Sestrin2 reduces the cancer stemness via the Wnt/β-catenin signaling in colorectal cancer

doi:10.21203/rs.3.rs-695161/v1

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Sestrin2 reduces the cancer stemness via the Wnt/β-catenin signaling in colorectal cancer

https://doi.org/10.21203/rs.3.rs-695161/v1

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published 11 Feb, 2022

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Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in both men and women in China. In previous studies, Sestrin2 was demonstrated to have functions in CRC. However, the relationship between Sestrin2 and cancer stemness has not been reported. To investigate the contribution of Sestrin2 in CRC, we performed bioinformatics analysis of The Cancer Genome Atlas datasets and found that Sestrin2 was downregulated in CRC. Using a lentivirus vector, we verified that Sestrin2 suppressed CRC cell proliferation, migration, and colony formation. Furthermore, sphere formation, flow cytometry, quantitative PCR, and Western blot analysis verified the effect of Sestrin2 on the cancer stem ness, including the expression of cluster of differentiation 44, octamer-binding transcription factor 4, sex-determining region Y-box 2, CXC chemokine receptor 4, and the Wnt pathway downstream factors β-catenin and c-Myc. Consistently, the Wnt pathway activator BML-284 partially rescued the effects of Sestrin2 on the expression of proteins related to the cancer stemness. Furthermore, in a mouse xenoplant model, tumors with Sestrin2 were significantly reduced in size with corresponding changes in the cancer stemness. Collectively, our results suggest that Sestrin2 inhibits CRC cell progression by downregulating the Wnt signaling pathway. Thus, Sestrin2 may be a promising therapeutic target for CRC.

Molecular Biology

Oncology

Sestrin2

Colorectal cancer

Wnt/β-catenin

cancer stemness

therapeutic target.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in both men and women in China [1]. Standard anti-cancer therapies for CRC include surgery and chemotherapy, but the survival rate is still poor. The failure of standard anti-cancer treatments is due to cancer stem cell (CSC) [2].CSCs are tumour cells that have properties of self-renewal, tumour initiation capacity, and long-term repopulation potential [3]. Targeting CSCs would be a promising therapeutic method. Therefore, investigating on CSCs to find new molecules and mechanisms in CRC would supply novel therapeutic targets.

At present, A variety of molecules are found to participate in CSC. Many cell surface proteins are used as CRC markers for different derived tumours, for example, cluster of differentiation 34-positive (CD34+)/CD38- for leukaemia cells, CD13/CD45/CD90 for liver cancer, CD117/CD90/epithelial cell adhesion molecule for lung cancer [4, 5], and CD44 for colon and gastric cancers [6]. The stem cell state is governed by stem cell factors such as the four Yamanaka factors: sex-determining region Y-box 2 (Sox2), octamer-binding transcription factor 4 (Oct4), Kruppel-like factor 4, and c-Myc [7].

Wnt signalling is one of the key pathways that regulate development and stemness in cancer [8]. The function of the Wnt pathway in CSCs is commonly accepted and depends on the amount of β-catenin in the cytoplasm [9, 10]. β-catenin is able to activate not only proliferation and transmission factors, such as c-Myc, c-Jun, CCND1 (the gene encoding cyclin D1), but also epidermal growth factor receptor, like CD44 and CD133.

Sestrin family members including Sestrin1, Sestrin2, and Sestrin3 can be induced by cellular stresses, such as ER stress, DNA damage and ROS accumulation[11–13]. Among sestrin family, Sestrin2 has shown regulating activities in some cancers [14]. Knocking down Sestrin2 in non-small cell lung cancer cells resulted in reduced lung cancer progression [15]. Down-regulation of Sestrin2 promoted cell proliferation, migration, and ROS production in endometrial cancer cells [16]. In CRC, our previous study demonstrated that there is a lower expression of Sestrin2 than in normal colorectal epithelial [17]. Other studies have shown that Sestrin2 has many other functions in CRC; for example, Sestrin2 is involved in inducing apoptotic process of HCT116 CRC cells [18], and its overexpression inhibits the migration, invasion, and growth of CRC [19]. However, the relationship between Sestrin2 and CRC stemness has not been previously reported.

