CRKL promotes hepatocarcinoma through enhancing glucose metabolism of cancer cells via activating PI3K/Akt

Abstract Abnormal glucose metabolism may contribute to cancer progression. As a member of the CRK (v‐crk sarcoma virus CT10 oncogene homologue) adapter protein family, CRKL (CRK‐like) associated with the development and progression of various tumours. However, the exact role and underlying mechanism of CRKL on energy metabolism remain unknown. In this study, we investigated the effect of CRKL on glucose metabolism of hepatocarcinoma cells. CRKL and PI3K were found to be overexpressed in both hepatocarcinoma cells and tissues; meanwhile, CRKL up‐regulation was positively correlated with PI3K up‐regulation. Functional investigations revealed that CRKL overexpression promoted glucose uptake, lactate production and glycogen synthesis of hepatocarcinoma cells by up‐regulating glucose transporters 1 (GLUT1), hexokinase II (HKII) expression and down‐regulating glycogen synthase kinase 3β (GSK3β) expression. Mechanistically, CRKL promoted glucose metabolism of hepatocarcinoma cells via enhancing the CRKL‐PI3K/Akt‐GLUT1/HKII‐glucose uptake, CRKL‐PI3K/Akt‐HKII‐glucose‐lactate production and CRKL‐PI3K/Akt‐Gsk3β‐glycogen synthesis. We demonstrate CRKL facilitates HCC malignancy via enhancing glucose uptake, lactate production and glycogen synthesis through PI3K/Akt pathway. It provides interesting fundamental clues to CRKL‐related carcinogenesis through glucose metabolism and offers novel therapeutic strategies for hepatocarcinoma.


| INTRODUC TI ON
Hepatocarcinoma (HCC) is the most common primary neoplasm of liver with the highest morbidity and mortality rate in the world. 1,2 Marked progress in hepatocarcinoma treatment has been achieved by the combination of surgical resection and chemotherapy. 3 However, the relative higher metastasis, recurrence and chemoresistance of hepatocarcinoma still leads to poorer prognosis of patients. 4 Therefore, improvement in the diagnosis and treatment of hepatocarcinoma depends on boosting our comprehending for the underlying molecular mechanisms controlling its development and progression. Identifying potential indicators for the progression and drug-tolerance of hepatocarcinoma will lead to better diagnosis and treatment of the patients.
Emerging as a novel hallmark of cancer, energy metabolism reprogramming is crucial for the proliferation, differentiation and metastasis of cells. 5 Even in the presence of enough oxygen, tumour cells enhance glucose consumption with increased lactate production through aerobic glycolysis instead of oxidative phosphorylation for their growths. 6 Cancer cells preferentially selected aerobic glycolysis over oxidative phosphorylation for glucose-dependent ATP production due to mitochondrial impairments. 7,8 Increased glycolysis benefits tumour cell growth and dissemination. Lactate accumulation results in an acidic extracellular microenvironment causing the death of surrounding normal cells, ECM remodelling, promoting EMT and cancer cells metastasis. [9][10][11] Uncovering the mechanisms underlying the Warburg effect is helpful for the development of novel therapeutic targets and strategies of human cancers.
As a member of the CRK (v-crk sarcoma virus CT10 oncogene homologue) adapter protein family, CRKL (CRK-like) is ubiquitously expressed in eukaryotic organisms. 12 CRKL has various linkages for combining to BCR-ABL, GAB, C3G, Pax, GEF and SOS to form complexes that are crucial for cell survival, proliferation, migration and adhesion. [13][14][15] Hence, CRKL can function in cell signalling pathway by either directly forming complex with downstream receptor molecules to mediate tyrosine kinase activity, or by acting as an upstream regulator for signal initiation. 15,16 The deregulation of CRKL associated with the development and progression of cancers. 17, 18 We previously found that CRKL deregulation was remarkably affecting the metastasis capacities of HCC cells. 19,20 However, the biological function and regulatory mechanism of CRKL in metabolism are unknown. Previously, we screened the differentially expressed proteins in K562 cells with CRKL knockdown by iTRAQ quantitative proteomics, Gene ontology (GO) analysis revealed that CRKL deregulation related to metabolic process ( Figure 1A). We speculate that CRKL may affect glucose metabolism of hepatocarcinoma cells.
In the present study, we report a novel biological function of CRKL in glucose metabolism of hepatocarcinoma. We found that CRKL and PI3K were up-regulated in HCC cells and tissues. Moreover, CRKL overexpression facilitated the Warburg effect and glycogen synthesis of hepatocarcinoma cells. Further investigation revealed that CRKL promoted glucose metabolism of hepatocarcinoma cells by activating the PI3K/AKT pathway. Our study has uncovered a novel CRKL-PI3K/AKT regulatory pathway in glucose metabolism of hepatocarcinoma and partially elucidated the molecular mechanism underlying glucose metabolism in hepatocarcinoma cells.

