Integrative Systems Biology Analysis of MicroRNA Regulation in Wnt Signaling Pathway During Breast Cancer Progression

Breast cancer, a disease of profound significance, stands as a global health challenge affecting millions of people in the world (1). Its prevalence and impact on individuals and communities underscore the pivotal role of understanding, addressing, and combating this formidable adversary. Using molecular and histological data, breast cancer can be classified into 3 main subtypes: Hormone receptor-positive breast cancer [expressing estrogen receptor (ER+) or progesterone receptor (PR+)], human epidermal growth factor receptor 2-positive breast cancer (HER2+), and triple-negative breast cancer (lacking expression of ER, PR, and HER2). Changes in different cellular pathways and biological processes (BPs) of the cell can be the main cause of breast cancer (2, 3).

The Wnt pathway, a critical cell growth and development regulator, is significantly implicated in breast cancer. Disruption of this pathway contributes significantly to the pivotal role in the initiation and progression of breast cancer, fostering uncontrolled cell proliferation. Interactions between the Wnt pathway and other signaling pathways amplify its impact on breast cancer, influencing tumor development and metastasis (4). Targeting the Wnt pathway emerges as a promising therapeutic strategy to impede breast cancer cell growth and spread. Additionally, Wnt signaling’s involvement in maintaining cancer stem cells, driving tumor initiation, recurrence, and therapy resistance, adds complexity to breast cancer biology (4). Crosstalk with hormone receptors, like estrogen receptors, further complicates the role of the Wnt pathway in breast cancer. This intricate relationship requires thorough exploration for the identification of biomarkers and the development of targeted therapies. Ongoing research efforts aim at unraveling the precise mechanisms underlying Wnt pathway influence in breast cancer, paving the way for more effective and personalized treatment approaches (5).

Network analysis is a pivotal tool in cancer treatment, offering a multifaceted approach to understanding and combating this complex disease. By delving into intricate interactions between genes, proteins, and pathways, network analysis aids in the identification of crucial biomarkers and therapeutic targets tailored to specific cancer types. This knowledge proves invaluable in the era of personalized medicine, allowing for more precise and effective treatment strategies. Moreover, network analysis is a key factor in deciphering the heterogeneous nature of tumors. This comprehensive understanding is essential for developing targeted therapies that consider the diverse characteristics of individual cancers (6). Crucially, network analysis unravels resistance mechanisms, shedding light on how cancers adapt and evade treatments (7). Armed with this knowledge, researchers can develop strategies to overcome resistance, a significant challenge in cancer therapy. Finally, network analysis fosters collaboration among researchers, clinicians, and bioinformaticians. By sharing data and insights, the scientific community can collectively advance our understanding of cancer biology and improve treatment strategies, ultimately bringing us closer to more effective and targeted cancer therapies (7).

MicroRNAs (miRNAs) are small, naturally occurring non-coding RNA molecules that play a vital role in controlling gene expression. Robust evidence indicates the disruption of pertaining to miRNA levels in cancers affecting humans, stemming from diverse processes including miRNA gene duplication as well as removal, combined with irregular transcriptional activity, disturbed epigenetic modifications, and flaws during miRNA synthesis. These dysregulated miRNAs can act as either tumor-promoting genes or tumor-restraining genes, influencing essential neoplasm characteristics like continuous proliferation pathways, evasion of expansion suppressors, avoidance of programmed cell death, initiation of tissue invasion and cancer dissemination, and stimulation of the formation of new blood vessels. An expanding body of research underscores miRNAs as promising indicators for cancer detection, prognosis, and therapeutic targets, necessitating further exploration and validation (8, 9).

The present study focuses on understanding the disruption in miRNA expression, pinpointing central genes, and uncovering the complex interactions between signaling pathways. By applying clustering techniques, we analyze the functional components within these networks, offering a comprehensive perspective on the interactions between critical molecular components. Additionally, a thorough investigation of promoter regions associated with hub genes highlights the regulatory components of transcription that propel breast cancer development and progression.

By integrating these multiple approaches, our research aims to provide detailed insights into the molecular framework of breast carcinoma, potentially informing diagnostic approaches and the identification of targets for therapy. While investigating the intricacies of miRNAs control networks, this examination signifies a significant step toward deciphering the molecular complexity that characterizes breast carcinoma.

Numerous studies underscore the potential of miRNAs serving as indicators for cancer detection, outcome prediction, and treatment strategies intervention, highlighting the importance of additional research and confirmation. The MiRNA aberrant regulation in human cancers, driven by processes including miRNA gene reinforcement or gene loss, irregular gene expression patterns, epigenetic alterations, and disruptions in miRNA biogenesis, contributes significantly to tumor initiation, progression, metastasis, and resistance to therapy (14). The miRNAs function as both cancer-promoting genes and tumor-inhibiting genes, influencing key carcinoma characteristics, encompassing uncontrolled cell proliferation, evasion of growth suppression, resistance to apoptosis, activation of invasion and metastasis, and stimulation of angiogenesis. Using computational resources, our research investigates the complex interplay between miRNAs that regulate the Wnt/β-catenin cellular signaling cascade and their role in breast carcinoma initiation and advancement. These miRNAs are critical due to their activation and interaction with other essential cellular pathways (15, 16).

