Study liver tumorigenesis in transgenic tumor zebrafish using chemical screens

STUDY LIVER TUMORIGENESIS IN TRANSGENIC TUMOR ZEBRAFISH USING CHEMICAL SCREENS ZHOU LI NATIONAL UNIVERSITY OF SINGAPORE 2011 STUDY LIVER TUMORIGENESIS IN TRANSGENIC TUMOR ZEBRAFISH USING CHEMICAL SCREENS ZHOU LI (B.Sc., XMU) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements   I would like to thank my supervisor, Professor Gong Zhiyuan, who offered me a valuable opportunity to pursue my Master degree in his lab Throughout my graduate studies, Professor Gong has been supportive and helpful in providing me guidance on my research project and my future career His passion, attitude in research and scientific advices would benefit me in the life long run I would like to give special thanks to Li Zhen, who is my young supervisor in this lab and plays a critical role in the project She was the first one who introduced me to the zebrafish research field from the zero start Li Zhen’s enthusiasm in scientific research has impressed me a lot I also own much gratitude to Xu Hongyan and Li Caixia, whom I bothered a lot and always liked to help me out of trouble Hongyan gave me a lot of valuable guidance on benchwork, without which the progression of the project would definitely become more difficult Caixia not only provided me lots of assistance on my project but also concerned me a lot about my daily life I would also like to thank my labmates including Tina, Grace, Weiling, Lili, Sahar, Xiaoqian, Xiaoyan, Shen Yuan, Euiyin, Li Yan, Yan Chuan, Anh Tuan, Hendrian, Zhengyuan, Lana, Choong Yong and Yin Ao They helped with my experiments and made the lab a nice and warm place to make me feel just like at home During these two years, we get along well with each other The friendship and happy time with them would be unforgettable in my mind i    In addition, I would like to thank Balan and Qinghua, without whose work on fish maintenance in the aquarium, the project would be hard to move on Last but not least, I would like to thank my family members, my father, mother, brothers and sisters, lacking of whose support and trust, the project would not be accomplished I would also like to give special thanks to my loyal friends, Jingjie, Yinwen, Jinling, Hongmei and etc Life would be no meaning without their love and company ii    Contents  Acknowledgements i Summary v List of Figures vi List of Tables vii List of Abbreviations viii Introduction 1.1  Introduction of HCC   2  1.2  Zebrafish   3  1.3  Recent development in chemical screens using zebrafish  . 4  1.4  Zebrafish models for liver cancer research   6  1.5  Overview of Signaling Pathways Implicated in HCC  . 9  1.5.1  MAPK pathway  . 10  1.5.2  JAK/STAT pathway   10  1.5.3  PI3K/AKT pathway  . 11  1.6  Main objectives and significance of the study  . 12  Materials and Methods 14 2.1  Zebrafish husbandry   15  2.2  TO(Myc), TO(kras) and Lipan transgenic zebrafish   15  2.3  Mating of zebrafish   15  2.4  Timing of exposure to chemicals  16  2.5  Doxycycline induction effect on cMyc and kras transgenic fish   16  2.5.1  Survival curve assay by Dox   16  2.5.2  Dox induction treatment   17  2.6  Inhibition effect of different inhibitors on cMyc and kras fish  . 17  2.6.1  Survival curve analysis   17  2.6.2  Inhibition treatment  . 17  2.7  Photography   18  2.8  Image analysis and statistical analysis   18  2.9  Criteria for ‘effective inhibitor’  . 19  2.10  Analysis of cell proliferation   19  2.10.1  Cryosection of zebrafish liver   19  2.10.2  Detection of proliferating cell in zebrafish liver by PCNA staining  . 20  2.10.3  Image analysis and statistical analysis   21  iii    2.11  Inhibitors used in the study   21  Results 23 3.1  fish  Dose-dependent induction of liver hyperplasia by doxycycline in cMyc and kras 24  3.2  Inhibitory effect of different pathway inhibitors on cMyc-dependent liver hyperplasia   29  3.2.1  Stat5 inhibitor   29  3.2.2  MEK1/2 inhibitor: PD0325901  . 31  3.2.3  MEK1/2 inhibitor: U0126  . 33  3.2.4  PI3K inhibitor: LY294002   35  3.2.5  cMyc inhibitor: 10058-F4   37  3.2.6  Proliferation analysis on liver from treated cMyc fish   39  3.3    Inhibitory effect of different pathway inhibitors on kras-dependent liver hyperplasia 41  3.3.1  Stat5 inhibitor   41  3.3.2  MEK1/2 inhibitor: PD0325901  . 