HDC downregulation induced by chronic stress promotes ovarian cancer progression via IL-6/STAT3/S100A9 pathway

doi:10.21203/rs.3.rs-4857053/v1

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HDC downregulation induced by chronic stress promotes ovarian cancer progression via IL-6/STAT3/S100A9 pathway

https://doi.org/10.21203/rs.3.rs-4857053/v1

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Depression is prevalent in ovarian cancer patients and contribute to the progression of the disease. However, the underlying mechanism remains unclear. In vivo, we established a comorbidity mouse model of ovarian cancer and depression. We found that chronic stress induced depression-like behaviors and promoted inoculated ovarian tumor growth in mice. Histidine decarboxylase (HDC) level was downregulated both in tumor tissue and in plasma of model mice. Exogenous histamine (HIS) treatment significantly alleviated chronic stress-induced depression-like behaviors and inhibited ovarian tumor growth, as well as decreased serum levels of inflammatory factors IL-6 and IL-17A, stress hormones norepinephrine (NE) and cortisol (COR), and 5-hydroxytryptamine (5-HT). Furthermore, HIS treatment regulated the immune response, particularly by increasing the percentage of CD3⁺ T cells, CD8⁺ cytotoxic T (Tc) cells, and decreasing the secretion of IL-17A. In vitro research of A2780 and ES-2 cell lines, NE and COR treatment down-regulated HDC expression and promoted cancer cells proliferation, migration, and invasion. HIS treatment reversed these effects. Preliminary mechanism research showed that chronic stress downregulated HDC expression and promoted ovarian cancer progression via IL-6/STAT3/S100A9 pathway. HIS may be a potential molecule for treating comorbidity of ovarian cancer and depression.

Biological sciences/Neuroscience/Diseases of the nervous system/Depression

Biological sciences/Cancer/Cancer therapy/Drug development

Ovarian cancer is the third most common gynecologic malignancy but accounts for the highest mortality rate among gynecological cancers worldwide[1]. According to the global cancer statistics published by the World Health Organization (WHO), there are 324398 new cases of ovarian cancer and 206839 deaths worldwide in 2022[2].

Chronic stress is a longterm psychological and physiological exposure process triggered by mainly negative environmental and/or psychosocial factors, which disrupts the internal homeostasis of the body[3]. Chronic stress contributes to the development and/or maintenance of mental health problems, and results in depression[4, 5]. The chronic unpredictable mild stress (CUMS) animal model, a widely used and reliable rodent model for depression, can effectively mimics human depression and is utilized extensively in research of depression[6–8].

Many clinical investigations show that cancer patients suffer from a high incidence of major depressive disorder. Ovarian cancer patients are reported an even higher depression incidence ranging from 17–21% [9, 10]. Comorbid depression in cancer patients may promote cancer incidence, progression, and metastasis, resulting in higher cancer-related mortality rates[3, 11, 12]. In fact, there is a interplay between chronic stress, depression, and cancer, which emphasizes the significant connection between psychological and physiological factors in cancer progression and management.

Histidine decarboxylase (HDC) is a key enzyme involved in endogenous HIS synthesis in the human body, which catalyzes the decarboxylation of L-histidine into histamine (HIS). Both HDC and HIS have pivotal roles in nervous system, such as regulating sleep-wakefulness, working memory and antidepressant-like effects[13–15]. In our previous studies, we found HDC mRNA significantly was decreased both in depression patients and in chronic stress model mice[16]. HDC and HIS are involved with the formation, progression, and metastasis of various tumors[17–19]. Depression and ovarian cancer have a mutually reinforcing relationship, however, there is lack of research to explore HDC expression profile in depression ovarian cancer comorbidity [20–22].

The IL-6/STAT3/S100A9 signaling pathway, drives the proliferation, survival, invitation, and metastasis of tumor cells, meanwhile strongly inhibits the antitumor immune response[23, 24]. This study aimed to investigate the impact of chronic stress on HDC expression in ovarian cancer progression, and the effect of HIS on the IL-6/STAT3/S100A9 pathway.