This study aimed to investigate the role of sestrin2 in cancer stemness of CRC. Firstly, the effects of up-regulating Sestrin2 on the survival, migration, and colony formation of CRC cells were studied and the expression of cancer stemness in high-expression Sestrin2 CRC cells were detected. Secondly, the relationship between Sestrin2 and Wnt signalling was explored. Finally, we observed that up-regulation of Sestrin2 inhibited tumor growth in CRC cells in vivo. Our results suggest that Sestrin2 inhibits CRC cell progression by downregulating the Wnt signalling pathway.

Cell culture

The human CRC cell lines were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). HCT-116 were cultured in McCoy’s 5A medium (Biological Industries, Beit HaEmek, Israel) and SW620 cells were cultured in RPMI 1640 medium (Biological Industries) supplemented with 10% fetal bovine serum (FBS) (Biological Industries). Cells were cultured in 37℃ with 5% of CO2 concentrations in incubators.

Infection with lentiviral con℃structs

Lentiviral constructs expressing Sestrin2 (LV-Sestrin2) and a non-targeted green fluorescence protein (GFP) virus (LV-GFP) were purchased from GeneChem (Shanghai, China). Cells (1.5´10⁴) were plated in 24-well plates on the day before transfection. Lentiviral constructs were transduced at multiplicity of infection 10 in HCT-116 and SW620 cells using HiTransG P transfection reagent (Genechem). The infected cells were maintained in fresh medium without FBS for 12 h and then washed with phosphate-buffered saline (PBS), after which the medium was replaced with fresh medium supplemented with 10% FBS. Puromycin (2 mg/mL; Beyotime, Beijing, China) was used to generate stable expression cell lines in HCT116 and SW620 cells.

Quantitative PCR

The RNAsimple Total RNA Kit (TIANGEN, Beijing, China) was used to extract total RNA according to the manufacturer’s instructions. All-in-One cDNA Synthesis SuperMix (Bimake, USA) was used to generate the first-strand cDNA. The expression level of cDNAs was normalized to that of β-actin by the comparative CT method. The primer sequences used in this study are provided in Table 1.

Cell proliferation assay

A total of 500 cells were plated in the wells of a 96-well plate. Cell viability was measured using Cell Counting Kit-8 (CCK-8) (Bimake) and the Thermo Scientific™ Varioskan™ LUX Multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm once a day for 7 days.

Sphere formation

Sphere cells were cultured in stem cell medium (SCM), which is a DMEM/F-12 medium supplemented with epidermal growth factor (20 ng/mL, MedChemExpress, MCE), basic fibroblast growth factor (20 ng/mL, MCE), and B-27 (2%; Invitrogen, Carlsbad, CA, USA). A total of 200 cells were plated into each 96-well with 200 µL SCM. The number of spheres was counted after 2 weeks. For second sphere assay, the sphere cells were separated using ACCUTASE (YEASEN). A total of 200 cells were plated into each 96-well with 200 µL SCM. The number of spheres was counted after 2 weeks.

Limiting dilution assay in vitro

The procedure is same as described before [20]. Briefly, cells were seeded in U-bottom 96-well at final cell concentrations 1000, 100, 10, 1 per well with 200 µL SCM. After 2 weeks, the wells contain cell spheres were counted and empty wells were excluded. The frequency of CSCs was calculated on website (http://bioinf.wehi.edu.au/software/elda/) [21].

Transwell assay

Total 1×10⁶ suspended cells were added in the upper chamber (0.8 um in 6 well) with serum-free culture medium of transwell chamber (JET, China). The lower chamber contained medium supplemented with 10% FBS. After 24 h cultured, cells on the upper side of the filter membrane were removed. The rest of the cells were fixed with 5% paraformaldehyde for 20 min and dyed using Crystal Violet Staining Solution (Beyotime, China), then photographed at 200× and counted in five random fields.

Flow cytometry

Cells were detached into single cells and washed with cold PBS, pre-incubated with Human TruStain FcX™ (422301; BioLegend, San Diego, CA, USA), and then incubated with CD44-APC (B265921; BioLegend) on ice in the dark. Cells were assayed by flow cytometry.

Soft agar colony formation assay

A concentration of 1×10⁴ cells was suspended in SCM containing 0.36% agar and poured on an agar bed (SCM with 0.75% agar). After 3 weeks, the sphere number was counted after 0.04% crystal violet staining.

