Introduction

Despite notable progress in the diagnosis and treatment of breast cancer in recent years, the post-surgery therapeutic effects remains highly uncertain1. Breast cancer is one of the most common malignant tumors among women, which mostly occurs in women aged 40–60 years old, before and after menopause2. About 3%~8% of the newly diagnosed breast cancer patients are advanced patients. Even for early diagnosed patients, after receiving standardized treatment, 30–40% will eventually progress to advanced stages. For patients with advanced breast cancer, the 5-year survival rate is only 20%, which greatly reduces the average 5-year survival rate of breast cancer. Consequently, identifying potential indicators that could impact patient outcomes has become a focal point in clinical and fundamental medical investigation. Rab10 (GTP binding protein RAB10), initially studied for its role in intracellular material transport as a small GTP enzyme, has more recently emerged as a significant contributor to cancer onset and progression. As members of the Ras superfamily, the Rab family plays important intracellular biological functions. Rab10, as a novel GTP binding protein, is involved in the formation, transport, anchoring, and fusion of intracellular vesicles. Abnormal expression of Rab10 may lead to various organelle defects and functional disorders. Studies have shown an increased presence of Rab10 in various cancers, correlating with disease progression, worsening condition, and unfavorable clinical outcomes3,4. TLR4 (Toll-like receptor 4), a member of the Toll-like receptor family, plays a crucial role in the body’s immune response, particularly in infection, inflammation, and autoimmune diseases. Nevertheless, recent research have highlighted TLR4’s atypical expression in numerous malignancies, strongly associated with tumor invasion, migration, and unfavorable therapeutic effects. The NF-κB (nuclear factor kappa-B) family consists of transcription factors that have a crucial function in regulating cell growth, inflammatory response, and immune response. Prior research has demonstrated that the persistent NF-κB stimulation is associated with the development, advancement, and resistance to chemotherapy in diverse malignant cancers. It is worth noting that most studies on these three proteins are focused on tumor tissues, with relatively few examining their presence in peripheral blood. Detecting these proteins in peripheral blood offers the benefits of being non-invasive, easy to collect, and repeatable5. Rab10 levels in peripheral blood of patients with liver cancer, breast cancer and pancreatic cancer are all increased, which can be used as an early diagnostic indicator of cancer and can also guide cancer treatment6. Hence, understanding the correlation between the expression of these proteins in the bloodstream and the therapeutic effects of breast cancer holds immense importance. This study aimed to investigate the correlation between Rab10, TLR4, and NF-κB expression in peripheral blood and the postoperative therapeutic effects of breast cancer patients. It also strived to offer fresh perspectives and a theoretical basis for enhancing breast cancer diagnosis, treatment, and therapeutic effects evaluation.

Materials and methods

Baseline data

The research sample consisted of 160 patients with stage I-III breast cancer who underwent surgery at our hospital’s Department of Breast Surgery and Oncology between January 2021 and June 2021. Based on their levels of Rab10, TLR4, and NF-κB in peripheral blood, participants were categorized into two groups: the low marker expression group (72 participants with relatively low expression of Rab10, TLR4, and NF-κB: Rab10 < 2.0ng/ml; TLR4 < 2.75ng/ml; NF-κB < 3.5ng/ml) and the high marker expression group (88 participants with relatively high expression: Rab10 ≥ 2.0ng/ml; TLR4 ≥ 2.75ng/ml; NF-κB ≥ 3.5ng/ml). This study was approved by the Ethics Committee of Affiliated Hospital of Hebei Engineering University and the study was performed in accordance with the Helsinki II declaration. In order to eliminate confounding factors between exposure and outcomes, strict inclusion and exclusion criteria were implemented during the study process. Moreover, the researchers involved in this study received unified training to avoid human bias in the receipt collection process. The patient enrollment process was shown in Fig. 1.

Fig. 1
figure 1

The flow diagram of patient enrollment.

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Inclusion criteria

Participants were required to be females’ ages between 18 and 70 who had undergone surgical intervention for breast cancer. They should not have been pregnant or breastfeeding and were required to actively engage in the study by providing written informed consent.

Exclusion criteria

Exclusion criteria included individuals with malignant tumors in different regions who were not suitable for surgery, radiotherapy, or chemotherapy; patients suffering from severe mental disorders that hindered their comprehension of the research material or effective communication; individuals with advanced stage IV breast cancer and a projected survival time of less than 6 months; and those who declined to sign the protocol or provide informed consent.

