Serum levels of IL-17A and cathelicidin antimicrobial peptide LL37 in females with breast cancer

Ahmed Abdullah Ahmed Al-Harbi¹, Asmaa Zaki Shetawi¹, Alya Abd Alaziz Najim Al Zobair2,3

Background

Breast cancer (BC) is a leading cause of morbidity and mortality in women. Interleukin (IL)-17 and the antimicrobial peptide LL37 have been implicated in BC pathogenesis. This study aimed to assess their levels in women with BC and evaluate their correlation with molecular subclasses, clinical, and laboratory parameters.

 

Methods

This study included 69 BC females and 35 healthy controls. Patients were classified based on metastatic status and molecular indicators. Serum IL-17A and LL37 concentrations were estimated using ELISA and compared across patient subgroups. The area under the curve (AUC) was analyzed to determine cut-off values, sensitivity, and specificity for their diagnostic potential in BC.

 

Results

IL-17A and LL37 levels were notably higher in patients with BC than in controls (p<0.0001). LL37 levels were also higher in the metastatic than in the non-metastatic group and controls. IL-17A levels were considerably higher in patients with metastatic than in controls (p<0.0001), but did not differ significantly between metastatic and non-metastatic patients (p = 0.5573). Regarding diagnostic performance, LL37 showed an AUC of 0.989 (p<0.001) at the best cut-off value of 16.79 ng/ml, LL37 exhibited 95% sensitivity and 94% specificity, indicating diagnostic potential. IL-17A showed an AUC of 0.87 (p<0.001), with a best cut-off value of 15.11 pg/ml, 78.81% sensitivity, and 100% specificity. Both LL37 and IL-17A levels were correlated with each other (r = 0.284, p = 0.018).

 

Conclusions

Patients with BC had increased serum levels of IL-17A and LL37. Their estimation is important for the follow-up of patients with metastatic.

 

Keywords

breast cancer, IL-17A, LL37 protein, metastasis

Breast cancer (BC) is one of the most common cancers and major causes of cancer-related mortality in women worldwide.1,2 The Iraqi Cancer Registry recently reported an increasing number of BC cases in Iraq in recent years.3,4 BC is caused by genetic and non-genetic factors, primarily hormonal imbalances, lifestyle, diet, and inflammation.5 Cytokines, key mediators of inflammation,6 have been widely studied due to their role in inflammation, including the contribution of inflammatory and anti-inflammatory cytokines to BC development.7

Interleukin (IL)-17A, a cytokine released by T helper 17 (TH17) cells, plays a crucial role in inflammation, immunological responses, and tumorigenesis.8 It promotes the development of autoimmune disorders, such as autoimmune encephalomyelitis, inflammatory bowel disease, rheumatoid arthritis, psoriatic arthritis, and inflammatory bowel diseases. IL-17A-blocking antibodies have shown promise in treating psoriatic arthritis and ankylosing spondylitis.9 In BC, IL-17A influences tumor growth through direct and indirect mechanisms; it directly affects angiogenesis and gene expression and indirectly promotes the growth of both primary and metastatic BC cells.9

LL37, a human cathelicidin, is a multifunctional antimicrobial peptide generated through the cleavage of the C-terminus of human cathelicidin antimicrobial peptide (hCAP-18) by a proteinase enzyme.10 In addition to its direct toxic effects on microorganisms, it also plays other roles in chemotaxis, tissue repair, wound healing, inflammation, and tumorigenesis. It also activates cytokine release by transactivating epidermal growth factors in immune cells.11 LL37 overexpression has been observed in BC tissues as well as in bronchogenic, melanoma, squamous cell, and prostatic carcinomas.12 It exhibits anti-malignant activity in some cancers owing to its cationic charge, which disrupts the anionic-charged lipid-phosphatidylserine on malignant cell membranes. For example, LL37 induces apoptosis in colonic and Jurkat T leukemia malignancies and promotes malignant cell degeneration in oral squamous cell carcinoma via DNA methylation. However, LL37 enhanced BC cell migration and invasiveness by stimulating insulin-like growth factor-1 receptors.13 This study aimed to investigate IL-17A and LL37 levels in patients with BC, their relationship with clinical and molecular markers, and their potential sensitivity, specificity, and utility in predicting disease occurrence.

