Restraint stress promotes lung cancer xenograft growth via the IL2-Ras-Erk pathway

doi:10.21203/rs.3.rs-2504507/v1

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Restraint stress promotes lung cancer xenograft growth via the IL2-Ras-Erk pathway

https://doi.org/10.21203/rs.3.rs-2504507/v1

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Objectives To assess the impact of restraint stress on lung cancer xenograft development, and explore the Molecular mechanism.

Methods We established a restraint stress model by placing nude mice in a ventilated 50 mL centrifuge tube and inoculating them with subcutaneous xenografts of the A549 lung cancer cell line on day 21. In order to verify the effects of IL2 on tumor growth, we treated A549 cells with IL2 (1 or 5 ng/mL) in vitro.

Results Compared with the non-stressed mice, the stressed mice exhibited lower body weight gain and larger tumors after 42 days of restraint stress treatment. The stressed mice also exhibited a higher level of proinflammatory cytokine IL2 in serum, tumor tissue, spleen and lymph nodes. The tumors from the stressed mice exhibited higher activity of the ras-extracellular signal-regulated kinase (Ras-Erk) signaling pathway. Compared with the control group (0 ng/mL), the cells treated with IL2 exhibited a higher rate of proliferation and migration, along with increased activity of the Ras-Erk pathway. Knockdown of the IL2 receptor by siRNA alleviated the increase of proliferation, migration and RAS-Erk pathway activity stimulated by IL2.

Conclusion Based on these findings, we conclude that restraint stress increased IL2 levels to promote tumor growth by activating the Ras-Erk pathway.

restraint stress

cancer

inflammation

IL2

Ras-Erk signaling pathway

Stress can induce psychological disorder, which has been extensively investigated since 1968[1]. In the past two decades, there has been growing evidence that stress is also associated with many physical diseases, from worsening of autoimmune diseases to increased risk of cardiovascular diseases [2], obesity [3], Alzheimer's disease [4], gastrointestinal disease [5], non-alcoholic fatty liver [6], and even certain types of cancer [7]. A clinical review based on 330 studies found that stress is related to poorer patient survival, and higher cancer-related mortality in 53 studies [8]. Animal models indicate that stress induces immune dysfunction, contributing to the destabilization of atherosclerotic plaques [9]. These observations suggest that stress induces an altered physiological state which results in the deterioration of both psychological and physical health.

Stress can induce the secretion of various hormones and cytokines [10], which increases the incidence and promotes the development of cancers [11, 12]. Epidemiological studies showed that distress might be associated with increased cancer progression and shortened survival time in cancer patients [8]. Studies in experimental animals also demonstrated the negative effects of stress on the progression of different malignancies, including pancreatic [13], gastric [14], breast and ovarian cancer. Mechanistically, stress can promote most disease hallmarks through specific stress hormones, their receptor systems and intracellular signaling cascades [15]. However, it remains unclear how stress affects a certain type of cancer and through which stress hormones.

In this study, we explored the effects of restraint stress on lung cancer using a xenograft model in nude mice. A dramatic increase in the levels of the pro-inflammatory cytokine IL2 were detected both in the serum and tumor tissues, which was associated with the activation of the Ras/Erk signaling pathway in tumor tissues. In vitro, cultured A549 cells treated with IL2 exhibited an increase of proliferation and migration. Activation of the Ras/Erk pathway was also detected. Taken together, we identified pro-inflammatory cytokine IL2 as the stress hormone, through which restraint stress promotes the malignancy of lung cancer cells.

2.1 Cell culture

The human lung cancer cell line A549 was preserved in our laboratory and cultured in DMEM high glucose medium containing 10% inactivated fetal bovine serum and 1% penicillin-streptomycin. The cells were cultured at 37°C in a humidified atmosphere comprising 5% CO₂.

2.2 Animals and Sampling

The animal experiments were approved by Ethics Committee of Medical College of Jiaxing University and the whole protocol was performed in accordance with institutional guidelines (JUMC2019-060). Specific pathogen-free (SPF) nude mice (BALB/c-nu) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China) and were housed in a temperature-controlled room (24 ± 2°C) with a 12 h light/dark cycle.

