Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Targeted therapy for prostate cancer may offer potential improvement over current conventional therapies because of its specificity. Although conventional treatments are effective, they are not curative and have several limitations. In prostate cancer, activation of both the epidermal growth factor receptor (EGFR) and the phosphatidylinositol 3 – kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway have been implicated in tumorigenesis and resistance to both conventional and targeted anticancer therapies. Having a better understanding of the molecular mechanisms involved in PCa development, progression and resistance to therapy, could assist in the design of novel therapeutic strategies. The objective of this study was to inhibit key molecular targets of the human epidermal growth factor receptor signalling pathway and expose prostate cell lines to doses of radiation, so as to establish potential therapeutic targets that may be amenable to combined modality therapy, and formulate a cocktail of inhibitors to evaluate its radiosensitising capability. The EGFR/PI3K/mTOR pathway plays an important role in the radiosensitivity of the human prostate carcinoma cell line (DU145) and the normal cell line (1542N). In our study we have shown that AG-1478, an EGFR inhibitor, and BEZ-235, a dual inhibitor of the PI3K/mTOR pathway, singly or in combination, at low and relatively high radiation doses, resulted in radiosensitisation of DU145 cells. Radio-protection was achieved in 1542N cells. AG-1478 had no effect on radiosensitivity. / AFRIKAANSE OPSOMMING: Geteikende terapies wens hul spesifisiteit teenoor konvensionele terapies vir prostaat kanker, mag potensieel verbetering offer. Konvensionele behandeling is wel effektief maar nie genesend nie wens ‘n aantal beperkings, sowel as die toksisiteit vir normale selle. In prostaat kanker is die aktivering van beide die epidermiese groei faktor reseptor (EGFR) en fosfatidielinositol 3-kinase/Akt/soogdier teiken vir rapamisien (mTOR) seingewing baan sterk betrek by tumor groeisel en weerstand teen konvensionele en geteikende anti-kanker terapies. Beter begrip van die molekulêre meganismes betrokke by prostaat kanker ontwikkeling, bevordering en weerstand teen terapie, kan die ontwerp van nuwe terapeutiese strategies ondersteun. Die doelwit van hierdie studie was om sleutel molekulêre teikens van die epidermiese groei faktor reseptor seingewing baan te inhibeer en om prostaat selle bloot te stel aan dosisse bestraling, om potensiële terapeutiese teikens te vestig wat vatbaar is vir gekombineerde modaliteit terapie, om ‘n mengsel van stremmiddels te formuleer, en om die straling gevoeligmaking bekwaamheid daarvan te evalueer. Die EGFR/PI3K/mTOR seingewingbaan speel ‘n belangrike rol in the radiosensitiwiteit van die menslike prostaat kanker sellyn (DU145) en die normale prostaat sellyn (1542N). Die studie bevind dat AG-1478, ‘n EGFR stremmer, en BEZ-235, ‘n tweevoudige beperker van die fosforinositied 3-kinase (PI3K) en soogdier teiken vir rapamisien (mTOR) seingewingbaan, enkel of in kombinasie die DU145 selle radiosensitiseer vir straling dosisse van 2 en 6 Gy. Stralings beskerming was verkry met die 1542N sellyn. AG-1478 het geen effek getoon op radiosensitiwiteit nie.

No description available.

