USP28 promotes aerobic glycolysis of colorectal cancer by increasing stability of FOXC1

Aerobic glycolysis is essential for cancer cell metabolism and growth. Deubiquitinase, USP28 (ubiquitin specific peptidase 28), could maintain stability of proteins involved in tumor progression. This study was performed to investigate the role of USP28 in aerobic glycolysis of colorectal cancer. Our data showed that USP28 mRNA and protein expressions were enhanced in colorectal cancer tissues and cells. Functional assays demonstrated that overexpression of USP28 promoted cell proliferation and aerobic glycolysis of colorectal cancer, while USP28 inhibition could reverse these effects. Protein expression of Forkhead Box C1 (FOXC1) was increased by USP28 over-expression, whereas knockdown of USP28 aggravated cycloheximide (CHX; protein synthesis inhibitor) stimulated decrease of FOXC1. Moreover, proteasome inhibitor, MG132, could rescue USP28 silence-induced degradation of FOXC1. Overexpression of FOXC1 counteracted the suppressive effects of USP28 interference on colorectal cancer cell viability and aerobic glycolysis. In conclusion, USP28 enhanced cell viability and aerobic glycolysis of colorectal cancer by stabilizing FOXC1, suggesting that USP28-FOXC1 might be a novel therapeutic avenue for colorectal cancer.


INTRODUCTION
Colorectal cancer is one of the most common cancers in the world (Yang, 2019). Although 5-year survival rate of colorectal cancer patients is improved via surgical resection, radiotherapy and systemic chemotherapy at an early stage, high recurrence rate caused by distant metastasis still threatens a large proportion of advanced stage patients (Prenen et al.,, 2013). Recently, efforts have been made to elucidate the genetic and molecular characteristics of colorectal cancer for better prognosis and response to targeted therapies (Fleshman & Smallwood, 2015). Understanding the mechanisms involved in cell proliferation and metastasis of colorectal cancer might also improve treatment of the patients.
Tumor cells undergo abnormal glycolysis, accompanied by increased glucose uptake and lactic acid production, that is known as aerobic glycolysis or Warburg effect (Lu, 2019). Aerobic glycolysis plays an essential role in tumor cell proliferation with the synthesis of nucleic acids, amino acids and fats, and the production of lactic acid in microenvironment that could enhance tumor metastasis and treatment resistance (Doherty & Cleveland, 2013). Activation of aerobic glycolysis has been shown to be associated with colorectal cancer metastasis and poor prognosis of the patients (Satoh et al., 2017). Therefore, inactivation of glycolysis might facilitate for suppression of colorectal cancer metastasis (Hu et al., 2020).
Ubiquitin specific peptidase 28 (USP28), member of USPs, contains a ubiquitin-recognition site and corresponding catalytic mechanism . With the ability to regulate physiological homeostasis of ubiquitination process, tune DNA-damage response and maintain cell cycle, targeting USP28 has shown clinical significance in cancer therapy . For example, USP28 could increase the characteristics of breast cancer stem cells by deubiquitinating lysine-specific demethylase 1 (Wu et al., 2013). Inhibition of USP28 promotes degradation of lysine-specific demethylase 1 to repress gastric cancer cell metastasis . In colorectal cancer, USP28 could regulate intestinal homeostasis and promote the progression of intestinal diseases and tumor progression (Diefenbacher et al., 2014;Diefenbacher et al., 2015). Inhibitor of USP28 could reduce cancer-related proteins, followed by suppressing colorectal cancer progression . However, the mechanism of USP28 in colorectal cancer aerobic glycolysis has not been reported yet.
This study was performed to explore the role and molecular mechanism of USP28 in aerobic glycolysis of colorectal cancer. Results showed that USP28 could promote aerobic glycolysis of colorectal cancer and promote the cell growth through enhanced stability of Forkhead Box C1 (FOXC1), thus providing a novel therapeutic avenue for colorectal cancer. incubated with horseradish peroxidase-labeled secondary antibody (Abcam, Cambridge, UK) followed by staining with diaminobenzidine and counterstaining with hematoxylin. Slides were visualized under a light microscope (Olympus, Tokyo, Japan).

