E3 ubiquitin protein ligase, E6-associated protein (E6-AP) regulates PI3K-Akt signaling and prostate cell growth

Abstract

This study elucidates the role of E6-associated protein, E6-AP (a dual function steroid hormone receptor coactivator and ubiquitin-protein ligase) in the regulation of PI3K-Akt signaling pathway, prostate gland growth and proliferation. Here, we report the generation of transgenic mice and prostate cancer cell line, LNCaP cells that overexpress E6-AP protein. Using these models we show that the levels of total Akt and phosphorylated Akt (active Akt) are increased in E6-AP overexpressing prostate gland and LNCaP cells suggesting that E6-AP regulates the PI3K-Akt signaling pathway. The prostate glands in our transgenic mice are ~20% larger and produce preneoplastic lesions at the age of 18 months. Our data also suggest that E6-AP modulates PI3K-Akt signaling pathway by both androgen-independent and -dependent mechanisms. In the androgen-independent mechanism, E6-AP modulates PI3K-Akt signaling by regulating the protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. Further, we show that E6-AP, a known coactivator of AR, amplifies the androgen-dependent activation of PI3K-Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased cell size and proliferation. Overall our data suggests that E6-AP regulates both the positive and negative modulators of the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and decreased apoptosis.This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!

Figure 1

Characterization of E6-AP transgenic mice. (A & B) Representative Western blots from two lines of E6-AP over expressing transgenic mice showing the expression levels of E6-AP in different tissues. E6-AP is specifically over expressed in the prostate glands of transgenic mice. (WT- Wild-type, Tr- Transgenic). (C) Wet weights of prostate glands from wild-type and E6-AP over expressing transgenic mice. The wet weights were normalized with body weights. Prostate glands from E6-AP overexpressing transgenics weigh ~20% more than wild-type prostate glands (*, P < 0.05, n=15). (D) Western blots showing levels of total and phospho Akt (p-Akt) are elevated in E6-AP over-expressing transgenic mice. (E) Western blots showing lobe specific expression of E6-AP, p-Akt and phospho GSK β (p-GSK β) in E6-AP over-expressing transgenic mice.

Figure 2

Histology of prostate glands from E6-AP transgenic mice. Representative sections from wild-type and transgenic mice stained for H & E. Precancerous lesions characteristic of PIN can be identified in transgenic sections (Arrows).

Figure 3

Over expression of E6-AP induces PI3K-Akt signaling pathway. (A) Western blots showing inducible expression of Flag-E6-AP in E6-AP-LNCaP stable clones. (B) Parental LNCaP cells and E6-AP-LNCaP cells were treated with or without Dox for 48 hours. Increased protein levels of total Akt and phosphorylated-Akt (p-Akt, Ser-473) were observed in E6-AP-LNCaP cells under E6-AP over expressing conditions (−Dox). β-actin was used as a loading control. (C) Parental LNCaP cells and E6-AP-LNCaP cells were treated with or without Dox for 48 hours. Increased protein levels of PI3K was observed in E6-AP-LNCaP cells under E6-AP over expressing conditions (−Dox). β-actin was used as a loading control. (D) Western blot analysis of p-Akt (Ser-473) levels from LNCaP cells and E6-AP-LNCaP stable cells grown in 5% charcoal stripped serum supplemented with or without synthetic androgen R1881 for 1h and 36h.

Figure 4

Involvement of RhoA in E6-AP-mediated regulation of Akt activity. (A) Western blots showing that the levels of RhoA are decreased under E6-AP overexpressing conditions (−Dox) in E6-AP-LNCaP stable cells compared to that of parental LNCaP cells. (B) To determine if RhoA is target of the ubiquitin-protesome pathway in prostate cells, LNCaP cells were treated with DMSO or proteasome inhibitor, MG132 and cell lysates were examined for RhoA levels. Western blots showing RhoA stabilization with MG132 treatment. (C) In vitro interaction of E6-AP with RhoA. The human RhoA protein was synthesized in vitro using the TNT-coupled reticulocyte lysate system. RhoA protein was then incubated with GST-E6-AP fusion protein that was bounded to glutathione-Sepharose beads. The glutathione-bounded proteins were separated on SDS-PAGE, followed by autoradiography. Twenty percent of the TNT reaction was used as input, and GST alone was used as a negative control.

Figure 5

Exogenous overexpression of E6-AP enhances cell growth, proliferation and decreases apoptosis. (A) Morphology of E6-AP-LNCaP cells compared with parental control LNCaP cells, indicating the difference in cell size and shape. (B) E6-AP-LNCaP cells exhibit increased cell cycle progression. Parental LNCaP cells and E6-AP-LNCaP cells were grown for 72 h, harvested, fixed with 70% ethanol, stained with propidium iodide, and processed for FACS analysis. Data are plotted as the percentage of S phase cells. (*, P < .05) (C) MTT assay to measure cell proliferation. Parental LNCaP cells and E6-AP-LNCaP cells were grown in 96 well plates in the presence of normal and charcoal stripped serum and MTT assay was carried out on each day for 5 days. Error bars represent standard error of mean from three different measurements. (**, P < .01) (D) siRNA mediated knock down of E6-AP reduces the proliferation of LNCaP cells. 1 × 10⁵ cells were plated in 6 well plates, transfected with either siScramble or siE6-AP, starved for 3 days without hormone and treated with R1881 for 7 days. Cell numbers were counted as a measure of proliferation. Error bars represent standard error of mean from three different experiments. (**, P < .01) (E) Overexpression of exogenous E6-AP in prostate cells protects cells against apoptosis. Parental LNCaP cells and E6-AP-LNCaP cells were grown in normal serum for twenty four hours and then treated with 100μM etoposide for another 6 hours. The cell lysates were analyzed by Western blots using anti-caspase 3 and anti-bax antibodies. E6-AP-LNCaP cells shows less amounts of proapoptotic proteins than parental LNCaP cells.

Figure 5

Exogenous overexpression of E6-AP enhances cell growth, proliferation and decreases apoptosis. (A) Morphology of E6-AP-LNCaP cells compared with parental control LNCaP cells, indicating the difference in cell size and shape. (B) E6-AP-LNCaP cells exhibit increased cell cycle progression. Parental LNCaP cells and E6-AP-LNCaP cells were grown for 72 h, harvested, fixed with 70% ethanol, stained with propidium iodide, and processed for FACS analysis. Data are plotted as the percentage of S phase cells. (*, P < .05) (C) MTT assay to measure cell proliferation. Parental LNCaP cells and E6-AP-LNCaP cells were grown in 96 well plates in the presence of normal and charcoal stripped serum and MTT assay was carried out on each day for 5 days. Error bars represent standard error of mean from three different measurements. (**, P < .01) (D) siRNA mediated knock down of E6-AP reduces the proliferation of LNCaP cells. 1 × 10⁵ cells were plated in 6 well plates, transfected with either siScramble or siE6-AP, starved for 3 days without hormone and treated with R1881 for 7 days. Cell numbers were counted as a measure of proliferation. Error bars represent standard error of mean from three different experiments. (**, P < .01) (E) Overexpression of exogenous E6-AP in prostate cells protects cells against apoptosis. Parental LNCaP cells and E6-AP-LNCaP cells were grown in normal serum for twenty four hours and then treated with 100μM etoposide for another 6 hours. The cell lysates were analyzed by Western blots using anti-caspase 3 and anti-bax antibodies. E6-AP-LNCaP cells shows less amounts of proapoptotic proteins than parental LNCaP cells.