Ioanna G. Maroulakou

Akt1 Ablation Inhibits, whereas Akt2 Ablation Accelerates, the Development of Mammary Adenocarcinomas in Mouse Mammary Tumor Virus (MMTV)-ErbB2/Neu and MMTV-Polyoma Middle T Transgenic Mice

Ample evidence to date links the phosphatidylinositol 3-kinase-regulated protein kinase Akt with the induction and progression of human cancer, including breast cancer. However, there are three Akt isoforms with limited information about their specificity during oncogenesis. This study addresses the role of the three isoforms in polyoma middle T (PyMT) and ErbB2/Neu-driven mammary adenocarcinomas in mice. The effects of ablation of Akt1, Akt2, and Akt3 on the induction and the biology of these tumors were dramatically different, with ablation of Akt1 inhibiting, ablation of Akt2 accelerating, and ablation of Akt3 having a small, not statistically significant, inhibitory effect on tumor induction by both transgenes. Whereas PyMT-induced tumors are all invasive, Akt1(-/-)Neu-induced tumors are more invasive than Akt2(-/-)Neu-induced tumors. Invasiveness, however, does not always correlate with metastasis. Ablation of individual Akt isoforms does not affect the development of the mammary gland during puberty or the expression of the transgenes. Akt ablation, therefore, influences tumor induction by modulating transgene-induced oncogenic signaling. Immunostaining for Ki-67 and cyclin D1 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays on tissue sections revealed that the delay of tumor induction in Akt1 knockout mice is due to the inhibitory effects of Akt1 ablation on cell proliferation and survival. Given that these animal models exhibit significant similarities to human breast cancer, the results of the present study may have significant translational implications because they may influence how Akt inhibitors will be used in the treatment of human cancer.

MicroRNAs Differentially Regulated by Akt Isoforms Control EMT and Stem Cell Renewal in Cancer Cells

Akt-dependent induction of a metastatic phenotype may depend on the balance of Akt1 and Akt2. Balance Is Everything Members of the Akt family of protein kinases, which are activated by growth factor signaling, have been implicated in human cancer; however, the different Akt isoforms appear to play distinct roles. Iliopoulos et al. used cells containing only a single Akt isoform—or cells in which the abundance of one or two of the three Akt isoforms was selectively reduced—to explore their specific contributions to tumor induction and invasiveness. They found that growth factor stimulation of the different isoforms led to distinct changes in the abundance of microRNAs, a class of molecules that regulate gene expression and can thereby promote or inhibit oncogenesis. Intriguingly, the effects of Akt signaling on the abundance of the miR-200 family of microRNAs—and thereby on the induction of a metastatic phenotype in human breast cancer cells—appeared to depend on the balance in abundance or activity of Akt1 and Akt2 rather than on overall Akt signaling per se. Although Akt is known to play a role in human cancer, the relative contribution of its three isoforms to oncogenesis remains to be determined. We expressed each isoform individually in an Akt1−/−/Akt2−/−/Akt3−/− cell line. MicroRNA profiling of growth factor–stimulated cells revealed unique microRNA signatures for cells with each isoform. Among the differentially regulated microRNAs, the abundance of the miR-200 family was decreased in cells bearing Akt2. Knockdown of Akt1 in transforming growth factor–β (TGFβ)–treated MCF10A cells also decreased the abundance of miR-200; however, knockdown of Akt2, or of both Akt1 and Akt2, did not. Furthermore, Akt1 knockdown in MCF10A cells promoted TGFβ-induced epithelial-mesenchymal transition (EMT) and a stem cell–like phenotype. Carcinomas developing in MMTV-cErbB2/Akt1−/− mice showed increased invasiveness because of miR-200 down-regulation. Finally, the ratio of Akt1 to Akt2 and the abundance of miR-200 and of the messenger RNA encoding E-cadherin in a set of primary and metastatic human breast cancers were consistent with the hypothesis that in many cases breast cancer metastasis may be under the control of the Akt–miR-200–E-cadherin axis. We conclude that induction of EMT is controlled by microRNAs whose abundance depends on the balance between Akt1 and Akt2 rather than on the overall activity of Akt.

Expression and function of Ets transcription factors in mammalian development: a regulatory network

The Ets transcription factor family is involved in a variety of mammalian developmental processes at the cellular, tissue and organ levels. They are implicated in cellular proliferation, differentiation, migration, apoptosis and cell–cell interactions. This article reviews recent studies that demonstrate the integral importance of Ets in the dosage dependent regulation of development. The expression of many Ets genes is associated with mesenchymal–epithelial interactions and changes in extracellular matrix proteins. These inductive processes contribute to tissue remodeling and integrity, particularly during embryonic development. Overlapping as well as unique patterns of Ets expression are evident in developing tissues, including development of the lymphoid and myeloid lineages, brain and central nervous system, bone and mammary gland. Integration of these data will allow the development of predictive models for the regulation of complex developmental processes.