43  3.3.3  MEK1/2 inhibitor: U0126  . 45  3.3.4  PI3K inhibitor: LY294002   47  3.3.5  cMyc inhibitor: 10058-F4   49  3.3.6  Proliferation analysis on liver from treated kras fish  . 51  3.4  Comparsion of inhibition efficacy of different inhibitors on liver hyperplasia between cMyc and kras fish   53  Discussion 55 4.1  Development of image-based phenotypic analysis in transgenic zebrafish to evaluate inhibition effects on liver tumorigenesis  . 56  4.2  Insights of inhibition mechanisms underlying different inhibitors on cMyc fish and kras fish during tumor development   58  4.2.1  JAK/STAT pathway and Stat5 inhibitor  . 58  4.2.2  MAPK pathway and PD0325901, U0126  . 60  4.2.3  PI3K pathway and LY294002  . 62  4.2.4  cMyc pathway and 10058-F4   64  4.3  Major conclusions and findings  . 65  4.4  Prospects   67  References 69   iv    Summary   Hepatocellular carcinoma (HCC) is one of the leading cancers in the world and this disease is often diagnosed at an advanced stage when potentially curative therapies are not feasible Understanding of the molecular mechanism of HCC is vital to develop therapeutical approaches to cure this disease In recent years, the zebrafish has become a popular model to study human diseases, particularly for small molecule screening in drug discovery In the current study, we employed two zebrafish tumor models previously established in our lab, TO(Myc) and TO(kras) transgenic line, which contain Myc and krasv12 oncogenes respectively in a tetracycline-inducible (tet-on) system and produced HCC by doxycycline induction To develop a rapid assay for potential anticancer drug screening, several chemical inhibitors which target a few signaling pathways involved in HCC, including MAPK pathway, JAK/STAT pathway, PI3K pathway and Myc transcription factor, were selected to treat larvae of the two transgenic lines Following the treatment, liver images were taken and analyzed by ImageJ for two-dimensional area quantification followed by cell proliferation analysis to further investigate the inhibition effect We observed that some inhibitors such as Stat5In and PD0325901 inhibited liver overgrowth in both TO(Myc) and TO(kras) larvae; U0126 was only effective in TO(kras) larvae but not in TO(Myc) larvae; LY294002 was able to reduce liver enlargement in TO(Myc) larvae but failed to so in TO(kras) larvae We conclude that inhibition of JAK/STAT pathway or MAPK pathway in both Myc and kras mediated oncogenesis suppresses tumor growth, and targeting PI3K pathway using LY294002 is a potential means to treat Myc driven oncogenesis v    List of Figures   Figure An overview of signal transduction pathways………………………… page 11 Image analysis with ImageJ program…………………………………… 19 Dose-dependent induction of liver hyperplasia by dox in cMyc fish……………………………………………………………………… 25 Dose-dependent induction of liver hyperplasia by dox in kras fish…… 27 Comparison of induced liver area in cMyc fish and kras fish under the same concentration of Dox……………………………………………… 28 Effect of stat5In on liver hyperplasia in cMyc fish…………………… 30 Effect of PD0325901 on liver hyperplasia in cMyc fish……………… 32 Effect of U0126 on liver hyperplasia in cMyc fish…………………… 34 Effect of LY294002 on liver hyperplasia in cMyc fish……………………………………………………………………… 36 10 Effect of 10058-F4 on liver hyperplasia in cMyc fish………………… 38 11 Proliferation analysis on liver from treated cMyc fish………………… 40 12 Effect of stat5In on liver hyperplasia in kras fish……………………… 42 13 Effect of PD0325901 on liver hyperplasia in kras fish………………… 44 14 Effect of U0126 on liver hyperplasia in kras fish……………………… 46 15 Effect of LY294002 on liver hyperplasia in kras fish………………… 48 16 Effect of 10058-F4 on liver hyperplasia in kras fish…………………… 50 17 Proliferation analysis on liver from treated kras fish…………………… 52 vi    List of Tables   Figure page Chemicals concentrations used in survival curve and inhibition treatment kras fish…………………………………………………… 22 Chemicals concentrations used in survival curve and inhibition treatment cMyc fish………………………………………………… 22 List of inhibitors……………………………………………………… 22 Different ‘inhibitors’ efficacy on cMyc and kras fish based on statistical analysis on liver area measurement and proliferation assays………………………………………………………………… 54   vii    List of Abbreviations HCC Hepatocellular carcinoma TGF Transforming growth factor FGF Fibroblast growth factor BCI 2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one Dusp6 dual-specificity phosphatase ERK extracellular signal-regulated kinase BMP Bone morphogenetic protein T-ALL T-cell acute lymphoblastic leukemia ERMS Embryonal rhabdomyosarcoma fabp10 fatty acid binding protein 10 Dox doxycycline PI3K Phosphatidylinositol 3-kinases STAT Signal Transducer and Activator of Transcription JAK Janus kinase MAPK Mitogen-activated protein kinase DNA Deoxyribonucleic acid Hpf hours post fertilization Dpf days post fertilization GFP green fluorescent protein RFP red fluorescent protein ElaA elastaseA PTU 1-phenyl-2-thiourea DMSO Dimethyl sulfoxide PFA Paraformaldehyde viii    4.2.2 MAPK pathway and PD0325901, U0126 MEK1/2 is a component of MAPK pathway MAPK is a downstream effector of RAS and MAPK phosphorylates cMyc, which regulates expression of genes involved in cell proliferation and cell apoptosis Once the MAPK pathway is disrupted, cMyc function would be affected Indeed, inhibition of MEK has the potential to prevent the subsequent downstream phosphorylation and activation of MAP kinase and consequently induces tumor regression and/or stasis in some contexts (Solit et al., 2006, Ji et al., 2007) It has been shown that administration of MEK inhibitor helps to prevent development of HCC and increase apoptosis in existing HCC tumors in mice (Wentz et al., 2008), and results in dose-dependent growth inhibition of HCC xenografts (Huynh et al., 2007) Inhibitory activity of U0126 is selective for MEK1/2, and does not inhibit other protein kinases like Raf, MEKK, ERK or JNK (Duncia et al., 1998) The specificity of U0126 as a MEK1/2 inhibitor has already been established by many studies both in vitro (Murakami et al., 2000) and in vivo (Bedogni et al., 2004), including one analysis in zebrafish (Hong et al., 2006) In one study using HCC cell line, administration of U0126 blocked ERK phosphorylation, and showed anticancer effects through inducing cell cycle arrest, increasing cell apoptosis, and decreasing tumorigenecity (Wiesenauer et al., 2004) Additionally, in one study using human muscle-derived Rhabdomyosarcoma (RD) cell line and other non muscle-derived human tumor cell lines, U0126 was found to induce cMyc de-phosphorylation, followed by expression level reduction in cMyc and other cMyc targets, such as cyclins E2, A and B and CDK2, all of which are regulators of cell cycle (Marampon 60 et al., 2006), and consequently resulted in blocked oncogenic phenotype in used cell lines PD0325901 and U0126 both effectively inhibit MEK activity, but the former is more potent than the latter in HCC cells based on the median inhibitory concentration lying in the nanomolar range (Ishizaki et al., 2010).Targeting MAPK pathway with PD0325901 was shown to decrease HCC growth in vitro and in vivo mouse model system by inhibiting proliferation and inducing apoptosis (Hennig et al., 2010) Moreover, PD0325901 recently emerged to be a new clinical candidate in treating cancer (Sebolt-Leopold, 2008) Additionally, inducing angiogenesis is a part of event of tumorigenesis (Hanahan and Weinberg, 2011), and PD0325901 has a potential role in anti-angiogenesis (Sosman and Puzanov, 2006) Thus we suspect that the effect observed for PD0325901 inhibition may be a result of inhibition of both cell proliferation and angiogenesis In our study, PD0325901 inhibited liver overgrowth in both cMyc and kras fish, supported by decreased cell proliferation (Figure 11A, B and Figure 17A, B), but U0126 was effective only in kras fish However, according to other research literatures, U0126 helped to block the oncogenic phenotypes (Wiesenauer et al., 2004, Marampon et al., 2006), we can not rule out the possibility that U0126 inhibits tumor progression in cMyc fish in a modest way due to the low concentration which provided only part suppression of MEK It is also possible that it may regulate other molecules that stimulate the cell proliferation