Wnt/β-catenin pathway inhibition assay

HCT-116 cells in the LV-Sestrin2 and LV-GFP groups were treated with 0.5 µM BML-284 (Wnt signaling activator; MedChemExpress, Monmouth Junction, NJ, USA) in dimethyl sulfoxide for 24 h and then collected for Western blot analysis. In the sphere formation assay, 0.1 µM BML-284 was mixed with cells and then plated in 96 wells with SCM. The number of spheres was counted after 2 weeks.

Western blot analysis

Cells were washed with PBS and lysed in RIPA buffer with Protease Inhibitor Cocktail (Bimake) on ice. Cell lysates were centrifuged (12,000 rpm) at 4°C for 20 min and then quantified using the BCA Protein Assay Kit (Beyotime). The lysate was denatured with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Sample Loading Buffer (Beyotime), followed by SDS-PAGE and electrotransfer to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). The membranes were incubated overnight at 4°C with anti-Sestrin2, anti-CD44, anti-Sox2, anti-Oct4, anti-CXC chemokine receptor 4 (Cxcr4), and anti-β-actin, (Proteintech Group, Wuhan, China) at the appropriate dilution, and incubated with secondary antibody at room temperature for 2 h. The bands were visualized using BeyoECL Plus (Beyotime).

Xenograft mouse model

A xenograft mouse model was determined by subcutaneously injection. One million HCT-116 cells in the LV-Sestrin2 and LV-GFP groups injected into the hips of 6-week-old female BALB/c-nu mice (Animal Center of Chongqing Medical University, Chongqing, China). All animal studies were approved by the Ethics Committee of Chongqing Medical University. The tumour size was measured every 3 days. The tumour volume (V= l×w2/2) was calculated by measuring the length (l) and width (w). Mice were euthanized by increasing volume of CO₂in a chamber, 21 days after cells injection.

Bioinformatics Analysis

The Colorectal cancer dataset used comprised mRNA-seq data from TCGA tumors ( https://tcga-data.nci.nih.gov/tcga/). The two-gene correlation map is realized by the R software package ggstatsplot, and the multi-gene correlation map is displayed by the R software package pheatmap. We used Spearman’s correlation analysis to describe the correlation between quantitative variables without a normal distribution. A p-value of less than 0.05 was considered statistically significant.

Statistical analysis

Except where otherwise noted, experiments were repeated at least three times. All statistical analyses were performed using GraphPad Prism Version 8. The t test was used to evaluate the significance of the differences between two groups of data. P <0.05 was considered statistically significant.

Sestrin2 inhibits CRC cell proliferation, migration, and colony formation

To evaluate the effects of Sestrin2 on CRC, we firstly investigated the mRNA expression of Sestrin2 compared to corresponding adjacent tissues in The Cancer Genome Atlas datasets using the UALCAN website (UALCAN: http://ualcan.path.uab.edu) (Fig. 1A) [22]. The results showed that Sestrin2 was significantly downregulated in both subtypes of CRC, which are colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). Secondly, we introduced LV-Sestrin2 and LV-GFP into HCT116 and SW620 cells, and proliferation, migration, and colony formation were evaluated. The CCK-8 assay showed that Sestrin2 inhibited the proliferation of CRC cells (Fig. 1B). The results from our colony formation assay demonstrated that the number of colonies formed was decreased in LV-Sestrin2-transduced CRC cells (Fig. 1C). Furthermore, the transwell showed that the migration of LV-Sestrin2-transduced CRC cells was inhibited (Fig. 1D) and wound healing assay also showed a consistent result (Fig. 1E). These results demonstrate that Sestrin2 is downregulated in CRC patients and has anti-cancer effects on CRC cells.