Animal

The study protocol was approved by the Institutional Experimental Animal Ethics Committee of Fengxian Hospital, Southern Medical University (No. 2022 − 0334). Female C57BL/6 mice (15 ~ 18 g, aged 5 weeks) were obtained from Guangdong DK Pharmaceutical Co Ltd (License No: SCXK 2022-0060). The mice were housed in cages collectively for control group, and individually for CUMS group at controlled temperature. A week-long acclimatization period was provided, during which the mice had ad libitum access to food and water.

Scheme of the animal experimental design was shown in Fig. 1A. At the endpoint, all mice were euthanized, and then blood serum, tumors, and spleens were collected. Tumors were photographed and weighed, with a portion of tumor fixed in formalin for histopathology slides, and the remaining tumor tissue utilized for Western blot and qRT-PCR experiments. The spleens were processed into single-cell suspensions for flow cytometry analysis.

CUMS Procedure

CUMS procedure was performed according to protocol[7]with slight modification. Mice in CUMS group were exposed to two different stressors daily. The detail of the specific interventions performed was shown in Supplementary Table S1

Behavioral Assessments

Behavioral assessments were conducted consistently in 6 days, including sucrose preference test (SPT), tail suspension test (TST), forced swimming test (FST), and open field test (OFT). Each behavioral evaluation was conducted with a 24-hour interval between tests. The detail of behavioral testing had been shown in our previous research[25].

Subcutaneous heterotopic transplantation ID8 model

ID8 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in the laboratory. Cells were cultured in DMEM high glucose medium (Gibco, California, United States) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, California, United States), penicillin (10 U/mL), and streptomycin (100 mg/mL). Mice were subcutaneously injected with 1×10⁶/ml ID8 cells in 100 µl of PBS to establish a subcutaneous heterotopic transplantation tumor model. Tumor volume was measured every week.

Immunohistochemistry (IHC)

Tumor tissues of mice were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin, and cut into 4µm sections and used for IHC staining with antibodies against Ki67 (1:1000, Abcam, United Kingdom) and HDC Polyclonal Antibody (Immunoway Biotechnology, United States). The representative images were acquired under a light microscope (Olympus IX73, Tokyo, Japan). Ki67 index was calculated as the number of immunopositive cells × 100% divided by the total number of cells in 7 random fields at ×400 magnification.

Blood sample analysis with Enzyme-linked immunosorbent assay (ELISA)

HIS (Elabscience, China), NE (Fine Test, China), COR (Fine Test, China), IL-6 (Fine Test, China), IL-17A (Elabscience, China), and 5-HT (Fine Test, China) in serum and in cell supernatant was analyzed using ELISA kits following the manufacturer's instructions.

Hematoxylin and eosin (H&E) staining

The tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The sliced sections were dewaxed using solvents, stained with hematoxylin and eosin, and examined under a microscope for photography.

Protein expression analysis by Western blottingThe expressions of HDC, STAT3, p-STAT3, S100A9 and GAPDH proteins were analyzed by Western blotting. The primary antibodies used include HDC Polyclonal Antibody (Immunoway Biotechnology, United States), Stat3 (124H6) Antibody (Cell Signaling, Massachusetts, United States), Phospho-Stat3 (Tyr705) (D3A7) XP® Antibody (Cell Signaling, Massachusetts, United States), S100A9 Antibody (Invitrogen California, Texas, United States) and GAPDH (ABclonal, China). The band density was analyzed using a gel imaging system and compared with an internal control.

mRNA Expression analysis by qRT-PCR

Total RNA was extracted with TRIzol (Invitrogen™, Carlsbad, United States). The extracted RNA was reverse transcripted to complementary DNA (cDNA) by using Evo M-MLV RT Mix Kit (ACCURATE BIOTECHNOLOGY CO., LTD, Changsha, China). For qRT-PCR, SYBR Green Premix Pro Taq HS qPCR Kit (ACCURATE BIOTECHNOLOGY CO., LTD, Changsha, China) was utilized following the manufacturer’s protocol. Relative gene expression was done using the 2^−ΔΔCT method. The list of primers was shown in Supplementary Table S2.

