PI3K/AKT pathway promotes keloid fibroblasts proliferation by enhancing glycolysis under hypoxia

Our previous study demonstrated altered glucose metabolism and enhanced phosphorylation of the PI3K/AKT pathway in keloid fibroblasts (KFb) under hypoxic conditions. However, whether the PI3K/AKT pathway influences KFb cell function by regulating glucose metabolism under hypoxic conditions remains unclear. Here, we show that when PI3K/AKT pathway was inactivated with LY294002, the protein expression of glycolytic enzymes decreased, while the amount of mitochondria and mitochondrial membrane potential increased. The key parameters of extracellular acidification rate markedly diminished, and those of oxygen consumption rate significantly increased after inhibition of the PI3K/AKT pathway. When the PI3K/AKT pathway was suppressed, the levels of reactive oxygen species (ROS) and mitochondrial ROS (mitoROS) were significantly increased. Meanwhile, cell proliferation, migration and invasion were inhibited, and apoptosis was increased when the PI3K/AKT pathway was blocked. Additionally, cell proliferation was compromised when KFb were treated with both SC79 (an activator of the PI3K/AKT pathway) and 2‐deoxy‐d‐glucose (an inhibitor of glycolysis), compared with the SC79 group. Moreover, a positive feedback mechanism was demonstrated between the PI3K/AKT pathway and hypoxia‐inducible factor‐1α (HIF‐1α). Our data collectively demonstrated that the PI3K/AKT pathway promotes proliferation and inhibits apoptosis in KFb under hypoxia by regulating glycolysis, indicating that the PI3K/AKT signalling pathway could be a therapeutic target for keloids.


| INTRODUCTION
Keloids are pathological scars characterised by uncontrolled proliferation of dermal fibroblasts and excessive deposition of extracellular matrix (ECM). 1 As a result of abnormal wound healing, keloids are clinically accompanied by itching, pain, cosmetic deformities and joint dysfunction. 2 Additionally, keloids exhibit cancer-like properties, including outgrowth beyond the boundary of the initial wound and invasion into adjacent normal skin. 3 Clinically, multiple approaches, including surgical removal combined with steroid injections, laser therapy, radiotherapy and compression therapy are available for the prevention and treatment of keloids. 2 However, the above therapeutic approaches are unsatisfactory and cannot prevent recurrence. In recent decades, genetics, inflammation, tumour-related factors and Qifei Wang and Xin Yang authors contributed equally to this work. immune cells have been shown to participate in the pathogenesis of keloids. [4][5][6][7] However, the mechanisms of keloid pathogenesis are poorly documented, and keloid scarring continues to be one of the most challenging skin problems.
Metabolic reprogramming is an emerging hallmark of cancer.
Altered glucose metabolism generally occurs in the form of the Warburg effect (aerobic glycolysis), which was first observed in cancer cells. It describes a phenomenon that occurs even in an oxygenenriched environment. Cancer cells are dependent on glycolysis to rapidly generate ATP from glucose. It has been demonstrated that aerobic glycolysis facilitates the autonomous proliferation and survival of cancer cells by providing substrates for macromolecule synthesis, apoptosis regulation and redox homeostasis. Furthermore, it has been established that the Warburg effect exists in various cells and disorders, including immune cells, 6 stem cells, 8 sepsis 9 and Alzheimer disease. 10 Interestingly, altered metabolism was also observed in keloids. Our previous studies revealed that KFb exhibited elevated glucose intake and lactate accumulation, enhanced mRNA and protein expression of glycolytic enzymes, and attenuated mitochondrial oxidative phosphorylation.
Additionally, an increase in the extracellular acidification rate (ECAR; indicating glycolytic level) and a decrease in oxygen consumption rate (OCR; indicating mitochondrial respiration) have been reported.
Tissue hypoxia exists in various solid tumours and is clinically related to resistance to therapy and poor prognosis. The hypoxic microenvironment in keloids results from massive collagen deposition and partially or completely occluded capillaries. Okuno et al. 11 reported that the keloid center was a hypoxic zone and exhibited higher expression of HIF-1α when compared with the margin zone. The hypoxic microenvironment in keloids facilitates epithelial-mesenchymal transition 12 and modulates apoptosis. 13 Additionally, our previous studies indicated that hypoxia significantly promoted KFb proliferation, migration, invasion and collagen synthesis. 14 Furthermore, insufficient oxygen supply in keloids profoundly alters glucose metabolism in KFb. Our previous work demonstrated enhanced glycolysis and compromised mitochondrial respiration in KFb under hypoxic conditions. 14 Moreover, hypoxia promotes the activation of metabolic pathways, including HIF-1α 15 and the PI3K/AKT signalling pathway. 16 The PI3K/AKT pathway is a major growth regulatory pathway in mammalian cells and is frequently dysregulated in human cancers. 17 The pathway plays a major role in critical physiological functions and multiple cellular processes, including survival, growth and proliferation. 18 Additionally, this signalling pathway promotes the reprogramming of glucose metabolism. 19,20 The stimulation of diverse oncogenes and growth factor receptors triggers PI3K activation, which has diverse downstream effects on mammalian target of rapamycin complex 1 (mTORC1), members of the forkhead boxO family of transcription factors and glycogen synthase kinase 3. Activated PI3K can phosphorylate phosphatidylinositol-4,5bisphosphate to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3), and the process can be inversed by phosphatase and tensin homologue deleted on chromosome ten (PTEN). PIP3 leads to AKT phosphorylation by activating pyruvate dehydrogenase kinase 1. These downstream effectors play a key role in cellular metabolic reprogramming. 21,22 Additionally, AKT phosphorylation directly regulates the phosphorylation and activation of specific glycolytic enzymes, including hexokinase 2 (HK2) and phosphofructokinase 1. In addition to the direct regulation of glycolytic enzymes, the PI3K-AKT signalling pathway also drives aerobic glycolysis through increased protein expression of glucose transporters and glycolytic enzymes modulated through the regulation of downstream transcription factors, including HIF-1α and MYC. [23][24][25] Accumulating evidence demonstrates that the PI3K-AKT signalling pathway plays a critical role in facilitating glucose intake and glycolysis under physiological conditions and in the context of the tumour microenvironment. However, further studies are required to determine whether the activation of PI3K/AKT signalling enhances mitochondrial respiration.
While accumulating studies have demonstrated the key role of the PI3K-AKT pathway in the regulation of glucose metabolism in a range of cancer cells, the mechanism of PI3K-AKT signalling in KFb is not clear. Keloids manifest some similar biological features as solid tumours, including metabolic reprogramming, and our previous study showed upregulated phosphorylation of the PI3K-AKT pathway in KFb. Herein, we postulated that the PI3K-AKT pathway modulates glucose metabolism, which further influences cell function in keloid fibroblasts (KFb) under hypoxic conditions. In the present study, we investigated the effects of the PI3K-AKT pathway on glycolysis and mitochondrial functions, and the subsequent role of glycolysis in regulating cell functions in KFb under hypoxia. In addition, the interactions between the PI3K/ AKT pathway and HIF-1α under hypoxia were also explored.

