Upstream Suppressor Research Articles

TRIM31 is upregulated in hepatocellular carcinoma and promotes disease progression by inducing ubiquitination of TSC1–TSC2 complex

Tripartite motif (TRIM) 31 is a member of the tripartite motif-containing protein family, and TRIM family proteins are involved in a broad range of biological and pathological processes. However, the role of TRIM31 in hepatocellular carcinoma (HCC) progression is not known. Here we demonstrated that TRIM31 expression was significantly upregulated in liver cancer tissues compared with paired distal non-cancerous liver tissues from HCC patients, and its overexpression was significantly correlated with advanced disease status. Both gain and loss of function assay verified that TRIM31 promoted the malignant behaviors of HCC cells through overactivation of mammalian target of rapamycin complex1 (mTORC1) pathway. We further demonstrated that TRIM31 exerted its oncogenic effect by directly interacting with the tuberous sclerosis complex (TSC) 1 and TSC2 complex, the upstream suppressor of mTORC1 pathway, and promoting the E3 ligase-mediated K48-linked ubiquitination and degradation of this complex. In conclusion, this study demonstrated TRIM31 could promote HCC progression by targeting TSC1-TSC2 complex for degradation and further overactivating mTORC1 pathway. Thus, it revealed a novel molecular mechanism of HCC progression and indicated a potential therapeutic strategy against HCC by targeting TRIM31.

Abstract MIP-058: YAP, VIA INTERACTIONS WITH THE FGF SIGNALING PATHWAY, REGULATES TUMORIGENESIS OF FALLOPIAN TUBE SECRETORY EPITHELIAL CELLS

Abstract INTRODUCTION: Accumulating evidence indicates that ovarian high-grade serous carcinoma (HGSC) originates from Fallopian tube secretory epithelial cells (FTSECs). Our recent studies indicated that the Hippo/YAP signaling pathway contributes to transformation of ovarian surface epithelial cells. However, whether this pathway is also involved in the initiation and progression of FTSEC-derived ovarian HGSC has not been examined. AIMS: The present study aims to determine whether the Hippo/YAP signaling pathway plays a role in initiation and progression of Fallopian tube-derived HGSC and the potential mechanism(s) by which YAP regulates FTSEC tumorigenesis. RESULTS: Immunohistochemical analysis showed that YAP expression was very low in normal human fallopian tube and ovarian tissues. However, YAP immunosignals significantly increased in ovarian HGSCs, cancerous and inflammatory fallopian tube tissues. Data extracted from multidimensional human cancer genomics datasets revealed that the YAP and TEADs (the major target transcription factors of YAP) genes are frequently amplified and/or up-regulated, while the upstream tumor suppressor genes such as NF1 and LATS1, are frequently deleted and/or mutated in ovarian HGSC tissues. Overexpression of YAP in immortalized non-tumorigenic FTSECs promoted cell proliferation in both 2D and 3D culture systems, induced colony formation in soft agar, and drove tumorigenesis in athymic nude mice. Pathohistological and biochemical analyses clearly indicated that tumors derived from the FTSECs resembled ovarian HGSC. In addition, our mechanistic studies showed that activation of YAP in human FTSECs upregulated the expression of fibroblast growth factors (FGF1, FGF2) and their receptors (FGFR1, FGFR2, FGFR3 and FGFR4). Intriguingly, we found that the activated FGF signaling in turn suppressed the Hippo pathway and activated YAP protein. Consistent with this result, treatment of FTSECs with YAP antagonist (Verteporfin) and/or FGFR inhibitor (BGI398) not only blocked YAP-induced FTSECs transformation, but also suppressed tumor cell growth and induced tumor cell death in a human HGSC xenograft model. CONCLUSION: Our results demonstrate that the disruption of the Hippo/YAP signaling pathway contributes to the carcinogenesis of the fallopian tube secretory epithelial cells. The Hippo/YAP and FGF signaling pathways formed an autocrine/paracrine positive feedback loop to drive the progression of the FTSECs-derived HGSC. Evidence in this study strongly suggests that combined targeting of YAP and FGFRs represents a novel therapeutic strategy for ovarian HGSCs. Citation Format: Chunbo He, Guohua Hua, Xiangmin Lv, Zhengfeng Wang, Ronny I. Drapkin, John S. Davis, Cheng Wang. YAP, VIA INTERACTIONS WITH THE FGF SIGNALING PATHWAY, REGULATES TUMORIGENESIS OF FALLOPIAN TUBE SECRETORY EPITHELIAL CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-058.

