Colorectal cancer (CRC), one of the leading causes of cancer-related death, is associated with poor survival and has a high mortality rate (1). Currently, surgery is an effective therapeutic approach for CRC at the early stages (2). In addition, radiotherapy and chemotherapy are the most common treatment methods for CRC, particularly at the advanced and metastatic stages (3). Paclitaxel (Taxol) is one of the most commonly used chemotherapeutic agents, and is the first-line treatment for multiple cancers (4). Taxol is known to directly target the microtubules of the mitotic spindle to impede chromosome processes, leading to the induction of apoptosis pathways (5). Moreover, Taxol is an inhibitor of cell replication and migration through arresting cells in the late G2/M phase of the cell cycle (6). Despite the fact that better outcomes have been achieved by chemotherapy over recent decades, a large fraction of CRC patients develop drug resistance, leading to disappointing survival rates of CRC (7). However, the molecular mechanisms for the development of Taxol resistance in CRC remain largely unknown. Thus, investigation of effective approaches against chemoresistance is an urgent task to improve the therapeutic outcomes of colon cancer patients.
F-box and WD40 domain protein 7 (Fbxw7), a conserved F-box WD40 protein, plays an important role in recognizing subunits of the SKP1/CUL1/F-box protein (SCF) E3 ubiquitin ligase (8). Accumulating evidence has revealed that Fbxw7 targets a network of oncoproteins which regulate tumorigenesis and progression for ubiquitination and proteasome degradation (9). Moreover, multiple cancer-associated mutations of Fbxw7 have been detected in cancers (10), leading to favoriting tumor progression. However, a precise understanding of the biological roles and molecular targets of Fbxw7 in Taxol sensitivity is still under investigation.
NOXs are a family of membrane-associated enzymes which catalyze the oxidation of NADPH or NADH to NADP+ or NAD+ (11). NADPH oxidase 1 (Nox1) belongs to the NOX family and is a multifunctional protein participating in diverse tumor processes, including tumorigenesis, invasion, metastasis, and drug resistance (12). Remarkably, accumulating studies have demonstrated that Nox1 functions as an oncogenic protein with frequent upregulation in diverse tumors (13), indicating that Nox1 could be a therapeutic target for anti-CRC treatments. This study aims to investigate the biological roles and molecular mechanisms of Fbxw7 in Taxol resistance of colon cancer. Further results demonstrated that Fbxw7 is a negative regulator of the Nox1 protein, which is positively associated with colon cancer and Taxol resistance by promoting glucose metabolism. Our observations uncovered that the Fbxw7-mediated Taxol sensitivity was through the Nox1-glucose metabolism axis. We present the following article in accordance with the MDAR reporting checklist (available at http://dx.doi.org/10.21037/atm-21-2076).
Patient sample collection
This study was approved by the Ethics Committee of The Fifth People’s Hospital of Shanghai, Fudan University. Forty human CRC specimens and the matched adjacent non-tumorous colon tissues were collected from CRC patients who underwent surgical removal in The Fifth People’s Hospital of Shanghai from March 2016 to October 2018. All patients were histopathologically examined as CRC and did not receive other surgery or chemo/radiotherapy. After dissection, specimens were immediately placed in liquid nitrogen and transferred to a –80 °C freezer for storage. All patients signed the informed consent. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).
Cell culture and reagents
Human normal colon cell lines, CRL-1790 and CCD-18co, and CRC cell lines, HT29, HCT116, DLD-1, and LoVo, were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA). The cells were cultured in RPMI medium (Hyclone, MA, USA) with 10% fetal bovine serum (FBS, Gibco, MA, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, MA, USA). Cells were maintained in a humidified incubator at 37 °C in an atmosphere with 5% carbon dioxide. The HCT-116 Taxol-resistant (TR) colon cancer cell line was established by exposing cells to increased concentrations of Taxol for 3 months to select surviving cells. Rabbit Fbxw7 antibody was purchased from Sigma-Aldrich (#AB10620, Shanghai, China). Rabbit Nox1 antibody was purchased from Abcam (#ab78016, Shanghai, China). Rabbit β-actin antibody was purchased from Cell Signaling Technology (#4970, Danvers, MA, USA). Paclitaxel (Taxol) and 2-DG were purchased from Sigma-Aldrich (Shanghai, China).