Flow cytometric analysis of T cell

Mouse spleen single cell suspensions were stained with specific antibody for 20 minutes, including PE anti-mouse CD4 (Biolegend, California, United States), FITC anti-mouse CD8a (Biolegend, California, United States), and PerCP/Cyanine5.5 anti-mouse CD3 (Biolegend, California, United States), then were analyzed using a flow cytometer (Canto; BD Biosciences, Shanghai, China) equipped with Cell-Quest software (BD Biosciences).

Cell lines, culture conditions, and cell transfection

A2780, ES-2 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in the laboratory. A2780 cells were cultured in DMEM high glucose medium (Gibco, California, United States) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, California, United States), penicillin (10 U/mL), and streptomycin (100 mg/mL). ES-2 cells were cultured in RPMI 1640 medium (Gibco, California, United States) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, California, United States), penicillin (10 U/mL), and streptomycin (100 mg/mL). All cell lines were cultured in a humidified atmosphere of 5% CO₂ at 37 ℃.

Drug treatment

For in vitro cell test, 4 groups were set up with different treatment: normal control (NC) group was cultured in de-hormone medium, NE + COR group with 10µM NE and 1.5µM COR (Sigma-Aldrich, Saint Louis, United States) in the medium, NE + COR + HIS group with 10µM NE, 1.5µM COR, and 10µM HIS (APExBIO, Houston, United States) in the medium, and BHOA group with 1mM BHOA (APExBIO, Houston, United States) in the medium.

Construction of HDC knockdown and overexpression cell lines

HDC knockdown or overexpression cell lines were generated with transiently transfected cells with HDC siRNA or HDC overexpression plasmid, respectively (Hanbio Tech, Shanghai, China). The vectors of OE-HDC were pcDNA3.1-CMV-MCS-3flag-EF1-ZsGreen-T2A-Puro. Specific HDC siRNA and HDC overexpression plasmid were transfected using Lipofectamine 3000 (Thermo Fisher, Massachusetts, United States) for 48 hours. The transfection efficiency was observed through a fluorescence microscope, and HDC expression was determined through Western blotting analysis. Cells transfected with non-targeting siRNA (siNC group) and empty plasmid vectors (Vector group) were used as controls. The transfected cells were then utilized to establish the siHDC group (HDC knockdown) and the OE-HDC group (HDC overexpression). Details of the siRNA and overexpression plasmid sequences used are listed in Supplementary Table S3.

Cell Counting Kit-8 (CCK-8) assay

Cells (3000 per well) were seeded into 96-well plates. 10 µL of Cell Counting Kit-8 solution (NCM Biotech, Suzhou, China) was added to each well after the cells were incubated at 37°C for 24–120 hours. The optical density (OD) was then measured at 450 nm. Cell Viability (%) = [(OD value of experimental group - OD value of blank control group) / (OD value of control group - OD value of blank control group)] × 100%.

Clone formation assay

Cells seeded in 6-well plates (1×10²/well) were serum-starved and cultured in hormone-depleted medium with various treatments for 10–14 days (medium changed every 3 days). Cells were fixed as the number of cells in a single clone exceeded 50, stained with crystal violet, dried, and photographed.

Scratch healing assay

Cells were seeded in 6-well plates (1×10⁶/well) and were serum-starved for 12h. Wounds were scratched using sterile tips, followed by PBS washes. Cell migration was observed and photographed at 0, 24, and 48h, and mobility was calculated.

Cell migration assay

Cells were resuspended in serum-free medium at 2.5×10⁵ cells/mL. Transwell chamber was placed in a 24-well plate, with 600 µL of 10% FBS and treatment solution (10 µM NE + 1.5 µM COR or 10 µM NE + 1.5 µM COR + 10 µM HIS) in the lower chamber and 200 µL of cell suspension in the upper chamber. After 12–24 hours of incubation at 37℃, cells on the underside of the membrane were fixed with 4% paraformaldehyde for 30 minutes, and stained with 0.1% crystal violet for 30 minutes. Cells residing at the bottom of the chamber were gently removed, and the chamber was washed. After drying, six random fields were photographed under a microscope (100×) per chamber, and the number of migrated cells was counted and recorded.