At 8 weeks of age, 18 male mice were randomly divided into two groups, the stress group and the non-stressed group. The nude mice in the stress group were restrained in a ventilated 50 mL centrifuge tube (the bottom of the tube was punched 3 mm to provide good ventilation). The nude mice did not squeeze their tails during the operation and could move before and after the regular operation. The mice were subjected to restraining stress for 8 h per day (10:00 AM-6:00 PM) and then put back into their original cage with the supplement of food drink. The mice were stressed for 5 days and allowed to rest for 2 days, for a total treatment duration of 42 days. The non-stressed mice stayed in their cages under normal conditions as the control group.

On the 21st day, actively growing A549 lung cancer cells were trypsinized, and the cell density was adjusted to 1×10⁶ / mL. Then 0.2 mL of the resulting cell suspension was injected subcutaneously into the back of each nude mouse. After inoculation, the nude mice in the stress group continued to receive stress treatment (Fig. 1A). During the experiment, the survival status of nude mice in each group were observed, and the body weight, food intake, water intake, and tumor size of all animals were measured. The long diameter (a) and short diameter (b) of the tumor were measured, and the tumor volume (mm³) was calculated using the empirical formula 0.52 a × b². The maximal tumour size/burden was not exceeded the permissible tolerance in institutional guidelines. The serum was collected by retro-orbital bleeding before sacrificing and frozen for further analysis. All mice were sacrificed via cervical dislocation on the 42nd day.

2.3 Determination of the tumor ratio and organ index

The tumor was removed entirely from the back of the nude mice and weighed to calculate the tumor/body weight ratio (%) using the formula: tumor weight/host body weight. The heart, liver, spleen, lungs, and kidneys were removed entirely and weighed to calculate the organ index (mg/g) using the formula: tissue weight/mouse body weight.

2.4 ELISA

The blood of nude mice was collected by retro-orbital extraction, stored overnight at 4°C, and centrifuged at 3 500 rpm and 4°C for 15 min. The serum was collected and stored at -80°C. The concentrations of IL-6 and IL-10 (H007-1-2 and H009-1-2 kits, Nanjing Jiancheng Biological Engineering Institute, China) in the serum were determined according to the instructions of the ELISA kits.

2.5 H&E staining

Fresh tumor tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into horizontal sections with a thickness of 6 µm. As previously reported, the sections were stained with H&E using standard pathohistological procedures. The sections were observed and photographed under a conventional optical microscope (Nikon, Tokyo, Japan).

2.6 Cell treatment with IL-2/IL-2rb-siRNA

A549 cells were seeded at a density of 2×10⁵ cells/mL in 12-well plates, 6-well plates and 96-well plates overnight and treated with IL-2 or IL-2rb-siRNA at final concentrations of 0, 1, and 5 ng/mL for 24 h, respectively. Then, the cells from 12-well plates were harvested for real-time PCR and western blotting, those form 6-well plates were harvested for the wound healing assay and transwell assay, while the cells from the 96-well cells were harvested for the CCK-8 assay and EdU incorporation assay.

2.7 CCK-8 Assay

The CCK-8 method was used to assess cell viability. Briefly, 100 µL of CCK-8 test reagent (CCK-8:DMEM = 1:9) was added to each well and incubated a 37°C for 2 h, after which the absorbance at 450 nm(A₄₅₀) was measured, with four replicates for each group.

2.8 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay

Cell proliferation was assessed using a Cell-Light™ EdU Apollo 488 In Vitro Imaging Kit (RiboBio, Guangzhou, Guangdong, China), according to the manufacturer’s instructions. A549 cells were treated with IL-2 and incubated with 1:1,000 EdU (5-ethynyl-2’-deoxyuridine) diluted in culture solution for 2 h at 37°C before harvesting. After several washes, the cells were fixed with 4% paraformaldehyde and then permeated with 0.5% Triton X-100 for 10 min, followed by staining with Apollo buffer (RiboBio) for 30 min at room temperature in the dark. The cells were then incubated with Hoechst 33342 dye at room temperature for 30 min in the dark. Images were captured using fluorescence microscopy (Leica, Germany)and the positive cells were counted using ImageJ software (n = 4).