The first Japanese case of central precocious puberty with a novel MKRN3 mutation

MKRN3, located on chromosome 15q11.2, encodes makorin ring-finger 3, which is an upstream suppressor of the hypothalamic-pituitary-gonadal axis. Mutation of this gene induces central precocious puberty (CPP). As MKRN3 is maternally imprinted, only the paternal allele is expressed. This is the first report of an 8-year-old Japanese girl with CPP caused by a novel frameshift mutation in MKRN3 (p.Glu229Argfs*3).

Potential role of the OVOL1-OVOL2 axis and c-Myc in the progression of cutaneous squamous cell carcinoma.

OVOL1 and OVOL2 are ubiquitously conserved genes encoding C2H2 zinc-finger transcription factors in mammals. They promote epithelial cell proliferation, differentiation, and mesenchymal-to-epithelial transition, coordinately mediated via the Wnt signaling pathway. We previously reported that human OVOL1 and OVOL2 were preferentially expressed in the normal epidermis and hair follicles as well as their tumors, and found that OVOL1 is upregulated in Bowen's disease and downregulated in cutaneous squamous cell carcinoma. The aims of this study were to elucidate the potential role of the OVOL1-OVOL2 axis in Bowen's disease and squamous cell carcinoma, and to reveal the relationship between OVOL and c-Myc, a proto-oncogene that plays a pivotal role in the malignancy of epithelial tumors. We investigated 20 Bowen's disease and 20 squamous cell carcinoma clinical samples and a human squamous cell carcinoma cell line (A431) using immunohistochemical staining and molecular biological approaches. Immunohistochemical analysis revealed that OVOL1 was upregulated in Bowen's disease and markedly downregulated in squamous cell carcinoma; conversely, c-Myc was downregulated in Bowen's disease and upregulated in squamous cell carcinoma. OVOL2 was markedly upregulated in the nucleus of Bowen's disease cells, but the distribution of OVOL2 expression in squamous cell carcinoma varied widely; OVOL2 was typically expressed in the cytoplasm, but only sporadically in the nucleus. Furthermore, knockdown of OVOL1 using a specific small interfering RNA increased the mRNA and protein levels of c-Myc and OVOL2. Knockdown of OVOL2 did not significantly affect the mRNA and protein levels of either c-Myc or OVOL1. These results suggest that OVOL1 is an upstream suppressor of c-Myc and OVOL2, and the OVOL1-OVOL2 axis is a modulator of c-Myc, coordinately regulating the invasiveness of cutaneous squamous cell carcinoma. Taken together, this study suggests that the OVOL1-OVOL2 axis is a key modulator of c-Myc expression in the shift from in situ epidermal malignancy (Bowen's disease) to invasive squamous cell carcinoma.

Statin suppresses Hippo pathway-inactivated malignant mesothelioma cells and blocks the YAP/CD44 growth stimulatory axis

Malignant mesothelioma (MM) frequently exhibits Hippo signaling pathway inactivation (HPI) mainly due to NF2 and/or LATS2 mutations, which leads to the activation of YAP transcriptional co-activator. Here, we show antitumor effects of statin on MM cells with HPI, through the interplay of the mevalonate and Hippo signaling pathways. Statin attenuated proliferation and migration of MM cells harboring NF2 mutation by accelerating YAP phosphorylation/inactivation. CD44 expression was decreased by statin, in parallel with YAP phosphorylation/inactivation. Importantly, we discovered that YAP/TEAD activated CD44 transcription by binding to the CD44 promoter at TEAD-binding sites. On the other hand, CD44 regulated Merlin phosphorylation according to cell density and sequentially promoted YAP transcriptional co-activator, suggesting that CD44 plays two pivotal functional roles as an upstream suppressor of the Hippo pathway and one of downstream targets regulated by YAP/TEAD. Moreover, the YAP/CD44 axis conferred cancer stem cell (CSC)-like properties in MM cells leading to chemoresistance, which was blocked by statin. Together, our findings suggest that YAP mediates CD44 up-regulation at the transcriptional level, conferring CSC-like properties in MM cells, and statin represents a potential therapeutic option against MM by inactivating YAP.