MTT and colony formation assays
T84 or SW480 transfections were seeded into plates at the density of 4 x 10 3 cells/well at indicated time (0, 24, 48, 72, 96, 120 hours). MTT reagent (20 µL; Sigma Aldrich) was added to the plates two hours before the measurement of absorbance at 490 nm by microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA). For colony formation assay, T84 or SW480 transfections were seeded into plates at the density of 1×10 2 cells/well, and then cultured for 12 days. Colonies in each well were measured under microscope (Olympus) following fixation in methanol and staining in 0.05% crystal violet.

Measurement of glucose uptake, lactate and ATP levels
For glucose uptake analysis, T84 or SW480 transfections were seeded into plates for 48 hours. The cultured medium was collected and the cells were harvested. Protein concentration in cells was determined by Pierce bicinchoninic acid protein assay kit (Thermo Scientific) and the glucose concentration was determined by glucose assay kit (Shanghai Rongsheng Biotech, Shanghai, China). The values of glucose uptake were normalized to the total protein number. For lactate and ATP levels determination, the cultured medium was conducted with lactate or ATP assay kits (Nanjing KeyGen Biotech, Nanjing, China). The values of lactate and ATP levels were also normalized to the total protein number.

Extracellular acidification rate (ECAR)
XF Glycolytic Rate Assay kit (Agilent Technologies, Santa Clara, USA) with Seahorse Bioscience XF Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, USA) was used to determine ECAR for the evaluation of glycolytic function. T84 or SW480 was seeded into XF24 plates for 24 hours. XF Base Media (5 mM HEPES, 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine) without phenol red was then used to detect the glycolytic rate under WAVE version 2.6.0 (Agilent Technologies). Oligomycin (1 μM) and 2-deoxyglycose (50 mM) were added as inhibitors into the plate.

Statistical analysis
Data were presented as mean ± SEM from at least three independent experiments. Statistical analysis was conducted with SPSS 22.0, and evaluated by Student's ttests or one-way analysis of variance. p < 0.05 was considered as statistically significant difference.

Enhanced USP28 in colorectal cancer
To evaluate the expression level of USP28 in colorectal cancer tissue, qRT-PCR and western blot analysis were performed. Results showed that mRNA (Fig. 1A) and protein ( Fig. 1B and 1C) expression of USP28 were enhanced in the tumor tissues. Similarly, up-regulation of USP28 was also validated in colorectal cancer cells compared to NCM460 (normal human colon mucosal epithelial cell line) (Fig. 1D), suggesting potential regulatory role of USP28 on colorectal cancer.

USP28 promoted colorectal cancer cell growth
T84 was transfected with pcDNA-USP28 ( Fig. 2A) and SW480 was transfected with shUSP28#1 or 2# (Fig. 2B) to exploit the functional role of USP28 on colorectal cancer. shUSP28#1 showed lower expression of USP28 than shUSP28#2 and was used for the following functional assays. Data from MTT and colony formation assays indicated that overexpression of USP28 promoted cell viability (Fig. 2C) and proliferation (Fig. 2D) of T84, while knockdown of USP28 reversed the promotive effects on cell viability (Fig. 2C) and proliferation (Fig. 2D) in SW480. These results indicated that USP28 promoted colorectal cancer cell growth. USP28/FOXC1 in aerobic glycolysis of colorectal cancer

Figure 1. Enhanced USP28 in colorectal cancer
Increased mRNA expression of USP28 in colorectal cancer tissues compared to normal adjacent tissues. Immunohistochemical analysis of colorectal cancer tissues compared to normal adjacent tissues indicated the upregulation of USP28 in tumor tissues. Induced protein expression of USP28 in colorectal cancer tissues compared to normal adjacent tissues. Protein expression of USP28 was enhanced in colorectal cancer cells compared to NCM460 (normal human colon mucosal epithelial cell line). *p<0.05; **p<0.01.

USP28 promoted aerobic glycolysis of colorectal cancer cells
The role of USP28 on colorectal cancer cell metabolism was then evaluated to examine the involvement of USP28 in colorectal tumor development. Energy production was induced by USP28 overexpression with increased glucose uptake, lactate and ATP levels in T84 (Fig. 3A). However, knockdown of USP28 decreased glucose uptake, lactate and ATP levels in SW480 (Fig. 3B). Moreover, USP28 overexpression enhanced the ECAR (Fig. 3C), while knockdown of USP28 reduced the ECAR (Fig. 3D), indicating the promotive effect of USP28 on aerobic glycolysis of colorectal cancer cell.

