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†These authors contributed equally.
Background: Rab10 is a small GTPase protein belonging to the Ras superfamily. It is expressed and plays a role in a variety of malignant tumours. However, the expression of Rab10 and its role in breast cancer (BC) prognosis remains unclear. The aim of this study was to analyse the differential expression and prognostic value of Rab10 in BC using bioinformatics techniques and immunohistochemistry in a clinical cohort. Methods: The TIMER2, GEPIA2, and UALCAN databases were used to analyse the correlation between the differential expression of Rab10 and BC. Rab10 and BC prognosis were correlated using the Kaplan–Meier Plotter and UALCAN databases. The expression of Rab10 in BC tissues was detected using immunohistochemistry, and its correlation with the BC clinical cohort was analysed using Chi-squared tests and logistic regression analysis. Results: The expression of Rab10 mRNA identified in BC patients using TIMER2, GEPIA2, and UALCAN databases was higher than that in para-cancerous tissues. Kaplan–Meier plotter and the UALCAN database revealed that increased Rab10 expression was associated with poor prognosis in BC patients. Immunohistochemistry showed that Rab10 was expressed on cell membranes and in cytoplasm of BC tissues. In a clinical cohort, Rab10 expression correlated with histological grade, (human epidermal growth factor receptor 2) HER2 status, and molecular typing. Conclusions: Rab10 can be used as an effective clinical prognostic biomarker for BC.
Breast cancer (BC) is the most common malignant tumour in women worldwide, with the highest rates of disability and death [1]. The global burden of BC is rapidly increasing, especially in China, where BC incidence and mortality rates are rapidly rising [2]. According to international consensus guidelines, the current treatment for BC mainly includes chemotherapy, radiotherapy, targeted therapy, immunotherapy, and endocrine therapy before and after surgery [3]. Traditional treatment modalities, such as surgery, chemotherapy, and radiotherapy, have improved the prognosis of patients with BC. However, tumour progression and metastasis in BC patients remains a great clinical challenge. Therefore, it is crucial to elucidate the pathogenesis of BC and identify new therapeutic targets.
The GTP-binding protein Rab10 belongs to the Ras superfamily of small GTPases, which are mainly distributed in cytosolic compartments and are used to regulate intracellular vesicle transport [4]. Rab10 resides on glucose transporter 4 (GLUT4) vesicles and coordinates with myosin Va to store GLUT4 in vesicles and deliver GLUT4 to the plasma membrane for glucose uptake in adipocytes and muscle tissue [5]. Rab10 is a substrate of leucine-rich repertoire kinase 2 (LRRK2) and is highly expressed in phagocytes in the immune system. Reducing Rab10 expression inhibits AKT (protein kinase B, PKB) activation and chemotaxis, and Rab10 phosphorylation mediated by LRRK2 impedes rapid vesicle cycling and promotes PI3K-Akt immune responses [6].
In recent years, an increasing number of studies have discovered that Rab10 is aberrantly expressed in a variety of malignancies, including liver cancer, glioma, and cervical cancer, and is associated with poor patient prognosis [7, 8, 9]. In addition, Rab10 plays an important role in tumour cell proliferation, apoptosis, invasion, and metastasis, as well as in tumour autophagy [7, 8, 9, 10, 11]. Studies have found that FAM49B promotes proliferation, metastasis, and chemoresistance in BC by regulating the downstream Rab10/TLR4 pathway [12]. However, the expression profile of Rab10 in BC and its association with clinical prognosis remain unclear. We analysed the differential expression of Rab10 in tumours and adjacent tissues using the GEPIA2 and TIMER2 databases and found that Rab10 expression was significantly higher in BC tissues than in normal breast tissues. In this study, we further investigated the expression of Rab10 in BC tissues, analysed its biological functions, and correlated it with prognosis of BC patients.