Sestrin2 Inhibits The Cancer Stemness In Crc Cells

CSCs play a significant role in cancer progression such as tumour growth, recurrence, and metastasis [23]. Therefore, we hypothesized Sestrin2 effects on CSCs in CRC cells. Self-renew and initiating tumor ability are basic characteristics of CSCs[24]. Soft agar assay is able to imitate vivo micro-environment[25]. We used the sphere assay and soft agar colony formation assay to investigate the self-renew ability of CRC cells. The results showed that Sestrin2 suppressed tumoursphere formation (Fig. 2A). Moreover, to determine the colorectal cancer initiating cells, we used ELDA assay and the results showed that LV-Sestrin2 reduced the CSCs in HCT-116 and SW620 cell lines (Table 1). In addition, molecules relating to CSCs were evaluated. Flow cytometry showed that the percentage of CD44 + cells was decreased in Sestrin2-transfected CRC cells (Fig. 2B). Expectedly, the stemness-related markers, including CD44, Oct4, Sox2, and Cxcr4, was downregulated at both the mRNA and protein levels in HCT-116 and SW620 cells (Fig. 2C-D) and similar protein expression trends can also be observed in sphere cells (Supplement Fig. 1). These results indicate the significant effects of Sestrin2 on reducing the cancer stemness in CRC cells.

Sestrin2 Reduces The Cancer Stemness Via The Wnt/β-catenin Pathway

As the Wnt/β-catenin pathway is one pathway that regulates stemness [26]. Activated Wnt/β-catenin pathway increases the number of CSCs by regulating self-renewal and homeostasis in cancer cell[27] .We observed the protein expression of β-catenin and the downstream target c-Myc. Our observations showed the expression of β-catenin and c-Myc was downregulated (Fig. 3A). Furthermore, we used the Wnt/β-catenin pathway activator, BML-284, to rescue the effects of Sestrin2 and found that BML-284 was able to partly rescue the effects of Sestrin2 using the sphere assay (Fig. 3B-C), consistent with the Western blot analyses in HCT-116 cells (Fig. 3D). In addition, to investigate whether sestrin2 affect cancer stemness by regulating Reactive Oxygen Species (ROS), Scavengers of ROS, Sodium pyruvate and Carboxy-PTIO, were used and we found Scavengers of ROS not affect the protein expression of CD44, no matter a single use or a joint use (Supplement Fig. 2). Our findings suggest that Sestrin2 regulates the cancer stemness via the Wnt/β-catenin pathway.

Sestrin2 Inhibits The Cancer Stmness In Vivo

To evaluate the effects of Sestrin2 in vivo, a subcutaneous xenotransplant tumour model was used. A concentration of 1×10⁷ cells from the LV-Sestrin2 or LV-GFP groups was injected into each of three female BALB/c-nu mice. The volume of tumour produced by HCT116-Sestrin2 cells was significantly reduced compared with that from HCT116-GFP cells when the weight of mice was not significantly different (Fig. 4A-E). Western blot analysis showed that the upregulation of Sestrin2 inhibited the protein levels of Sox2, c-Myc, and β-catenin (Fig. 4F). Further, in order to confer our finding in CRC patients, the mRNA expression correlations between Sestrin2 and CD44, Oct4, Sox2, Cxcr4, c-Myc and β-catenin were detected by R using TCGA data. The mRNA expression of CD44, β-catenin, Cxcr4 and c-Myc were negatively related to Sestrin2, which consistent with our results in vitro (Fig. 4G). The efficiency of tumour formation and cancer stemness was decreased by Sestrin2 in vitro.

Previous studies have introduced the role of Sestrin2 in CRC. In this study, we further investigated the role of Sestrin2 in CRC and found Sestrin2 inhibits the cancer stemness both in vivo and in vitro. Using primary and secondary sphere formation assay to detect the self-renew of CRCs, we found Sestrin2 up-regulating inhibited sphere formation of CRC cells. Consistently, The ELDA suggested that Sestrin2 inhibited the initial ability. Further, the expression of CD44, a key marker for CRC, and Oct4, Sox2, and Cxcr4, the stem cell factors, was also decreased. Overall, our data suggested that Sestrin2 not only reduce self-renew but also the initial ability of CRCs.

Sestrin2 may be a therapeutic target. First of all, CSC plays a very important role in cancer including maintenance, self-renewal, division, and tumour development [28]. As tumour propagation initiators, CSCs are considered to be a promising therapeutic method [29]. Besides, in this study, Sestrin2 targeting the Wnt/β-catenin pathway reduced the cancer stemness of CRC cells and Sestrin2 inhibited the proliferation, migration, and colony formation of CRC cells. If a medicine targeting on Sestrin2 would affect some tumor biological characteristics of CRC. Thus, Sestin2 may be a promising therapeutic target for CRC.