General information

The patients’ age, height, weight, BMI [BMI = weight (kg)/height (m) ^2], pathological type (catheter/lobular/other), tumor size, tumor stage, maternal history, hypertension, diabetes, smoking and drinking history, lymph node metastasis and recurrence within 3 years after surgery were analyzed. Survival at 3 years after surgery (including median overall survival and median progression-free survival).

Detection of Rab10, TLR4 and NF-κB levels in peripheral blood

To detect Rab10, TLR4, and NF-κB levels in peripheral blood, specific antibodies for these proteins were used to coat a 96-well plate. Each well was loaded with 100µL of the sample and incubated at 4 °C overnight. On the following day, unbound antibodies were removed by washing the sample three times with a washing buffer. Next, 300 µl of a blocking solution were added, and the plate was left to incubate for one hour to prevent any non-specific binding. Following another round of washing, patients’ blood samples from the outer regions, with varying concentrations, were added to each well, 100µL per well. The samples were then incubated with previously prepared protein standards for duration of 2 h at room temperature. The samples were then treated with biotinylated secondary antibodies specific to the target proteins, 100µL per well, and incubated for an additional hour. After another washing step to remove non-specific binding, 100µL of t3,3’, 5,5’ – tetramethylbenzidine (TMB) substrate was added, and the mixture was incubated for 15 minutes. The reaction was stopped by adding 50µL of termination solution. The absorbance at 450nm was measured using a microplate reader, and the quantities of Rab10, TLR4, and NF-κB in the sample were assessed based on the established protein standard curve.

Correlation analysis between protein expression and therapeutic effects

Kendall’s tau-b correlation analysis method was used to evaluate whether the relationship between the expression level of Rab10, TLR4 and NF-κB protein and therapeutic effects (whether breast cancer recurred, distant metastasis, and death) was statistically significant.

Statistical analysis

SPSS 20.0 (SPSS, Tokyo, Japan) was utilized to perform the data analysis. Normally distributed measurement data were expressed as mean ± standard deviation (SD), while non-normally distributed measurement data were expressed as median (interquartil range), and the comparisons were examined by Student-t test and Mann-Whitney test (non-parametric distribution). The categorical data were expressed as n (%), and the differences between the two groups were examined by chi-square analysis or Fisher’s exact test. Kendall’s tau-b correlation coefficient was used to analyze the correlation between the expression of Rab10, TLR4 and NF-κB and the therapeutic effects of patients, and P < 0.05 was considered statistically significant.

Results

General data statistics

According to the statistical data, the low marker expression group had an average age of 55.47 ± 6.23 years and an average BMI of 22.75 ± 2.16 kg/m2. The majority of the patients had ductal and lobular pathological types, and the average tumor size was 3.37 ± 1.22 cm. The majority of the patients had a history of pregnancy, including 11 cases of high blood pressure, 8 cases of diabetes, 6 cases of smoking, and 13 cases of alcohol consumption. The high marker expression group had an average age of 57.05 ± 5.18 years and an average BMI of 23.64 ± 1.98 kg/m2. Ductal and lobular types were predominant in the pathological categories, and an average tumor size was approximately 3.28 ± 1.14 cm. A significant number of patients in the high marker expression group had a history of pregnancy, including 15 cases of high blood pressure, 10 cases of diabetes, 9 cases of smoking, and 17 cases of alcohol consumption. Importantly, general data showed no significant differences between the two groups (P > 0.05) (Table 1).

Table 1 General statistics.
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Expression analysis of Rab10, TLR4, and NF-κB in peripheral blood

The expression of Rab10, TLR4, and NF - κ B in peripheral blood of patients was detected by enzyme-linked immunosorbent assay (ELISA). The levels of Rab10, TLR4, and NF - κ B in peripheral blood of the high marker expression group were higher than those of the low marker expression group (Rab10: 1.87 ± 0.18 vs. 3.15 ± 0.24 ng/ml; TLR4: 2.17 ± 0.20 vs. 3.26 ± 0.25 ng/ml); NF-κB: 2.68 ± 0.27 vs. 4.63 ± 0.30 ng/ml; P < 0.05) (Table 2).

