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. 2001 Aug;159(2):431-7.
doi: 10.1016/s0002-9440(10)61714-2.

AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells

M Sun  1 , G Wang, J E Paciga, R I Feldman, Z Q Yuan, X L Ma, S A Shelley, R Jove, P N Tsichlis, S V Nicosia, J Q Cheng
Affiliations

AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells

M Sun et al. Am J Pathol. 2001 Aug.

Abstract

Extensive studies have demonstrated that the Akt/AKT1 pathway is essential for cell survival and inhibition of apoptosis; however, alterations of Akt/AKT1 in human primary tumors have not been well documented. In this report, significantly increased AKT1 kinase activity was detected in primary carcinomas of prostate (16 of 30), breast (19 of 50), and ovary (11 of 28). The results were confirmed by Western blot and immunohistochemical staining analyses with phospho-Ser473 Akt antibody. The majority of AKT1-activated tumors are high grade and stage III/lV (13 of 16 prostate, 15 of 19 breast, and 8 of 11 ovarian carcinomas). Previous studies showed that wild-type AKT1 was unable to transform NIH3T3 cells. To demonstrate the biological significance of AKT1 activation in human cancer, constitutively activated AKT1 (Myr-Akt) was introduced into NIH3T3 cells. Overexpression of Myr-Akt in the stably transfected cells resulted in malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice. These data indicate that AKT1 kinase, which is frequently activated in human cancer, is a determinant in oncogenesis and a potential target for cancer intervention.

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Figures

Figure 1.
Figure 1.
Activation of AKT1 in human cancers. A: Western blot. HEK293 cells were transiently transfected with HA-AKT1, HA-AKT2, or HA-AKT3 expression construct, lysed, immunoprecipitated with anti-HA antibody, and detected with anti-HA (top) or anti-AKT1 antibody (bottom). The top panels of B and C are in vitro kinase assays of AKT1 immunoprecipitates from frozen tumor specimens of prostate and ovary (B), and breast (C). AKT1 kinase levels were highly elevated in cases indicated by arrows. Normal tissue lysates (N) from prostate, ovary, and breast were used as controls. The second panels of B and C are Western blot analyses of AKT1 immunoprecipitates probed with anti-phospho-Ser473 Akt antibody. The cases exhibiting elevated AKT1 kinase activity displayed phosphorylation bands. The third and fourth panels of B and bottom panel of C are Western blot analyses with anti-AKT1 and anti-β-actin antibodies.
Figure 2.
Figure 2.
Phospho-AKT1 was detected in tumor cells and located only in cytoplasm. Immunochemical staining of the paraffin sections prepared from serum-starved (1), EGF-stimulated MCF7 cells (2), and adenocarcinomas of ovary (3), prostate (4), and breast (5 and 6) with phospho-S473 Akt antibody. Strong staining was observed in EGF-stimulated MCF7 cells and ovarian, breast, and prostate tumor cells, indicated by black arrows. No immunoreaction was detected in unstimulated MCF7 cells (1) normal prostate gland and ductal epithelial cells (4 and 5, white arrow), and the breast tumor without elevated AKT1 activity (6).
Figure 3.
Figure 3.
Activation of PI 3-kinase or down-regulation of PTEN in human tumors. A: In vitro PI 3-kinase assay of the anti-p85 immunoprecipitates from nine tumor specimens that exhibit elevated AKT1 kinase activity. Elevated levels of PI 3-kinase activity were detected in cases 4, 6, and 7. B: Immunostaining of paraffin sections of prostate adenocarcinomas with anti-PTEN (left) and anti-phospho-Ser473 Akt (right) antibodies. PTEN is negative in tumor cells (black arrows), but positive in hyperplastic glands (arrowhead). However, phosphorylation of Akt was only detected in tumor cells (black arrow).
Figure 4.
Figure 4.
Constitutively activated AKT1 transforms NIH3T3 cells. A: Western blot (top) and in vitro kinase assay (bottom) analyses of Akt expression and kinase activity in stably transfected clones and a tumor sample from nude mice. For Western blot analysis, the lysates were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to membrane, and detected with anti-HA antibody. For assay of kinase activity, immunoprecipitation was performed with anti-HA antibody and the HA-Akt immunoprecipitates were subjected to in vitro kinase assay using histone H2B as substrate. B: The morphology of constitutively active Akt-transfected NIH3T3 cells (2) is more rounded and larger than WT-Akt-transfected cells (1). Constitutively activated Akt-transfected cells grew on soft agar (3) and formed tumors in nude mice (4).
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