| Western blotting (WB) assay
Protein was extracted from cells and tissues using ice-cold RIPA buffer as previously described. 20

| Co-immunoprecipitation (Co-IP) assay
The interaction between CRKL and PI3K was determined by Co-IP assay as previously described. 20 Protein was extracted from 5 × 10 6 HCCLM3, HCCLM3-PCDH or HCCLM3-PCDH-CRKL cells by RIPA buffer; then, protein was incubated with control anti-rabbit IgG (Santa Cruz Biotechnology) or anti-CRKL (Santa Cruz Biotechnology), PI3K (Sanying) for 1 hour at 4°C. The immune complexes were pulled down by protein A/G magnetic beads (Santa Cruz Biotechnology) overnight at 4°C. After washing with RIPA buffer for 3 times, the immunoprecipitated proteins were detected by WB.

| Glucose oxidase-peroxidase (GOD-POD) assay
The effect of CRKL up-regulation on glucose uptake of hepatocarcinoma cells was detected by glucose detection kit (Nanjing Jiancheng Bioengineering Institute). Each group 1 × 10 4 cells in 200 μL 10% FBS supplemented with DMEM from each group were seeded into a separate well of 96-well plate, incubated at 37°C with 5% CO 2 for 24 hours, removed into a 0.6 mL Eppendorf tube and centrifugated with 560 g for 10 minutes. Then, 3 μL of supernatant from each group was mixed well with 300 μL of working solution already loaded in a 96-well pate and incubated at 37°C for 15 minutes. The absorbances at 505 nm were measured using a microplate reader (Thermo). Meanwhile, 3 μL of calibrator (5.55 mmol/L) was used for calibration. Results were the averages from triplicate measurements.

F I G U R E 2
The different expressions and correlationship of PI3K and CRKL in HCC tissues and cells. WB assays indicated that PI3K was overexpressed in HCC tumourous tissues compared with paracancerous normal liver tissues (A) and in HCC HepG2, HCCLM3 and HuH7 cell lines compared with normal liver LO2 cells (B). C, The expression level of CRKL was positively correlated with PI3K expression in both HCC tissues and cells

| Lactate oxidase (LOD) assay
The effect of CRKL overexpression on lactate production of hepatocarcinoma cells was measured using a lactate detection kit (Nanjing Jiancheng Bioengineering Institute). Each group 1 × 10 4 cells in 200 μL 10% FBS supplemented with DMEM from each group were seeded into a separate well of 96-well plate, incubated at 37°C with 5% CO 2 for 6, 12, 18 and 24 hours, removed into a 0.6 mL Eppendorf tube and centrifugated with 560 g for 10 minutes. Then, 2 μL of supernatant from each group was mixed well with 100 μL enzyme working liquid and 20 μL chromogenic reagent in a 96-well pate, and incubated at 37°C for 10 minutes. Then, 200 μL stop reagent was added to each well for absorbance assay at 530 nm using a microplate reader (Thermo). Meanwhile, 2 μL of calibrator (3 mmol/L) was used for calibration. Results were the averages from triplicate measurements.

