The Relationship Between Cathepsins and Breast Cancer

Cancer remains a critical global health issue, ranking as the second leading cause of death worldwide. According to the 2022 Cancer Statistics, breast cancer (BC) represents nearly one-third of all cancer diagnoses among women, with particularly high mortality rates in certain populations due to disparities in treatment access and underlying health conditions [1]. While multiple factors, including genetic predispositions and environmental exposures, contribute to BC's complex etiology, the proteolytic enzyme family known as cathepsins has recently gained attention for their crucial roles in tumor biology. Cathepsins play essential roles in cellular metabolism, proliferation, and tissue homeostasis. Among this family, enzymes such as cathepsin B, S, and L have been implicated in the progression and metastasis of various malignancies. However, their specific involvement in BC, particularly regarding subtype distinctions such as HER-2 or estrogen receptor (ER) status, remains unclear. Clarifying these associations could significantly enhance our understanding of BC pathophysiology and inform therapeutic strategies. Therefore, we conducted a Mendelian randomization (MR) study to investigate causal relationships between genetically predicted levels of nine cathepsins (H, E, F, B, L2, O, G, S, and Z) and BC risk across multiple subtypes. We used publicly available genome-wide association study (GWAS) datasets to derive robust genetic instruments. Genetic variants strongly associated with circulating cathepsins levels were extracted from the INTERVAL study (n = 3301), while BC outcome data were obtained from the FinnGen database (15,680 cases; 167,189 controls) and the Breast Cancer Association Consortium (BCAC) GWAS database, enabling subtype-specific analyses (HER-2 positive, HER-2 negative, ER-positive [ER+], and ER-negative [ER−] cases). We selected instrumental variables using rigorous criteria, including a genome-wide significance threshold (p < 5 × 10−6) and an F-statistic > 10 to reduce weak instrument bias. We then performed fixed-effects inverse-variance weighted (IVW) analyses, complemented by MR-Egger, weighted median, and mode-based approaches to confirm robustness. Horizontal pleiotropy was carefully evaluated using MR-Egger intercept and MR-PRESSO tests, ensuring reliability. Reverse MR analysis was conducted to evaluate potential reverse causality by treating BC as the exposure and cathepsins as outcomes. Additionally, multivariable MR accounted for potential interactions among cathepsins, refining causal estimates specific to BC subtypes. Analyses were performed using the “MendelianRandomization” package. The detailed methods were described in Supporting Information S1. Survival analyses based on cathepsin E (CTSE) expression levels were conducted using data from The Cancer Genome Atlas (TCGA) to further validate findings. Additionally, four human BC cell lines (SKBR-3, MCF-7, ZR-75-1, and MDA-MB-231 cells) and breast epithelial cell line (MCF-10A) were cultured at 37°C in 5% CO2 for experimental validation. All cell lines were undergone authentication by short tandem repeat (STR) analysis. CTSE expression was measured using RT-PCR, western blot analysis, and immunofluorescence to confirm its role in BC. Each cell line was divided into three experimental groups for consistency. RNA quality was assessed by a NanoDrop spectrophotometer (ND-2000, Thermo Fisher Scientific). cDNA was synthesized using AMV reverse transcriptase (Takara, Japan), and real-time PCR was performed to amplify the cDNA with SYBR Premix Ex Taq (Takara, Japan). The antibodies used in the analysis included Mouse polyclonal to Cathepsin E (Origene, #TA309616) and mouse polyclonal GAPDH (BioTechne, AF5718). To investigate CTSE expression in clinical samples, human BC tissues were compared with fibroadenoma tissues. Slides were stained using an anti-CTSE antibody and DAPI nuclear dye, and images were captured using Olympus cellSens imaging software (Olympus Corporation). Figure 1A summarizes the study design. The outcomes of the univariable MR analysis (Supporting Information S1: Table S1) showed that heightened levels of CTSE were linked to an increased risk of overall BC (p = 0.0211, odds ratio [OR] = 1.064) by the IVW and MR-Egger method. In addition, there was a weaker but still significant association between elevated cathepsin F levels and the overall BC (IVW: p = 0.0384, OR = 1.0664), while cathepsin H levels were associated with a lower risk of ER- BC (IVW: p = 0.0198, OR = 0.9663). Neither MR-Egger intercept nor MR-PRESSO global tests indicated directional pleiotropy, supporting the causal nature of these associations (Supporting Information S1: Table S2). Reverse MR analysis suggested potential reverse causality, where BC increased CTSE levels (p = 0.0362, OR = 1.0561) while decreasing cathepsin H (p = 0.0187, OR = 0.9095) and Z levels (p = 0.0489, OR = 0.9177). The p-values of the sensitivity analysis showed no directional pleiotropy (Supporting Information S1: Table S2). The above results showed that a causal