Table 2 Analysis of Rab10, TLR4, and NF-κB expression in peripheral blood.
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Relationship between patient staging and expression of Rab10, TLR4, NF-κB

Analyzing the relationship between patient staging and Rab10, TLR4, and NF - κ B expression, the number of patients in high marker expression group III-IV increased compared to the low marker expression group (54.55% vs. 36.12%; P < 0.05), while the number of patients in high marker expression group I-II decreased compared to the low marker expression group (45.45% vs. 63.88%; P < 0.05) (Fig. 2; Table 3).

Fig. 2
figure 2

Relationship between patient staging and expression of Rab10, TLR4, and NF-κ B.

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Table 3 Relationship between patient staging and expression of Rab10, TLR4, and NF-κB.
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Relationship between therapeutic effects and expression of Rab10, TLR4, and NF-κB

Analyzing the relationship between the 3-year outcome of patients and the expression of Rab10, TLR4, and NF - κ B, it was found that the number of patients with no recurrence or metastasis in the high marker expression group decreased compared to the low marker expression group (56.81% vs. 73.61%; P < 0.05), while the number of patients with recurrence or metastasis in the high marker expression group increased compared to the low marker expression group (43.19% vs. 26.39%; P < 0.05) (Fig. 3, Table 4).

Fig. 3
figure 3

Relationship between therapeutic effects and expression of Rab10, TLR4, and NF-κ B.

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Table 4 Relationship between prognosis and expression of Rab10, TLR4, and NF-κ B.
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Relationship between survival rate and the expression of Rab10, TLR4, and NF-κB

Analyzing the relationship between the 3-year survival rate of patients and the expression of Rab10, TLR4, and NF - κ B, the median overall survival and median progression free survival in the high marker expression group were shorter than those in the low marker expression group (median overall survival: 21.45 ± 2.68 months vs. 28.38 ± 3.44 months; median progression free survival: 15.25 ± 2.37 vs. 20.72 ± 2.58 months; P < 0.05) (Table 5).

Table 5 Relationship between survival rate and expression of Rab10, TLR4, and NF-κ B.
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Kendall’s tau-b correlation analysis of rab10, TLR4, NF-κB expression and therapeutic effects

Kendall’s tau-b correlation indicated a positive correlation between the expressions of Rab10, TLR4, and NF-κB and a poor therapeutic effects (P < 0.05), suggesting that elevated levels of Rab10, TLR4, and NF-κB may lead to a worsened therapeutic effects (Table 6).

Table 6 Pearson correlation analysis.
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Discussion

Breast cancer remains the most prevalent malignant tumor among women worldwide, making the investigation of therapeutic effects an essential aspect of enhancing treatment outcomes and patient well-being7,8,9. Recurrence, metastasis, and death are typically associated with negative postoperative therapeutic effects for breast cancer. Hence, discovering biomarkers related to therapeutic effects is crucial for timely interventions and enhancing patient outcomes. Once activated RABGTPases reach the site of action, they interact with specific effector proteins to regulate vesicle formation, movement, tethering, and fusion. Many RAB GTPases have been identified as part of LD surface proteins. As lipophagy is a process dependent on coordination membrane transport activity, RAB GTPases play an important role in lipophagy. Under starvation conditions, RAB10 on LD surface is activated and triggers the degradation of LD’s lipophagy dependent pathway. Knockout of RAB10 can lead to abnormal accumulation and proliferation of LD in HCC cells. Therefore, Rab10 is associated with the recurrence rate of breast cancer after surgery. The expression of Rab10, TLR4, and NF - κ B are all correlated. This study was aimed to investigate the effect of Rab10, TLR4, NF-κB levels in peripheral blood on the therapeutic effects of patients with breast cancer after surgery. Associating these three proteins with breast cancer therapeutic effects offers new insights into comprehending the biological characteristics of this disease. The findings of this research indicate that the presence of these three proteins in the bloodstream strongly correlates with the therapeutic effects of breast cancer.