Regulatory Differences in Natal Down Development between Altricial Zebra Finch and Precocial Chicken

Birds can be classified into altricial and precocial. The hatchlings of altricial birds are almost naked, whereas those of precocial birds are covered with natal down. This regulatory divergence is thought to reflect environmental adaptation, but the molecular basis of the divergence is unclear. To address this issue, we chose the altricial zebra finch and the precocial chicken as the model animals. We noted that zebra finch hatchlings show natal down growth suppressed anterior dorsal (AD) skin but partially down-covered posterior dorsal (PD) skin. Comparing the transcriptomes of AD and PD skins, we found that the feather growth promoter SHH (sonic hedgehog) was expressed higher in PD skin than in AD skin. Moreover, the data suggested that the FGF (fibroblast growth factor)/Mitogen-activated protein kinase (MAPK) signaling pathway is involved in natal down growth suppression and that FGF16 is a candidate upstream signaling suppressor. Ectopic expression of FGF16 on chicken leg skin showed downregulation of SHH, upregulation of the feather growth suppressor FGF10, and suppression of feather bud elongation, similar to the phenotype found in zebra finch embryonic AD skin. Therefore, we propose that FGF16-related signals suppress natal down elongation and cause the naked AD skin in zebra finch. Our study provides insights into the regulatory divergence in natal down formation between precocial and altricial birds.

TRIM29 overexpression is associated with poor prognosis and promotes tumor progression by activating Wnt/β-catenin pathway in cervical cancer.

Dysregulation of TRIM29 has been reported to be involved in tumorigenesis, but the role of TRIM29 in cervical cancer is unclear. In this study, we first examined TRIM29 expression and found that TRIM29 mRNA and protein expression was upregulated in cervical cancer tissues when compared with the matched adjacent cervical tissues. We further detected TRIM29 protein with immunohistochemistry in 150 paraffin-embedded samples from early-stage cervical cancer patients. The results showed that high expression of TRIM29 was significantly associated with pelvic lymph node metastasis (p=0.002), advanced FIGO stage (p=0.026) and post-operative recurrence (p<0.001). Patients with high expression of TRIM29 had a shorter overall survival (HR 5.042, p<0.001) and disease-free survival (HR 4.260, p<0.001). TRIM29 was proven to be an independent prognostic factor for cervical cancer patients. When endogenous TRIM29 expression was knocked down by siRNAs, cell proliferation, colony formation, migration and invasion in cervical cancer cell lines HeLa and SiHa were obviously inhibited. Meanwhile, TRIM29 knockdown increased E-cadherin expression but decreased the expression of N-cadherin and β-Catenin, which indicated that TRIM29 could promote epithelial-mesenchymal transition (EMT). Mechanically, knockdown of TRIM29 enhanced GSK-3β protein expression and inhibited the expression of β-Catenin and C-myc proteins. GSK-3β is a key upstream suppressor of β-Catenin and c-myc expression is an indicator of Wnt/β-Catenin activity. Therefore, these results demonstrate that TRIM29 promotes tumor progression by activating Wnt/β-Catenin signaling. In conclusion, TRIM29 is overexpressed and associated with survival of early-stage cervical cancer, indicating that TRIM29 may be a potential prognostic biomarker and therapeutic target for cervical cancer.

Unexpected requirement for a binding partner of the syntaxin family in phagocytosis by murine testicular Sertoli cells.

Testicular phagocytosis by Sertoli cells (SCs) plays an essential role in the efficient clearance of apoptotic spermatogenic cells under both physiological and pathological conditions. However, the molecular mechanism underlying this unique process is poorly understood. Herein, we report for the first time that α-taxilin protein (TXLNA), a binding partner of the syntaxin family that functions as a central player in the intracellular vesicle traffic, was dominantly expressed in SCs. Induction of apoptosis in murine meiotic spermatocytes and haploid spermatids by busulfan treatment stimulated a significant increase of TXLNA in SCs at day (d) 14 and d 24 after busulfan treatment, respectively. Consistently, TXLNA expression was steadily upregulated when SCs were co-cultured with apoptotic germ cells (GCs). Moreover, using siRNA treatment, we found that ablation of endogenous TXLNA significantly impaired the phagocytotic capacity of SCs and thereby resulted in defective spermiogenesis and reduced fertility during the late recovery after testicular heat stress. Mechanistically, upregulation of TXLNA expression by apoptotic GCs was associated with the stabilization of ATP-binding cassette transporter 1 (ABCA1), a transporter-mediated lipid efflux from SCs and influencing male fertility. TXLNA acted as an upstream suppressor of ABCA1 ubiquitination and thus promoted ABCA1 stability and accumulation following GC apoptosis. We further provide in vitro evidence that epidermal growth factor receptor (EGFR)-mediated phosphorylation regulated ABCA1 ubiquitination and was enhanced by TXLNA deficiency during testicular phagocytosis. Taken together, the TXLNA/ABCA1 cascade may serve as an important feedback mechanism to modulate the magnitude of subsequent phagocytotic process of SCs in response to testicular injury.