2.9 Wound healing assay

The cell migration ability was detected using the cell monolayer scratch method. The A549 cells were seeded into 6-well plates and grown until 90–100% confluence. The medium was changed to fresh medium containing 0, 1, and 5 ng/mL IL-2 or IL-2rb-siRNA. Sterilized 200 µL pipette tips were used to generate the wound across the cell monolayer. After the detached cells and debris were washed away with PBS, images at 0 h were recorded using a conventional optical microscope (Leica) at 100× magnification. Migration of cells into the wound was photographed after incubation for 24 h. The scratch width of the cell monolayer was measured, to calculate the migration rate.

2.10 Transwell assay

The upper chambers of the transwell plate were treated with precooled matrigel (DMEM : Matrigel = 8:1) and seeded with 2×10⁵/mL A549 cells in 200 µL serum-free medium. The bottom chamber of the transwell plate contained 600 µL 10% FBS medium. After 24 h of incubation at 37 ℃ in an atmosphere with 5% CO2, the cells that penetrated to the bottom chamber were fixed with 4% paraformaldehyde and stained with crystal violet. Images were recorded using an inverted microscope, and the numbers of invading cells was counted.

2.11 Quantitative real-time PCR Analysis

TRIzol reagent (Takara, Shiga, Japan) was used to extract total RNA from the hypothalamus, tumor, spleen, and lymph nodes, according to the manufacturer’s recommendations. Reverse transcription was performed using the HiFiScript gDNA Removal cDNA Synthesis Kit (CWbio, Beijing, China). SYBR Green mix (BioTek, Beijing, China) was used for quantitative RT-PCR assays.

2.12 Protein isolation and western blot analysis

RIPA Lysis Buffer (CWbio, Beijing, China) supplemented with protease inhibitor (Pierce, Bradenton, Florida, USA) was used to extract the total proteins from the xenograft tumors of the nude mice. After centrifugation at 12 000 g and 4°C for 15 min, the supernatants were collected and stored at -80°C for further analysis. Proteins were separated by SDS-PAGE on a 12% acrylamide gel and transferred onto a PVDF membrane (Millipore, Massachusetts, USA). The following antibodies were used for western blotting: Ras (3965, Cell Signaling, USA), p-Erk (9101, Cell Signaling, USA), Erk (9102, Cell Signaling, USA), Cyclin D1 (ab16663, Abcam, UK), p-AKT (9271, Cell Signaling, USA), AKT (9272, Cell Signaling, USA), p-NF-κB (3033, Cell Signaling, USA), NF-κB (6956, Cell Signaling, USA), GAPDH (ARG10112, Arigobio, China), β-Tubulin (KM9003, Tianjin Sungene Biotech, China). A ChemiDoc XRS + image analyzer system (Bio-Rad, USA) was used to photograph the blots. ImageJ software (National Institutes of Health, USA) was used to quantify the protein levels via densitometric analysis of the western blots.

2.13 Statistical Analysis

The experimental data were analyzed using Prism 8.0 (GraphPad, USA) and expressed as means ± standard error of the mean (SEM). The significance of differences was assessed using Student’s t-test. Differences with P < 0.05 were considered statistically significant.

3.1 Restraint stress reduced weight gain and intensified inflammation

To investigate the effect of stress on cancer development, we established a 42-day restrain stress model with BALB/c-nude mice. During the experimental process, the stressed mice showed observable anxiety and lower food and water intake (Fig. 1B, C), which led to a consistently lower body weight, compared with the non-stressed mice (Fig. 1D). We also found that the liver, lung, and kidney indexes were markedly increased in the stressed mice (Fig. 1E).