In modern medical research, identifying disease-related biomarkers has emerged as a key focus, particularly in the field of cancer research. Biomarkers, as substances that reflect disease status, therapeutic effects, or treatment response, provide clinicians with more accurate treatment options and improved methods for disease monitoring10,11. In this research, we conducted a thorough examination of the levels of Rab10, TLR4, and NF-κB in the peripheral blood of individuals diagnosed with breast cancer. Our findings revealed a noteworthy elevation in their expression within the high marker expression group. The discovery holds great significance for understanding the causes, predicting outcome, and devising breast cancer treatment strategies. Rab10 belongs to the RAS superfamily of small GTPases and is a GTP binding protein that regulates the transport of intracellular vesicles through continuous binding and hydrolysis of ATP, facilitating protein transport between different organelles. It may affect colon cancer cell proliferation through the tumor microenvironment. For a considerable time, Rab10, a diminutive GTP enzyme, has been known for its crucial role in various biological activities, including internalization and endocytosis processes. In the context of oncology, Rab10’s expression has been associated with the development, metastasis, and therapeutic effects of various tumors. The findings of this research indicate a substantial increase in Rab10’s presence in the bloodstream of individuals diagnosed with breast cancer, suggesting a potential correlation with disease progression and worsening. This novel perspective suggests that Rab10 could serve as both a potential breast cancer biomarker and a promising treatment target. TLR4, a pattern recognition receptor of the immune system, plays a crucial role in the body’s inflammatory and immune responses. Recent research has indicated that the atypical expression of TLR4 in certain malignancies might be associated with tumor immune evasion, the establishment of chronic inflammatory conditions, and tumor progression. Our data corroborate this perspective, indicating that TLR4 may contribute to an unfavorable therapeutic effects in breast cancer cases, which warrants further investigation. NF-κB, a nuclear factor, regulates the transcription of numerous genes associated with inflammation, immune response, cellular proliferation, and viability. Previous research has uncovered NF-κB’s involvement in various types of cancer, particularly its connection with the inflammatory microenvironment during tumor progression. The study further highlights the potential involvement of NF-κB in breast cancer.

The accurate determination of breast cancer stage is a crucial for devising treatment strategies and forecasting patient outcomes12,13. Traditionally, clinicians have relied on factors such as tumor size, lymph node metastasis, and distant metastasis to stage breast cancer. However, as medical research advances, an increasing number of biological markers are being recognized for their connection to breast cancer progression and therapeutic effects. The elevated levels of Rab10, TLR4, and NF-κB in this research are strongly associated with advanced stages of breast cancer, suggesting their potential as crucial indicators of breast cancer advancement. Rab10, TLR4, and NF-κB play essential roles in cellular processes as significant regulatory factors. Rab10 is closely related to cell signal transduction, endocytosis, and endocytosis processes. TLR4 serves as a pattern recognition receptor of the immune system, involved in inflammatory reactions and immune responses. NF-κB, a nuclear transcription factor, plays a role in inflammation, as well as cell survival and proliferation. Furthermore, the presence of an inflammatory response in the tumor microenvironment is believed to be linked to tumor progression and metastasis. Inflammation cells can provide tumor growth factors, promote the formation of new blood vessels, and aid in the evasion of immune surveillance by tumor cells14,15. TLR4 and NF-κB are crucial in this process. Therefore, their elevated expression in individuals with breast cancer might indicate a heightened tumor microenvironment susceptible to spreading. Clinicians can gain additional information about patients’ therapeutic effects and treatment response through the elevated expression of these biomarkers in clinical practice.

Breast cancer patients face the grave dangers of recurrence and metastasis, leading to complex treatment plans, increased medical expenses, and a low chance of survival. Hence, clinicians must identify patients prone to recurrence and metastasis early, enabling them to provide tailored treatment and follow-up plans. In this regard, the indications from Rab10, TLR4, and NF-κB expressions offer significant insights. The high marker expression group exhibited an evident increase in the levels of Rab10, TLR4, and NF-κB, accompanied by a notable rise in the incidence of recurrence and metastasis. This correlation suggests that these proteins could be associated with the invasion and movement capability of breast cancer cells and their interaction with the adjacent microenvironment. Several research studies have indicated a strong correlation between tumor recurrence and metastasis and the capability for cell migration and invasion16,17,18. Thus, Rab10, TLR4, and NF-κB could potentially serve as pivotal controlling elements in this particular mechanism. Moreover, these three proteins could potentially be associated with various biological traits of cancer cells, including cellular growth, suppression of cell death, keratin production, and the formation of new blood vessels. These traits have a significant impact on the progression and spread of tumors19,20. It has been suggested that NF-B is a key regulator of the inflammatory response and immune regulation, playing a role in the development of many cancers, which promotes tumor growth and metastasis. Furthermore, the ability to anticipate the possibility of recurrence and metastasis beforehand holds immense importance for both patient treatment plans and prognoses. Additionally, it aids clinicians in optimizing medical resources by prioritizing high-risk patients and offering them more comprehensive follow-up and examinations, enabling the timely detection and management of recurrence and metastasis. The recurrence and metastasis of breast cancer could be significantly influenced by Rab10, TLR4, and NF-κB. Elevated expression is strongly linked to a higher likelihood of recurrence and metastasis, offering a fresh approach to the treatment and control of breast cancer.

