CD44 modulates metabolic pathways and altered ROS-mediated Akt signal promoting cholangiocarcinoma progression

CD44 is a transmembrane glycoprotein, the phosphorylation of which can directly trigger intracellular signaling, particularly Akt protein, for supporting cell growth, motility and invasion. This study examined the role of CD44 on the progression of Cholangiocarcinoma (CCA) using metabolic profiling to investigate the molecular mechanisms involved in the Akt signaling pathway. Our results show that the silencing of CD44 decreases Akt and mTOR phosphorylation resulting in p21 and Bax accumulation and Bcl-2 suppression that reduces cell proliferation. Moreover, an inhibition of cell migration and invasion regulated by CD44. Similarly, the silencing of CD44 showed an alteration in the epithelial-mesenchymal transition (EMT), e.g. an upregulation of E-cadherin and a downregulation of vimentin, and the reduction of the matrix metalloproteinase (MMP)-9 signal. Interestingly, a depletion of CD44 leads to metabolic pathway changes resulting in redox status modification and Trolox (anti-oxidant) led to the recovery of the cancer cell functions. Based on our findings, the regulation of CCA progression and metastasis via the redox status-related Akt signaling pathway depends on the alteration of metabolic profiling synchronized by CD44.


Introduction
Cholangiocarcinoma Cluster of differentiation 44 (CD44) is a family of transmembrane glycoproteins containing extracellular transmembrane and intracellular cytoplasmic domains [1]. Members of this family are alternatively spliced into several variant exon products of the extracellular domains as variant isoforms [2]. CD44 can be used as a cell surface marker in order to identify cancer stem-like cells (CSCs) in many cancer types [3][4][5][6]. The functional relevance of CD44 expression is associated with features of CSCs that are shared with normal stem or

Gene knockdown and establishing stable cell lines
The CD44 genes in KKU-213 and KKU-156 cells were silenced using lentivirus (Sigma-Aldrich) containing shRNAs for human CD44. Briefly, 1.6 x 10 4 cells of KKU-213 and KKU-156 were seeded into a 48-well plate. A lentiviral construct was then transduced into the cell lines with DMEM containing hexadimethrine bromide (Sigma-Aldrich). A lentivirus containing shRNAs for a non-targeting gene was used as a control (Sigma-Aldrich). After 24 h transduction, KKU-213 and KKU-156 cells were treated with 1 μg/ml of puromycin (Sigma-Aldrich) for two weeks to establish stable clones of CD44 KD, and the expression of CD44 was confirmed using flow cytometry.

Flow cytometry analysis
For flow cytometry (FACS) analysis, single-cell suspensions of control and CD44 KD cells were incubated with antibodies to CD44 (Invitrogen, CA, USA) for 15 min at 4˚C. Apoptotic cells were excluded during flow cytometry by elimination of cells staining positive with propidium iodide (Sigma-Aldrich). Flow cytometry analysis was conducted with FACSCanto II (BD Bioscience, Khon Kean, Thailand) and analyzed with BD FACSDiva™ software.

Western blot analysis
CCA cell lines with either transduced controls or with CD44shRNA (5x10 5 cells per well) were seeded into cell culture dishes for 24 h and then the pellets of these cells were collected. The method was performed as previously described [25]. In brief, equal amounts of protein (15 μg) with SDS sample buffer were separated on 10% polyacrylamide gels by electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies against total-Akt, phospho-Akt, total-mTOR, phospho-mTOR, p21, Bax, Bcl-2, E-cadherin, vimentin, and β-actin, overnight at 4˚C. They were then incubated with secondary antibody for 1 h.

The Trolox treatment assay
The KKU-213 CCA cell line with either transduced control or CD44shRNA (2x10 3 cells per well) was treated with Trolox for 24 h. The number of viable cells was evaluated with a Cell Titer-Glo luminescence cell viability kit (Promega, Madison, WI). The luminescence signal was detected on a SpectraMaxL microplate reader. In addition, the CCA KKU-213 cell line with either transduced control or CD44shRNA (2x10 4 cells per well) with serum free DMEM with the absence or present of Trolox was placed on the upper layer of a cell permeable membrane and a solution containing 10% fetal bovine serum was placed below the cell permeable membrane for 24 h. Next, migrated cells were fixed with methanol and then stained with hematoxylin (Sigma-Aldrich). The number of migrated cells were counted as cells per field under a Microscope Axio Imager A2 (Carl-Zeiss, Oberkochen, Germany, located in Khon Kaen, Thailand).