or blood vessel growth in fish development, since we observed that U0126 alonetreated normal fish also had liver enlarged comparing with vehicle-treated control fish 61 In summary, our test implicated MAPK signaling pathway is activated in both cMyc and kras oncogenesis and targeting MAPK pathway could be useful in treating HCC While inhibitor PD0325901 was effective in both cMyc and kras carcinogenesis, U0126 was effective in kras carcinogenesis Anti-apoptotic proteins Bcl-Xl and survivin have been shown to be highly upregulated in human HCC tissues (Ikeguchi et al., 2002, Watanabe et al., 2002); and MEK inhibition by U0126 decreased the expression of these two proteins (Wiesenauer et al., 2004), suggesting these two proteins potential playing roles as MEK-dependent apoptosis mediators It might be interesting to perform further apoptotic assay and western blot examining the ERK (downstream of MEK) and BclXl and survivin so as to better understand the mechanism 4.2.3 PI3K pathway and LY294002 Initially, validation of PI3Ks as drug targets has been attempted using wortmannin and LY294002, both of which target a broad range of PI3K-related enzymes (Arcaro and Wymann, 1993, Wymann et al., 2003) LY294002 is competitive for ATP binding site on PI3K, thus leading to the kinase out of function We selected LY294002 in our current study also due to its superior chemical stability Our study showed that LY294002 was able to reduce liver hyperplasia in cMyc fish but not in kras fish One study demonstrated that cMyc accumulation was controlled by Ras activities either via Raf/ERK pathway or via PI3K/Akt-mediated inactivation of Glycogen synthase kinase-3 (GSK-3) activities (Sears et al., 2000) In this study, the treatment of PD98059 (an inhibitor of MEK1/2) and Wortmannin (an inhibitor of PI3K) impairs the Ras-mediated stabilization of cMyc protein, thus both ERK and 62 PI3K involve in the regulation of cMyc levels Thus, if PI3K was inhibited, Akt activity would be declined and GSK-3 would be activated which subsequently results in cMyc protein degradation, which then down regulate the cell proliferation However, on the controversial side, the overexpression of kras results in cMyc accumulation via Raf/ERK pathway and may cause subsequent increased cell proliferation Therefore, there must be a competition between the cMyc degradation and accumulation under the treatment of PI3K inhibitor LY294002 in kras-dependent liver hyperplasia In our study, the anti-tumor effects of LY294002 in kras fish was not achieved, which might be the result of overwhelming accumulation of cMyc; another reason might be low concentration of this inhibitor used (5 µM), comparing with higher concentration used in other works (50 µM in (Perkinton et al., 2002); 10 µM in (Nguyen et al., 2012) It is required to look into the protein levels of ERK, Akt, GSK-3 and cMyc to determine whether the targeted protein PI3K is inhibited and the downstream effectors are affected so as to dig out the mechanism As in cMyc fish, we observed reduced liver enlargement when treated with LY294002 The result obtained here seems contradictory to one recent study, where elevation of cMyc drives tumors to become independent of PI3K pathway, suggesting the cMyc-driven tumors resistant to current PI3K-targeted therapies (Liu et al., 2011) As cMyc is a downstream effector of PI3K pathway (Sears et al., 2000), it can be hypothesized that cMyc-driven carcinogenesis is independent of PI3K inhibition Our result is not coincide with this hypothesis, thus further investigation into the protein level of Akt, GSK-3 and cMyc would be helpful to address this controversy It is also interesting to note that LY294002 alone treated fish also showed enlarged liver comparing with control fish While it is unknown if LY294002 has other potential roles in regulating other cellular activities, it is likely that an increased 63 angiogenesis leads to enlarged liver Arkaitz et al showed that inhibition of mTOR results in activation of MAPK pathway via PI3K-dependent negative feedback loop stemming from S6, a downstream effector of mTOR (Carracedo et al., 2008) Moreover, a recent study carried out by Anne and colleagues on human umbilical