Transfection of plasmid DNA or siRNA into colon cancer cells was performed using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. Plasmid DNA was transfected at 1 µg/mL, and siRNA was transfected at 50 nM. After 48 h, cells were collected for downstream experiments. Transfection was performed in triplicate.
RNA isolation and qRT-PCR
Total RNA was isolated using the Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The quality and concentrations of RNA samples were examined using a NanoDropTM 2000/2000c Spectrophotometer (Thermofisher, Waltham, MA, USA). The cDNA was synthesized from total RNA using the SuperScript II Reverse Transcriptase (Invitrogen, USA). The qRT-PCR reactions were performed using the SYBR Green PCR Master Mix (Thermofisher, Waltham, MA, USA). The reaction conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 sec and 60 °C for 30 sec. Primer sequences were as follows: Fbxw7 forward: 5'-CGAACTCCAGTAGTATTGTGGACCT-3' and reverse: 5'-TTCTTTTCATTTTTGTTGTTTTTGTATAGA-3'; Nox1 forward: 5'-GCCTGTGCCCGAGCGTCTGC-3' and reverse: 5'- ACCAATGCCGTGAATCCCTAAGC-3'; GAPDH forward: 5'-GCCGCATCTTCTTTTGCGTCGC-3' and reverse: 5'-TCCCGTTCTCAGCCTTGACGGT-3'. The relative gene expression was calculated by the 2–ΔΔCt method. Experiments were performed in triplicate and repeated three times.
Cell viability assay
After treatment with Taxol, cell viabilities were examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the manufacturer’s instructions (Sigma-Aldrich Co., St. Louis, MO, USA). Briefly, cells (8×103/well) were seeded in a 96-well plate for 24 h. After treatment, 0.025 mL of MTT solution [5 mg/mL in phosphate-buffered saline (PBS)] was added and incubated for 4 h. Cells were then lysed with DMSO for 2 h at 37 °C. The optical density (OD) value was detected at 570 nm on a 96-well multi-scanner plate reader (Biotek, USA). Experiments were performed in triplicate and repeated three times.
Colon cancer cells (1×103/well) were seeded into six-well plates. After treatment with Taxol for 48 h, the medium was replaced, and cells were cultured for 7 days in drug-free medium. Cells were fixed by 4% paraformaldehyde (PFA) and stained with a 10% crystal violet solution for 10 min at room temperature. After washing by PBS, colonies were photographed under microscopy. Experiments were repeated three times.
Cell apoptosis analysis
Cell apoptosis/death was examined using an Annexin V apoptosis Detection Kit I from BD Biosciences (San Jose, CA, USA) following the manufacturer’s protocols. Colon cancer cells (2×105/well) were cultured in 6-well plates for 24 h. After treatment with indicated concentrations of Taxol or 2-DG, cells were collected and washed with PBS followed by adding the binding buffer (500 µL) to resuspend the cells. The Annexin-FITC (5 µL) and propidium iodide (PI) (5 µL) solutions were added to cells which were then incubated with the mixture for 15 min at room temperature. The assay was performed with FACS Calibur flow cytometry (Becton Dickinson, San Jose, CA, USA) and analyzed by Flowjo software. Experiments were performed in triplicate and repeated three times.
Measurements of glucose metabolism
The glucose metabolism of colon cancer cells was determined by the glucose uptake assay and the lactate production assay. The glucose uptake assay was performed using the Glucose Uptake Colorimetric Assay Kit (#MAK083, Sigma-Aldrich, Shanghai, China). The lactate production assay was performed according to previous reports (14). Experiments were performed in triplicate and repeated three times. Results were normalized by cell numbers of each reaction.
Proteins from colon cancer cells were extracted by adding 100 µL RIPA lysis buffer with 1× protease inhibitor cocktail (Beyotime, Shanghai, China) on ice for 15 min. Lysates were then centrifuged at 10,000 ×g at 4 °C for 10 min. The protein concentration from each sample was determined by the Bradford method. Subsequently, 20 µg protein of each sample was resolved by electrophoresis with 10% polyacrylamide gels. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MO, USA), which were blocked by 4% non-fat milk in PBS with Tween 20 (PBST). The PVDF membranes were incubated with specific primary antibodies (1:1,000) at 4 °C for overnight. Membranes were washed by PBST for 3 times with 10 min each and incubated with HRP-conjugated secondary antibody at 1:3,000 at room temperature for 1 h. The enhanced chemiluminescent substrate (Millipore, MO, USA) was applied to visualize the protein bands. Experiments were repeated three times.