The therapeutic effects of cancer patients relies on two fundamental factors: overall survival and progression-free survival. Overall survival indicates the period from diagnosis until death, whereas the progression-free survival signifies the time from the initiation of a specific treatment until disease progression or death21. Clinical research frequently utilizes these two measures to assess the efficacy of a specific therapeutic approach or strategy. Our study revealed that the survival rates in the high marker expression group were significantly lower compared to the low marker expression group, highlighting a concerning situation. This finding suggests that the elevated levels of Rab10, TLR4, and NF-κB could serve as crucial markers for predicting a negative outcome in breast cancer patients. When evaluating patients’ risk, clinicians can take this factor into consideration, aiding in treatment planning and long-term management. The elevated expression of these three proteins could be associated with various biological mechanisms linked to cancer progression and spread. For instance, they may be involved in key processes such as tumor development, cell cycle regulation, programmed cell death, new blood vessel formation, cell adhesion, and cell movement. Furthermore, the elevated levels of Rab10, TLR4, and NF-κB could potentially be associated with inflammation, immune response, and intercellular signal transmission within the tumor microenvironment, thereby impacting tumor progression and spread. The significance of comprehending these proteins and their roles in breast cancer is underscored by these discoveries. In the coming years, additional experimental investigations may be required to explore their distinct functions in breast cancer development. Simultaneously, these may also emerge as novel therapeutic targets, offering enhanced and tailored treatment regimens for individuals diagnosed with breast cancer.

Kendall’s tau-b correlation analysis is a statistical technique employed to assess the magnitude and orientation of the linear relationship between two variables. In this study, we utilized it to measure the association between the expression of Rab10, TLR4, and NF-κB and the therapeutic effects of breast cancer. The findings of our study depicted a notable and adverse association between the presence of these three proteins and the unfavorable therapeutic effects, providing valuable insights into their contributions to breast cancer progression and therapeutic effects. When one variable increases, another variable also increase, indicating a positive correlation. In this context, as the expression of Rab10, TLR4, and NF-κB raised, the likelihood of a adverse outcome also escalated. This relationship suggests the potential involvement of these proteins in tumor biology, particularly their role in tumor progression, invasion, and metastasis. The discovery enhances our comprehension of the significance of Rab10, TLR4, and NF-κB in breast cancer.

This discovery is consistent with the conclusions of relevant studies, these proteins are involved in various cell signal transduction pathways and biological processes, including but not limited to inflammatory reaction, cell proliferation, migration, and apoptosis22,23. TLR4, found on the surface of macrophages and dendritic cells, plays a pivotal role in both inflammation and immune response, while NF-κB serves as a transcription factor regulating genes associated with inflammation, immune response, and cellular viability. Furthermore, these proteins have the capacity to interact with other cells and factors within the tumor microenvironment, thereby facilitating tumor progression24,25. The increased expression of Rab10, TLR4, and NF-κB may strengthen the communication between cancer cells and neighboring cells, promoting modifications in the extracellular matrix. Consequently, this process establishes a conducive environment for tumor invasion and metastasis. The results of our Kendall’s tau-b correlation analysis unequivocally demonstrated that the expressions of Rab10, TLR4, and NF-κB were significantly associated with an unfavorable therapeutic effects in breast cancer.

The study still has some limitations. First, although we included a total of 160 patients after breast cancer surgery, as an observational study, the sample size was still limited, which may lead to problems of bias, limiting the reliability and comprehensiveness of the results. Secondly, although the results of this study clearly showed that the expressions of Rab10, TLR4 and NF-κB in peripheral blood were associated with poor therapeutic effects of breast cancer, their correlation indexes were all less than 0.5, which required more verification with large samples.

To conclude, there is a substantial correlation between the levels of Rab10, TLR4, and NF-κB in the peripheral blood of breast cancer patients and their postoperative therapeutic effects. Elevated levels of Rab10, TLR4, and NF-κB are linked to an increased risk of recurrence and metastasis, reduced overall survival, and limited progression-free survival. These findings underscore the significance of Rab10, TLR4, and NF-κB as potential biomarkers for breast cancer, opening up new avenues for research and potential targets for future therapeutic strategies.