Abstract
Lymphovascular invasion (LVI) is a common phenomenon in breast cancer (BC), and it is correlated to poor outcome. However, the biomarkers that influence the development of LVI remain to be defined. Through rigorous bioinformatics analyses, high mobility group protein 3 (HMGB3) was revealed as a driver gene that is associated with the presence of LVI. The purpose of this study was to further investigate the role of HMGB3 in the pathogenesis of LVI in BC. In vitro functional assays were performed to investigate the effect of HMGB3 silencing on cell proliferation, migration, adherence and transmigration of BC cell lines with dermal lymphatic endothelial cells (DLECs) and human vascular endothelial cells (HUVECs). The correlation of HMGB3 expression with clinicopathological parameters was also assessed at the transcriptomic and the proteomic levels using large BC cohorts with well-characterised LVI status. Silencing HMGB3 reduced cell proliferation, migration, adherence and transmigration across endothelial cell lines. At the mRNA and protein levels, high HMGB3 expression was significantly correlated with LVI-positivity, higher tumour grade, lymph nodal stage, hormone receptor negativity, HER2 positivity and poor outcome. Moreover, high HMGB3 expression was an independent predictor of shorter breast cancer-specific survival. HMGB3 plays an oncogenic function and contributes to the development of LVI in BC. Results warrant further investigation as a potential target to inhibit LVI in BC.
Keywords: Breast cancer, HMGB3, prognosis, progression, LVI
Introduction
Patients with early-stage breast cancer (BC) have experienced better outcomes as a result of early detection, enhanced diagnostic accuracy and targeted drug therapies [1]. Despite these improvements, metastasis remains the leading cause of BC-related mortality, affecting more than 20% of patients [2]. Various histopathological characteristics, such as tumour size, lymph node status and tumour grade, are strongly correlated with mortality [3,4]. Lymphovascular invasion (LVI) is considered an early event in the development of tumour metastasis and represents a significant predictor of poor outcome [5]. While the molecular profiles involved in tumour differentiation, such as histological type, grade, and the development of lymph node metastasis, have been well studied [6,7], the molecular mechanisms underlying LVI, which may serve as potential predictor biomarkers or therapeutic targets, remain unknown. Targeting LVI and its associated genes is a promising approach for inhibiting tumour dissemination in early-stage BC.
High mobility group (HMG) proteins are the second most predominant proteins in the cell, and they are involved in the global assembly of chromatin domains. HMG enhances transcriptional fine-tuning in response to abrupt environmental changes by interacting with nucleosomes, transcription factors, nucleosome remodelling complexes and histone H1 [8]. The high mobility group box (HMGB) family comprises chromosomal proteins that participate in DNA replication, transcription and repair [9,10]. Abnormal expression of HMGB is correlated with various cancer hallmarks, including uncontrolled replicative capacity, resistance to apoptosis, tissue invasion and metastasis [8,11]. The HMGB family includes HMGB1, HMGB2, HMGB3 and HMGB4 [12]. These members have 80% amino acid homology; however, their biological roles in cells are distinct [8,11]. Several studies have revealed that HMGB1, HMGB2 and HMGB3 play a significant role in a variety of malignancies, including hepatocellular carcinoma [13], pancreatic cancer [14] and colon cancer [15].
Among the HMGB family members, HMGB3, is one of the highly expressed genes associated with LVI positivity in BC as determined in the weighted average difference (WAD) bioinformatics analyses [16]. In normal tissue, HMGB3 expression is high during embryogenesis, with low or no expression in normal adult tissue [17,18]. High expression of HMGB3 is strongly associated with the occurrence of numerous tumours and poor prognosis of advanced tumours of the lung [19], bladder [20] and in prostate cancer [21]. To enhance tumour development, HMGB3 can control the cell cycle and stimulate the proliferation and invasion of cancer cells via the Wnt/beta-catenin, MAPK and other signalling pathways. HMGB3 can also increase the reactive oxygen species (ROS) formation and tumour cell growth by stimulating the expression of HIF-1α [22]. However, the exact role of HMGB3 in LVI in BC is unclear as it has not been previously investigated. This study aimed to assess the in vitro mechanistic role of HMGB3 in BC cell lines, with emphasis in its role in LVI development, and to investigate the clinicopathological significance of HMGB3 at the transcriptomic and proteomic levels using large BC cohorts with long-term follow-up.
Materials and methods
Pre-clinical studies
In vitro studies of HMGB3
Pre-clinical investigations using the following cells and assays were conducted to determine the possible involvement of HMGB3 in LVI and other biological functions.