 

 

Participants

A total of 104 women participated in the study between August 2023 and August 2024, including 69 patients with BC attending the oncology clinic, who were randomly selected using random patient ticket selection. Patients aged 18 years or older with histopathologically verified BC were included. Thirty-five age-matched healthy caregivers at the University of Mosul, Teaching Research Centers with no family history of BC were included as controls. Participants with a history of autoimmune diseases, chronic inflammatory disorders, recent or ongoing antibiotic medication or infection, pregnancy or lactation, a history of other cancers, or those taking vitamin D supplements within the last three months were excluded. Oncologists assessed the patient data based on metastasis and molecular classification. Patients were classified as metastatic or non-metastatic and further categorized according to the results of immunohistochemical staining techniques for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and antigen Kiel (KI-67). Patients with metastasis were categorized into progressive and controlled non-progressive disease groups.

 

Molecular staining and classification

Tissue samples were fixed with formaldehyde, embedded in paraffin, and stained for immunohistochemical markers according to the Dako Cytomation protocol.14 After alcohol and xylene were used to deparaffinize the tissue sections, the samples were rehydrated at 95°C for 40 min using an antigen retrieval solution. After treatment with 3% peroxide for 5 min, followed by a 30-min incubation with anti-ER or anti-Ki-67 primary antibodies, or anti-PR or anti-v-erb-b2 avian erythroblastic leukemia viral oncogene homolog-2. The horseradish peroxidase solution was added, and 3,3’-diaminobenzidine (DAB) staining was performed for 10 min before background staining with hematoxylin. All immunohistochemical assays included both positive and negative controls. Positive controls comprised external normal breast tissues and internal tumor-adjacent normal tissues to assess the staining intensity.

 

Slide examination

Two independent histopathologists (W and N) assessed each tissue slide. For evaluation of ER and PR expression, 100 tumor cells were analyzed with percentages representing total cell ratio, and staining was deemed positive if ≥1% of invasive cells were stained, as per The American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) guidelines15. Staining intensity was categorized as strong, moderate, or weak.16 HER2 staining was graded as 0, +1, +2, or +3 according to the ASCO/CAP instructions: 0 or +1 represented HER2-negative, +3 indicated HER2-positive, and +2 was considered equivocal (requiring fluorescent in situ hybridization [FISH] testing for confirmation).17 For Ki-67 measurements, approximately 500 malignant cell nuclei were examined, and the data are presented as the percentage of positive cells.16,18

Based on these marker levels, patients were classified into molecular sub-classes: luminal A (ER+, PR+, HER2−, and Ki-67 ≤20), luminal B (ER+, PR+, HER2−, and Ki-67 >20), luminal B/HER2+ (ER+, PR+, and HER2+); and HER2-enriched (ER−, PR−, and HER2+), and triple-negative breast cancer (TNBC) (ER−, PR−, and HER2-).19

 

Blood sampling

After obtaining written consent, five milliliters of venous blood was collected aseptically from all participants using anticoagulant-free collection tubes. Then, samples were centrifuged to separate the sera, which were stored at −20°C for subsequent laboratory tests.

 

Estimation of serum LL37 and IL-17A levels

Serum concentrations of IL-17A and LL37 were measured using enzyme-linked immunosorbent assay kits (Catalog No: RE2867H for IL-17A, RE2459H for LL37; Reed Biotech, China) according to the manufacturer’s protocol. Concentrations were calculated after plotting standard curves with IL-17A in pg/ml and LL37 in ng/ml.

 

Statistical analysis

Data visualization and analysis were performed using Microsoft Excel 2019 (Microsoft, USA), GraphPad Prism version 9.5 (GraphPad Software Inc., USA), and MedCalc software (MedCalc Software Ltd, Belgium). Data were presented as the minimum, maximum, mean, median, standard deviation (SD), standard error of the mean (SEM), and 95% confidence interval (CI). Appropriate statistical tests were applied, including the Mann-Whitney test for non-parametric comparisons, one-way analysis of variance, the Kruskal-Wallis test, Dunnett’s multiple comparison test, and Spearman’s correlation, as appropriate. The area under the receiver operating characteristic (AUC-ROC) curve was used to assess the diagnostic potential of LL37 and IL-17A levels, with p≤0.05 set as the statistical significance level.