Differential expression of Rab10 mRNA in BC and paraneoplastic tissues was analysed using the TIMER2 (http://timer.cistrome.org/), GEPIA2 (http://gepia2.cancer-pku.cn/), and UALCAN (http://ualcan.path.uab.edu/) databases. Correlation between Rab10 and BC overall survival (OS) and recurrence-free survival (RFS) was explored using the Kaplan–Meier Plotter website (https://kmplot.com/analysis/) and the UALCAN databases.
This study included 106 patients with invasive BC who underwent breast surgery
at the Affiliated Hospital of Hebei Engineering University between January 2014
and December 2015. Patient inclusion criteria were as follows: (1) none of the
patients had neoadjuvant treatment, such as radiotherapy or chemotherapy before
surgery; (2) specimens were all analysed pathologically for diagnosis; (3) all
patients were treated with standard adjuvant therapy following radical surgery.
Exclusion criteria were as follows: (1) distant metastasis of the tumour; (2)
combination of other malignant tumours; (3) combination of immune system-related
diseases; (4) presence of severe liver, kidney or cardiac defects; (5) mental
disorders; (6) not meeting the indications for breast cancer surgery; (7) age
Our study used the 2013 St. Gallen International Expert Consensus Report to identify four subtypes of BC: luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) overexpression, and triple-negative.
Our study and all experimental methods were approved by the Ethics Committee of the Affiliated Hospital of Hebei Engineering University.
Immunohistochemistry (IHC) was performed as follows. Paraffin-embedded mammary
sections were first placed in fresh xylene solution for 15 min, and then in
100%, 95%, 90%, 80%, and 70% ethanol for 5 min each, washed three times (3
min each) in phosphate-buffered saline (PBS), after which the sections were
placed in a 3% H
Rab10 expression in breast tissue was analysed separately and semi-quantitatively by two senior pathologists.
Clinical data were analysed using SPSS v.26.0 (IBM Corp., Armonk, NY, USA), and
the
Analysis of the TIMER2 database revealed that Rab10 mRNA was expressed in a variety of tumour tissues, and the expression levels of Rab10 mRNA in BC and paraneoplastic tissues were statistically different, as shown in Fig. 1A. Analysis of the GEPIA2 database revealed differential expression of Rab10 in a variety of tumours, and BC included a total of 1376 samples (tumour = 1085; normal = 291) with much higher Rab10 expression than in paraneoplastic tissues (Fig. 1B).
Differential expression of Rab10 in BC cells and BC prognosis.
(A) Determination of Rab10 expression levels in different tumour types using
TIMER (*p
Similarly, analysis of 1211 BC samples from the UALCAN database revealed that Rab10 mRNA was significantly upregulated in BC tissues (Fig. 1C), consistent with the GEPIA2 data.
Analysis of the UALCAN database showed that BC patients with high Rab10 mRNA
expression had worse OS, and the difference was statistically significant
(p
A total of 106 female patients with BC with a mean age of 54.93 years (range, 26–82 years) were included in this study.
IHC results showed that Rab10 was mainly present in BC glandular cells and myoepithelial cells, localised in the cell membrane and cytoplasm, and the cytoplasmic staining was stronger than the cell membrane staining (Fig. 2).
Immunohistochemical analysis of Rab10 expression in BC
tissues. (A) Brown indicates Rab10 positively expressed in BC tissue
(magnification 400
A clinical cohort analysis revealed that Rab10 positivity in BC tissue was
27.4% (29/106), and Rab10 positivity in HER2+ BC was as high as 48.15% (13/27).
