Mechanism of Akt1 inhibition of breast cancer cell invasion reveals a protumorigenic role for TSC2

Abstract

Akt1 is frequently up-regulated in human tumors and has been shown to accelerate cell proliferation and to suppress programmed cell death; consequently, inhibition of the activity of Akt1 has been seen as an attractive target for therapeutic intervention. Paradoxically, hyperactivation of the Akt1 oncogene can also prevent the invasive behavior that underlies progression to metastasis. Here we show that overexpression of activated myr-Akt1 in human breast cancer cells phosphorylates and thereby targets the tumor suppressor tuberous sclerosis complex 2 (TSC2) for degradation, leading to reduced Rho-GTPase activity, decreased actin stress fibers and focal adhesions, and reduced motility and invasion. Overexpression of TSC2 rescues the migration phenotype of myr-Akt1-expressing tumor cells, and high levels of TSC2 in breast cancer patients correlate with increased metastasis and reduced survival. These data indicate that the functional properties of genes designated as oncogenes or tumor suppressor genes depend on the context of the cell type and the tissues studied, and suggest the need for caution in designing therapies targeting the function of individual genes in epithelial tissues.

Fig. 1.

Activated Akt1 promotes growth and anchorage independence in T4-2 cells but suppresses motility and invasion. Cells expressing myr-Akt1 showed increased tumor volume in nude mouse xenografts (A), increased colony number in methyl cellulose (B), and increased colony size in soft agar (C). However, myr-Akt1 inhibited cell migration (D) and motility (E), as well as invasion (F). All graphs display averages ± SEM. ∗, P < 0.05.

Fig. 2.

Activated Akt1 alters cell-substratum adhesion. Cells expressing myr-Akt1 show reduced cell spreading (A), increased cortical distribution of actin (B), and fewer paxillin-containing focal adhesions (C). (Scale bars: A and C, 10 μm; B, 20 μm.)

Fig. 3.

Rho GTPases are selectively inhibited by Akt1. Cells were plated for the indicated times, then lysates were analyzed for activity by pull-down with GST-Rhotekin (A) or GST-PAK-CD (B). (C) Inhibition of Rho by pretreatment with recombinant C3 transferase (10 μg/ml) reduced migration of vector control cells in scratch assay to levels of myr-Akt1-expressing cells. The graph displays the average ± SEM. ∗, P < 0.05.

Fig. 4.

TSC2 mediates the effects of Akt1 on motility and invasion. (A) TSC2 shows increased phosphorylation but decreased total levels in cells expressing myr-Akt1. (B) Decreased levels of TSC2 in tumor xenografts from myr-Akt1 cells. (Scale bar: 50 μm.) (C) TSC2 shows increased association with 14-3-3 in cells expressing myr-Akt1. (D) Expression of functional TSC2 in myr-Akt1 cells as shown by reduced phosphorylation of p70S6K. (E) Expression of TSC2 rescues cell invasiveness and motility in myr-Akt1 cells. The graphs display averages ± SEM. ∗, P < 0.05; ∗∗, P < 0.02; ∗∗∗, P < 0.001.

Fig. 5.

Analysis of pathway implication on human breast cancer metastasis. (A) Pathway model. a, Active Akt1 phosphorylates TSC2, stimulating down-modulation of TSC2 levels; b, TSC2 activates Rho; c, Active Rho promotes cytoskeletal rearrangements permissive for cell invasion. (B) Metastasis-free survival of human breast tumors stratified by expression of Akt1 and TSC2. Higher expression of TSC2 together with low expression of Akt1 is predictive of decreased time to metastasis in breast tumors.