| Isolation and culture of fibroblasts
The study was approved by the Ethics Committee of the Peking University Third Hospital (IRB00006761-2014173). The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. Keloids were diagnosed based on clinical and histological evidence, and keloid samples were obtained from subjects who underwent surgical procedures. None of the participants received treatment for keloids before surgical removal. Basic information of the keloid specimens is described in Table 1. Fibroblast extraction and culture were performed as described previously with slight modifications. 14 Briefly, primary KFb were extracted using the collagenase digestion method and were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone, USA), with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/ streptomycin (HyClone, USA), in an incubator conditioned at 37 C and 5% CO 2 . Cells derived from six keloid patients and six normal participants. Four to six passages of fibroblasts were used in the experiments. Proliferation was determined using change in cell number relative to inoculation number (measured cell number/inoculation number).

| Migration and invasion assays
The inhibition of LY294002 on KFb migration was assessed by trans-

| Apoptosis assay
The apoptotic level of KFb was determined with an Annexin

| Western blotting
Western blotting analysis was performed as described in our previous study. 14  and blocked with 5% bull serum albumin blocking buffer at room temperature for 1 h. Then, the PVDF membranes were incubated overnight at 4 C with primary antibodies ( Table 2). The PVDF membranes were then washed and incubated with HRP-conjugated secondary antibody and washed with TBST (0.1%). Finally, the signals were visualised using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, UK). Densitometric analysis was performed using Image J software.