B lymphocytes are an important type of immune cell that contributes to pathogen clearance. When dysregulated, these cells contribute significantly to diseases such as autoimmunity, allergy and B cell cancers. Here we examine an important regulatory circuit that involves the lipid phosphatase SHIP, a key regulator of the PI3K signaling pathway. SHIP was first described as the major effector of inhibitory IgG receptor FcγRIIB, which downregulates B cell antigen receptor (BCR) signaling pathways when co-engaged. However, it is also known to inhibit signaling downstream of several other receptors, both activating and inhibitory. Here we examine the regulation and function of SHIP in B cells, focusing on the inter-related influences of binding partners, tyrosine phosphorylation and subcellular localization dynamics. First, we assess interaction of SHIP to selected candidate binding partners using an in vitro screening approach. The two most robust interactions were further characterized with respect to dissociation constant. These were: a novel interaction between SHIP phospho-Tyr944 and the SH2 domain of Nck, and a known interaction between the SH2 domain of SHIP and FcγRIIB phospho-Y292. Next, we perform the first examination of SHIP Tyr944. We provide evidence that it contributes to interaction with Nck after BCR engagement and is required for inhibition of actin turnover by SHIP. Finally, we perform the first detailed examination of the mechanisms controlling SHIP localization in human B cells stimulated through the BCR with and without co-engagement of FcγRIIB. We discover that SHIP is recruited to the plasma membrane equally in both stimulation contexts, however FcγRIIB co-ligation results in reduced mobility of SHIP molecules at the cell periphery. We identify a novel and essential role for Syk kinase in promoting SHIP membrane localization, tyrosine phosphorylation, and interaction with known binding partner Shc1. Together, these results provide significant and exploitable insight into the molecular control of a clinically important regulator of B cell responses. / February 2017

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23568号 / 医博第4782号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 小川 誠司, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody cross-reactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. IMALDI, which combines immuno-enrichment with analysis using a benchtop MALDI-Time-of-Flight (TOF) mass spectrometer, is an especially well-suited method for translating mass-spectrometry based assays into the clinical lab. We systematically optimized the iMALDI workflow regarding sensitivity, robustness, and throughput while developing highly flexible automation protocols using a Bravo 96LT liquid handling robot. We further developed custom R scripts to improve data visualization and analysis. One hour digestion using a protein to trypsin ratio of 1:2, followed by direct immuno-enrichment for 1 h yielded high and consistent peptide recoveries. We demonstrated that the PTEN and p110α iMALDI assays can be multiplexed using both simultaneous and sequential enrichment, reducing the amount of required sample material as well as simplifying the workflow. The PTEN+p110α iMALDI assay was validated and demonstrated high accuracy for both target proteins (90-112% recovery of known spiked-in concentrations) as well as high precision and 5-day reproducibility (overall CVs of 9%) across the linear range of the assay (0.6 to 20 fmol). Lower limits of quantitation below 1 fmol were achieved. Endogenous PTEN and p110α were quantified in cell lines as well as fresh-frozen tumor tissue samples. A novel two-point internal calibration strategy (2-PIC) was developed, based on spiking two peptide isotopologues into the sample as internal standards, avoiding the need for an external calibration. We quantified endogenous PTEN in a Colo-205 cell line using the PTEN iMALDI assay, as well an orthogonal PTEN immuno-multiple reaction monitoring (immuno-MRM) method to demonstrate this technique. Excellent agreement was shown between both calibration approaches (residual standard deviation between 2-PIC and external calibration of 1.6-5.8%), as well as high correlation between PTEN iMALDI and PTEN immuno-MRM (R²= 0.9966) and good agreement between quantified amounts (0.48±0.01 and 0.29±0.02 fmol/µg of total protein). Finally, we analysed a set of patient samples from a AKT inhibitor AZD5363 drug trial using a multi-site workflow combining the developed PTEN+p110α assay with established AKT1+AKT2 iMALDI assays and untargeted proteomics. We demonstrated how the combination of targeted and untargeted proteomics approaches may be used to gain novel insights into the tumor biology of patient tissue samples. Further, we showed that the PTEN iMALDI assay has good correlation with a comparable immunohistochemistry method (R²=0.86), and that our assays can be further multiplexed, reducing the required amount sample material. Thus, we showed that iMALDI is promising tool for biomarker quantitation. / Graduate / 2022-08-12