The collection of cellular metabolites
A total of 15x10 6 cells of KKU-213 were counted, collected and washed with 1xPBS pH 7.4. Before homogenization, the cells was dipped into liquid nitrogen and then slowly thawed on ice. The cellular metabolites were extracted with methanol and chloroform; the extract was separated into 2 phases, a polar phase and a lipophilic phase after centrifugation at 1000 g for 15 min. The solvents were removed from the samples using a speed vacuum concentrator and the extracts were kept at -80˚C. Prior to NMR data acquisition, the polar cell extract was resuspended in 580 μl of D 2 O containing 0.1mM 3-trimethysilypropionic acid (TSP) (Cambridge Isotype Laboratories, Massachusetts, USA) as a chemical shift reference (δ = 0 p.p.m.). A total of 550 μl of the supernatant was transferred into an NMR tube with mm diameter after vortexing and centrifuging at 12000 g for 5 min.

The collection of extracellular metabolites
The conditioned media of KKU-213 cells was collected after the cells were cultured for 48 h at 37˚C under 5% CO 2 in a humidified incubator. The free water samples were concentrated using a speed vacuum concentrator and the extracted samples kept at -80˚C. Before NMR acquisition, polar cell extraction was resuspended in 580 μl of D 2 O containing 0.1mM TSP (Cambridge Isotype Laboratories) as a chemical shift reference (δ = 0 p.p.m.). A total of 550 μl of the supernatant was transferred into an NMR tube after vortexing and centrifuging at 12000 g for 5 min. 1 H NMR analysis was performed using Bruker AVANCE III 400 MHz spectrometer at 300 K. All samples were analysed using standard 1-dimension pulse sequence (recycle delay-90˚-t1-90˚-tm-˚-acquisition) with t1 and to 3 ms, tm to 10 ms, abd 90˚pulse to 10 μs in 64 scans.

Spectral data processing
The phase and baseline of all NMR spectra were adjusted in MATLAB software, and the TSP peak was set to 0 ppm. Data points within 0.6-9.0 ppm were kept and the water peak interval (4.5-5.0 ppm) was removed. The data were calculated according to a control spectrum after probabilistic quotient normalization (PQN).

Metabolite identification and network analysis
Pseudo two-dimensional spectra were drawn based on preprocessed data using statistical total correlation spectroscopy (STOCSY) which indicates correlation between factors among each chemical shift [31,32]. The ratio of false positives and the threshold of the correlation factor r were calculated. P-value was adjusted with Bonferroni false discovery rate (FDR) correction. The area under peaks was also integrated and used as relative concentration. The relative concentrations of metabolites were analyzed using log2 fold-changes using GraphPad Prism 5 (GraphPad Software, Inc., CA, US). The metabolites with log2 fold-changes were analyzed using Spearman correlation using R programming. The names of the metabolites and their HMDB ID were matched and network analysis was performed using MetaboAnalyst (www. metaboanalyst.ca/) [26]. The MBROLE2 system, accessible at http://csbg.cnb.csic.es/mbrole2 [27], was used for pathway analysis including metabolite-metabolite interactions.

Statistical analysis
SPSS software version 17.0 (IBM Corporation, Armonk, NY) was used for statistical analysis of clinical and baseline data. The differences among each group of samples were analyzed using a t-test. The data were expressed as a graph of mean ± S.D. using Graph Pad prism 5. All analyses were two-tailed and P-values < 0.05 were considered statistically significant.