| Periodic acid Schiff reaction (PAS) assay
The effect of CRKL overexpression on glycogen synthesis of hepatocarcinoma cells was measured by a glycogen staining kit (Wanlei Biotechnology). The 25 mm diameter cover glasses were placed into 6-well plates, 4 × 10 4 cells in 100 μL 10% FBS supplemented with DMEM from each group were loaded into a well of 6-well plate with a 25 mm cover glass and incubated at 37°C for 24 hours. Then, the cover glass was washed with PBS, fixed with ice-cold acetone, incubated with periodic acid and washed with distilled water. Then, the cover glasses were incubated in 100 μL Schiff for 15 minutes, stained with haematoxylin, clarified by xylene, dehydrated by gradient ethanol and imaged by an upright microscope (Olympus) with 400× and 1600×. Amaranth spherical particle in cytoplasm refers to the PAS-positive reaction.

| Statistical analysis
All data analysis was completed using GraphPad Prism 5.0 software (GraphPad Software Inc). Student's t test was used for comparison between two groups. The correlation between the expression level of CRKL and PI3K was analysed by the Spearman's rank correlation coefficient. P ≤ .05 was considered statistically significant. All experiments were performed in triplicate for quantitative comparison.

| The expression patterns and correlation of CRKL and PI3K in HCC tissues and cells
We found CRKL expression was up-regulated in HCC tumour tissues by IHC and WB assays. 19,20 Consistently, CRKL was more F I G U R E 3 CRKL promotes Warburg effect of hepatocarcinoma cells. A and B, CRKL were stably overexpressed in HCCLM3 and HuH7 cells. C, CRKL enhanced glucose uptake of hepatocarcinoma cells by GOD-POD assay. D and E, CRKL enhanced lactate production of hepatocarcinoma cells by LOD assay We detected PI3K was overexpressed in HCC tissues and cells.
WB assay showed that PI3K expression was up-regulated by 74.5% (P = .0422) in the tumour tissues from 10 HCC patients ( Figure 2A).
Meanwhile, PI3K was expressed in all the tested cells, and a com-

| CRKL promotes the Warburg effect of hepatocarcinoma cells
Warburg effect is a hallmark of cancer cell growth and metastasis.
LOD assay showed CRKL enhanced lactate production of hepatocarcinoma cells ( Figure 3D,E, Table 2). No lactate production differences were observed between HCCLM3 and HCCLM3-PCDH ( Figure 3D), or in HuH7 and HuH7-PCDH cells ( Figure 3E). In comparison with HCCLM3-PCDH or HuH7-PCDH, the lactate pro- Taken together, the above findings strongly support the notion that CRKL enhances the Warburg effect of hepatocarcinoma cells.

| CRKL promotes glycogen synthesis of hepatocarcinoma cells
Glycogen synthesis is another important indicator of glucose metabolism. PAS assay showed CRKL overexpression promoted glycogen synthesis of hepatocarcinoma cells (Figure 4). Compared to
The results further indicate that CRKL promotes glucose metabolism of hepatocarcinoma cells.

| CRKL promotes glucose metabolism of hepatocarcinoma cells by activating the PI3K/AKT pathway
The underlying molecular mechanism of CRKL in glucose metabolism is unclear. We found a remarkable positive relationship F I G U R E 6 The schematic underlying mediation mechanism of CRKL on glucose metabolism in hepatocarcinoma. CRKL promotes glycolysis and glycogen synthesis through the PI3K/Akt pathway by increasing GLUT1 expression, potentiating HKII activity and inactivating GSK3β activity between the expression levels of CRKL and PI3K in HCC tissues and cells. Meanwhile, KEGG analysis revealed that CRKL deregulation is related to the PI3K/AKT pathway ( Figure 1B). CRKL overexpression activated the PI3K/AKT pathway ( Figure 5A). CRKL overexpression increased the protein expression levels of PI3K  Table 4). Moreover, the glycogen synthesis was weakened after blocking the PI3K/AKT pathway in HCCLM3-PCDH-CRKL and HuH7-PCDH-CRKL cells, the PAS-positive reaction was weakened and the content of glycogen was obviously reduced after blocking the PI3K/AKT pathway ( Figure 5F). These results indicate that CRKL regulates glucose metabolism of hepatocarcinoma cells by mediating PI3K/AKT pathway.
A previous study reported that the SH3N domain of CRKL could binding to the p85 PxxP motif of PI3K and resulted in activated PI3K. 46 We performed a Co-IP experiment to confirm the direct interaction between CRKL and PI3K in HCCLM3 cells. Our results obviously demonstrated that CRKL or PI3K protein band presented in the immunoprecipitated CRKL-PI3K complexes with antibodies against PI3K or CRKL. Moreover, the amount of CRKL-PI3K complex was more in HCCLM3-PCDH-CRKL cells than in HCCLM3-PCDH cells ( Figure 5G). Our results illuminate that CRKL directly binds to PI3K to positively mediate the expression of PI3K.