Cell invasion assay

Cells were coated with Matrigel (Corning, New York, United States) 4 hours in advance and the other steps were the same as the cell migration assay.

Statistical Analysis

All the data are presented as mean ± standard deviation (SD). The GraphPad Prism 9.5 software package was used for statistical analysis and visualization. Three independent experiments were carried out with similar results in qRT-PCR, Western blotting, ELISA, clone formation assay, scratch healing assay, cell migration assay, and cell invasion assay. Data were analyzed with an unpaired Student’s t test and one-way analysis of variance. P-value < 0.05 was considered statistically significant.

CUMS induced depression-like behaviors

To explore the effects of CUMS on depression-like behaviors in mice, The SPT, TST, FST and OFT were performed before and after CUMS to verify the depression-like behaviors induced by CUMS (Fig. S1). Specifically, the sucrose preference was significantly lower in the CUMS group in SPT compared with the Normal Control (NC) group (p < 0.01). Immobility time in the CUMS group were increased significantly in TST and FST compared with the NC group (p < 0.01). Mice exposed to CUMS tended to move less in OFT, with significant reductions in total distance, distance in central area, time in central area, and number of entries to the central area compared with the NC mice (p < 0.01, respectively). These behavior results confirm that the depression model was successfully built.

HIS treatment alleviated depression-like behaviors induced by CUMS

To investigate the impact of histamine treatment on depression-like behaviors in mice, we compared the behavioral outcomes of mice before and after histamine treatment. We found that HIS treatment effectively alleviated depression-like behaviors induced by CUMS in the ovarian cancer comorbid depression mouse model. Specifically, HIS increased sucrose preference in SPT (Fig. 1B), decreased immobility time in TST (Fig. 2C) and FST (Fig. 2D), and decreased total distance, distance in central area, time in central area, and number of entries to the central area in OFT (Fig. 2H, I) compared with the Tumor + CUMS group.

CUMS promoted ovarian cancer progression, while HIS inhibited CUMS-induced tumor growth in vivo

To investigate the effects of chronic stress and histamine treatment on tumor progression in vivo, we measured diverse tumor features. The image of tumors in different groups were shown in Fig. 2A. Compared with the Tumor group, tumor volume and weight of the Tumor + CUMS group were increased significantly (p < 0.01 respectively), while tumor volume and weight of the Tumor + HIS group were decreased with no significant difference. Compared with the Tumor + CUMS group, tumor volume (p < 0.05) and weight (p < 0.01) of the tumor + CUMS + HIS group decreased significantly (Fig. 2B, C). Additionally, H&E staining of tumor tissue showed that comparing with the Tumor group, the Tumor + CUMS group exhibited an increasing in the nucleus-to-cytoplasm ratio, distinct nucleoli, and pathological nuclear division. In contrast, compared with the Tumor + CUMS group, the Tumor + CUMS + HIS group showed a decrease in tumor cells and an increasing in stroma (Fig. 2D). Histopathological examination suggested that CUMS enhanced the Ki67 index, a marker of cell proliferation, while HIS treatment reduced Ki67 index (p < 0.01, respectively, Fig. 2E).

Exogenous HIS treatment reversed the CUMS-induced alterations in mice serum in vivo

In exploring the interplay between chronic stress and histamine in tumor progression, we observed that CUMS profoundly altered key biochemical markers in mice serum. Compared with the tumor group, CUMS significantly decreased HDC and HIS levels while increased IL-6, IL-17A, NE, COR, and 5-HT levels. Exogenous HIS treatment significantly increased HIS levels and decreased IL-6, IL-17A, COR, and 5-HT, with no significant effect on NE. Our findings indicated that CUMS changed the levels of HDC, HIS, inflammatory factors, stress hormones, and 5-HT, and HIS treatment reverses these changes, except HDC (Fig. 3A).