CD44 depletion effects on cell proliferation
To study the function of CD44 on CCA cells, we carried out stable knockdown of CD44 and examined cell proliferation, migration and invasion. The evaluation of CD44 level using flow cytometry shows a high CD44 expression on cell surfaces in both CCA cell lines. Furthermore, stable knockdown in KKU-213 and KKU-156 CCA cell lines was performed using CD44shRNA to target two different sequences allowing the exploration of cellular functions including cell proliferation. Fig 1A shows that the level of CD44 was decreased in KKU-213 and KKU-156 after the knocking down by CD44shRNA in both targets. The levels of CD44 expression of KKU-213 were decreased to 45% and 18% of control in CD44shRNA target 1 (p = 0.0577) and 2 (p = 0.0263), respectively, and KKU-156 were reduced to 48% and 15%in both CD44shRNA target 1 (p = 0.0114) and target 2 (p = 0.0002), respectively.
In addition, the depletion of CD44 reduced proliferation at 72 and 96 h in both CCA cell lines (Fig 1B and 1C). The cell proliferationin percentage of CCA cells with transduction of CD44shRNA targeting sequence 2 was significantly reduced in KKU-213 cells at 72 h (p = 0.017) and 96 h (p = 0.035). Additionally, cell viability of CCA cells with transduction of CD44shRNA targeting both sequences 1 and 2 was significantly decreased in KKU-156 cells at 72h (p = 0.005 for sequence 1, p = 0.011 for sequence 2). The cell viability in KKU-156 CCA cells with transduction of CD44shRNA targeting sequence 2 was also significantly diminished at 96 h (p = 0.047).

Trolox recovers cell proliferation and migration after CD44 depletion via Akt activation
The intracellular ROS level was increased in CD44 knockdown CCA cell (Fig 2A). We, therefore, tested the effect of Trolox (ROS scavenger) on cell viability and migration to confirm the reverse effect on these factors in silencing of CD44. The result demonstrates that Trolox could recover cell viability ( Fig 2B) and migration in CD44 knockdown cells ( Fig 2C). Interestingly, the phosphorylation of Akt was increased after Trolox treatment in CD44shRNA silencing cells, while, control cells were not changed (Fig 2D and 2E). The phospho-Akt protein was not altered after treatment with 100 μM hydrogen peroxide in the control, whereas CD44 knockdown cells was slightly upregulated. These findings indicate that CD44-mediated Akt activation and its downstream targets is associated with intracellular ROS change.

The mechanisms of CCA progression is regulated by CD44 protein through the Akt signaling pathway
Our findings demonstrated that CD44 protein appeared to play important roles in cancer progression. We next examined the molecular mechanisms of CD44 contributing to cell

PLOS ONE
CD44-regulated metabolic pathways promoted CCA progression proliferation (Akt, mTOR, p21, CDK4, cyclin D1, Bax, Bcl-2), migration (E-cadherin, vimentin) and invasion (MMP-9) using the western blotting analysis and the localization of protein expression was also conducted using immunofluorescence staining. Our results revealed the downregulation of phosphorylation of Akt and its downstream target (mTOR) in KKU-213 and KKU-156 cells, with CD44 depletion in both targets (Fig 3A and 3B). Consequently, p21 cell cycle inhibitor was upregulated and translocated to the nucleus after CD44 depletion (Fig 3C), potentially leading to cell cycle arrest possibly via CDK4 and cyclin D1 depletion ( Fig 3A). These circumstances might trigger apoptosis pathway through an increase of proapoptotic protein (Bax) and a decrease in pro-survival protein (Bcl-2) (Fig 3A and 3B). Moreover, the investigation of epithelial mesenchymal transition (EMT) markers using immunoblotting showed that E-cadherin was increased while vimentin was decreased after CD44 depletion of KKU-213 and KKU-156 cells (Fig 3A and 3B). In addition, the co-localization of E-cadherin and vimentin revealed that the upregulation of E-cadherin and the downregulation of vimentin are found in CD44 silencing of KKU-213 and KKU-156 cells (Fig 3D). Furthermore, the immunofluorescence of MMP-9 shows that silencing of CD44 in both CCA cell lines was slightly decreased (Fig 3E). Therefore, the progression of CCA cells, including proliferation, migration and invasion, was potentially driven by CD44-mediated Akt activation and its downstream targets. The alteration of metabolic profiling in CD44-suppressed CCA cells CD44 protein plays important roles in cancer progression and metastasis possibly via Akt activation and its downstream targets. These alterations might derive from metabolic changes in both intracellular and extracellular metabolites. Therefore, we next identified the representative metabolites of CCA cells and conditioned media after interrupting CD44, and determined the changes in metabolic profiles using the NMR spectroscopic analysis. The metabolite assignment was confirmed with ChenomxNMR Suite software, STOCSY and HMDB. Fig 4 shows the spectra of cellular metabolites and conditioned media, while chemical shifts and corresponding groups in cells and conditioned media are summarized in Tables 1 and 2. By integrating the area under the peaks corresponding to signals of intrerest, the relative concentration of metabolites between the control sample and CD44 knockdown samples was determined as log2 fold changes (S1 and S2 Tables). Our results revealed intra-cellular thirtythree metabolites, whereas conditioned media contained twenty-five metabolites. We found that relatrive concentrations of acetate, citrate, creatine, cysteate, cysteine, glutamate, glutamine, homoserine, isoleucine, l-homocysteic acid, succinate, tryptamine, tryptophan and uracil were significantly altered. Interestingly, decreased levels of intracellular cysteine, glutamine, and homoserine after CD44 depletion were inversely associated with the level of extracellular metabolites. Therefore, the alteration of metabolic profiles, especially amino acid related-glutathione synthesis, was driven by CD44 and may contribute to CCA progression.