Statistical analysis was performed using the GraphPad Prism 6.0 software. Data were expressed as mean ± SD. The unpaired Student’s t-test and one-way analysis of variance (ANOVA) were applied to analyze the differences between the two groups and among three groups, respectively. A statistical difference of P<0.05 was considered significant.
Fbxw7 is downregulated in CRC and correlates with Taxol sensitivity
Previous studies have revealed a positive association between Fbxw7 and improved prognosis and survival rates in diverse cancers (9,10), suggesting that Fbxw7 functions as a tumor suppressor in colon cancer. To evaluate the biological roles of Fbxw7 in colon cancer and chemosensitivity, we compared the expression of Fbxw7 in CRC tumors and their adjacent normal tissues. Expectedly, qRT-PCR results showed that Fbxw7 mRNA expression was significantly attenuated in colon tumor tissues (Figure 1A). Moreover, expression levels of Fbxw7 in four human colon cancer cell lines, HT-29, HT116, DLD-1, and LoVo, were significantly downregulated compared with the two normal colon cell lines, CRL-1790 and CCD-18co (Figure 1B). To assess the roles of Fbxw7 in regulating Taxol sensitivity of CRC cells, Fbxw7 was overexpressed in HCT-116 and HT-29 cells by transfection with a control or Fbxw7 overexpression plasmid (Figure 1C). Colon cancer cells were treated with gradually increased concentrations of Taxol. Results from Figure 1D,E consistently showed that HCT-116 cells with higher Fbxw7 expression were more sensitive to Taxol. The IC50s of HCT-116 and HT-29 cells were 9.2 and 8.7 nM, respectively. CRC cells with Fbxw7 overexpression had decreased IC50s at 3.9 and 4.1 nM, respectively (Figure 1D,F). The Fbxw7-promoted Taxol sensitivity of HT-29 cells was further validated by the Annexin V/FITC cell apoptosis assay (Figure 1G).
Fbxw7 attenuates glucose metabolism of colon cancer cells
Metabolic reprogramming in response to cellular stresses as well as chemotherapeutic drugs is a known hallmark of cancer cells (15). Bioinformatics analysis and the glucose metabolism assay validated that compared with normal colon tissues and cells, CRC displayed significantly upregulated glucose metabolism enzyme expression and glucose metabolism rate (Figure S1A,B,C,D). We further characterized the roles of Fbxw7 in cellular glucose metabolism of CRC cells. As we expected, overexpression of Fbxw7 effectively inhibited glucose uptake and lactate production (Figure 1H,I), two key steps of glucose metabolism in colon cancer cells. Taken together, the above results demonstrated a tumor-suppressive role of Fbxw7 in colon cancer.
TR colon cancer cells display elevated glycolysis rates and suppressed Fbxw7 expression
We further revealed Fbxw7-mediated Taxol sensitization and glucose metabolism suppression. To investigate the molecular mechanisms of Fbxw7 in Taxol sensitivity, a TR colon cancer cell line was established from HCT-116 cells though treating cells with gradually increased concentrations of Taxol. Consequently, surviving cells were collected and characterized. As shown in Figure 2A,B, HCT-116 TR cells displayed higher tolerance under Taxol treatment compared with HCT-116 parental cells. The IC50 value of HCT-116 TR cells was 43.6 nM, which was approximately 5-fold higher than that of parental cells (Figure 2A,B). Consistently, results from the clonogenic assay demonstrated that the viability of HCT-116 TR cells was less inhibited by Taxol treatment than that of parental cells (Figure 2B). We then evaluated the correlation between Taxol resistance and glucose metabolism in colon cancer cells. HCT-116 TR cells exhibited markedly elevated glucose uptake (Figure 2C), lactate production (Figure 2D), and glycolysis key enzymes (GLUT1, HK2, and LDHA) expression (Figure 2E), suggesting that targeting dysregulated glucose metabolism could effectively overcome chemoresistance. Moreover, with the observation that Fbxw7 was remarkedly downregulated in TR colon cancer cells (Figure 2E), we assessed whether overexpression of Fbxw7 could reverse the Taxol resistance. Expectedly, HCT-116 TR cells with Fbxw7 overexpression displayed significantly elevated Taxol sensitivity compared with control cells (Figure 2F). Taken together, the above results clearly demonstrated that Fbxw7 contributes to anti-Taxol treatments.