 

Ethical approval

This study was approved by the Medical Research Ethics Committee of the College of Medicine, University of Mosul, Brazil (Ref: UOM/COM/MREC/22-23/JL1 and UOM/COM/MERC/22-23/JL2). This study followed the Declaration of Helsinki for Medical Research. Prior to their involvement, all participants provided written informed consent.

 

 

Table 1 shows the clinical and molecular laboratory characteristics of the participants: 69 females with BC (37 and 32 with and without metastasis, respectively) and 35 healthy controls. The ages of the patients and controls were not significantly different (p = 0.1076). According to the results of the molecular investigations (ER, PR, HER2, and Ki-67 [50%]), the patients were classified into molecular classes. Luminal B/HER2 constituted the highest percentage of patients with metastasis, followed by the Luminal B, TNBC, and Luminal A subtypes. In contrast, most patients with no metastasis lay in the Luminal A class, followed by TNBC and Luminal B-enriched molecular classes. No patients belonged to the HER2-enriched class.

 

 

Table 2 compares the serum levels of LL37 and IL-17A among the patient groups. A significant increase in the serum concentrations of LL37 and IL-17A in patients with BC compared with those in the control group was observed (p<0.0001). A comparison of the serum concentrations of LL37 or IL-17A between patients with metastasis, patients without metastasis, and healthy controls was conducted. LL37 expression was significantly higher among those who had metastasis than those who did not show any type of metastasis (p<0.01). Both patient groups had significantly higher concentrations of LL37 than the healthy controls (p<0.0001). This comparison showed that IL-17A levels among patients with metastasis were significantly higher than those of the controls (p<0.0001) but not those of patients without metastasis (p = 0.5573).

 

 

Patients with BC were classified into molecular subclasses based on four molecular indicators. The serum levels of LL37 and IL-17A varied among patients in these classes. Despite these variations, the statistical analysis revealed no significant differences in the levels of IL-17A or LL37 among these classes (Figure 1a and 1b). Higher levels of LL37 were found in patients with progressive metastatic BC, although there were no notable differences between these levels and those of patients with controlled metastatic cancer. In contrast, IL-17A expression was lower in patients with progressive disease; however, this difference was not statistically significant (Figure 2a and b).

 

 

 

 

Receiver operating characteristic (ROC) curve analysis revealed that LL37 levels may effectively distinguish women with BC and healthy controls, with an AUC of 0.96 (p<0.001). At the optimal cutoff of 21.5 pg/ml, LL37 demonstrated a sensitivity of 87% and specificity of 97%, yielding a Youden index (J) of 0.841 (Figure 3a). Similarly, the ROC curve for IL-17A showed an AUC of 0.86 (p<0.001), indicating a good diagnostic performance. Using a cutoff value of 15 pg/mL, IL-17A achieved 77% sensitivity and 97% specificity, with a Youden index of 0.739 (95% CI: 0.78–0.90) (Figure 3b). Spearman’s correlation analysis demonstrated a significantly positive relationship between LL37 and IL-17A levels (r = 0.28, p = 0.018) (Figure 4).

 

 

 

 

 

 

This study focused on two key immunological factors: LL37, an antimicrobial peptide, and IL-17A, a Th17 cytokine. Previous studies focused on gene expression and immunohistochemical staining of LL37 and IL-17 family members across various malignancies.13,20 In this study, the serum levels of both LL37 and IL-17A were considerably greater in patients with BC than those in the controls. The results of previous studies support our findings that LL37 expression is significantly upregulated in BC cells.20 This increase in expression may lead to higher serum concentrations of LL37 in patients with BC. Similarly, IL-17A is upregulated in BC, and our results revealed significantly higher levels of IL-17A in patients with BC compared with those in the controls. A study in Egypt by EL-Batal et al21 demonstrated a mean serum IL-17A concentration of 79.2818 pg/ml in patients with BC, which was a significant elevation compared with that in the control groups. Avalos-Navarro et al22 studied Th17 cytokines in BC and found that IL-17A levels averaged 14.8 pg/ml in patients with BC and only 2.5 pg/ml in healthy women. Although the mean IL-17A concentrations in that study were lower than ours, this confirms our results regarding the significant differences in IL-17A levels between patients and healthy women. In 2023, Popovic et al23 studied patients with early invasive BC and found elevated serum IL-17A levels in early malignancy compared to healthy controls.23 These results support our findings of elevated levels in IL-17A patients with BC.

