human complement. The combination of DAF and CD59 would be an effective strategy to help overcome host complement-induced hyperacute rejection in xenotransplantation. We constructed a dicistronic mammalian expression vector pcDNA3-CD59IRESDAF by using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV). RT-PCR, Western blotting and immunofluorescence microscopic analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hCD59 and hDAF on the surface of NIH/3T3 cells transfected stably with pcDNA3-CD59IRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and CD59 proteins could provide more significant protection against complement-mediated cytolysis than either hCD59 or hDAF alone. These results suggest that IRES containing polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery and that the construct pcDNA3-CD59IRESDAF vector has potential therapeutic value for effectively controlling complement activation and for preventing hyperacute rejection in clinical gene therapy.