vein endothelial cells (HUVEC) demonstrated that on the one hand, mTOR inhibitors effectively block mTOR activity and on the other hand increase MAPK phosphorylation (Dormond-Meuwly et al., 2011) These works inspire us to hypothesize that although LY294002 blocks PI3K activities, it also increases MAPK phosphorylation which induces cell proliferation through negative feedback loop To confirm this hypothesis, western blot analysis about the level of S6, ERK and Akt proteins and CD31 immunohistochemistry (evaluation of the degree of tumor angiogenesis) can be performed to unravel the veil of the mechanism 4.2.4 cMyc pathway and 10058-F4 It is known that activation of cMyc contributes to the cancer development The cMyc protein is a transcription factor, plays critical roles in cell proliferation, growth, cell cycle progression, genomic stability, differentiation, and apoptosis (Yin et al., 2003) cMyc contains a basic helix-loop-helix/leucine zipper motif (bHLHZ) that drives heterodimer formation with the bHLHZ domain of the Max protein (Amati et al., 1993, Henriksson and Luscher, 1996, Oster et al., 2002, Yin et al., 2003) Myc/Max heterodimers function by binding E-box DNA (CACGTG) in the target gene, thereby regulating transcription Recent work confirms that Myc inhibitors including some small molecules hinder tumor cell growth One class of small molecule inhibitors disrupts the formation of the Myc/Max heterodimer The disruption of the dimer results in inhibition of the transcriptional function of cMyc 64 10058-F4 is among the first compounds found to disrupt the association between cMyc and Max Gomez-Curet et al reported that 10058-F4 not only blocked cMyc function through the mechanism of cMyc/Max heterodimer dissociation, but also decreased cMyc mRNA levels in lymphoma cells (Gomez-Curet et al., 2006) In our study, we observed that both cMyc and kras fish showed no obviours change in liver enlargement after exposure to 10058-F4, indicating that 10058-F4 by inhibiting cMyc failed to fully reduce the liver hyperplasia in both cMyc and krasdriven carcinogenesis One of the reasons could be due to the low concentration of this inhibitor used in current fish study, only 0.5/0.8 µM which is far lower than those used in cell based assay (IC50=60 µM); another reason might be that inhibition of cMyc alone may not be sufficient to block tumorigenesis since there are many pathways that may lead to tumorigenesis; the third reason for the case in kras-driven carcinogenesis may be that the kras-dependent liver carcinogenesis is not via cMyc pathway, thus inhibition of cMyc could not help to reverse the kras role in cancer 4.3 Major conclusions and findings In our current work, we employed two transgenic zebrafish tumor models, cMyc and kras line, to study the early tumorigenesis in liver We administered several chemical inhibitors targeting the HCC signaling pathways including MAPK pathway, JAK/STAT pathway, PI3K pathway and cMyc transcription factor, in the two transgenic larval zebrafish We initially assessed the morphological change in liver size by quantification analysis and further we confirmed the inhibition effects by analyzing the cell proliferation in the treated fish liver Our findings include: 1) By employing the ImageJ program to analyze the images, we developed a sensitive and simple way for evaluating the chemical effects in larval fish, 65 complementing the advantages of transgenic approach and transparency of zebrafish larvae, and overcoming the problem that some delicate morphological alterations that are not easy to be distinguished by direct visual examination 2) Stat5 inhibitor (Stat5In) significantly inhibited liver hyperplasia in induced cMyc and kras fish, suggesting that JAK/STAT pathway participate in both cMyc-mediated and kras-mediated tumor development and targeting this pathway is helpful to delay and hinder tumor growth 3) MEK1/2 inhibitor PD0325901 significantly inhibited liver hyperplasia in both induced cMyc and kras fish Unlike PD0325901, U0126 stimulated liver overgrowth in normal fish Consequently, we found that U0126 only significantly inhibited liver overgrowth in kras fish but not in cMyc fish The contradictory role of