Correlation of metabolite profile in CD44-silencing CCA cells
As the alteration in metabolites occurred after CD44 depletion, the association between each metabolite might be of importance. Hence, we next examined the metabolite inter-correlations using heatmap. The graphical output containing the metabolome view is presented in Fig 5A  and 5B. Our figures show the heatmap correlation of each intracellular and extracellular metabolite from CCA cells. The association of metabolites after CD44 suppression was found in both the intra-and extra-cellular metabolic pathways, especially in glutathione synthesis and energy metabolism such as amino acid metabolism, and TCA cycle. For example, CD44-knockdown cells showed a decreased level of cysteine, a precursor of glutathione synthesis, directly associates with reduction of energy metabolism related-metabolites including acetate, carnitine, and succinate. In addition, this may trigger amino acid catabolism and decreased levels of amino acid and derivatives including creatine, alanine, glutamate,  glutamine, homoserine, and tryptophan were clearly observed. Extracellularly, several compounds are inversely associated, for example, an increase in glutamine was related to the reduction of amino acid and its intermediate metabolites including glutamate, homoserine, pyruvate, alpha-hydroxybutyrate, and tryptamine. A high level of cysteine was correlated with reducing of cystease, formate and threonine. Moreover, a decrease in glutamate was associated with a low level of isoleucine.

Suppression of the CD44 altered metabolic pathway in CCA cells
Based on the relationship between the metabolites, network analysis and pathway enrichment analysis using MBRole showed significantly related pathways (Fig 5 and Table 3): these were alanine, aspartate and glutamate metabolism, pyruvate metabolism, cysteine and methionine metabolism, glyoxylate and dicarboxylate metabolism, citrate cycle (TCA cycle), sulfur metabolism, glycine, serine and threonine metabolism, D-glutamine and D-glutamate metabolism,   The compounds from all metabolites in the pathway are listed; the set is the actually matched number from the user uploaded data; the p-value is the original p-value calculated from the enrichment analysis. https://doi.org/10.1371/journal.pone.0245871.t003 and tryptophan metabolism (Fig 6A and 6B; Table 3). Fig 7A and 7B show the network pathway for intracellular compounds that were significantly associated with extracellular compounds, including cysteine, glutamine, and homoserine. Taken together, we conclude that the depletion determined by CD44 altered extra-and intra-cellular metabolic profiles of CCA cells, leading to increased levels of ROS, particularly due to the alteration of amino acids such as cysteine, glutamate, and glutamine, that is driven by CD44-mediated Akt activation and its downstream targets contributing to CCA progression (Fig 8).

