FHIT gene expression is repressed by mitogenic signaling through the PI3K/AKT/FOXO pathway

Abstract

The Fragile Histidine Triad gene or FHIT functions as tumor suppressor in many epithelial cell types. Although its tumor suppressive mechanism is the subject of intense study, less is known about how FHIT gene expression itself is regulated. Here we show that PI3 kinase and its downstream target AKT suppress FHIT gene expression in response to growth factor stimulation in actively cycling cells. Upon removal of mitogens from the culture environment, FHIT mRNA and protein levels are observed to increase as a result of derepression from these protooncogenic kinases. AKT signaling through the FOXO transcription factors appears to be the basis for FHIT gene regulation. Increases in FHIT gene expression are directly dependent on endogenous FOXO3a in MCF7 breast carcinoma cells as evidenced by experiments with RNAi targeting FOXO transcription factor family members. Thus, this is the first report demonstrating that FHIT gene expression is normally repressed in actively cycling cells through the PI3K/AKT/FOXO3a axis.

Keywords: AKT; FHIT; FOXO; PI3K; mitogen signaling.

Figure 1

FHIT expression increases in response to mitogen deprivation. (A) MCF7 breast carcinoma cells were treated with serum containing media (0) or serum starved for 48 and 72 hours. FHIT protein and mRNA levels were determined by Western blotting (upper panel) or RT-PCR (lower panel). The Western blot was reprobed for beta-actin to demonstrate equivalent loading. Error bars represent 95% confidence intervals in the fold change of FHIT expression. (B) MCF10A immortalized mammary epithelial cells were grown with (0) or without (96) EGF for 96 hours. Western (top panel) and RT-PCR (bottom panel) were determined as described in (A).

Figure 2

Inhibition of PIP3 kinase activity with Wortmannin leads to upregulation FHIT gene expression. MCF7 cells were treated for increasing periods of time (hours) with 1 μM Wortmannin, a specific inhibitor of PIP3 kinase activity and then analyzed for FHIT mRNA expression by RT-PCR. (Inset) Western blot demonstrating a decrease in AKT phos-phorylation relative to total AKT following exposure of MCF7 cells to Wortmannin.

Figure 3

Inhibition of PIP3 kinase with LY-294002 leads to an upregulation FHIT gene expression. MCF7 cells were treated with 50 μM LY-294002, a specific PIP3K inhibitor for increasing amounts of time and then analyzed for FHIT (black bars) and p27 (grey bars). Gadd45a mRNA expression was also shown to increase with LY-294002 treatment (data not shown). Error bars represent 95% confidence intervals.

Figure 4

Overexpression of a constitutively active AKT impedes the serum deprivation-dependent increase in FHIT expression. MCF7 cells were trans-fected with expression plasmids encoding no protein (CMV), constitutively active AKT (AKT) or kinase dead AKT (KD-AKT) prior to being serum deprived or remaining in complete media. Cells were serum starved for 72 hours and treated with 1 μM Wortmannin for 24 hour prior to analysis of FHIT gene expression by RT-PCR. Error bars represent 95% confidence intervals.

Figure 5

FHIT protein expression increases in response exogenous expression of the FOXO1 or FOXO3a transcription factors. Top panel: MCF7 cells were transfected with no DNA (mock), CMV plasmid (Vector), FLAG -FOXO1 TM plasmid or FLAG-FOXO3a plasmid. Forty-eight hours post-transfection RNA was isolated and used in RT-PCR to determine FHIT gene expression in the various cell transfections. Error bars represent 95% confidence intervals. Bottom panel: MCF7 cells were transfected with the indicated plasmids. Forty-eight hours later, whole cell extracts were analyzed by SDS-PAGE and probed with the indicated antibodies.

Figure 6

PIP3K-dependent FHIT gene repression requires FOXO3a. MCF7 cells were infected with lenti-shRNA expression constructs targeting no human gene (shControl) or FOXO3a (shFOXO3a). Seventy-two hours post-infection, 1 μM Wortmannin was applied to cells for twenty-four hours. RNA was isolated and FHIT (dark gray bars) and FOXO3a (light gray bars) gene expression analyzed by RT-PCR. Error bars represent 95% confidence intervals.