To determine whether restraint stress influenced inflammation, we measured the expression levels of inflammatory cytokines in the serum, tumors, spleen, and lymph nodes. Compared with the non-stressed mice, the stressed mice showed a high level of inflammatory cytokine IL2 in the serum and all the other analyzed tissues. Other inflammatory cytokines (IL6 and TNG-α) were higher in some tissues and anti-inflammatory cytokines (IL10 and TGF-β1) tended to be lower with the restrain stress treatment (Fig. 2A-D). Taken together, the data showed an obvious negative effect of restraint stress on the mice.

3.2 Restraint stress promoted the growth of tumor xenografts via the Ras/Erk signaling pathway

To investigate the effects of restraint stress on tumor growth, tumor size was monitored since 21st day by calculating the approximate volume. Finally the intact tumors were removed and measured after the mice were sacrificed on the 42nd day. The results showed that the tumors of the stressed mice were larger than those of the non-stressed mice (Fig. 3A-C). And a similar trend was observed from the tumor/body weight ratio (Fig. 3D). Histologically, tumors from stressed mice showed a higher frequency of pathological nuclear fission, indicating that tumor cells may have been transformed into multinucleated giant cells (Fig. 3E). These data suggested that restraint stress promoted the growth of tumor xenografts in vivo.

To further investigate the effects on restraint stress at the molecular level, we analyzed the signaling pathways related to inflammation and tumor growth. The toll-like receptor 4 (TLR4)/NF-κB pathway showed no difference in activity between the two groups at both the mRNA and protein levels in tumors, spleen, and lymph nodes (Fig. 4A, B). However, k-Ras (Ras) and Erk were found to be upregulated in tumor tissues of the stressed group according to qRT-PCR analysis (Fig. 4C). Western blot analysis confirmed the increased Ras and Erk phosphorylation levels, which reflect the activation of the Erk signaling pathway (Fig. 4D). Accordingly, we analyzed the downstream gene expression and found that AKT1, Cyclin D1, and CDK4 were upregulated in the tumors of stressed mice (Fig. 4E). Western blot analysis confirmed the up-regulation of Cyclin D1 expression (Fig. 4F). Based on literature reports on the effects of IL2 on cells [16], these results indicated that restraint stress possibly promoted tumor growth through the IL2-Ras/Erk signaling pathway and upregulation of Cyclin D1.

3.3 IL2 induced proliferation and migration in A549 lung cancer cells

We used lung cancer cell line A549 to explore the effects of IL2 up-regulation on cancer cells. According to pre-experiments, 0, 1 or 5 ng/mL IL2 were added into the medium to stimulate the A549 cells. EdU staining indicated that 1 or 5 ng/mL IL2 promoted cell proliferation significantly at 24 hours (Fig. 5A, B). The CCK-8 assay showed that 1 or 5 ng/mL IL2 increased the numbers of viable cells (Fig. 5C). Gene expression analysis showed that 1 or 5 ng/mL IL2 upregulated the cell cycle marker genes cyclin D1 and CDK4, while downregulated CDKN2B (Fig. 5D, E and F). All these data are consistent with the results of IL2 elevation promoting the growth of tumor xenografts in vitro.

In order to evaluate the malignancy of cancer cells following IL2 treatment, migration and infiltration assays were conducted. The cell monolayer scratch test proved that 1 or 5 ng/mL IL2 increased the migration of A549 cells (Fig. 6A, B). However, the transwell assay showed no difference between IL2 treatment (1 or 5 ng/mL) and control cells (0 ng/mL), which suggested that IL2 may not affect the infiltration ability of A549 cells (Fig. 6C, D).

Three siRNAs were used to knock down the expression of IL2 receptor in A549 cells, and siRNA-2/3 exhibited the highest inhibition effects. At 48 hours after transfection with siRNA-2/3, the expression of IL-2 receptor decreased to about 40% of the original value (Fig. 7A). 0 or 5 ng/mL IL2 were added into the medium and the proliferation and migration were analyzed after 24 hours. As expected, The EdU (Fig. 7B, C) and CCK8 (Fig. 7D) assays showed no difference between IL2 treatment (5 ng/mL) and control cells (0 ng/mL), and the same trend was observed in the cell-monolayer scratch test (Fig. 7E, F).