Brain-expressed X-linked 2 Is Pivotal for Hyperactive Mechanistic Target of Rapamycin (mTOR)-mediated Tumorigenesis

Frequent alteration of upstream proto-oncogenes and tumor suppressor genes activates mechanistic target of rapamycin (mTOR) and causes cancer. However, the downstream effectors of mTOR remain largely elusive. Here we report that brain-expressed X-linked 2 (BEX2) is a novel downstream effector of mTOR. Elevated BEX2 in Tsc2(-/-) mouse embryonic fibroblasts, Pten(-/-) mouse embryonic fibroblasts, Tsc2-deficient rat uterine leiomyoma cells, and brains of neuronal specific Tsc1 knock-out mice were abolished by mTOR inhibitor rapamycin. Furthermore, BEX2 was also increased in the liver of a hepatic specific Pten knock-out mouse and the kidneys of Tsc2 heterozygous deletion mice, and a patient with tuberous sclerosis complex (TSC). mTOR up-regulation of BEX2 was mediated in parallel by both STAT3 and NF-κB. BEX2 was involved in mTOR up-regulation of VEGF production and angiogenesis. Depletion of BEX2 blunted the tumorigenesis of cells with activated mTOR. Therefore, enhanced STAT3/NF-κB-BEX2-VEGF signaling pathway contributes to hyperactive mTOR-induced tumorigenesis. BEX2 may be targeted for the treatment of the cancers with aberrantly activated mTOR signaling pathway.

Epidemiological and genetic clues for molecular mechanisms involved in uterine leiomyoma development and growth.

Uterine leiomyomas (fibroids) are highly prevalent benign smooth muscle tumors of the uterus. In the USA, the lifetime risk for women developing uterine leiomyomas is estimated as up to 75%. Except for hysterectomy, most therapies or treatments often provide only partial or temporary relief and are not successful in every patient. There is a clear racial disparity in the disease; African-American women are estimated to be three times more likely to develop uterine leiomyomas and generally develop more severe symptoms. There is also familial clustering between first-degree relatives and twins, and multiple inherited syndromes in which fibroid development occurs. Leiomyomas have been described as clonal and hormonally regulated, but despite the healthcare burden imposed by the disease, the etiology of uterine leiomyomas remains largely unknown. The mechanisms involved in their growth are also essentially unknown, which has contributed to the slow progress in development of effective treatment options. A comprehensive PubMed search for and critical assessment of articles related to the epidemiological, biological and genetic clues for uterine leiomyoma development was performed. The individual functions of some of the best candidate genes are explained to provide more insight into their biological function and to interconnect and organize genes and pathways in one overarching figure that represents the current state of knowledge about uterine leiomyoma development and growth. In this review, the widely recognized roles of estrogen and progesterone in uterine leiomyoma pathobiology on the basis of clinical and experimental data are presented. This is followed by fundamental aspects and concepts including the possible cellular origin of uterine fibroids. The central themes in the subsequent parts are cytogenetic aberrations in leiomyomas and the racial/ethnic disparities in uterine fibroid biology. Then, the attributes of various in vitro and in vivo, human syndrome, rodent xenograft, naturally mutant, and genetically modified models used to study possible molecular mechanisms of leiomyoma development and growth are described. Particular emphasis is placed on known links to fibrosis, hypertrophy, and hyperplasia and genes that are potentially important in these processes. Menstrual cycle-related injury and repair and coinciding hormonal cycling appears to affect myometrial stem cells that, at a certain stage of fibroid development, often obtain cytogenetic aberrations and mutations of Mediator complex subunit 12 (MED12). Mammalian target of rapamycin (mTOR), a master regulator of proliferation, is activated in many of these tumors, possibly by mechanisms that are similar to some human fibrosis syndromes and/or by mutation of upstream tumor suppressor genes. Animal models of the disease support some of these dysregulated pathways in fibroid etiology or pathogenesis, but none are definitive. All of this suggests that there are likely several key mechanisms involved in the disease that, in addition to increasing the complexity of uterine fibroid pathobiology, offer possible approaches for patient-specific therapies. A final model that incorporates many of these reported mechanisms is presented with a discussion of their implications for leiomyoma clinical practice.

Molecular Evaluation of Transformation‐Induced Methylation of the High Mobility Group A1 (HMGA1) Chromatin Binding Proteins

Aberrant expression of high mobility group A1 (hmga1), resulting from inactivating defects in upstream tumor suppressor genes or upregulation of oncogenic transactivators, induces the initiation of cancer progression pathways. The protein products of hmga1, HMGA1a and HMGA1b, are characterized by the presence of three DNA-binding motifs that possess a central RGR sequence and preferentially bind to the minor-groove of AT-rich stretches of target DNA. Importantly, R25 of the RGR sequence within the first DNA-binding motif bears covalent modifications that may recruit or repel specific transcriptional regulators in cancer cells. Despite the implication of hmga1 and arginine methylation in oncogenic function, the role of epigenetic regulation of hmga1 by arginine methylation has not been evaluated. Here, we investigate the impact of R25 substitutions on HMGA1b binding DNA targets using fluorescence spectroscopy and functional cellular assays. R25 substitutions disrupt DNA binding to various targets and suppress HMGA1-dependent oncogenic transformation. Together, our data reveals new potential anti-cancer strategies by targeting HMGA1 protein methylation and downstream targets.