Additionally, Rab10 expression was associated with the histological grade
(
Clinicopathologic characteristics | N | Rab10(+) | Rab10(–) | p | ||
Nationality | ||||||
Han | 106 | 29 | 77 | |||
Year | ||||||
35 | 7 | 28 | 1.42 | 0.23 | ||
71 | 22 | 49 | ||||
Grading | ||||||
1 | 22 | 3 | 19 | 8.39 | 0.02 | |
2 | 35 | 6 | 29 | |||
3 | 49 | 20 | 29 | |||
pTNM stage | ||||||
I | 31 | 10 | 21 | 2.02 | 0.36 | |
II | 52 | 11 | 41 | |||
III | 23 | 8 | 15 | |||
T stage | ||||||
T1 | 38 | 12 | 26 | 1.39 | 0.71 | |
T2 | 50 | 11 | 39 | |||
T3 | 15 | 5 | 10 | |||
T4 | 3 | 1 | 2 | |||
N stage | ||||||
N0 | 57 | 15 | 42 | 0.91 | 0.82 | |
N1 | 34 | 9 | 25 | |||
N2 | 14 | 5 | 9 | |||
N3 | 1 | 0 | 1 | |||
Subtype | ||||||
LA | 30 | 5 | 25 | 8.45 | 0.04 | |
LB | 32 | 8 | 24 | |||
HER2+ | 27 | 13 | 14 | |||
TN | 17 | 3 | 14 | |||
ER status | ||||||
Positive | 60 | 13 | 47 | 2.25 | 0.13 | |
Negative | 46 | 16 | 30 | |||
PR status | ||||||
Positive | 55 | 12 | 43 | 1.77 | 0.18 | |
Negative | 51 | 17 | 34 | |||
HER2 status | ||||||
Positive | 59 | 21 | 38 | 4.54 | 0.03 | |
Negative | 47 | 8 | 39 |
pTNM stage, refer to AJCC 8th edition; T, tumer size; N, lymph node involvement; ER, estrogen receptor; PR, progesterone receptor; HER2, Human epidermal growth factor receptor 2; LA, Luminal A; LB, Luminal B; HER2+, HER2 over expression; TN, Triple-negative.
The bold: p
To exclude the influence of confounding factors, we conducted a logistic analysis of the above factors. Multifactorial analysis showed that Rab10 expression was positively correlated with HER2 status (Exp (B) = 3.31, p = 0.03) and histological grade (Exp (B) = 2.66, p = 0.01). Unfortunately, Rab10 expression was not associated with BC molecular typing (Exp (B) = 0.54, p = 0.22) (Table 2), which could be related to the number of clinical samples.
Characteristic | B | Exp (B) | 95% CI for Exp (B) | p |
Year | –0.87 | 0.42 | 0.13–1.32 | 0.14 |
Grading | 0.98 | 2.66 | 1.28–5.55 | 0.01 |
pTNM stage | –0.76 | 0.47 | 0.10–2.26 | 0.35 |
T stage | 0.00 | 1.00 | 0.35–2.90 | 1.00 |
N stage | 0.29 | 1.34 | 0.44–4.05 | 0.60 |
Subtype | –0.63 | 0.54 | 0.20–1.47 | 0.22 |
ER status | –1.71 | 0.18 | 0.02–1.86 | 0.15 |
PR status | 0.42 | 1.52 | 0.24–9.62 | 0.66 |
HER2 status | 1.20 | 3.31 | 1.14–9.66 | 0.03 |
ER, estrogen receptor; PR, progesterone receptor; HER2, Human epidermal growth factor receptor 2; CI, Confidence interval.
The bold: p
This is the first report of a study involving Rab10 and a BC clinical cohort. Rab10 has been reported in recent years to be found in a variety of tumours. Our previous study showed that Rab10 can promote BC progression by regulating the TLR4 pathway [12]. However, a correlation between Rab10 and BC has not yet been extensively studied. Therefore, in the present study, we found that Rab10 expression was much higher in BC than in paraneoplastic tissues using bioinformatics analysis, which suggests that Rab10 may be involved in BC development and progression.
To demonstrate whether Rab10 offers potential as a clinical prognostic biomarker, we used the UALCAN database and Kaplan–Meier Plotter website to analyse the correlation between Rab10 and BC patient prognosis. The results showed that Rab10 was associated with RFS and OS in BC cases at 5 years. Unfortunately, the relationship between Rab10 and survival in clinical cohorts was not confirmed due to the lack of clinical data.
