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. 2007 May 1;104(18):7438-43.
doi: 10.1073/pnas.0605874104. Epub 2007 Apr 25.

Akt1 governs breast cancer progression in vivo

Xiaoming Ju  1 , Sanjay Katiyar, Chenguang Wang, Manran Liu, Xuanmao Jiao, Shengwen Li, Jie Zhou, Jacob Turner, Michael P Lisanti, Robert G Russell, Susette C Mueller, John Ojeifo, William S Chen, Nissim Hay, Richard G Pestell
Affiliations

Akt1 governs breast cancer progression in vivo

Xiaoming Ju et al. Proc Natl Acad Sci U S A. .

Abstract

The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Here, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1, in the mouse demonstrates a requirement for Akt1 in ErbB2-induced mammary tumorigenesis. Akt1 deficiency delayed tumor growth and reduced lung metastases, correlating with a reduction in phosphorylation of the Akt1 target, tuberous sclerosis 2 (TSC2) at Ser-939. Akt1-deficient mammary epithelial tumor cells (MEC) were reduced in size and proliferative capacity, with reduced cyclin D1 and p27(KIP1) abundance. Akt1 deficiency abrogated the oncogene-induced changes in polarization of MEC in three-dimensional culture and reverted oncogene-induced relocalization of the phosphorylated ezrin-radixin-moesin proteins. Akt1 increased MEC migration across an endothelial cell barrier, enhancing the persistence of migratory directionality. An unbiased proteomic analysis demonstrated Akt1 mediated MEC migration through paracrine signaling via induction of expression and secretion of CXCL16 and MIP1gamma. Akt1 governs MEC polarity, migratory directionality and breast cancer onset induced by ErbB2 in vivo.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Akt1-deficient mice are resistant to ErbB2-induced mammary tumorigenesis. (A) The percentage of mice free of ErbB2-induced mammary tumors is shown in Akt1+/+, Akt1+/−, and Akt1−/− transgenic mice. (B) Histopathology of tumor cells migrating (asterisk) into the lung parenchyma from hematogenous spread of ErbB2 mammary tumors in Akt1+/+mouse. (Inset) Higher power magnification of migrating tumor cells penetrating the basement membrane. Similar histologic features of the mammary gland tumors in Akt1 wild type and Akt1 knockout mice (S1A) are shown. (C) Lung metastasis in 30 separate mice in which mammary tumors had grown to a similar size. (D) Tumor metastasis in the lungs of ErbB2/ART+/+ mice. ErbB2 (brown) marks metastasis tumor cells. (E) Immunohistochemical staining [von Willebrand] as a marker of blood vessel endothelium (SI Fig. 6B). DAPI staining identified nuclei. Data are mean ± SEM of separate fields of tumors (vessels/mm2). (F) Western blot analysis of MMTV-ErbB2 mammary tumors for mice of each Akt1 genotype (G and H) with densitometry quantified (D).
Fig. 2.
Fig. 2.
Akt1 reduced cellular proliferation and cellular size of Akt1-deficient mammary tumor cells. (A) Cellular morphology of MEC lines of MMTV-ErbB2 transgenic mice of either Akt1+/+ or Akt1−/− genotype. (B–D) Western blot (B), cellular diameter (C), and cellular proliferation (D) of MEC in response to the growth factor (FGF), comparing F12 with 10% FBS (5 μg/ml insulin/10 ng/ml EGF) vs. F12 with growth factor-deleted (charcoal stripped) media. (E) Western blot analysis of cell cycle control proteins. GDI is a protein loading control. (F) Western blot analysis of Akt1−/− MEC transduced with viral vectors expressing GFP or Akt1 with accompanying phase contrast and fluorescent images. (G and H) Cellular proliferation assessed by Ki67 (G) and BrdU staining, quantitated by FACS analysis (H).
Fig. 3.
Fig. 3.
Akt1 deficiency reduces cellular migration and migratory directionality. (A) ErbB2 MEC were assessed for cellular adhesion at 2 and 6 h by OD550. (B and C) ErbB2 MEC transwell migration assays (B) and the cellular migration determined by wounding assay of ErbB2 MECs at the time points indicated (C).
Fig. 4.
Fig. 4.
Akt1 regulates mammary acini formation and cellular polarity in 3D matrigel. (A) Mammary epithelial cells derived from ErbB2 tumors of transgenic mice were fixed and stained for F-actin (phalloidin) and DAPI. (B) Distribution of paxillin and centripetal distribution of tyrosine-phosphorylated paxillin (Y-118). (C) MEC isolated from ErbB2 littermates of each Akt1 genotype shown were subjected to 3D culture on reconstituted basement membrane for 25 days. Colonies were fixed and stained with hematoxylin and eosin to reveal histology. At 25 days of culture, cells were stained for DAPI (blue), hDlg (red), and p-ezrin–radixin–moesin (p-ERM) (green). (D) Schematic representation designating the role of Akt1 and the ErbB2 oncogene in mammary epithelial cell polarity and acini development.
Fig. 5.
Fig. 5.
Akt1 governs secretion of promigratory factors from MEC. (A and B) ErbB2-MEC were analyzed for directional migration (D/T) in the presence of either native supernatant or coculture with medium from Akt1+/+ MEC (B). (B–D) Analysis of ErbB2-MEC migratory activity in the presence of native or heterologous conditioned media with quantitation of either distance traveled (B), cellular velocity (C), or PMD (D) (D/T ratio was quantitated for n > 20 cells over 6 h). Cellular migratory distance, velocity, and PMD (D/T) are shown as mean ± SEM for n > 3 separate experiments. (E) Cellular migration of Akt1−/− MEC transduced with pBABE vectors encoding either GFP, mAKT1, or cAKT1. (F) Proteomic analysis showing relative abundance of secreted cytokines and growth factors in the media of ErbB2 MEC (3 days, n > 3 separate experiments). Cytokines and growth factor proteins produced differentially (two-fold) between Akt1−/− or Akt1+/+ MEC. Quantitation of abundance of secreted proteins determined by ELISA and RT-PCR quatititation of the receptors for the secreted ligands. (G and H) Transwell migration assays were conducted to determine the functional significance of migratory and antimigratory secreted proteins identified by cytokine array. Assays were conducted by using either immunoneutralizing antibody (α) or the addition of ligands (H). Data are shown as mean ± SEM of n > 3 Boyden chamber assays.
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