Akt2 regulates all Akt isoforms and promotes resistance to hypoxia through induction of miR-21 upon oxygen deprivation

The growth and survival of tumor cells in an unfavorable hypoxic environment depend upon their adaptability. Here, we show that both normal and tumor cells expressing the protein kinase Akt2 are more resistant to hypoxia than cells expressing Akt1 or Akt3. This is due to the differential regulation of microRNA (miR) 21, which is upregulated by hypoxia only in Akt2-expressing cells. By upregulating miR-21 upon oxygen deprivation, Akt2 downregulates PTEN and activates all three Akt isoforms. miR-21 also targets PDCD4 and Sprouty 1 (Spry1), and the combined downregulation of these proteins with PTEN is sufficient to confer resistance to hypoxia. Furthermore, the miR-21 induction by Akt2 during hypoxia depends upon the binding of NF-κB, cAMP responsive element-binding protein (CREB), and CBP/p300 to the miR-21 promoter, in addition to the regional acetylation of histone H3K9, all of which are under the control of Akt2. Analysis of the Akt2/miR-21 pathway in hypoxic MMTV-PyMT-induced mouse mammary adenocarcinomas and human ovarian carcinomas confirmed the activity of the pathway in vivo. Taken together, this study identifies a novel Akt2-dependent pathway that is activated by hypoxia and promotes tumor resistance via induction of miR-21.

Unequal Contribution of Akt Isoforms in the Double-Negative to Double-Positive Thymocyte Transition

Pre-TCR signals regulate the transition of the double-negative (DN) 3 thymocytes to the DN4, and subsequently to the double-positive (DP) stage. In this study, we show that pre-TCR signals activate Akt and that pharmacological inhibition of the PI3K/Akt pathway, or combined ablation of Akt1 and Akt2, and to a lesser extent Akt1 and Akt3, interfere with the differentiation of DN3 and the accumulation of DP thymocytes. Combined ablation of Akt1 and Akt2 inhibits the proliferation of DN4 cells, while combined ablation of all Akt isoforms also inhibits the survival of all the DN thymocytes. Finally, the combined ablation of Akt1 and Akt2 inhibits the survival of DP thymocytes. Constitutively active Lck-Akt1 transgenes had the opposite effects. We conclude that, following their activation by pre-TCR signals, Akt1, Akt2, and, to a lesser extent, Akt3 promote the transition of DN thymocytes to the DP stage, in part by enhancing the proliferation and survival of cells undergoing β-selection. Akt1 and Akt2 also contribute to the differentiation process by promoting the survival of the DP thymocytes.

VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase

The growth factor, vascular endothelial growth factor (VEGF), induces angiogenesis and promotes endothelial cell (EC) proliferation. Affymetrix gene array analyses show that VEGF stimulates the expression of a cluster of nuclear‐encoded mitochondrial genes, suggesting a role for VEGF in the regulation of mitochondrial biogenesis. We show that the serine threonine kinase Akt3 specifically links VEGF to mitochondrial biogenesis. A direct comparison of Akt1 vs. Akt3 gene silencing was performed in ECs and has uncovered a discrete role for Akt3 in the control of mitochondrial biogenesis. Silencing of Akt3, but not Akt1, results in a decrease in mitochondrial gene expression and mtDNA content. Nuclear‐encoded mitochondrial gene transcripts are also found to decrease when Akt3 expression is silenced. Concurrent with these changes in mitochondrial gene expression, lower O2 consumption was observed. VEGF stimulation of the major mitochondrial import protein TOM70 is also blocked by Akt3 inhibition. In support of a role for Akt3 in the regulation of mitochondrial biogenesis, Akt3 silencing results in the cytoplasmic accumulation of the master regulator of mitochondrial biogenesis, PGC‐1α, and a reduction in known PGC‐1α target genes. Finally, a subtle but significant, abnormal mitochondrial phenotype is observed in the brain tissue of AKT3 knockout mice. These results suggest that Akt3 is important in coordinating mitochondrial biogenesis with growth factor‐induced increases in cellular energy demands.—Wright, G. L., Maroulakou, I. G., Eldridge, J., Liby, T. L., Sridharan, V., Tsichlis, P. N., Muise‐Helmericks, R. C. VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase. FASEB J. 22, 3264–3275 (2008)