U0126 in promoting normal liver growth and potential suppression of induced liver hyperplasia requires further investigation 4) The PI3K inhibitor LY294002 was able to hinder the liver overgrowth in treated cMyc fish but not in treated kras fish, indicating that PI3K pathway, which reflects angiogenesis, a hall mark of cancer, is a more striking event in cMyc driven oncogenesis than kras driven carcinogenesis and thus antiangiogenesis is more effective in cMyc-mediated tumorigenesis 5) 10058-F4, a specific inhibitor of cMyc, showed no obvious inhibition effect in both cMyc and kras fish, which might indicate that higher concentration of this inhibitor is needed to achieve better inhibition effect, and targeting cMyc in kras driven oncogenesis may not be fully effective 66 6) Based on the above analysis, these two inducible tumor lines share common signaling pathways during early HCC tumor initiation though difference exists in determining which pathway is the leading role Collectively, we have proved that larval zebrafish systems are ideal for chemical screening in preclinical drug discovery for liver cancer And we have confidence in believing that our two inducible zebrafish models are suitable for further studying and delineating the respective signaling pathways in cMyc driven and kras driven liver oncogenesis The limitations of this study include insufficient evidences to demonstrate the inhibition mechanism, e.g lacking of information such as apoptosis analysis, the protein or mRNA level of the targeted molecules or downstream effectors In consequence, further study need to be carried out to fill in this gap 4.4 Prospects From above analysis, it seems that sometimes targeting only one pathway is not so efficacious 10058-F4 targeting cMyc transcription factor in kras fish and U0126 targeting MEK1/2 in cMyc fish both failed to inhibit tumor growth, it might be possible that if combining 10058-F4 with U0126 in kras fish or cMyc fish, the result would be different In fact, there is a strong therapeutic rationale for attempting to inhibit several important signaling pathways simultaneously in patients with HCC It was found that PKI-587 a highly potent dual PI3K/mTOR kinase inhibitor was able to enhance antitumor efficacy when combined with PD0325901 (Mallon et al., 2011) Therefore, simultaneous inhibition of both the MAPK pathway and the PI3K pathway may also offer therapeutic advantages over inhibiting either pathway alone Thus using different inhibitors targeting different pathways may yield better effect and may 67 be useful to develop a cocktail approach for anti-cancer drug discovery Preliminary data about the suppression of induced liver hyperplasia by chemical inhibitors were obtained and the next step is to look into the underlying mechanism of this inhibition It is very intriguing to perform western blots to examine the proteins level of the targeting genes and their downstream effectors, which may help to validate the inhibition mechanism and to reveal novel perturbed protein during tumorigenesis More compounds targeting specific signaling pathways will be identified for cancer treatment in the future For example, FGF signaling pathway and Wnt pathway are of great interest to us, and SU5402 and IWR-1 have been reported to target these two pathways respectively; hence, in the near future, we would like to test these two inhibitors on our transgenic zebrafish It is very interesting to include xmrk fish, where liver carcinogenesis is driven by xmrk overexpression (Li et al., 2012), in our study to apply the same inhibitors as those in cMyc and kras fish, so that we can get more and comprehensive understanding about the HCC progression in different genetic background By crossing our existing tumor lines, 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DEGREE... phenotypic analysis in transgenic zebrafish to evaluate inhibition effects on liver tumorigenesis? ? . 56  4.2  Insights of inhibition mechanisms underlying different inhibitors on cMyc fish... as well as tumors by taking the advantage of embryonic and transparent strain, the casper zebrafish (White et al., 2008) 1.3 Recent development in chemical screens using zebrafish The zebrafish