CUMS downregulated HDC protein expression and activated IL-6/STAT3/S100A9 pathway, HIS reversed the overactivation of IL-6/STAT3/S100A9 pathway induced by CUMS in vivo

Given that chronic stress promoted tumor growth while histamine treatment alleviated this effect, yet the regulatory mechanisms of HDC remain unclear, we analyzed mouse tumor gene expression using immunohistochemistry, Western blot, and qRT-PCR. Immunohistochemical staining experiments specifically indicated that CUMS significantly reduced the expression of HDC in mouse tumors (Fig. 3B).

Western blot analysis indicated that compared with the Tumor group, the expression of HDC protein in the Tumor + CUMS group was significantly decreased (p < 0.01), In contrast, the level of p-STAT3 and S100A9 proteins were significantly increased (p < 0.01), while there was no significant difference in STAT3 protein expression. When comparing the Tumor + CUMS group with the Tumor + CUMS + HIS group, the expression of p-STAT3 (p < 0.01) and S100A9 (p < 0.01) were significantly decreased in the Tumor + CUMS + HIS group, with no significant difference in HDC and STAT3 proteins (Fig. 3C, D). Similar results were observed for the changes in the mRNA expression levels (Fig. 3E). The above results indicate that CUMS-induced downregulation of HDC lead to increased secretion of IL-6, phosphorylation of STAT3, and expression of S100A9, thereby activating IL-6/STAT3/S100A9 pathway, whereas HIS effectively reversed the overactivation of this pathway induced by CUMS.

HIS enhanced cellular immunity in tumor-bearing mice in vivo

To explore the effects of CUMS and HIS treatment on mouse immunity, we utilized flow cytometry to measure the proportions of various immune cell (Fig. 4A). Specifically, the proportion of CD3⁺ T cells (Fig. 4B) was significantly reduced (p < 0.01) in the Tumor group compared with the NC group. HIS treatment significantly increased the levels of CD3⁺CD8⁺ Tc cells (Fig. 4C) and CD3⁺ T cells compared with the Tumor group (p < 0.05). Additionally, the proportion of CD3⁺ T cells in the Tumor + CUMS + HIS group was significantly higher than that in the Tumor + CUMS group (p < 0.05). The proportion of CD3⁺CD4⁺ Th (Fig. 4D) cells remained stable. Furthermore, compared with the NC group, the spleens weight of mice in the Tumor group were much heavier (p < 0.05), while HIS treatment reduced the spleen weight of mice in the Tumor + HIS group compared with the Tumor group (Fig. 4E).

Stress hormones promoted cells proliferation, migration, and invasion, while HIS treatment inhibited it in vitro

To further validate the impact of chronic stress on ovarian cancer progression, we employed in vitro approach by treating A2780 and ES-2 cell lines with stress hormones and HIS. CCK-8 analysis showed that 100 µM and 500 µM HIS suppressed A2780 and ES-2 cells proliferation at 12h, 24h and 48h (p < 0.01, respectively) while 10µM not. Thus, 10 µM HIS was chosen as a non-toxic concentration to study its effects on the IL-6/STAT3/S100A9 pathway, excluding direct tumor growth interference (Fig. 5A).

To further explore the function of HIS in modulating the effects of stress hormones, A2780 and ES-2 cells were stimulated with the stress hormone NE and COR, then subsequently treated with HIS. We found that HIS treatment inhibited cells proliferation, migration, and invasion induced by stress hormones. Specifically, NE and COR treatment increased colony numbers, and promoted wound healing, cell migration and invasion (p < 0.01, respectively). However, 10 µM HIS treatment decreased the number of colonies formed, promoted scratch healing, cell migration and invasion (p < 0.01, respectively, Fig. 5B-D).

HDC regulated ovarian cells' proliferation, migration, and invasion in vitro

Based on our previous results that chronic stress induced downregulation of HDC, we further explored the impact of HDC on tumor progression by constructing knockdown and overexpression of HDC in ovarian cell lines. Functionally, knockdown of HDC promoted cells proliferation, migration, and invasion in A2780 and ES-2 cells with siHDC compared with the NC groups (Fig. 6A-C), while overexpression of HDC inhibited cells proliferation, migration, and invasion (Fig. 6D-F).