Les résultats des projets actuels apportent une information, sur différents aspects des rôles de la voie PI3K, dans le développement du cancer du sein, et la réponse au traitement. Les projets particuliers couvrent des sujets liés à la voie aux niveaux concernant les récepteurs de la famille HER, activant la voie PI3K, ainsi que PI3K et les effecteurs en découlant. Les effets pronostic et prédictif de la dérégulation de PI3K sont les sujets centraux de la recherche décrite ici. Une baisse d’expression de PI3KR1 est associée à une survie réduite dans notre cohorte de patients. Une attention particulière a été portée aux mutations de PIK3CA communes dans le cancer du sein. Tandis que les mutations de PIK3CA agissent comme des marqueurs de bon pronostic chez les patients anti-HER2-naïfs, ces mutations agissent au contraire comme prédicteurs négatifs de la réponse au traitement par trastuzumab. Les résultats décrits mènent un peu plus vers l’implication de plusieurs voies moléculaires altérées, en particulier la voie de signalisation Wnt, dans la tumorigénèse des cancers du sein PIK3CA mutés. De plus, nous avons testé les taux de lapatinib plasmatique montrant une augmentation pertinente dans les périodes d’état d’équilibre du traitement. Par ailleurs, nous avons démontré des incohérences dans l’évaluation de l’EGFR et proposé des approches pour l’interprétation des comptages d’immunohistochimie et de FISH. Tous ces sujets sont connectés par la 170 voie PI3K, et le besoin d’approfondir les connaissances actuelles, et d’apporter de nouvelles informations utiles applicables dans le futur dans les pratiques cliniques / Results of the presented research projects bring information about several aspects of the PI3K signaling pathway roles in breast cancer development and treatment response. The particular projects covered the subjects connected with the signaling pathway, ranging from the HER family receptors activating the pathway, and PI3K to the downstream levels of signalisation. The prognostic and predictive effect of PI3K deregulation was the central subject of the described research. The decreased expression of PIK3R1 associated with reduced survival of our patients. A special focus was put on the PIK3CA mutations which are common in breast cancer. Whereas the PIK3CA mutations act as a good prognostic marker in patients non-treated with the HER2 inhibitors, these mutations predict a negative response to trastuzumab treatment. The described results, furthermore, draw attention to the role of several altered molecular signaling pathways in breast cancer development, especially to the Wnt signaling pathway. The lapatinib plasma levels showing the relevant increase in comparison with the already described efficient steady-state levels were also described in one of the projects. Moreover, various modifications to EGFR status assessment were compared and showed that EGFR FISH and IHC count interpretation depended significantly on method and thresholds used. All these subjects are connected by the PI3K pathway, the need to deepen current knowledge and bring new useful information applicable in future clinical practice.

O carcinoma epidermóide representa mais de 90% das neoplasias malignas de cabeça e pescoço, apresentando taxas elevadas de morbi-mortalidade, porém pouco se sabe sobre as vias de sinalização que estão envolvidas na progressão tumoral. Têm sido relatado na literatura, que alguns estímulos podem ativar a holoenzima PI3K que, por sua vez, desencadeia um processo que induz a fosforilação da proteína Akt (pAkt) que leva a ativação e a translocação do NF-B do citoplasma para o núcleo, onde ocorre a transcrição de genes envolvidos na proliferação e invasão celular. Assim, esse estudo analisou, através dos métodos de Imunofluorescência e Western Blot, a expressão das proteínas pAkt, NF-B e Ciclina D1 em três linhagens de células de carcinoma epidermóide de cabeça e pescoço (HN6, HN30 e HN31) submetidas a cultivo tridimensional e ensaio de invasão (gerando clones invasivos HN6.1, HN30.1 e HN31.1), ambos realizados com Matrigel®. O pAkt apresentou