In a clinical cohort study, we found that Rab10 was expressed in human BC tissues and was associated with histological grade, molecular typing, and HER2 status. After excluding confounding factors, Rab10 expression was positively correlated with clinicopathological variables, including histological grade and HER2 status. Higher Rab10 expression levels implied higher tissue grading and HER2 expression. Histological grading can reveal the degree of differentiation of tumour cells. HER2 is amplified or overexpressed in approximately 20% of BCs and indicates a poor prognosis in the absence of systemic therapy [13]. This evidence further suggests that Rab10 may be a potential prognostic biomarker for BC or a molecular target for BC.
Since this was a retrospective study with a small sample size, there may have been potential selection bias. In addition, given the small tissue volume of BC samples from surgical punctures in neoadjuvant patients and the altered pathological characteristics of the samples after neoadjuvant therapy, neoadjuvant BC patients were not included in the study, so any correlation between Rab10 and BC molecular typing still needs to be validated in a larger clinical cohort for confirmation.
Due to the lack of tissue specimens and limitations inherent to IHC, only the location of Rab10 expression in BC tissues was confirmed in this study, and it was difficult to assess the differential expression of Rab10 protein in BC and paracancerous tissues. In addition, the results may be more convincing if the survival status analysis of Rab10 is performed using the BC clinical cohort.
HER2 is a type I transmembrane growth factor receptor localised to the cell membrane, and its biological activity is influenced by such cell membrane localisation [14]. In addition, activation of HER2 can cause cascade activation of AKT and MAPK signalling pathways, and the termination of these signalling cascades is mainly dependent on epidermal growth factor receptor (EGFR) endocytosis and its corresponding ligand-forming complexes recycled to the cell surface or degraded by various enzymes [15]. We note that Rab10, as a small GTPase, is a key regulator in eukaryotic membrane exocytosis and endocytosis pathways, involved in a wide range of membrane transport events [16]. Furthermore, dysfunction of Rab10 is able to activate AKT and MAPK signalling pathways [17]. We therefore hypothesised that Rab10 may influence HER2 expression and signal transduction at the cell membrane by participating in HER2 transport and localisation and may also participate in the co-regulation of AKT or MAPK signalling pathways for interactive functions. In addition, upregulation of Rab10 protein was observed in extracellular vesicles released from HER2+ BC cells resistant to trastuzumab, suggesting a possible association of Rab10 with HER2+ BC resistance [18]. Knockdown of Rab10 in hepatocellular carcinoma inhibits the proliferative capacity of hepatocellular carcinoma cells, induces G0/G1 phase arrest, and increases the level of apoptosis in hepatocellular carcinoma cells [7]. HER2 gene amplification in BC is associated with increased tumour cell proliferation, cell cycle progression, and decreased apoptosis [19]. The mechanism of action of Rab10 in BC, especially in HER2-positive BC, deserves further in-depth study.
In conclusion, this study indicates that Rab10 mRNA and protein are highly expressed in BC tissues, which is related to histological grading, molecular subtypes, and HER2 status. High expression of Rab10 reflects poor prognosis in BC patients and can be used as a potential clinical biomarker.
The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.
JZ took part in the data curation, formal analysis, investigation and drafting the article. YZ and JH were involved in clinical resources. HL, RH, YY, LD and AS analyzed the data. XY was responsible for methodology and project administration. WL made conceptualization, funding acquisition and final review of the manuscript. All authors contributed to editorial changes in the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors read and approved the final manuscript.
All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki and the Ethics Committee of the Affiliated Hospital of Hebei Engineering University approved study number 2021[K]019 for approval.
Not applicable.
This work was supported by the Natural Science Foundation of Hebei Province (H2021402015) and the Medical Science Research Project of Hebei Province (20231527).
The authors declare no conflict of interest.
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