| D ISCUSS I ON
Metabolism involves the biological processes that allow healthy cells to maintain energy balance. 21 Malignant cells reprogram their metabolism and energy production for their rapid proliferation and survival in severe environments due to the mutation of oncogenes and the inactivation of tumour suppressor genes. 22 The dysregulated metabolism represents an adaptive advantage that facilitates growth and metastasis of tumour cell during tumorigenesis. 23 Most cancer cells rely on the Warburg effect as a source of ATP. The change of tumour metabolism, particularly glucose metabolism, is regarded as a target for anti-cancer therapy. 24,25 Better understanding of the molecular mechanism underlying energy metabolism is urgently needed in order to discover novel therapeutic targets and strategies to fight cancers.
CRKL deregulation is associated with various cancers, which is an interesting biomarker for diagnosis, therapy and prognosis of tumours. 13 Previously, we found that CRKL was overexpressed in hepatocarcinoma patient tumour tissues and cells, indicating CRKL overexpression potentially e promoted hepatocarcinogenesis. 19,20 We also found that CRKL affected the migration and invasion potentials of HCCLM3, HuH7 and HepG2 cells. 19,20 Dysregulated metabolism of tumour cell provides an acidic microenvironment that facilitates tumour cell migration and invasion, CRKL deregulation associates with metastasis, and we speculate that CRKL may affect the energy metabolism of tumour cells. So, in the current study, we investigated the potential role of CRKL in energy metabolism of hepatocarcinoma.
Warburg effect is one of the best characterized metabolic disorders during cancer development and progression, which increases glucose uptake and lactate production. 5 Glucose uptake and lactate production are important biological processes of energy metabolism. 10 We measured the effect of CRKL overexpression on Warburg effect of hepatocarcinoma cells. We found that CRKL enhanced glucose uptake of HCCLM3 and Huh7 cells ( Figure 3C, Table 1). Glucose is not able to across the plasma membrane on its own due to the hydrophilic composition of glucose; therefore, to overcome this condition, cancer cells induce GLUTs expression. 26 GLUTs are transmembrane proteins responsible for facilitating the transport of extracellular glucose across the plasma membrane into cells during the glucose metabolism process. 27,28 GLUT1 is one of the most widely expressed isoforms in a variety of cells and has been reported to be up-regulated in various cancers; it is mostly relevant to the glucose metabolism. 29, 30 We found CRKL overexpression increased the protein expression level of GLUT1 in HCCLM3 and Huh7 cells ( Figure 5A). Meanwhile, we found CRKL enhanced lactate production of HCCLM3 and Huh7 cells ( Figure 3D,E, Table 2). The glycolytic pathway contains a series of 10 reactions, and the enzymes involved in the glycolysis are all potential targets for inhibitors used in anti-cancer therapy. 31 During the first step of glycolysis, glucose is transformed into glucose-6 phosphate through the phosphorylation of the 6-hydroxyl group of glucose by the enzyme hexokinase (HK). 32,33 The HK family contains four isoforms: I, II, III and IV. HKII is frequently overexpressed in a variety of cancers and is the major enzyme which is closely involved in glycolysis. [34][35][36] We found that CRKL overexpression increased the protein expression level of HKII in HCCLM3 and Huh7 cells ( Figure 5A). Taken together, our findings strongly support the notion that CRKL enhances the Warburg effect of hepatocarcinoma cells.