| Reactive oxygen species and mitochondrial ROS determinations
Reactive oxygen species (ROS) and mitochondrial ROS (mitoROS) levels

| Analysing ECAR and OCR
The XF96 Extracellular Flux Analyser (Seahorse Bioscience, USA) was used to evaluate glycolysis and mitochondrial oxidative phosphorylation using ECAR and OCR, respectively. Briefly, 6 Â 10 5 KFb were seeded into 10 cm cell culture plates and cultured in complete culture medium containing phosphate-buffered saline

| PI3K inhibition altered mitochondrial mass and MMP
The PI3K-AKT pathway is indispensable for sustaining mitochondrial Interestingly, we observed that KFb showed attenuated OCR when the PI3K-AKT pathway was activated by SC79 ( Figure 5G-M).
Importantly, KFb treated with SC79 coupled with 2-DG exhibited higher key OCR parameters compared with those in the SC79 group. Finally, we investigated the potential mechanism underlying the regulation of PI3K activation during proliferation. Cell proliferation was inhibited or promoted when KFb were cultured with LY294002 or SC79 ( Figure 5N,O). This observation suggests that the PI3K-AKT pathway promotes KFb proliferation. Additionally, the rate of proliferation of KFb treated with SC79 and 2-DG was remarkably inhibited compared with that in the SC79 group ( Figure 5N,O). These results collectively indicate that the PI3K-AKT pathway promotes proliferation by enhancing glycolysis.  The results showed elevated total ROS ( Figure 6A,B) and mitoROS ( Figure 6C,D) levels after PI3K inhibition. Additionally, the promotion of ROS generation by PI3K inactivation under hypoxia was more obvious than that under normoxia. These findings robustly suggest that the PI3K-AKT pathway is crucial for maintaining redox homeostasis.

| The PI3K-AKT pathway interacted with HIF-1α through a positive feedback mechanism
The PI3K-AKT signalling network and HIF-1α are two principal pathways for the modulation of glucose metabolism under hypoxia. However, the relationship between the two pathways in KFb remains to be elucidated. F I G U R E 6 Inhibition of the PI3K-AKT pathway induced reactive oxygen species (ROS) generation. After keloid fibroblasts (KFb) were treated with LY294002 (0, 5 and 25 μM) under normoxia or hypoxia for 24 h, ROS and mitoROS were determined by flow cytometry. PI3K inhibition increased ROS (A,B) and mitoROS (C,D) generation. In Figure 6A, the area of bright green represents the percentage of fluorescence intensity (ROS) greater than a value (6 x 10 4 ), and the area of dark green represents the percentage of fluorescence intensity less than the value. ROS content was detected using a Reactive Oxygen Species Assay Kit containing DCFH-DA solution. The fluorescence intensity of DCF (green) represents ROS content. In Figure 6D, the area of deep red represents the percentage of fluorescence intensity (mitoROS) greater than a value (3 Â 10 4 ), and the area of dark red represents the percentage of fluorescence intensity less than the value (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group under the same oxygen condition. #p < 0.05, ###p < 0.001 compared with the normoxia group at the same LY294002 concentration.

| DISCUSSION
Keloid scarring is a natural human disease model involving chronic inflammation, fibrosis and tumours, and is characterised by uncon-   The conclusion that the PI3K-AKT pathway promotes proliferation by enhancing glycolysis is supported by recent studies focusing on tumour metabolism. Hussain et al. 40 reported that inhibition of glycolysis and lipogenesis by the PI3K inhibitor, 3-dihydro-2-(naphthalene-1-yl) quinazolin-4(1H)-one represses angiogenesis and decreases proliferation of colon cancer cells. In a recent study by Wang et al.,41 FoxA2 inhibited the proliferation of hepatic progenitor cells by attenuating PI3K-AKT-HK2-mediated glycolysis. Additionally, glycolysis induced by the PI3K-AKT pathway also plays a key role in invasion and apoptosis in other cancer cells, such as breast cancer 42 and paediatric osteosarcoma. 43 Keloid features with tumour-like physiological functions. Accordingly, we postulated that the PI3K-AKT network can also regulate invasion and apoptosis by glycolysis.
The PI3K-AKT signalling network and HIF-1α are considered the two main pathways for glucose metabolic regulation under hypoxia. 44 However, the relationship between these two pathways remains con- inhibitor YC-1 inhibits activation of the PI3K/AKT/mTOR pathway during hypoxia in prostate cancer cells. 48 These results collectively suggest that the PI3K-AKT pathway interacts with HIF-1α through a positive feedback mechanism under hypoxia.
One issue to consider with the results of the current study is that