Stress hormones induce HDC downregulation, promoting ovarian cancer cell proliferation via the IL-6/STAT3/S100A9 pathway in vitro

In our preliminary experiments, we found that cells exposed to NE and/or COR downregulated the level of HDC protein, increased the phosphorylation of STAT3 and the expression of S100A9 (Fig. S2). IL-6/STAT3/S100A9 pathway is a potential downstream target of HDC[16]. To elucidate the regulatory role of HDC in this signaling pathway, we conducted western blot assay and ELISA to quantify protein expression and cytokine secretion in A2780 and ES-2 cell lines following various drug treatments, HDC knockdown, and HDC overexpression.

Following various drug treatments, IL-6/STAT3/S100A9 pathway expression in A2780 and ES-2 cell lines were detected by western blot (Fig. 7A) and ELISA (Fig. 7B, C). NE + COR decreased HDC but increased IL-6, p-STAT3(Tyr 705), and S100A9 (p < 0.01, respectively), while HIS treatment induced opposite changes except HDC. HDC inhibitor BHOA decreased HDC but increased IL-6, p-STAT3(Tyr 705), and S100A9 (p < 0.01, respectively).

Knockdown of HDC in A2780 and ES-2 cells reduced HIS but increased IL-6, p-STAT3, and S100A9 (p < 0.01, respectively), activating the IL-6/STAT3/S100A9 pathway (Fig. 7D-F). Conversely, HDC overexpression increased HIS and reduced IL-6, p-STAT3, and S100A9 (p < 0.01, respectively), inhibiting IL-6/STAT3/S100A9 pathway (Fig. 7G-I).

Overall, these results indicated that HDC downregulation induced by stress hormones promoted the proliferation, migration, and invasion of ovarian cancer cells through activating IL-6/ STAT3/S100A9 signaling pathway.

Chronic stress-induced ovarian cancer progression is a complex multistep process that requires the regulation of diverse cellular pathways and functions, which resulted to few studies on chronic stress promoting ovarian cancer. In the present research, we revealed that HDC expression was downregulated by chronic stress in vivo and in vitro experiments. The downregulation of HDC led to the activation of the IL-6/STAT3/S100A9 pathway, stimulating Th17 cell to secrete of IL-17A and thereby promoting ovarian cancer progression, which suggests that HDC may be a potential therapeutic target for comorbidity of ovarian cancer and depression.

Although many studies over the past several years have demonstrated that chronic stress and multiple genetic alterations of tumor can promote progression and metastasis of many cancers, such as breast, lung, gastric, and colorectal cancer[3, 26–28], the contribution of this information to the improvement of therapy is still small. In this research, we noted that CUMS promoted tumor growth in a successfully established the comorbidity of ovarian cancer and depression in vivo mouse model, consistent with previous studies[29], and provide a novel therapeutic target.

The HDC, HIS and histaminergic system had been widely discussed in different neoplasia, including gastric, colorectal, esophageal, oral, pancreatic, liver, lung, skin, blood and breast cancer[30]. Some researches hold opposing views to ours, considering HDC as an oncogene to drive pro-angiogenic tumor microenvironment remodeling[31] or to enhance autocrine loop[32]. However, increasing research suggests that HDC may function as a tumor suppressor gene to increase myeloid maturation and accumulation of CD11b + Ly6G + immature myeloid cells[17], to inhibit growth of myc-dependent dedifferentiation tumors[18]. It has also been shown to inhibit myeloid maturation and CD8⁺ activation[19]. Yet, few studies have examined the role of HDC in ovarian cancer. Our findings have successfully established a connection between chronic stress and HDC, further strengthening the hypothesis that HDC downregulation contributes to cancer development.