Abstract LB-61: Inhibiting “Dedifferentiation” in breast cancer cells using Honokiol - a plant derived polyphenol

Abstract Introduction: Dedifferentiation involves terminally differentiated cells reverting back to less differentiated cells such as precursor cells or stem cells within their own lineage allowing the cells to proliferate again before re-differentiation. Tumorigenesis involves uncontrolled cell proliferation and many host-related factors aid in dedifferentiation of tumor cells into stem cell like phenotypes hence providing a mechanism leading to tumor heterogeneity and aggressiveness. Pluripotency factors Oct4, Nanog and Sox2 have been implicated in dedifferentiation and are highly expressed in tumors. The importance of active constitutive agents in natural products has become increasingly apparent owing to their potential cancer chemopreventive and therapeutic properties. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our laboratory demonstrated that HNK has suppressing effects on different aspects of cancer progression. The present study was designed to specifically examine the potential of HNK to inhibit pluripotency related transcription factors and reverse the process of dedifferentiation associated with uncontrolled cell proliferation in tumorigenesis. Results: Here, we provide evidence that HNK inhibits the ability of breast cancer cells to form mammospheres. HNK treatment inhibits dedifferentiation/pluripotency markers Oct4, Nanog, Sox2 in MCF-7, MDA-MB-231, T47D, MDA-MB-468 breast cancer cells. An analysis of underlying signaling mechanism reveal that HNK inhibits phosphorylation of Stat3 and the downregulation of pluripotency factors is mediated via inhibition of Stat3. Stat3 inhibitor, Stattic potentiates the effect of HNK while overexpression of constitutively active Stat3 interferes with HNK-mediated inhibition of Oct4, Nanog and Sox2. It is interesting to note that HNK treatment also increases the expression of upstream kinase and tumor suppressor LKB1. Utilizing LKB1-null cells and gain-of-function strategies, we show that HNK-mediated inhibition of Oct4, Nanog and Sox2 is regulated by tumor suppressor LKB1. The ability of HNK to inhibit mammosphere formation is abrograted in breast cancer cells stably-silenced for LKB1. In vitro and in vivo analyses show novel functional interactions between HNK, Stat3, LKB1 and dedifferentiation/pluripotency markers. Conclusions: Taken together, these studies provide evidence for a previously unrecognized cross-talk between HNK and dedifferentiation/pluripotency markers Oct4, Nanog, Sox2 via tumor suppressor LKB1-Stat3 axis in inhibiting “dedifferentiation” in breast cancer. Citation Format: Sonali Sengupta, Michael Y. Bonner, Jack L. Arbiser, Neeraj K. Saxena, Dipali Sharma. Inhibiting “Dedifferentiation” in breast cancer cells using Honokiol - a plant derived polyphenol. [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 LB-61. doi:10.1158/1538-7445.AM2014-LB-61

Abstract LB-187: Novel mechanistic insights into the bioactive compound honokiol-mediated inhibition of epithelial to mesenchymal transition in breast cancer: Therapeutic modulation of miR-34a via tumor suppressor LKB1