Suppression of Neu-induced mammary tumor growth in cyclin D1 deficient mice is compensated for by cyclin E

Amplification and/or overexpression of the receptor tyrosine kinase HER2/Neu and the cell cycle regulatory gene cyclin D1 are frequently associated with human breast cancer. We studied the functional significance of cyclin D1 in Neu-induced mammary oncogenesis by developing mice overexpressing either wild-type or mutant Neu in a cyclin D1 deficient background. The absence of cyclin D1 suppresses mammary tumor formation induced by the wild-type or activated mutant form of Neu, which promote multi- and single-step progression of tumorigenesis, respectively. These data indicate that cyclin D1 is preferentially required for Neu-mediated signal transduction pathways in mammary oncogenesis. Significantly, 35% of mutant Neu/cyclin D1−/− mice regained mammary tumor potential due to compensation by cyclin E. Thus, shared targets of cyclins D1 and E are important in modulating Neu function in mammary tumorigenesis. Our results imply that the combinatorial inhibition of cyclins D1 and E might be useful in the treatment of malignancies induced by Neu.

Distinct Roles of the Three Akt Isoforms in Lactogenic Differentiation and Involution

The three Akt isoforms differ in their ability to transduce oncogenic signals initiated by the Neu and PyMT oncogenes in mammary epithelia. As a result, ablation of Akt1 inhibits and ablation of Akt2 accelerates mammary tumor development by both oncogenes, while ablation of Akt3 is phenotypically almost neutral. Since the risk of breast cancer development in humans correlates with multiple late pregnancies, we embarked on a study to determine whether individual Akt isoforms also differ in their ability to transduce hormonal and growth factor signals during pregnancy, lactation and post‐lactation involution. The results showed that the ablation of Akt1 delays the differentiation of the mammary epithelia during pregnancy and lactation, and that the ablation of Akt2 has the opposite effect. Finally, ablation of Akt3 results in minor defects, but its phenotype is closer to that of the wild type mice. Whereas the phenotype of the Akt1 ablation is cell autonomous, that of Akt2 is not. The ablation of Akt1 promotes apoptosis and accelerates involution, whereas the ablation of Akt2 inhibits apoptosis and delays involution. Mammary gland differentiation during pregnancy depends on the phosphorylation of Stat5a, which is induced by prolactin, a hormone that generates signals transduced via Akt. Here we show that the ablation of Akt1, but not the ablation of Akt2 or Akt3 interferes with the phosphorylation of Stat5a during late pregnancy and lactation. We conclude that the three Akt isoforms have different roles in mammary gland differentiation during pregnancy and this may reflect differences in hormonal signaling. J. Cell. Physiol. 217: 468–477, 2008.

Defective Activation of ERK in Macrophages Lacking the p50/p105 Subunit of NF-κB Is Responsible for Elevated Expression of IL-12 p40 Observed after Challenge with Helicobacter hepaticus

Helicobacter hepaticus is an enterohepatic Helicobacter species that induces lower bowel inflammation in susceptible mouse strains, including those lacking the p50/p105 subunit of NF-κB. H. hepaticus-induced colitis is associated with elevated levels of IL-12 p40 expression, and p50/p105-deficient macrophages express higher levels of IL-12 p40 than wild-type macrophages after challenge with H. hepaticus. However, the molecular mechanisms by which the p50/p105 subunit of NF-κB suppresses IL-12 p40 expression have not yet been elucidated. In this study we have demonstrated that H. hepaticus challenge of macrophages induces ERK activation, and this event plays a critical role in inhibiting the ability of H. hepaticus to induce IL-12 p40. Activation of ERK requires both p50/p105 and the MAPK kinase kinase, Tpl-2. Inhibition of the induction of IL-12 p40 by ERK was independent of c-Rel, a known positive regulator of IL-12 p40. Instead, it was linked to the induction of c-Fos, a known inhibitor of IL-12 p40 expression. These results suggest that H. hepaticus induces ERK activation by a pathway dependent upon Tpl-2 and p105, and that activation of ERK inhibits the expression of IL-12 p40 by inducing c-Fos. Thus, a defect in ERK activation could play a pivotal role in the superinduction of IL-12 p40 observed after challenge of macrophages lacking the p50/p105 subunit of NF-κB with H. hepaticus.

Development and characterization of a mouse prostate adenocarcinoma cell line: ductal formation determined by extracellular matrix.

Tumor vaccines show promise as a new approach for treating cancer. We have developed a murine prostate cancer cell line which can be used to study growth factor and extracellular matrix regulation of prostate differentiation and will be useful for generating tumor vaccines using the C3(1)/TAG transgenic model of prostate cancer.