However, few studies have examined role of HDC in ovarian cancer. Previous study had shown that chronic stress promotes tumor progression through the activation of the β-adrenergic receptor-mediated cAMP-PKA signaling pathway, which enhances tumor angiogenesis in ovarian cancer[29]. In this study, we provide a novel mechanism by which chronic stress enhances ovarian cancer progression: chronic stress-induced HDC downregulation and alterations in histidine metabolism serve as critical factors regulating cancer progression. We confirmed this via IHC, western blot, and qRT-PCR on tumor tissues. Additionally, HIS treatment counteracted CUMS-induced tumor growth, highlighting importance of HDC. This study broadens understanding of depression's role in tumor progression, especially in ovarian cancer.

Moreover, HIS treatment reversed the elevated serum levels of inflammatory factors (IL-6 and IL-17A), stress hormones (NE and COR), and 5-HT, all of which have been shown to be upregulated under chronic stress and are promoting tumor progression, consistent with previous research [33–36]. For example, chronic stress is known to trigger inflammatory responses, disrupts gut microbiota, activates IL-6/STAT3 signaling, and enhances immune responses, including DSS-induced colitis[33]. Moreover, chronic stress elevates IL-6 production in rheumatoid arthritis (RA), reducing glucocorticoids' anti-inflammatory effects in RA patients[37], and promotes breast cancer metastasis by accumulating myeloid-derived suppressor cells via activation of β-adrenergic signaling and IL-6/STAT3 pathway[38]. Notably, HIS treatment reversed the CUMS-induced upregulation of 5-HT, which was proven to inhibit tumor growth and enhance immune checkpoint blockade therapy[39]. While antidepressants like fluoxetine and ketamine have been found to alleviate depressive symptoms and inhibit tumor growth[39–41], few non-antidepressant medications have exhibited both anti-depression and anti-tumor therapeutic effects. This research firstly reported that HIS alleviated depression-like behaviors exacerbated by CUMS. This study underscores the effects of HIS in anti-inflammatory, alleviating depression-like behaviors, and potentially enhancing immune checkpoint blockade therapy. These findings provide both theoretical and experimental evidence for the use of HIS in the treatment of depression comorbid with ovarian cancer.

Functional investigations indicated that ovarian cancer progression via IL-6/STAT3/S100A9 pathway. IL-6 induces S100A9 expression in colonic epithelial cells through STAT3 activation has already been reported in recent studies about acute myeloid leukemia (AML), biliary tract cancer, and psoriasis-like skin and joint disease[24, 42, 43]. This research confirmed that Chronic stress downregulated HDC in ovarian tumor models, upregulating IL-6, p-STAT3 and S100A9. HIS reversed these changes, inhibiting stress hormones-induced wound healing, migration, and invasion. Furthermore, knockdown or overexpression of HDC activated or inhibited the expression of IL-6, p-STAT3 and S100A9. These findings strengthened that HDC, as an upstream cancer suppressor gene, catalyzing the formation of histamine, inhibiting ovarian cancer cell proliferation, migration, and invasion via IL-6/STAT3/S100A9 pathway.

Furthermore, studies reported that IL-6/STAT3 pathway may regulates the differentiation of naive CD4⁺T cells into Th17 cells[44, 45]. We attempted to explain the influence of CUMS and HIS treatment on tumor progression from the perspective of cellular immune, and found that HIS treatment increased the proportion of T and Tc cells in ovarian cancer mouse model, which suggested that HIS inhibited tumor progression. Tumor inoculation decreased T cell proportion in mice spleen, which further deteriorated by CUMS, and patients with depression have reduced circulating T cell. Depression promote the proliferation and activation of Th17 cells, thereby exacerbating the inflammatory response and produce the inflammatory cytokine IL-17A[46]. We detected that IL-17A increase induced by tumor and CUMS, which was reversed by HIS treatment. These findings confirm that IL-17A, secreted by Th17 cells, can promote tumor growth through the IL-6/STAT3 signaling pathway, as previously described[47]. Specifically, chronic stress downregulated HDC, reducing HIS production and triggering excessive IL-6 secretion in cancer cells. This activated STAT3 and S100A9, suppressing T cell formation, promoting Tc differentiation, and enhancing IL-17A secretion. Further studies are needed to elucidate the CUMS-cellular immunity-tumor interplay.