DISCUSSION
Cycles between ubiquitination and deubiquitination are essential for various biological processes, and the dysregulation of deubiquitination is involved in tumorigenesis (Sacco et al., 2010). For example, USP13 functioned as tumor-suppressor through stabilization of PTEN (Zhang et al., 2013). USP7 could stabilize Mdm2 to promote degradation of p53 (Sheng et al., 2006). Recently, deubiquitination by USP37 has been reported to regulate aerobic glycolysis of tumors (Pan et al., 2015). Considering the promotive effect of USP28 on colorectal (Diefenbacher et al., 2014;Diefenbacher et al., 2015), the role of USP28 on aerobic glycolysis of colorectal cancer was then evaluated in this study.
Previous study has shown that USP28 was enhanced in colorectal cancer tissues, and USP28 could form a positive feedback loop with oncoprotein, c-MYC, to promote colorectal cancer progression (Diefenbacher et al., 2014). Our results also showed that up-regulation of USP28 in colorectal cancer led to cell proliferation. Since cancer cells in aerobic glycolysis increased lactate production during the growth (Soga, 2013), the aerobic glycolysis in colorectal cancer was promoted by USP28 with increased glucose uptake, lactate and ATP production. As is well known, c-MYC is a transcriptional factor to regulate metabolic enzymes, such as hexokinase 2, phosphofructokinase and lactate dehydrogenase A, involved in aerobic glycolysis and tumor progression (He et al., 2015). Inhibition of c-MYC could suppress aerobic glycolysis in colorectal cancer (Xu et al., 2015). USP28 could interact with ubiquitin ligase, F-box and WD repeat domain containing 7 and further stabilize c-MYC (Popov et al., 2007;Chakravorty et al., 2020). Silencing USP28 destabilized c-MYC in esophageal cancer cells (Weili et al., 2019). Therefore, the downstream target of USP28 involved in colorectal cancer progression needs to be further investigated.
FBP1, key glycolytic enzyme in aerobic glycolysis, was a target gene of FOXC1 to enhance aerobic glycolysis in colorectal cancer (Li et al., 2019). Moreover, FOXC1 could bind to integrin α7 or fibroblast growth factor receptor 4 to promote colorectal cancer metastasis , and stabilization of FOXC1 could enhance the metastasis of colorectal cancer . Therefore, we suggested that USP28 might promote aerobic glycolysis in colorectal cancer by stabilizing FOXC1. Knockdown of USP28 decreased expression of FOXC1 in colorectal cancer cells post protein synthesis inhibitor (cycloheximide) treatment. Moreover, proteasome inhibitor MG132 attenuated USP28 interference-induced decrease of FOXC1. These results suggested that USP28 regulated stability of FOXC1 depending on inhibition of proteasome-mediated degradation in colorectal cancer cells. Functional assays further indicated that over-expression of FOXC1 counteracted the suppressive effects of USP28 knockdown on cell viability and aerobic glycolysis of colorectal cancer. Moreover, in line with previous report that FOXC1 could down-regulate FBP1 in colorectal cancer cells (Li et al., 2019), our results revealed that knockdown of USP28 decreased FOXC1 and increased FBP1, while over-expression of FOXC1 attenuated promotive effect of USP28 knockdown on protein expression of FBP1. Thus, USP28 could sustain growth and aerobic glycolysis of colorectal cancer cells through up-regulation of FOXC1 as well as down-regulation of FBP1. The present study for the first time showed that the deubiquitination of FOXC1 through USP28 could increase aerobic glycolysis in colorectal cancer. However, cycles of ubiquitination and deubiquitination have been shown to mediate activation of AKT , which regulated aerobic glycolysis in tumors (Elstrom et al., 2004), and phosphorylation of ser241 and ser272 of FOXC1 when p38 suppressed the ubiquitination and degradation of FOXC1 . Therefore, whether AKT or p38 pathways were involved in USP28/FOXC1/FBP1 axis-mediated colorectal cancer aerobic glycolysis would be further investigated in further research.

CONCLUSION
In summary, the present study illustrated that USP28, overexpressed in colorectal cancer tissues, could promote cell proliferation and enhance the aerobic glycolysis of colorectal cancer cells. Mechanistically, USP28 could block the degradation of FOXC1 to down-regulate FBP1, thereby promoting aerobic glycolysis. Our data demonstrated that USP28 and FOXC1 might be the potential therapeutic targets in colorectal cancer due to the biological significance of USP28/FOXC1/FBP1 axis.