Abstract Introduction: An emerging hypothesis is that epithelial-mesenchymal-transition (EMT) bestows metastatic potential to epithelial cancer cells enabling them to invade, migrate and subsequently disseminate to form distant metastases resulting in non-curable disease. Pharmacological inhibition of EMT can potentially lead to reversion of aggressive breast cancer cells to a more-differentiated epithelial phenotype by inducing mesenchymal-epithelial transition (MET) to prevent metastasis and diminish distant recurrence. The importance of active constitutive agents in natural products has become increasingly apparent owing to their potential cancer chemopreventive and therapeutic properties. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our lab demonstrated that HNK has suppressing effects on different aspects of cancer progression. The present study was designed to specifically examine the potential of HNK to inhibit EMT and elucidate the molecular mechanism by which HNK inhibits EMT in breast cancer cells. Aim: To elucidate the mechanism by which HNK inhibits EMT in breast cancer cells. Results: Here, we provide molecular evidence that HNK inhibits EMT in breast cancer cells resulting in significant downregulation of mesenchymal marker proteins and concurrent upregulation of epithelial markers. In vitro and in vivo analyses show functional interactions between HNK, STAT3, and EMT-signaling components. Mechanistically, HNK inhibits recruitment of STAT3 on mesenchymal transcription factor ZEB1 promoter resulting in decreased ZEB1 expression and nuclear translocation. We also discover that HNK increases E-cadherin expression via STAT3-mediated release of ZEB1 from E-cadherin promoter. Since ZEB1 is a transcriptional repressor of miR-34a, we next examined the effect of HNK on miR-34a expression. Intriguingly, we found that HNK upregulates the levels of miR-34a in a time- and dose-dependent manner. It is interesting to note that HNK treatment also increases the expression of upstream kinase and tumor suppressor LKB1. Utilizing LKB1-null cells and gain-of-function strategies, we found that HNK-mediated increase in miR-34a is regulated by tumor suppressor LKB1. Conclusions: Taken together, these studies provide evidence for a previously unrecognized cross-talk between HNK and miR-34a via tumor suppressor LKB1 in inhibiting EMT in breast cancer. Citation Format: Dimiter B. Avtanski, Michael Y. Bonner, Timothy P. Tiutan, Jack L. Arbiser, Neeraj K. Saxena, Dipali Sharma. Novel mechanistic insights into the bioactive compound honokiol-mediated inhibition of epithelial to mesenchymal transition in breast cancer: Therapeutic modulation of miR-34a via tumor suppressor LKB1. [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 LB-187. doi:10.1158/1538-7445.AM2014-LB-187

Gefitinib resistance resulted from STAT3-mediated Akt activation in lung cancer cells

Hyperactivation of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase is prevalent in human lung cancer and its inhibition by the tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, initially controls tumor growth. However, most patients ultimately relapse due to the development of drug resistance. In this study, we have discovered a STAT3-dependent Akt activation that impairs the efficacy of gefitinib. Mechanistically, gefitinib increased association of EGFR with STAT3, which de-repressed STAT3 from SOCS3, an upstream suppressor of STAT3. Such a de-repression of STAT3 in turn fostered Akt activation. Genetic or pharmacological inhibition of STAT3 abrogated Akt activation and combined gefitinib with STAT3 inhibition synergistically reduced the growth of the tumor cells. Taken together, this study suggests that activation of STAT3 is an intrinsic mechanism of drug resistance in response to EGFR TKIs. Combinational targeting on both EGFR and STAT3 may enhance the efficacy of gefitinib or other EGFR TKIs in lung cancer.

The Single-Stranded DNA-Binding Protein WHIRLY1 Represses WRKY53 Expression and Delays Leaf Senescence in a Developmental Stage-Dependent Manner in Arabidopsis

Leaf senescence in plants involves both positive and negative transcriptional regulation. In this work, we show evidence for the single-stranded DNA-binding protein WHIRLY1 (WHY1) that functions as an upstream suppressor of WRKY53 in a developmental stage-dependent manner during leaf senescence in Arabidopsis (Arabidopsis thaliana). The why1 mutant displayed an early-senescence phenotype. In this background, the expression levels of both WRKY53 and the senescence-associated protease gene SAG12 increased. WHY1 bound to the sequence region that contains an elicitor response element motif-like sequence, GNNNAAATT, plus an AT-rich telomeric repeat-like sequence in the WRKY53 promoter in in vivo and in vitro mutagenesis assays as well as in a chromatin immunoprecipitation assay. This binding to the promoter of WRKY53 was regulated in a developmental stage-dependent manner, as verified by chromatin immunoprecipitation-polymerase chain reaction assay. This direct interaction was further determined by a transient expression assay in which WHY1 repressed β-GLUCURONIDASE gene expression driven by the WRKY53 promoter. Genetic analysis of double mutant transgenic plants revealed that WHY1 overexpression in the wrky53 mutant (oeWHY1wrky53) had no effect on the stay-green phenotype of the wrky53 mutant, while a WHY1 knockout mutant in the wrky53 mutant background (why1wrky53) generated subtle change in the leaf yellow/green phenotype. These results suggest that WHY1 was an upstream regulator of WRKY53 during leaf senescence.