marcação citoplasmática e nuclear nas linhagens celulares HN6, HN30, HN6.1 e na HN30 submetida ao cultivo tridimensional, todavia, as linhagens celulares HN31, HN30.1, HN31.1 e HN6/HN31 cultivadas tridimensionalmente, apresentaram positividade predominantemente nuclear. No caso do NF-B, a localização foi marcadamente citoplasmática em todas as linhagens celulares cultivadas bi ou tridimensionalmente, porém, com exceção da HN31.1, o padrão de marcação foi citoplasmático e nuclear nos clones invasivos. Por fim, a Ciclina D1 exibiu imunopositividade apenas nuclear. A técnica de Western Blot mostrou diminuição nos níveis de expressão das proteínas analisadas quando as células foram cultivadas em Matrigel®, sendo, na maioria das vezes, essa redução estatisticamente significante, exceto no caso da linhagem celular HN6, que apresentou um aumento significativo de Ciclina D1. Os clones invasivos HN6.1 e HN30.1 exibiram elevação significativa dos níveis de expressão de pAkt e NF-B porém, a HN31.1 apresentou aumento de pAkt e discreta diminuição de NF-B, mas ambos os valores não estatisticamente significantes. Em relação à Ciclina D1, houve um aumento dos seus níveis em todas as linhagens invasivas, sendo que apenas na HN6.1 foi estatisticamente significante. Esses resultados mostraram que, nas linhagens celulares avaliadas, quando cultivadas em ambiente tridimensional, a significativa redução dos níveis de expressão de proteínas da via de sinalização PI3K ocorreu devido a uma possível fase adaptativa dessas células ao Matrigel® que seria seguida provavelmente por uma etapa de aumento do potencial proliferativo e posterior transdiferenciação de algumas células para ganho de fenótipo mais agressivo. Além disso, este estudo mostrou a participação da via de sinalização Akt/NF-B/Ciclina D1 no processo de invasão das células de carcinoma epidermóide de cabeça e pescoço (HN6.1 e HN30.1). / Squamous cell carcinoma represents more than 90% of the head and neck malignant tumors, with high mortality rates. Nevertheless, the knowledge about signaling pathways involved in tumor progression remains unclear. The relationship between pAkt and NF-B is well established in carcinogenesis. It is known that the activation of PI3K can be induced by some factors what leads to Akt phosphorilation (pAkt). This further event starts an activation cascade that induces NF-B translocation from the cytoplasm into the nucleus where the transcription of genes enrolled in cellular proliferation and invasion will be done. Therefore, this study analyzed the status of pAkt, NF-B and Cyclin D1 proteins by Immunofluorescence and Western Blot methods in three head and neck squamous cell carcinoma cell lines (HN6, HN30 and HN31) submitted to three-dimensional culture model and an in vitro invasion assay (invasive clones HN6.1, HN30.1 e HN31.1), both with Matrigel®. pAkt expression was detected in cytoplasm and nucleus in HN6, HN30, HN6.1 and HN30 cultured with Matrigel®, however, HN31, HN30.1, HN31.1 cell lines and HN6/HN31 submitted to three-dimensional culture model showed nuclear expression. NF-B localization was strongly cytoplasmatic in all cell lines cultured with or without Matrigel®, but, regarding HN31.1, NF-B protein expression was nuclear and cytoplasmatic in the invasive clones. Cyclin D1 was nuclear in all cell lines analyzed. Western blot assays showed a decrease in pAkt, NF-B and Cyclin D1 expression levels in all cells lines in three-dimensional culture model, most of them statistically significant, but It was detected a considerable increase in Cyclin D1 expression in HN6 cultured in threedimensional model. Moreover, HN6.1 and HN30.1 showed a significant enhance in pAkt and NF-B levels but, HN31.1 demonstrated a raise in pAkt and a slight decline in NF-B, both were not statistically significant. Finally, an increase in Cyclin D1 expression levels was illustrated in all cell lines but, only HN6.1 showed a statistic signal. These results suggest that, in the cell lines studied, when cultured in threedimensional model, a noteworthy reduction in expression levels of PI3K signaling pathway proteins happened for the reason that a possibly adaptation phase of these cells to Matrigel® was occurring, in the first moment, but it would be probably followed by an increase proliferative potential stage and subsequent transdifferentiation of some cells that could show, consequently, a more aggressive phenotype. At last, this study revealed the participation of Akt/NF-B/Cyclin D1 signaling pathway in invasion process of head and neck squamous cell carcinoma (HN6.1 and HN30.1).