3.4 Activation of the IL2-Ras-Erk pathway increased the malignancy of A549 lung cancer cells

We explored the activity of the Ras-Erk pathway after cells were treated with 0, 1 or 5 ng/mL IL2. At 1 or 5 ng/mL, IL2 increased the levels of p-ERK1/2 and Ras (Fig. 8A, B and C), which was consistent with the in vivo results. Next, we analyzed the effects of IL2 receptor knockdown using siRNA-3. Compared with the NC group, siRNA-3 attenuated the increase in the levels of Ras-Erk induced by IL2(Fig. 8D, E and F). Together with the proliferation and migration data, we concluded that activation of the Ras-Erk pathway is necessary for the proliferation and migration of lung cancer A549 cells induced by IL2.

It is generally accepted that stress is a negative factor with adverse effects on human health that can also affect the incidence and progression of cancer [17, 18]. However, epidemiological studies and clinical trials produced inconsistent results, or indicated insignificant effects of stress on cancer progression. Therefore, preventing stress responses is not included as a means to improve cancer survival in clinical practice. In the present study, we used physical restraint, a commonly used stress model, to evaluate the physiological response of stressed animals and clearly proved that restraint stress promoted lung cancer xenograft growth by enhancing inflammation. Firstly, we have shown that restraint stress decreased body weight gain and induced the expression of the pro-inflammatory cytokine IL2. Secondly, restraint stress promoted tumor growth and changed the histological structure of the xenografted tumors. Thirdly, we found that restraint stress promoted the activity of the Ras/Erk signaling pathway in tumor tissue. Finally, we confirmed that IL2 can promote the proliferation of cancer cell line A549 in vitro and that the Ras/Erk pathway is necessary for this process.

It has been known for a decade that stress increases the susceptibility to inflammatory disorders [19, 20, 21], and great efforts are directed towards identifying the precise pathways mediating this link. The inflammatory microenvironment is associated with the release of various pro-inflammatory and oncogenic mediators such as nitric oxide (NO), cytokines [IL-1β, IL-2, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)], growth factors, and chemokines. These mediators make the inflammatory microenvironment more vulnerable toward tumorigenesis [22]. Here, we observed that restraint stress promoted tumor progression and enhanced inflammation across different tissues of mice. The pro-inflammatory cytokine IL2 was found to be highly expressed in the serum and multiple tissues. As a growth factor, IL-2 was discovered in 1976[23] due to its ability to support the growth of T cells in vitro. Subsequently, IL-2 receptor (IL2R) was identified in both CD4⁺ and CD8⁺ T cells as well as other cell types [24, 25, 26]. IL-2 exerts crucial functions during immune homeostasis by triggering IL2R and intercellular signals. In the cancer context, high-dose IL-2 (600, 000–720, 000 IU per kg body weight) has been used for immunotherapy against metastatic cancer and positive results were reported in both mouse models and human patients [27]. However, the effects of low-dose IL2 on cancer development remain unclear. Our finding identified the connection between stress and inflammation via the upregulation of pro-inflammatory cytokine IL2, which may be the key factor through which stress promotes tumor growth.