MiR-31 regulates interleukin 2 and kinase suppressor of ras 2 during T cell activation

MicroRNA (miRNA) has an important role as a master regulator of gene expression in immune system and is upregulated during T cell differentiation, however its function is not clear yet. In this study, the contribution of miR-31 in T cell activation was investigated. miR-31 was upregulated during the activation of primary T lymphocytes upon T-cell receptor (TCR) stimulation. Ectopic expression of miR-31 increased the expression of interleukin (IL)-2, while knockdown of endogenous miR-31 decreased the IL-2 expression. To gain more insights into the regulatory mechanism, we performed a bioinformatic analysis and found miR-31 potentially targeted kinase suppressor of ras 2 (KSR2), a repression factor of Ras2 kinase. Using reporter gene and western blotting assays, we confirmed that miR-31 could inhibit KSR2 by directly targeting its 3' untranslated region (UTR). Moreover, miR-31 enhanced nuclear factor of activated T cells (NF-AT) activity in Jurkat T cells, and increased transcription activity of IL-2 promoter in primary T cells. In conclusion, our study demonstrated that miR-31 upregulated IL-2 expression via reduction of its up-stream kinase suppressor, KSR2, and is a component of T cell activation.

An osteoclastic protein-tyrosine phosphatase regulates the β3-integrin, syk, and shp1 signaling through respective src-dependent phosphorylation in osteoclasts

This study utilized the glutathione transferase (GST) pull-down assay to identify novel substrates of an osteoclastic protein-tyrosine phosphatase, PTP-oc. Consistent with the previous findings that the phosphorylated tyr-527 (pY527) of Src is a substrate of PTP-oc, the major protein pulled down with the phosphatase-deficient (PD)-PTP-oc-GST trapping mutant in RAW264.7 cells was Src. The GST-PD-PTP-oc also pulled down pY-Syk and pY-β(3)-integrin, but not after PP2 pretreatment. However, PTP-oc transgenic osteoclasts or PTP-oc-overexpressing RAW264.7 cells had elevated, and not reduced, levels of pY525/526-Syk and pY759-β(3) integrin, and the PTP-oc siRNA treatment drastically reduced levels of pY525/526 Syk and pY759-β(3)-integrin in RAW264.7 cells. These findings are incompatible with the premise that they are substrates of PTP-oc. The PTP-oc-dependent increases in pY525/526-Syk and pY759-β(3)-integrin levels were completely blocked by PP2, indicating that these effects are secondary to PTP-oc-mediated activation of the Src protein-tyrosine kinase (PTK). Overexpression of PTP-oc increased, and siRNA-mediated suppression of PTP-oc reduced, pY160-Vav1, pY173-Vav3, and pY783-PLCγ levels, and Rac1 activation, which are downstream mediators of the ITAM/Syk signaling. Overexpression of PTP-oc also increased, and PTP-oc siRNA treatment decreased, the pY-Shp1 levels, which were blocked by PP2. Since Shp1 is a negative regulator of osteoclast activity and is a key mediator of the ITIM signaling, these findings suggest that PTP-oc is an upstream suppressor of the ITIM/Shp1 signaling through PTP-oc-induced Src-dependent Shp1 phosphorylation. In summary, PTP-oc plays a central regulatory role in the concerted regulation of the β(3)-integrin, the ITAM/Syk, and the ITIM/Shp1 signaling indirectly through activation of Src PTK.

ASK3 responds to osmotic stress and regulates blood pressure by suppressing WNK1-SPAK/OSR1 signaling in the kidney

Changes in the osmolality of body fluids pose a serious danger to cells and living organisms, which have developed cellular systems to sense and respond to osmotic stress and to maintain homoeostasis of body fluid. However, these processes are incompletely understood in mammals. Here we show that apoptosis signal-regulating kinase 3 (ASK3) is predominantly expressed in the kidney and alters its kinase activity bidirectionally in response to osmotic stress. We further demonstrate that ASK3 interacts with WNK1, mutation in which causes an inherited form of hypertension in humans. Knockdown of Ask3 by short interfering RNA enhances the activation of the WNK1-SPAK/OSR1 signalling pathway. Moreover, Ask3 knockout mice exhibit a hypertensive phenotype, in addition to hyperactivation of SPAK/OSR1 in renal tubules. Our results suggest that ASK3 is a unique bidirectional responder to osmotic stress and that it has a role in the control of blood pressure as an upstream suppressor of the WNK1-SPAK/OSR1 signalling pathway.