Fbxw7 interacts with Nox1 and downregulates Nox1 in colon cancer cells
Fbxw7 is known to exert its tumor-suppressive roles mainly through mediating oncoprotein degradation (8,9). To identify potential substrates which are recognized by Fbxw7 and involved in cellular metabolism, we performed literature searches and noticed previous reports which uncovered that Nox1, a member of the Nox enzyme family which is involved in the production of ROS (11), is critical for supporting the elevated glycolysis of cancer cells. We then tested whether Fbxw7 could regulate Nox1 expression. Interestingly, overexpression of WT-Fbxw7 significantly downregulated Nox1 expression in two colon cancer cell lines, HCT-116 and HT29 (Figure 3A). However, overexpression of the inactive mutant Fbxw7 (Fbxw7 ΔF) did not affect Nox1 expression (Figure 3A). The qRT-PCR results showed that Nox1 mRNA was not regulated by neither WT- nor mutant-Fbxw7 overexpression in colon cancer cells (Figure 3B), suggesting that Fbxw7 regulates Nox1 at the protein level. We further performed co-immunoprecipitation (Co-IP) experiments on HCT-116 cells using the Fbxw7 specific antibody or IgG control antibody. IP results from Figure 3C clearly illustrate that Fbxw7 could specifically bind with the Nox1 protein. Given the known functions of Fbxw7 in mediating protein ubiquitination and degradation, the above results uncovered that Fbxw7 interacts with Nox1 and downregulates Nox1 through the ubiquitin protein degradation pathway.
Nox1 is positively associated with Taxol resistance and promotes glycolysis of colon cancer cells
We then investigated the biological roles of Nox1 in the Taxol sensitivity of colon cancer. Bioinformatics analysis showed that Nox1 was significantly upregulated in CRC tissues (Figure S2). Consistent with this, Nox1 was apparently overexpressed in CRC tissues from our 40 CRC patient specimens (Figure 4A). To evaluate the roles of Nox1 in Taxol sensitivity, Nox1 was overexpressed in HCT-116 and HT29 cells (Figure 4B), followed by treatment with Taxol at increased concentrations. Expectedly, overexpression of Nox1 significantly decreased Taxol sensitivity of CRC cells as determined by the cell viability assay (Figure 4C,D) and clonogenic assay (Figure 4E,F). Nox1 was significantly upregulated in TR colon cancer cells compared with HCT-116 parental cells (Figure 5A). Meanwhile, silencing Nox1 effectively sensitized HCT-116 TR cells to Taxol (Figure 5B), suggesting that Nox1 could be a therapeutic target for overcoming Taxol resistance.
To assess the correlation between Nox1-promoted Taxol resistance and the upregulated cellular glucose metabolism in TR colon cancer cells, we evaluated the roles of Nox1 in glucose metabolism. Expected results in Figure S3A,B demonstrated that overexpression of Nox1 significantly promoted glucose uptake and lactate production in CRC cells. Moreover, glycolysis key enzyme expression was effectively downregulated by Fbxw7 (Figure S4A) and upregulated by Nox1 (Figure S4B). We then compared the glucose metabolism rates of HCT-116 TR cells with or without Nox1 silencing. Expected results showed that TR cells with lower Nox1 maintained a relatively lower glucose uptake (Figure 5C) and lactate production (Figure 5D). In summary, these results revealed that Nox1 is positively associated with Taxol resistance and the glucose metabolism of colon cancer cells.
The Fbxw7-promoted Taxol sensitivity is partially through the Nox1-glycolysis axis
Finally, we summarized the above results and examined whether Fbxw7 sensitizes colon cancer cells to Taxol via the Nox1-glycolysis pathway. HCT-116 TR cells were transfected with a control vector, the Fbxw7 overexpression vector alone, or plus the Nox1 overexpression vector. Western blot results from Figure 6A showed successful rescue of Nox1 in Fbxw7-overexpressed cells. Glucose uptake (Figure 6B) and lactate production (Figure 6C) were consistently suppressed by Fbxw7 overexpression. Expectedly, this regulation by Fbxw7 was recovered by Nox1 restoration in HCT-116 TR cells (Figure 6B,C). Cells with the above transfections were treated with control, Taxol alone, or plus the glycolysis inhibitor 2-DG. As we expected, restoration of Nox1 in Fbxw7-overexpressed HCT-116 TR cells effectively recovered Taxol resistance (Figure 6D), which could be further overridden by glucose metabolism inhibition (Figure 6D). Taken together, our rescue experiments revealed that the Fbxw7-promoted Taxol sensitivity was partially through targeting the Nox1-glycolysis axis.