Discussion
In the present study, we demonstrated that the molecular targets of CD44 are related to redox status regulation and inhibition of CCA cell proliferation, migration and invasion via activation of the Akt pathway. Although the functions of CD44 and its variants in cancer development have been well studied, the obvious molecular mechanism of CD44 related to redox status regulation contributing to CCA progression and metastasis is yet to be elucidated. Previously, it was shown that RNA interference-mediated depletion of CD44 and its variants, causing the reduction of xCT, leads to a decrease of intracellular glutathione and an increase in the level of intracellular ROS in CCA cells. Signaling pathways are frequently regulated by ROS through the interactions with key signaling molecules affecting a variety of cellular processes such as proliferation, metabolism, differentiation and survival, including PI3 kinase (PI3K)protein kinase B (Akt) serine/threonine kinases [28]. ROS does not only activate PI3K directly to amplify its downstream signaling, but also concurrently inactivates the phosphatase and tensin homolog (PTEN), which inhibits the activation of Akt via oxidizing cysteine residues within the active center [29]. Furthermore, the phosphorylation of the CD44 intracellular domain can directly trigger intracellular signaling, particularly tyrosine kinase, including the Akt protein, for supporting growth, motility and invasion in many types of cancer [18], including breast [30,31], prostate [32], and colon cancers [33]. The Akt pathway, or PI3K-Akt pathway, is a signal transduction pathway that promotes survival and growth in response to extracellular signals. Akt successively promotes the activation and transcription of its target genes (GSK3, FOXO, BAD, mTOR1, and p53), which are involved in growth, motility and invasion [34][35][36][37]. Thus, we hypothesize that the CD44 function is driven by Akt activation and its downstream effects, potentially via stabilizing xCT on membranes for regulating ROS signaling and phosphorylation of the CD44 intracellular domain.
Our data demonstrated that the RNA interference-mediated depletion of CD44 and its variants caused a decrease in cell proliferation, cell migration and cell invasion. Moreover, the inhibited CCA cell proliferation after silencing of CD44 can be recovered using the Troloxvitamin E analog (data not shown). This indicates that the regulation of cell proliferation is associated with CD44 via the ROS-mediated cell signaling pathway. Moreover, the deletion of CD44 reduces the phosphorylation of Akt and mTOR, resulting in an increase of p21 (cell cycle inhibitor). Consequently, the pro-apoptotic proteins Bax was increased, while the antiapoptotic protein Bcl-2 was reduced. Hence, we conclude that the inhibition of cell proliferation is possible through the ROS-Akt signaling pathway mediated p21 cell cycle inhibition, leading to an alteration in apoptosis related-proteins. Although the expression of Akt phosphorylation was increased in H 2 O 2 treatment of CD44 silencing cells that might be involved with the imbalance of the redox status, the activation of Akt in CCA was regulated not only by the redox system, but also many other factors such as cytokines, growth factors, and bile acid [38]. Previously, it was demonstrated that the PI3K/AKT pathway in CCA is also stimulated by PTEN inactivation [39]. Currently, cell cycle progression is regulated by cyclin-dependent  kinase (CDK) inhibitors such as p21Cip1, which are indirectly and directly controlled by Akt [40]. In addition, the cAMP response element binding protein (CREB; promoting cell survival) is phosphorylated by Akt at Ser133, stimulating recruitment of CREB-binding protein (CBP) to the promoter of target genes, especially Bcl-2 [41].
In addition to its role in cell growth, our results demonstrated that cell migration and invasion are decreased after CD44 silencing in CCA cells. Previous studies suggested that CCA patients with high expression of CD44 had a poor prognosis [14,42], and an in vitro study of the depletion of CD44 showed that an inhibition of cell invasion and cell migration was seen in CD44 depleted CCA cells [43]. Importantly, this study links CD44 function and the alteration of EMT-related proteins with an increase of E-cadherin and a decrease of vimentin after the RNA interference-mediated reduction of CD44. EMT is characterized by the loss of epithelial characteristics including E-cadherin expression, and the gain of mesenchymal attributes such as vimentin signaling. It is a key phenomenon in wound healing, tissue repair and several diseases including cancers [44,45]. During EMT, epithelial cells downregulate cell-cell adhesion systems, lose their polarity, and acquire a mesenchymal phenotype associated with an increased interaction with the extracellular matrix, as well as an enhanced migratory capacity [46]. Previously, CD44 has been reported to promote