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 O(2) 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-1alpha, and a reduction in known PGC-1alpha 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.

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.

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.

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.

The study of HOX gene function in hematopoietic, breast and lung carcinogenesis.

HOX transcription factors regulate basic and cell type-specific activities throughout life. The combinatorial patterns of HOX gene expression along anterior-posterior and proximal-distal axes are relatively tightly-defined during embryogenesis and key remnants of such positional memory persist through adulthood. These normal patterns of HOX gene expression can be compared to a growing body of work on their dysregulation during carcinogenesis. In this review, simple and complex changes in HOX gene expression patterns will be considered using examples from hematopoietic, breast and lung cancers. Changes in individual and combinatorial patterns of HOX gene expression, co-factor expression and chromatin structure will be considered in a discussion of potential roles for dysregulated HOX genes in target gene regulation and various aspects of cancer progression. Collectively, studies indicate that, although a variety of factors must be delineated to assess the roles of individual HOX genes in particular cancers, approaches that modulate HOX gene expression and monitor both changes in the regulation of key target genes and cellular activities are making the greatest initial advances in this assessment.

MicroRNAs differentially regulated by Akt isoforms control EMT and stem cell renewal in cancer cells.

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-beta (TGFbeta)-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 TGFbeta-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.