CRC, the third most common cancer worldwide, is associated with poor survival and has a high mortality rate (1,2). Currently, despite the fact that improved outcomes have been achieved by chemotherapy, the aquired drug resistance is becoming the major challenge for cancer treatments (3). Therefore, investigation into the molecular mechanisms of chemoresistance is essential to improve therapeutic effects. In this study, we report a Fbxw7-mediated Taxol sensitization in colon cancer. Based on an in vitro TR CRC cell model, we demonstrated that Fbxw7 was significantly downregulated in HCT-116 TR cells, suggesting that enhancing the Fbxw7 signaling pathway could contribute to overcoming Taxol resistance.
Fbxw7 functions as a tumor suppressor and is negatively associated with tumor progression through targeting a cluster of oncoproteins, such as c-Myc (16), cyclin E (16), mTOR (17), and Aurora A (18). Despite the fact that diverse Fbxw7 substrates have been validated, novel oncoproteins of potential Fbxw7 targets and the molecular mechanisms underlying the functions of Fbxw7 in chemosensitivity remain largely unknown. In this study, we detected Nox1, an enzyme which is capable of oxidizing NADPH or NADH to NADP+ or NAD+ to generate superoxide, as a substrate of Fbxw7. We showed that Fbxw7 interacts with Nox1, leading to a significant downregulation of the Nox1 protein in colon cancer cells. Expectedly, overexpression of Fbxw7 did not affect the mRNA level of Nox1, indicating that Fbxw7 markedly promotes Nox1 protein degradation. Although a previous study described Fbxw7-mediated Nox1 degradation in smooth muscle cells (19), our results for the first time demonstrate that Nox1 is a Fbxw7-interacting protein and is downregulated by Fbxw7 in colon cancer cells. Importantly, this study integrated the Fbxw7-Nox1 interaction with Taxol sensitivity of colon cancer, potentiating the Fbxw7-Nox1 interaction as a novel therapeutic target against TR CRC.
In contrast to normal cells, tumor cells show increased glycolysis rate rather than oxidative phosphorylation for energy supply and fast proliferation, a phenomenon called the “Warburg effect” (15). Moreover, this dysregulated cellular metabolism reprograming provides tumor cells with a survival advantage under a stressful microenvironment or chemotherapeutic agents (15). In particular, we observed that Nox1, which was shown to be positively associated with CRC progression, had the capacity to activate the glucose metabolism of CRC cells. As an NADPH oxidase, Nox1 has been implicated in the regulation of gene expression and ROS generation (11). In addition, a recent study uncovered that upregulation of Nox1 was critical for elevated glycolysis by providing additional NAD+ in cancer cells with mitochondrial dysfunction (12,13). Here, we showed that Nox1 was upregulated in TR CRC cells, which showed apparently elevated glucose metabolism. Silencing Nox1 effectively sensitized TR cells by blocking glucose metabolism. Thus, our data linked Nox1-mediated glucose metabolism and Taxol resistance in CRC cells, presenting a novel therapeutic target for overcoming Taxol resistance. The present study has limitations, in that an in vivo xenograft model is needed to validate the above in vitro cellular mechanisms. Moreover, the precise mechanisms of Nox1-promoted glucose metabolism are still under investigation.
In summary, this study unveils the Fbxw7-Nox1-glucose metabolism axis in regulating the Taxol sensitivity of CRC. Our conclusions will enlighten researchers to develop Fbxw7-Nox1-based anti-chemoresistant drugs which can effectively enhance the clinical applications of Taxol against colon cancers.
Funding: The project was supported by the Medical System of Shanghai Minhang District (No. 2020MWDXK02), Natural Science Research Funds of Minhang District, Shanghai (No. 2018MHZ025, No. 2018MHZ037).
Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at http://dx.doi.org/10.21037/atm-21-2076
Data Sharing Statement: Available at http://dx.doi.org/10.21037/atm-21-2076
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-21-2076). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by institutional ethics committee of The Fifth People’s Hospital of Shanghai, China. Written informed consent was taken from all individual participants included in the study.
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
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