EMT through the activation of signaling by the Akt pathway in breast cancer [19]. Akt phosphorylation enhances cell migration and invasion in a variety of cancers [47,48]. The functional mechanism can possibly be explained by the Akt signaling pathway reactivating NF-κB to induce the expression of Snail, and this in turn leads to the downregulation of E-cadherin expression [47]. Furthermore, type III intermediate filament vimentin is phosphorylated by Akt at Ser39, preventing its degradation and promoting cell migration [48]. Vimentin organization within the cell has important effects on the formation and function of invadopodia and lamellipodia during cellular invasion and migration [49]. In addition, GSK-3b degradation after PI3K/Akt activation leads to Slug overexpression followed by the induction of EMT [50]. We conclude that CD44-driving EMT processes via the Akt pathway plays a vital role in cancer metastasis in CCA by promoting cell migration.
Our work also shows that the depletion of CD44 affected cell invasion via the reduction of the matrix metalloproteinases (MMP)-9 signal, which is a family of zinc-dependent endopeptidases with important functions in extracellular matrix remodeling during development, and in inflammation and wound repair processes [51,52]. Recently, MMP-9 has been shown to be required for invasion in breast cancer [53] and osteosarcomas [54]. The expression of MMP-9 is regulated by the Akt signaling pathway, and promotes cell invasion in many types of cancers, such as head and neck squamous cell carcinoma [55], gastric cancer [56] and renal cancer [57].
Based on these findings, the mechanism of action of the CD44 molecule involves the redox regulation contributing to CCA progression via activation of the ROS-Akt signaling pathway. In order to determine how CD44 is involved in redox regulation, we investigated the metabolic profile alteration of CCA cells with/without CD44 silencing using NMR-based metabolomics. The suppression of CD44 altered both the intra-and extra-cellular metabolic pathways, especially those involving metabolites related to glutathione synthesis and the TCA cycle. Their alteration was associated with various compounds including amino acids, glucose, and other intermediate metabolites. Previous reports showed that some cancers can escape from this mechanism via CD44v8-10 expression stabilizing xCT, thus reducing the ROS signal against cell death [12]. In addition, CD44 protein changed methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides in human colon cancer cells, promoting cancer growth [58]. As it is widely known, glutamate and cysteine are the key amino acids involved in glutathione synthesis, and increasing levels of glutathione are one of the most important mechanisms for cancer drug resistance [59,60]. Moreover, glutamine is one of the metabolites which is dysregulated in many cancers, and which is essential for cell proliferation through many pathways [61]. Importantly, it has been recently reported that the disruption of the glutamine metabolic pathways improves the efficacy of gemcitabine treatment via the alteration of protein glycosylation, expression and functional effects such as EGFR signaling, including downstream AKT-mTOR pathways, the MAPK pathway, as well as redox enzymes [62]. Furthermore, glutamine is hydrolyzed by glutaminases to become glutamate and inorganic ammonia [63]. Suppression of glutaminases has shown anti-tumor effects in several cancer models, and is currently under clinical investigation [64,65]. Intracellular glutamine is commonly known as a key substrate required for cell growth and metabolism, as it serves as a carbon source to energize the TCA cycle, and transfers nitrogen for proteins, nucleotides and hexosamine synthesis [66][67][68]. A current report indicates that cisplatin-resistant cells show an increased consumption of glutamine [69]. In the current study, CD44 silencing promoted an accumulation of glutamine extracellularly, while intracellular glutamine was reduced. These circumstances might improve sensitivity to cancer treatment via regulation of the AKT-mTOR pathways, as well as redox homeostasis. In this study, we elucidated, for the first time, the metabolic profile changes after CD44 silencing which led to increased levels of ROS accompanied with decreased levels of essential compounds for glutathione synthesis, including cysteine and glutamate, as well as glutamine. This resulted in the activation of the Akt pathway. These events could promote CCA progression and metastasis.

Conclusion
We present the first evidence that the molecular mechanisms of CCA proliferation, migration and invasion is regulated by the CD44-mediated Akt signaling pathway through metabolic profile alteration. Collectively, CD44 plays a crucial role in CCA progression, and therapy targeted to CD44-positive cells that can potentially improve the efficiency of CCA treatment.
Supporting information S1 Table. The