IL-2 affects T cells mainly through the Janus kinase (JAK)-STAT, extracellular signal-regulated kinase (Erk), and phosphoinositide 3-kinase (PI3K) pathways [25].In general, any cell type can be influenced by IL2 as long as it expresses IL-2R receptor (IL-2R), including cancer cells. However, intracellular signaling pathways triggered by IL2 were mostly studied in T cells, with few studies on the effects of IL2 in other cell types. In this study, we analyzed the activity of the TLR4/NF-κB pathway which is closely related to inflammatory status, but no difference was observed between stressed and non-stressed mice. We then examined the downstream cascades Ras/Raf/Mek/Erk signaling pathways and found elevated activity of Ras and Erk [28, 29]. The RAS-RAF-MEK-ERK pathway, also known as the MAPK cascade, has been studied in detail and found to play an important role in cell proliferation, differentiation, and fate decision. Abnormal activity resulting from mutations in components of this pathway, especially in the upstream protein RAS, are closely related with tumorigenesis [30]. The Ras family of oncogenes was one of the first to be identified as mutated in human cancers [16, 31]. These oncoproteins were found to induce IL-6 and IL-8, which in turn promote tumor-associated inflammation [32, 33]. In this study, we detected elevated IL6 levels in the tumors of stressed mice compared to the unstressed controls. All these findings were confirmed by rescue experiments in cultured cells in vitro. At the same time, we found that IL2 can up-regulate AKT1 and the cell cycle marker gene cyclin D1. Akt plays pivotal roles in cell proliferation and metabolism, while Akt hyperactivation may leads to dysregulated cell cycle progression, which is a cancer hallmark [34, 35, 36, 37]. Here, we detected higher mRNA levels of AKT1, Cyclin D1 and CDK4, as well an elevated trend of protein abundance in stressed mice. However the link between stress and Akt activity remains elusive at the moment. Together with these earlier studies, our research indicates that restraint stress may affect the inflammatory response to promote tumor growth via the Ras/Erk signal pathway.

Although our study offers valuable insights, whether restraint stress induces immune suppression in animals still needs to be investigated. Stress may regulate immune system function in part via direct sympathetic innervation of peripheral lymphoid tissues, including the thymus, spleen, lymph nodes, and bone marrow, to regulate peripheral immune processes [38]. The spleen weight and immune-related splenocytes were found to be affected by psychological stress in earlier studies [39, 40]. Our study found corresponding changes in inflammatory responses when analyzing the spleen and lymph node indexes, but the activity of the TLR4/NF-κB and Ras/Erk signaling pathways in the spleen and lymph nodes was unchanged in the stressed mice. It should also be noted that our results were mainly based on xenografted tumors, indicating that stress affects processes that occur after tumor occurrence, but its role in tumorigenesis remains unknown. Therefore, whether stress has a promoting effect on tumorigenesis needs to be investigated in future studies.

In summary, our findings provide suggestive evidence that restraint stress can promote tumor progression by priming molecular regulation in the tumor microenvironment accompanied by an inflammatory response, which may be meditated by elevated IL2 levels. Restraint stress exacerbates inflammation, which makes it difficult for the organism to maintain homeostasis and optimal health. To bring the stressed body back into balance, we should normalize the immune system and fight against related diseases based on molecular, cellular, and physiological mechanisms. Thus, emerging stress-reduction measures need to be administered to help the body cope with the adverse effects of stress.

Funding

This work was supported by the Natural Science Foundation of Zhejiang Province (LZ23C170002 & LY22C180005) and the Jiaxing Public Welfare Research Plan Project (2021AY30014 & 2021AD30125).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The study protocol was approved by the Ethics Committee of Jiaxing University College of Medicine.

Competing interests

The authors have no conflicting interests.

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No competing interests reported.

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Restraint stress promotes lung cancer xenograft growth via the IL2-Ras-Erk pathway

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Cell culture

2.2 Animals and Sampling

2.3 Determination of the tumor ratio and organ index

2.4 ELISA

2.5 H&E staining

2.6 Cell treatment with IL-2/IL-2rb-siRNA

2.7 CCK-8 Assay

2.8 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay

2.9 Wound healing assay

2.10 Transwell assay

3 Results

3.1 Restraint stress reduced weight gain and intensified inflammation

3.2 Restraint stress promoted the growth of tumor xenografts via the Ras/Erk signaling pathway

3.3 IL2 induced proliferation and migration in A549 lung cancer cells

3.4 Activation of the IL2-Ras-Erk pathway increased the malignancy of A549 lung cancer cells

4 Discussion

5 Conclusion

Declarations

References

Additional Declarations

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