O carcinoma epidermóide representa mais de 90% das neoplasias malignas de cabeça e pescoço, apresentando taxas elevadas de morbi-mortalidade, porém pouco se sabe sobre as vias de sinalização que estão envolvidas na progressão tumoral. Têm sido relatado na literatura, que alguns estímulos podem ativar a holoenzima PI3K que, por sua vez, desencadeia um processo que induz a fosforilação da proteína Akt (pAkt) que leva a ativação e a translocação do NF-B do citoplasma para o núcleo, onde ocorre a transcrição de genes envolvidos na proliferação e invasão celular. Assim, esse estudo analisou, através dos métodos de Imunofluorescência e Western Blot, a expressão das proteínas pAkt, NF-B e Ciclina D1 em três linhagens de células de carcinoma epidermóide de cabeça e pescoço (HN6, HN30 e HN31) submetidas a cultivo tridimensional e ensaio de invasão (gerando clones invasivos HN6.1, HN30.1 e HN31.1), ambos realizados com Matrigel®. O pAkt apresentou marcação citoplasmática e nuclear nas linhagens celulares HN6, HN30, HN6.1 e na HN30 submetida ao cultivo tridimensional, todavia, as linhagens celulares HN31, HN30.1, HN31.1 e HN6/HN31 cultivadas tridimensionalmente, apresentaram positividade predominantemente nuclear. No caso do NF-B, a localização foi marcadamente citoplasmática em todas as linhagens celulares cultivadas bi ou tridimensionalmente, porém, com exceção da HN31.1, o padrão de marcação foi citoplasmático e nuclear nos clones invasivos. Por fim, a Ciclina D1 exibiu imunopositividade apenas nuclear. A técnica de Western Blot mostrou diminuição nos níveis de expressão das proteínas analisadas quando as células foram cultivadas em Matrigel®, sendo, na maioria das vezes, essa redução estatisticamente significante, exceto no caso da linhagem celular HN6, que apresentou um aumento significativo de Ciclina D1. Os clones invasivos HN6.1 e HN30.1 exibiram elevação significativa dos níveis de expressão de pAkt e NF-B porém, a HN31.1 apresentou aumento de pAkt e discreta diminuição de NF-B, mas ambos os valores não estatisticamente significantes. Em relação à Ciclina D1, houve um aumento dos seus níveis em todas as linhagens invasivas, sendo que apenas na HN6.1 foi estatisticamente significante. Esses resultados mostraram que, nas linhagens celulares avaliadas, quando cultivadas em ambiente tridimensional, a significativa redução dos níveis de expressão de proteínas da via de sinalização PI3K ocorreu devido a uma possível fase adaptativa dessas células ao Matrigel® que seria seguida provavelmente por uma etapa de aumento do potencial proliferativo e posterior transdiferenciação de algumas células para ganho de fenótipo mais agressivo. Além disso, este estudo mostrou a participação da via de sinalização Akt/NF-B/Ciclina D1 no processo de invasão das células de carcinoma epidermóide de cabeça e pescoço (HN6.1 e HN30.1). / Squamous cell carcinoma represents more than 90% of the head and neck malignant tumors, with high mortality rates. Nevertheless, the knowledge about signaling pathways involved in tumor progression remains unclear. The relationship between pAkt and NF-B is well established in carcinogenesis. It is known that the activation of PI3K can be induced by some factors what leads to Akt phosphorilation (pAkt). This further event starts an activation cascade that induces NF-B translocation from the cytoplasm into the nucleus where the transcription of genes enrolled in cellular proliferation and invasion will be done. Therefore, this study analyzed the status of pAkt, NF-B and Cyclin D1 proteins by Immunofluorescence and Western Blot methods in three head and neck squamous cell carcinoma cell lines (HN6, HN30 and HN31) submitted to three-dimensional culture model and an in vitro invasion assay (invasive clones HN6.1, HN30.1 e HN31.1), both with Matrigel®. pAkt expression was detected in cytoplasm and nucleus in HN6, HN30, HN6.1 and HN30 cultured with Matrigel®, however, HN31, HN30.1, HN31.1 cell lines and HN6/HN31 submitted to three-dimensional culture model showed nuclear expression. NF-B localization was strongly cytoplasmatic in all cell lines cultured with or without Matrigel®, but, regarding HN31.1, NF-B protein expression was nuclear and cytoplasmatic in the invasive clones. Cyclin D1 was nuclear in all cell lines analyzed. Western blot assays showed a decrease in pAkt, NF-B and Cyclin D1 expression levels in all cells lines in three-dimensional culture model, most of them statistically significant, but It was detected a considerable increase in Cyclin D1 