MiRNAs Function As Biomarker in Pediatric AML and Regulate Gene-Silencing in AML-Relevant Pathways

Abstract 570microRNAs (miRNAs) are small (21-24 nt), non-coding and highly conserved molecules, which are involved in several important regulatory processes like cell growth, proliferation, differentiation, immune response and apoptosis. Thus, their involvement in the pathogenesis of several diseases, including acute myeloid leukemia (AML) is not surprising. Several studies address the miRNA expression changes in adulthood AML, however, comprehensive studies in AML of children and adolescents are missing so far. We investigated the miRNA expression profiles of different AML subtypes from pediatric patients, in order to identify differentially expressed miRNAs. Subsequently, appropriate cell line models were used for global biochemical identification of miRNA targeting structures.miRNA expression profiles of 102 pediatric AML patient samples were identified using microarray technology, and analyzed by unsupervised hierarchical cluster analysis and statistical testing. AML subtypes with translocations t(8;21) and t(15;17) can be separated from each other, solely based on their miRNA expression profile, while other translocations involving mixed-lineage leukemia (MLL) rearrangements are interspersed and lack a characteristic miRNA signature. Only six and seven miRNAs are differentially expressed between AML samples with translocations t(8;21) and t(15;17), respectively, and all other AML subtypes. This is surprising, since patients of different AML subtypes, investigated in this study, differ greatly in their clinical presentation. Differentially expressed miRNAs contain lineage specific miRNAs (miR-223), oncogenic miRNAs (miR-21) and more ubiquitously expressed miRNAs (let-7b/c, miR-100, −125b and −181a/b) with no designated characteristics. Furthermore, these differentially expressed miRNAs were not described as abundant in adult AML patients.To gain further insights into the function of differentially expressed miRNAs, we established a modified PAR-CLIP method termed PAR-CLIP-Array (Photo-activatable-Ribonucleoside-Enhanced Crosslinking-Immunoprecipitation and Microarray Hybridization) for global identification of Ago-associated miRNAs and their mRNA-targets. On average 25% of mRNAs in AML cell lines bearing the AML1/ETO or PML/RARα translocation were identified in Argonaute complexes and carry at least one miRNA binding site and thus are under miRNA control. 60% and 27% of miRNAs and mRNAs, respectively, overlap between the four analyzed Argonaute proteins, while 50% and 52% (46 miRNAs and 241 mRNAs) were associated with one Argonaute protein specifically. However, pathway classification of Ago-associated target-mRNAs indicate more than 90% overlap between the Argonaute proteins and thus are indicative of a concerted action of these four proteins in 150 pathways identified. Moreover, miR-181a/b, up-regulated in t(15;17)-positive AML patients, were detected in association with the four human Argonaute proteins in NB4 cells and show binding sites for the protein kinase PDPK1 potentially leading to inhibition of AKT, whereas eight other Ago-associated miRNA sequence families (seqgrp-miR-98, −130a, −19a, −25, −27a, −301a, −361 and −320) in association with Ago3 are able to repress the upstream tumor suppressor TSC1 leading to activation of the mTOR pathway and increased cell growth. In addition, the repression of the MAP kinase phosphatase DUSP6 by four Ago-associated miRNA sequence families (seqgrp-miR-29a, −17, −125a and −98) leads to activation of proliferative genes in the MAPK pathway of both, t(8;21)- and t(15;17)-positive AML.In summary, miRNAs represent suitable biomarkers for differentiation of AML subtypes of pediatric AML patients. Furthermore, our studies show that the four human Argonaute proteins cooperate in the regulation of AML-relevant signaling pathways providing new insights into AML biology and may present the starting point for novel therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

Unexpected functional similarities between gatekeeper tumour suppressor genes and proto-oncogenes revealed by systems biology

Familial tumor suppressor genes comprise two subgroups: caretaker genes (CTs) that repair DNA, and gatekeeper genes (GKs) that trigger cell death. Since GKs may also induce cell cycle delay and thus enhance cell survival by facilitating DNA repair, we hypothesized that the prosurvival phenotype of GKs could be selected during cancer progression, and we used a multivariable systems biology approach to test this. We performed multidimensional data analysis, non-negative matrix factorization and logistic regression to compare the features of GKs with those of their putative antagonists, the proto-oncogenes (POs), as well as with control groups of CTs and functionally unrelated congenital heart disease genes (HDs). GKs and POs closely resemble each other, but not CTs or HDs, in terms of gene structure (P<0.001), expression level and breadth (P<0.01), DNA methylation signature (P<0.001) and evolutionary rate (P<0.001). The similar selection pressures and epigenetic trajectories of GKs and POs so implied suggest a common functional attribute that is strongly negatively selected-that is, a shared phenotype that enhances cell survival. The counterintuitive finding of similar evolutionary pressures affecting GKs and POs raises an intriguing possibility: namely, that cancer microevolution is accelerated by an epistatic cascade in which upstream suppressor gene defects subvert the normal bifunctionality of wild-type GKs by constitutively shifting the phenotype away from apoptosis towards survival. If correct, this interpretation would explain the hitherto unexplained phenomenon of frequent wild-type GK (for example, p53) overexpression in tumors.