The PPI networks provide a mathematical representation of physical interactions between proteins in cellular environments. These interactions are highly specific, taking place in well-defined binding domains, and hold significant biological relevance by fulfilling distinct functional roles. In recent years, scientific focus has shifted toward analyzing protein substructures, employing iterative interaction algorithms to predict protein interactions and elucidate their functional roles. This methodology allows for the scrutiny of critical proteins vital for optimal targeting. In Figure 1, proteins directly associated with aberrantly miRNAs play crucial contributions in fundamental signaling cascades involved in cellular growth and division in subsequent sections, our objective is to offer a more detailed classification and comprehension of these proteins, pinpointing those with the highest number of interactions (17, 18).

RHOA is involved in cell migration and invasion. Dysregulation of RHOA has been linked to enhanced metastatic potential in cells of breast cancer, indicating a potential role in the spread of cancer. COL5A3 is a collagen gene (19). Altered collagen expression is associated with changes in the tumor microenvironment, impacting cancer cell behavior, including migration and invasion (20).

Again COL19A1, COL1A1, COL5A1, COL2A1, COL3A1, COL25A1, and COL13A1 role in the extracellular matrix suggests a potential impact on tissue remodeling, which could influence the advancement and invasion cells of breast cancer (21). ESR1 encodes estrogen receptor alpha protein. Mutations or alterations in ESR1 are crucial in hormone receptor-positive breast cancers, influencing treatment response to hormone therapies (22). H3F3B encodes a histone variant. Alterations in histones can affect chromatin structure, potentially influencing gene expression and contributing to breast cancer development (23). PLOD2 is associated with collagen modification. Changes in collagen stability may influence the extracellular matrix in breast cancer and play a role in tumor advancement (24). STAT3 is a signaling protein involved in cellular viability and proliferation. Its activation has been associated with breast cancer development and progression, positioning it as a promising candidate for therapeutic intervention (25). PTEN acts as a tumor suppressor gene. Absence of PTEN activity, often due to genetic alterations, is linked to breast cancer development, as PTEN regulates cell growth and division (26).

The functional enrichment results suggest that the identified hub proteins play critical roles in several key mechanisms associated with breast cancer development and progression. Their involvement in the regulation of cellular and metabolic processes, as well as multicellular development, reflects their potential impact on fundamental aspects of tumor biology, including proliferation, differentiation, and survival. Localization to the nucleoplasm and chromatin emphasizes their possible functions in gene expression regulation and epigenetic control, which are frequently altered in cancer cells. Moreover, the diversity of MFs, particularly their capacity for protein and transcription factor binding, highlights their regulatory complexity and potential to influence multiple signaling axes. The enrichment in PI3K-Akt and TNF signaling pathways supports their likely involvement in oncogenic signaling that governs cell viability, immune responses, and tumor microenvironment interactions. Notably, their association with stem cell-related pathways underscores a potential link to cancer stemness, a feature associated with tumor initiation, resistance to therapy, and recurrence. The unexpected connection to protein digestion and absorption pathways may also reflect an adaptive metabolic reprogramming in cancer cells, enabling efficient nutrient uptake to sustain rapid growth. Overall, these results provide valuable insights into the molecular mechanisms driven by hub proteins and reinforce their relevance as potential biomarkers or therapeutic targets in breast cancer (Figure 3).

CytoCluster provides a glimpse into the intricate molecular interactions within downregulated miRNA-targeted genes, potentially influencing key processes associated with breast cancer. The prominence of the PI3K-Akt pathway suggests a central influence on breast cancer-related signaling cascades, known for its involvement in cell survival and proliferation. The enrichment of pathways beyond breast cancer implies a broader regulatory network, potentially shared across multiple cancers. These findings highlight potential interplay and shared mechanisms between breast cancer and other malignancies, offering new perspectives for future research. In conclusion, the results of the CytoCluster analysis offer a nuanced understanding of the network organization of downregulated miRNA-targeted genes, emphasizing their direct relevance to breast cancer and providing valuable insights into potential therapeutic targets and mechanisms of cancer progression. Further experimental validations and functional studies are warranted to unravel the specific roles of these clusters and their implications for targeted therapeutic interventions in breast cancer and potentially other cancer types (Table 3).

Predicted motifs and functional annotations indicate that hub proteins may play key regulatory roles in diverse cellular processes, particularly in signal transduction and development. The consistent identification of transcription factor-related motifs highlights their potential involvement in transcriptional control mechanisms. Further insights from MF predictions, such as dimerization activities and ion-binding capabilities, suggest the formation of protein complexes and participation in cellular signaling and homeostasis. Localization patterns, including associations with dendritic structures, reinforce their functional importance in specialized cellular compartments. Overall, these findings broaden the understanding of the regulatory framework influencing hub protein expression and function. The results provide a compelling basis for future experimental validation and investigation into the biological relevance of these computational predictions (Figure 4).

The identification of approved, investigational, and experimental compounds targeting hub proteins such as STAT3, ESR1, PTEN, and RHOA reinforces the therapeutic relevance of these molecules in breast cancer management. The presence of both synthetic agents (e.g., acitretin, raloxifene, alpelisib) and natural compounds (e.g., epigallocatechin gallate, quercetin) highlights the diversity of pharmacological strategies aimed at modulating critical signaling pathways. Notably, the absence of drug associations for several hub proteins underscores a need for continued drug discovery efforts. These insights emphasize the translational potential of network-based drug screening and the importance of validating predicted interactions in preclinical and clinical studies (Table 4).