association between CTSE and overall BC (Figure 1B). Multivariable MR confirmed that elevated CTSE levels increased HER-2 positive BC risk, while higher cathepsin F levels were linked to HER-2 negative BC. Elevated cathepsin B levels correlated with ER−BC, while cathepsin Z levels appeared protective against HER-2 negative BC. The detailed information was summarized in Supporting Information S1: Figure S1. The Cancer Genome Atlas (TCGA) data analysis identified 145 patients with high and 845 patients with low CTSE expression. Disease-free survival (DFS) was significantly longer in patients with lower CTSE expression (log-rank test, p = 2.781 × 10⁻³), although overall survival showed no significant differences (Figure 1D). There is no significant difference in overall survival between the two groups (Figure 1D). To validate MR findings experimentally, we analyzed CTSE expression in vitro across four human BC cell lines (SKBR-3, MCF-7, ZR-75-1, and MDA-MB-231) and one normal breast epithelial cell line (MCF-10A). Both mRNA and protein assays confirmed significantly elevated CTSE expression in cancerous cells compared to noncancerous controls, with HER-2 positive SKBR-3 cells exhibiting the highest levels (Figure 1C). Immunofluorescence analysis further demonstrated elevated CTSE expression in BC tissue relative to fibroadenoma tissue (Figure 1D). These experimental findings support the biological relevance of the MR-derived evidence, strengthening the proposed role of CTSE in BC pathogenesis. Our study identifies CTSE as a promising biomarker and potential therapeutic target, aligning with STROBE-MR checklist requirements. Previous literature highlights CTSE's role in tissue remodeling, protein hydrolysis, and pancreatic cancer detection, though its function in breast carcinogenesis remains debated [2]. Kawakubo et al. noted CTSE's involvement in mammary gland development, indicating broader biological implications beyond cancer. Future studies should clarify how elevated CTSE expression contributes specifically to BC progression, aggressiveness, and subtype-specific risks [3]. Our MR analyses also highlighted other proteases deserving attention. Elevated cathepsin F was associated with overall BC incidence; its roles in tumor differentiation and metastasis remain understudied and thus represent critical directions for future inquiry [4]. Similarly, although cathepsin S has previously been implicated in triple-negative BC invasion, we did not observe positive associations in our analyses, further highlighting the protease family's complexity and the importance of exploring subtype-specific roles [5]. In conclusion, our analyses and subsequent experimental validations suggest a potential causal association between elevated CTSE levels and increased BC risk. Although our findings indicated a stronger association in HER-2 positive BC, further research with larger sample sizes and functional studies is necessary to conclusively establish CTSE's subtype-specific role, especially in HER-2 positive BC. Nevertheless, our results underscore CTSE as a promising biomarker candidate and justify its further exploration as a therapeutic target. Xin Wang: conceptualization, data curation, formal analysis, visualization, writing – original draft, writing – review and editing. Zihan Xu: data curation, formal analysis, resources, funding acquisition, software, supervision, writing – review and editing. Yiyao Zeng: formal analysis, methodology, project administration, writing – review and editing. Jie Chen: conceptualization, funding acquisition, methodology, project administration, resources, validation, visualization, writing – review and editing. All authors have read and approved the final manuscript. We express our gratitude to the Core Facilities of West China Hospital. This study was supported by the funding from the West China Hospital, Sichuan University (2022HXFH021), National Natural Science Foundation of China (No. 32371539), China Postdoctoral Science Foundation (2023M742486); the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation (GZC20231826). All participants provided written informed consent, and the study was approved by the Ethics committee of the West China Hospital, Sichuan University (20231675). The GWAS summary data were derived from published studies; therefore, no additional ethical approval was required. The authors declare no conflicts of interest. The raw data analyzed during the current study were available in public databases https://gwas.mrcieu.ac.uk and https://www.ebi.ac.uk/gwas. The gene expression and clinical data used in this study were obtained from The Cancer Genome Atlas (TCGA). We did not include the original sequencing files or individual level genotype data. All data accessed before March 29, 2025. No datasets analyzed here were subject to NlH-controlled access policies or embargoes prior to this cutoff date. Further information is available from the corresponding author upon request. All packages for data analysis used in this study were open source in R software. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.