Our data showed that patients with metastatic BC had significantly higher serum LL37 levels than those with non-metastatic BC, who had elevated levels compared to healthy controls. LL37 is highly expressed in BC cells and promotes tumor cell growth, creating a vicious cycle between increases in LL37 expression in BC cells and tumor growth, which increases the risk of metastasis.23 Pan et al13 observed LL37 overexpression in patients with BC and distal lymph node metastasis. Additionally, an in vitro study by Weber et al11 demonstrated that LL37 exposure activated cell migration, leading to a scattered morphology and indicating an increased metastatic tendency. Another in vitro study confirmed that LL37 exposure enhanced BC cell migration and metastasis.24 The growth of BC increases LL37 expression, and LL37 exposure further promotes the chance of metastasis, leading to a continuous cycle in which tumor growth and increased metastatic risk continually enhance each other. These studies support our findings that patients with metastatic BC exhibit higher LL37 levels and increased serum LL37 levels. Although IL-17A levels were elevated in metastatic cases, the elevation was not statistically significant in non-metastatic cases compared with the controls. Similarly, Ayari et al25 investigated the importance of cytokines as prognostic factors in BC and reported higher IL-17 levels in the metastatic group; however, there was no significant difference (p = 0.151).25 Notably, their study did not analyze all IL-17 analogs (A–F).

The patients were classified into molecular classes based on HER2, ER, PR, and Ki-67 expression. Patients with TNBC had the highest serum concentrations of LL37 and IL-17A, although there were no significant differences in these levels between the molecular classes of patients in this study. Subsequently, the expression of these two factors was studied in vitro. A Russian study on BC microenvironments analyzed biopsies and white blood cells from 117 patients with BC. They found that IL-17 release was higher in triple-negative molecular classes, whether from mitogenactivated cultured invasive carcinoma cells or patient-derived white blood cells, than from carcinoma or white blood cells from other molecular classes.26 Cultured TNBC cells express higher levels of HCAMP, which regulates LL37 production, leading to increased LL37 secretion into the culture medium. Furthermore, the inhibition of LL37 release decreases the invasiveness of TNBC cells.27 The comparison between IL-17A and LL37 levels in patients with progressive and controlled metastatic disease revealed no significant differences, likely because of the limited number of cases, particularly in the progressive disease group.

A comparison of the serum levels of LL37 and IL-17A revealed that LL37 had a higher AUC than IL-17A. Although LL37 showed a higher sensitivity than IL-17A, it had similar specificity. Regarding IL-17, Liu et al28 obtained a lower AUC (0.656) with a higher sensitivity (91.7%) and lower specificity (40%), likely because they detected every IL-17A family member, whereas our study focused solely on IL-17A. Our results suggested that LL37 is superior to IL-17A as an indicator of BC development.

Our study showed a positive correlation between LL37 and IL-17A levels among patients with BC. In contrast, studies on different diseases, such as guttate psoriasis, found a positive but insignificant correlation,29 whereas a negative correlation was observed for airway inflammatory diseases.30 This may be due to differences in the pathogenesis and immune response patterns of these diseases.

A key limitation of this study is the lack of patient information on early BC detection or prior to receiving different treatment regimens. In conclusion, patients with BC had higher concentrations of IL-17A and LL37 than controls, with increasing concentrations in metastatic cases. Measuring these factors may help decision-making in patient follow-up. Future prospective cohort studies may aid the evaluation of the utility of these indicators in the diagnosis and follow-up of patients with BC. Further studies are required to assess the importance and drawbacks of targeting and blocking these factors in BC treatment.

 

 

 

 

 

 

 

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