In conclusion, this study provides important insights into the effects of chronic stress on animal behavior, physiology, cellular functions and immune system, as well as the underlying molecular mechanisms. Specifically, chronic stress elevates stress hormones levels, stimulates relative receptors, and subsequently decreases HDC expression, promoting ovarian cancer progression via the IL-6/STAT3/S100A9 pathway. A mechanism diagram is presented in Fig. 7J. Histamine therapy may serve as a potential strategy to counteract the downregulation of HDC induced by chronic stress, providing crucial insights and establishing a theoretical foundation for further research on the treatment of ovarian cancer comorbid with depression.

Ethics statement

This research received approval from the Experimental Animal Ethics Committee of Fengxian Hospital, Southern Medical University and was conducted under the oversight of the hospital's Review Committee (No. 2022 − 0334).

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Funding Information

This work received financial support from the and by Shanghai Fengxian Science and Technology Commission [grant number 20201607].

Author Contributions

Conceptualization: Feng Xu (lead), Ting Zhou (supporting). Data acquisition: Zhicong Chen, Jinming Cao, Zhijun Xiao, Zhen Yang, Yuanchi Cheng, Jingjing Duan. Formal analysis: Zhicong Chen, Ting Zhou. Writing − original draft: Zhicong Chen, Ting Zhou. Writing − review & editing: Feng Xu. All authors read and approved the final manuscript.

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Fig.S1MicebehavioraltestinginNCgroupn21andCUMSgroupn14beforeafterCUMSprocedure.tif
Fig. S1 Mice behavioral testing in NC group (n =21) and CUMS group (n =14) before/after CUMS procedure. A Sucrose preference in SPT. B Time of immobility in TST. CTime of immobility in FST. D Activity tracks in OFT. E Locomotion activity scores in OFT. Data are shown as the mean ± SD. *p<0.05, **p<0.01.
Fig.S2StresshormonesdownregulatedHDCproteinexpressionincreasedthephosphorylationofSTAT3andtheexpressionofS100A9.tif
Fig. S2 Stress hormones downregulated HDC protein expression, increased the phosphorylation of STAT3 and the expression of S100A9. A The HDC, STAT3, p-STAT3 and S100A9 protein expression in A2780 and ES-2 with various treatments of stress hormones NE or COR detected by Western blot, adjusted by GAPDH. B The HDC, STAT3, p-STAT3 and S100A9 protein expression in A2780 and ES-2 with various treatments of stress hormones NE and COR detected by Western blot, adjusted by GAPDH.
TableS1ChronicunpredictablemildstressCUMSprocesses.xlsx
Table S1 Chronic unpredictable mild stress (CUMS) processes
TableS2Theprimerssequences.xlsx
Table S2 The primers sequences
TableS3siRNAandoverexpressionplasmidsequences.xlsx
Table S3 siRNA and overexpression plasmid sequences

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HDC downregulation induced by chronic stress promotes ovarian cancer progression via IL-6/STAT3/S100A9 pathway

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Animal

CUMS Procedure

Behavioral Assessments

Subcutaneous heterotopic transplantation ID8 model

Immunohistochemistry (IHC)

Blood sample analysis with Enzyme-linked immunosorbent assay (ELISA)

Hematoxylin and eosin (H&E) staining

mRNA Expression analysis by qRT-PCR

Flow cytometric analysis of T cell

Cell lines, culture conditions, and cell transfection

Drug treatment

Construction of HDC knockdown and overexpression cell lines

Cell Counting Kit-8 (CCK-8) assay

Clone formation assay

Scratch healing assay

Cell migration assay

Cell invasion assay

Statistical Analysis

RESULTS

CUMS induced depression-like behaviors

HIS treatment alleviated depression-like behaviors induced by CUMS

DISCUSSION

Declarations

Ethics statement

Conflicts of Interest

Funding Information

Author Contributions

References

Additional Declarations

Supplementary Files

Status:

Version 1