Vertical inhibition of PI3K/Akt/mTOR signaling demonstrates in vitro and in vivo anti-fibrotic activity

Highlights

• BEZ235 leads to superior inhibitory effect to rapamycin for dermal fibrosis in two complementary systemic sclerosis mouse models.

• BEZ235 attenuates dermal fibrosis by vertical inhibiting PI3K/Akt/mTOR signaling via suppression of fibroblast activation.

• Feedback induction of phosphorylation of the Akt on Ser473 by single mTOR inhibitor rapamycin might hamper its anti-fibrotic effect.

• The strategy with BEZ235 overcomes the limitation of rapamycin in feedback activation of p-Akt on Ser473 following TGF-β treatment.

Abstract

Background

The mammalian target of rapamycin (mTOR) regulates cellular activity in many diseases, but the complex interplay with PI3K/Akt pathway may hampers its function.

Objective

This study was undertaken to determine the activity of PI3K/Akt/mTOR signaling in the fibroblasts from systemic sclerosis (SSc) patients, and compare the effects of vertical inhibiting PI3K/Akt/mTOR by BEZ235 and inhibiting mTOR alone by rapamycin in fibroblast activation and in two complementary established mouse model of SSc.

Methods

Pharmaceutical specific inhibitors BEZ235 and rapamycin were used to vertical inhibit PI3K/Akt/mTOR signaling and mTOR signaling alone in cultured fibroblasts and in mice. SSc mouse model was established by daily injecting bleomycin subcutaneously or by overexpression of constitutively active type I TGF-β receptor (TβRI^ca). To delineate the mechanisms underlying the antifibrotic effects of BEZ235 and rapamycin, activity of PI3K/Akt/mTOR signaling was analyzed by determining the expressions of phosphorylated Akt, GSK-3β, mTOR and S6 ribosomal protein (S6).

Results

Primary dermal fibroblasts demonstrated hyperactivity of PI3K/Akt and mTOR signaling. mTOR inhibitor rapamycin failed to inhibit dermal fibrosis in an established SSc mouse model. However, administration of a dual inhibitor for PI3K/Akt and mTOR signaling BEZ235 attenuated dermal fibrosis by reversing increased dermal thickness and collagen deposition in two SSc mouse models. Furthermore, BEZ235 showed superior inhibitory effect on fibroblast activation relative to rapamycin in vitro. Also both BEZ235 and rapamycin could prevent the phosphorylation of mTOR and S6 completely. BEZ235 also blocked the activation of Akt and GSK-3β dramatically, whereas rapamycin has been shown to increase further upregulation of phosphorylated Akt on Ser473 both in vitro and in vivo.

Conclusion

These data show that blocking PI3K/Akt/mTOR with BEZ235 leads to superior inhibitory effect for dermal fibrosis, suggesting that vertical inhibition of PI3K/Akt/mTOR signaling may have therapeutic potential for SSc.

Introduction

The mammalian target of Rapamycin (mTOR) is a 289-kDa serine–threonine kinase that regulates cellular activity [1]. mTOR kinases form two distinct multiprotein complexes mTORC1 and mTORC2. Pretreatment with mTORC1 inhibitor rapamycin could prevent skin fibrosis in a bleomycin-induced SSc model [2]. In the present study, daily treatment of rapamycin from week 3 to week 6 has not shown a protective effect on two established SSc mouse model. Many lines of evidence show that inhibition of mTOR can activate Akt by deactivating the mTOR-S6-IRS1-negative feedback loop [3], [4]. mTORC2 phosphorylates Akt on Ser473, increasing its enzyme activity up to 10-fold [5], [6]. Hyperactivation of Akt is associated with resistance to apoptosis, increased cell growth, cell proliferation, and cell metabolism [7]. Akt activity was upregulated in the skin fibroblasts of SSc patients [8] and it has been implicated to be hyperactivated in the experimental SSc models under different stimulations, such like TGF-β [9], [10], [11]. So targeting mTOR by repamycin lead to the alleviation of this feedback loop resulting in the activation of PI3K and subsequent activation of Akt, which may explain the limited success by using mTOR inhibitor in the established SSc model. Therefore simultaneously target of both PI3K/Akt and mTOR has a rationale to inhibit both upstream and downstream signaling in this pathway.

SSc is an autoimmune disease with skin and multiple organs involved. The mechanism of SSc is far from clear and current pharmacological therapies are still insufficient. SSc is characterized of activated fibroblasts. Under multiple stimulation, fibroblasts express α-smooth muscle actin (α-SMA), transform to myofibroblasts, and produce extracellular matrix (ECM). It is well documented that one of the most critical profibrotic mediators is transforming growth factor (TGF)-β, which could drive Smad-dependent and non-Smad-dependent signaling and ultimately leading to the fibrosis [12], [13]. We found the activity of mTOR was upregulated in SSc fibroblasts. The functional significance of mTOR signaling in SSc needs further clarification.

We hypothesized that vertical PI3K/Akt/mTOR blockade would overcome the limitations of single mTOR inhibition. We tested this hypothesis with BEZ235, a novel dual PI3K/mTOR inhibitor that has recently entered clinical development. To our knowledge, this strategy has not been studied or applied in the field of the treatment for fibrotic diseases. First of all, we demonstrated the activation of PI3K/Akt/mTOR signaling in the fibroblasts from SSc patients, and then explored the functional significance of PI3K/Akt/mTOR signaling by inhibiting mTOR alone or using a dual inhibitor BEZ235 in two complementary established SSc mouse model induced by daily injecting bleomycin subcutaneously or by overexpression of constitutively active type I TGF-β receptor (TβRI^ca). We describe herein a remarkable protective effect for skin fibrosis by vertical inhibition of PI3K/Akt/mTOR signaling.