It is wildly accepted that the epithelial-mesenchymal transition (EMT) and CSCs are two closely related processes in tumour progression and therapeutic resistance [30]. In studies form other teams, Sestrin2 was shown to inhibit the EMT [31, 32]. These results can become evidence for our results which is Sestrin2 regulating the cancer stemness.

Sestrin2 is an anti-cancer molecule. In CRC, the decreased expression of Sestrin2 in patients predicts unfavorable outcomes, and Sestrin2 is also an important facilitator of the p53-mediated control of cancer cell growth [17, 33]. Moreover, Sestrin2 has been shown to function as a tumour suppressor in lung cancer, hepatocellular carcinoma, and melanoma [34–36]. Anti-tumour molecules such as tanshinone IIA, fangchinoline, and nelfinavir work together with Sestrin2 to carry out these functions [37–39]. This study also showed that Sestrin2 also has anti-cancer effects.

The effects of Sestrin2 on Wnt pathway may be via AKT signal pathway. Usually, Sestrin2, known as a p53 target gene, inhibits mTORC1 signaling and induction of autophagy [40, 41]. Interestingly, our results indicated that Sestrin2 also downregulates the Wnt/β-catenin signaling pathway. The reason why our results differ from others may be that SENS2 activates AKT and AMPK signaling [34] and PI3K/Akt/Wnt/β-catenin signaling, which controls the levels of EMT-related proteins [32]. Thus, Sestrin2 may affect β-catenin by interacting with AKT.

In conclusion, the results of this study showed that LV-Sesrin2 inhibits cancer stemness through the Wnt pathway in HCT-116 and SW620 cells, suggesting that Sesrin2 may be a promising therapeutic target for CRC.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the first affiliated hospital of chongqing medical university.

Consent for publication

All authors consent for publication.

Competing interests

The authors declare that no competing interests exist.

Funding

This study was funded by the Chongqing Science and Technology Commission (No. cstc2017jcyjAX0127).

Author contributions

JW and ZF designed the experiments. ZF supervised the whole project. XZ performed the major research and wrote the paper. WL and XL provide their professional guidance. XZ and WL provided the technical support.

Acknowledgments

All authors thank professor Weiyin Zhou from the College of Pharmacy, Chongqing Medical University, for research equipment and support.

Availability of data and materials

The data and materials can be obtained from the first author and corresponding author.

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TABLE 1

Limiting dilution data showing the effect of Sestrin2 expression on the frequency of CSCs

Cells seeded		1	10	100	1000	Estimated Frequency of CSCs	p value
HCT-116	LV-GFP	62/78	152/162	89/92	24/24	1 in 6 cells	0.087
HCT-116	LV-Sstrin2	78/105	136/170	86/91	24/24	1 in 7 cells	0.087
SW620	LV-GFP	45/90	101/145	76/94	24/24	1 in 19 cells	4.24×10-8
SW620	LV-Sstrin2	51/128	96/172	74/96	22/24	1 in 35 cells	4.24×10-8

SupplementTable1.docx
supplimentfigure1.tif
SUPPLEMENT FUGURE 1 Representative Western blot images of the effect of LV-Sestrin2 on the expression levels of Sestrin2, Sox2, Oct4, and Cxcr4 in HCT-116 and SW620 sphere cells. β-actin was used as a loading control.
supplimentfigure2.tif
SUPPLEMENT FUGURE 2 Representative Western blot images of the effect of LV-Sestrin2 with or without 48h treatment of scavengers of ROS, Sodium pyruvate and Carboxy-PTIO, on the expression levels of CD44 in HCT-116 and SW620 cells. β-actin was used as a loading control.

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Sestrin2 reduces the cancer stemness via the Wnt/β-catenin signaling in colorectal cancer

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Abstract

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Background

Materials And Methods

Results

Sestrin2 inhibits CRC cell proliferation, migration, and colony formation

Sestrin2 Inhibits The Cancer Stemness In Crc Cells

Sestrin2 Reduces The Cancer Stemness Via The Wnt/β-catenin Pathway

Sestrin2 Inhibits The Cancer Stmness In Vivo

Discussion

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