Liver is a crucial organ in glycogen synthesis, glucose metabolism and blood glucose maintenance. 37 Glycogen is thought to be 'a store of glucose'. 38 Hepatic glycogen synthesis plays a crucial role in maintaining normal glucose homeostasis. 39 We found CRKL overexpression promoted glycogen synthesis of HCCLM3 and HuH7 cells ( Figure 4). Glycogen synthase kinase 3 (GSK3) is the primary regulatory kinase for glycogen synthase (GS) through phosphorylating and inactivating GS, including GSK3α and GSK3β, 40,41 GSK3β plays an important role in the mediation of blood glucose homeostasis. 42,43 We also found that CRKL overexpression increased the protein expression level of GSK3β in HCCLM3 and Huh7 cells ( Figure 5A). Our findings strongly support the notion that CRKL enhances the glycogen synthesis of hepatocarcinoma cells.
The PI3K/Akt pathway plays a crucial role in the mediation of glucose metabolism through its downstream effector molecules, including promotion of glycolysis, glucose uptake, glycogen synthesis and inhibition of gluconeogenesis in the liver. 44 Akt increases the glycolysis rate by promoting transcription and plasma membrane localization of GLUT1. 45 Meanwhile, AKT can enhance HKII activity to promote glycolysis, at least in part, by increasing its association with a voltage-dependent anion channel at the outer mitochondrial membrane. 46 In addition, AKT can directly phosphorylate GSK3β, thereby rendering the kinase inactive and thus promoting glycogen synthesis. 47 Previously, our proteomic results showed that CRKL deregulation related to the PI3K/ Akt pathway ( Figure 1B), PI3K was up-regulated in HCC tissues ( Figure 2A) and cells ( Figure 2B). Meanwhile, CRKL up-regulation was positively correlated with PI3K overexpression ( Figure 2C).
We speculate that CRKL may mediate glucose metabolism of hepatocarcinoma cells via PI3K/AKT pathway. In confirmation of the hypothesis, our results showed that CRKL overexpression enhanced PI3K and p-AKT expression levels in HCCLM3 and Huh7 cells ( Figure 5A). Furthermore, blocking the PI3K/AKT pathway resulted in decreased protein expression levels of GLUT1 and HKII, and increased protein expression level of GSK3β ( Figure 5B,C).
Meanwhile, the promotion effects of CRKL overexpression on the glucose uptake ( Figure 5D), lactate production ( Figure 5E) and glycogen synthesis ( Figure 5F) of HCCLM3 and Huh7 cells were induced drug resistance and increased tumour metastasis risk, limit their future applications. 53 To overcome these challenges, combination therapy of cancer starvation with molecule-targeted therapy will be an efficient way, which can maximize the therapeutic efficiency.
As illustrated in Figure 6, the direct binding of SH3N domain of CRKL to the p85 PxxP motif of PI3K result in activated PI3K for generating the PIP2 and PIP3 at the inner membrane. Then, Akt binds to PIP3 via PH domain, simultaneously get phosphorylated at Thr308 by PDK1 binding to PIP3. Subsequently, p-Akt promotes transcription and translocation of GLUT1 from the endomembrane to the cell surface; then, GLUT1 promotes the transport of extracellular glucose across the plasma membrane into the cells.
In addition, p-Akt promotes transcription and potentiates activity of HKII; then, glucose is transformed into glucose-6-P by HKII and undergoes glycolysis followed by the rapid conversion of pyruvate into lactate. Meanwhile, p-AKT directly phosphorylates GSK3β lead to the inactivation of the kinase, the inactivated GSK3β is unable to phosphorylate and inactivate GS, subsequently glycogen is synthesized by GS.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

PA RTI C I PATE
This study has been conducted in accordance with ethical standards and has been approved by the authors' institutional review board.
The study protocol was approved by the Medical Ethics Committee of Dalian Medical University.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data support the findings of this study are available from the corresponding author upon reasonable request.