expression in HN6 cultured in threedimensional model. Moreover, HN6.1 and HN30.1 showed a significant enhance in pAkt and NF-B levels but, HN31.1 demonstrated a raise in pAkt and a slight decline in NF-B, both were not statistically significant. Finally, an increase in Cyclin D1 expression levels was illustrated in all cell lines but, only HN6.1 showed a statistic signal. These results suggest that, in the cell lines studied, when cultured in threedimensional model, a noteworthy reduction in expression levels of PI3K signaling pathway proteins happened for the reason that a possibly adaptation phase of these cells to Matrigel® was occurring, in the first moment, but it would be probably followed by an increase proliferative potential stage and subsequent transdifferentiation of some cells that could show, consequently, a more aggressive phenotype. At last, this study revealed the participation of Akt/NF-B/Cyclin D1 signaling pathway in invasion process of head and neck squamous cell carcinoma (HN6.1 and HN30.1).

The phosphatidylinositol 3' kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival, and glucose transport, and is implicated in various disease states such as cancer and diabetes. Though there have been numerous studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenicity, PI3K activity might also be important for self-renewal of ES cells. Paling et al. (2004) reported that the inhibition of PI3K led to a reduction in the ability of leukemia inhibitory factor (LIF) to maintain self-renewal causing cells to differentiate. Studies in our lab have revealed that ES cells completely lacking GSK-3 remain undifferentiated compared to wildtype ES cells. GSK-3 is negatively regulated by PI3K suggesting that PI3K may play a vital role in maintaining pluripotency in ES cells through GSK-3. By using a modified Flp recombinase system, we expressed activated alleles of PDK-1 and PKB to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate determination. In vitro characterization of the transgenic cell lines revealed a strong tendency towards maintenance of pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. To assess growth and differentiation capacity in vivo, the ES cell lines were grown as subcutaneous teratomas. The constitutively active PDK-1 and PKB ES cell lines were able to form all three germ layers when grown in this manner – in contrast to ES cells engineered to lack GSK-3. The resulting PI3K pathway activated cells exhibited a higher growth rate which resulted in large teratomas. In summary, PI3K signaling is sufficient to maintain self-renewal and survival of stem cells. Since this pathway is frequently mutationally activated in cancers, its effect on suppressing differentiation may contribute to its oncogenicity.

The phosphatidylinositol 3' kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival, and glucose transport, and is implicated in various disease states such as cancer and diabetes. Though there have been numerous studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenicity, PI3K activity might also be important for self-renewal of ES cells. Paling et al. (2004) reported that the inhibition of PI3K led to a reduction in the ability of leukemia inhibitory factor (LIF) to maintain self-renewal causing cells to differentiate. Studies in our lab have revealed that ES cells completely lacking GSK-3 remain undifferentiated compared to wildtype ES cells. GSK-3 is negatively regulated by PI3K suggesting that PI3K may play a vital role in maintaining pluripotency in ES cells through GSK-3. By using a modified Flp recombinase system, we expressed activated alleles of PDK-1 and PKB to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate determination. In vitro characterization of the transgenic cell lines revealed a strong tendency towards maintenance of pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. To assess growth and differentiation capacity in vivo, the ES cell lines were grown as subcutaneous teratomas. The constitutively active PDK-1 and PKB ES cell lines were able to form all three germ layers when grown in this manner – in contrast to ES cells engineered to lack GSK-3. The resulting PI3K pathway activated cells exhibited a higher growth rate which resulted in large teratomas. In summary, PI3K signaling is sufficient to maintain self-renewal and survival of stem cells. Since this pathway is frequently mutationally activated in cancers, its effect on suppressing differentiation may contribute to its oncogenicity.