Breast cancer cells
The protein expression levels of HMGB3 were used to select BC cell lines (American Type Culture Collection (ATCC, Manassas, VA, USA)) for in vitro investigations. The BC cell lines MCF-7 (luminal oestrogen receptor (ER+)/progesterone receptor (PR+)/human epidermal growth factor 2 (HER2-)), SK-BR-3 (HER2+, ER-/PR-) and MDA-MB-231 (triple negative, ER-/PR-/HER2-) were used in this study. MCF-7 and MDA-MB-231 were cultured in Roswell Park Memorial Institute (RPMI 1640) medium with L-glutamine (Cytiva, SH30027.01, UK) supplemented with 10% foetal bovine serum (FBS) (Sigma, F9665, UK), while the SK-BR-3 cell line was grown in McCoy’s 5A medium modified with L-glutamine and sodium bicarbonate liquid (Sigma, M9309, UK) supplemented with 10% FBS. Western blot (WB) was performed to detect relative protein expression in all BC cell lines, and the LI-COR Odyssey machine was used for quantification.
Endothelial cells
To investigate the impact of HMGB3 on LVI, primary human umbilical vein endothelial cells (HUVECs) and dermal lymphatic endothelial cells (DLECs) were used as in vitro models of tumour-endothelial interactions. HUVECs and DLECs were purchased from Promocell (C12218, Heidelberg, Germany), and cultured in the endothelial cell growth medium MV2 (Promocell, C-22022, Germany).
All cell lines were tested for Mycoplasma monthly, cultured under a sterile condition in a class II cabinet and incubated with 5% CO2 at 37°C.
Silencing HMGB3 using siRNA
A siRNA-based approach was used to study the potential functional consequences of HMGB3 knockdown, and its role in BC progression and LVI. Two independent pre-validated Silencer Select siRNA constructs, mainly for HMGB3, or scrambled negative control siRNA (Silencer® Select siRNA, AM4611, ThermoFisher Scientific), that did not target HMGB3, MCF-7, SK-BR-3 and MDA-MB-231, were transfected using the forward transfection method using Opti-MEM medium, 25 pmol siRNA, and LipofectamineTM RNAiMAX (13778150; ThermoFisher Scientific, Loughborough, UK). The sequences of HMGB3-siRNA were as follows: 5’-GCACCCUGAAACUGUAUCAtt-3’ and 5’-CCGAGACAAACCCUUGAUGtt-3’. Similar knockdown was observed on both siRNA targeting HMGB3, so siRNA with 5’-GCACCCUGAAACUGUAUCAtt-3’ sequence was prioritised for the following in vitro studies (Supplementary Figure 1).
MTS assay
The effect of HMGB3 knockdown on the proliferation of tumour cells was evaluated via the AQueous Non-radioactive Cell Proliferation Assay assay (Promega (G3580); CellTitre 96 Aqueous One Solution Cell Proliferation Assay) according to the manufacturer’s protocol.
Colony formation assay
BC cell lines were seeded and grown in culture medium in an incubator for 14 days. Following incubation, colonies were washed with phosphate-buffered saline (PBS), fixed with methanol for 30 min, stained with crystal violet and counted using a microscope.
Wound healing assay
In this assay, the ability of tumour cells to migrate was assessed by measuring the wound repair rate of HMGB3 knockdown and control at the following time points after transfection: T0h, T24h, and T48h. A Culture-Inserts 2 wells (Thistle Scientific Ltd., IB-81176) with a built-in gap was used according to the manufacture protocol. The wounds were observed by taking images at 10× microscopic magnification several times via light microscopy (Leica Microsystems, Lecia DMI 3000B, Germany). The wound area was measured, and the percentage of wound closure was calculated using Image J software (1.52 version).
Static adhesion assay
In a 24-well plate, endothelial cells (HUVECs and DLECs) were seeded to confluence. Tumour cell adhesion was determined after cells were labelled with 1 μM Cell Tracker Green CMFDA (Invitrogen, C2925), and incubated for 30 min at 37°C. Following labelling, the tumour cells were resuspended in medium supplemented with serum, and incubated for 35 min at 37°C with endothelial cell monolayers. Non-adherent cells were washed away with tumour cell medium, and adherent tumour cells were counted using a fluorescent microscope (Lecia DMI 3000B, Leica Microsystems, Germany) at a 10× magnification. The findings were represented as the absolute number of cells adhering to the endothelial layer as well as the percentage of cells adhering in comparison to the control.
Transmigration assay
A confluent endothelial cell monolayer was grown on hanging transwell inserts (Sigma, MCEP24H48). Tumour cell transmigration was determined following labelling with 1 μM Cell Tracker Green CMFDA (Invitrogen, C2925). To ensure the confluency and integrity of the endothelial cell barrier, lucifer yellow leakage was used (Invitrogen, L453). After 16 hours, transmigration was observed using a fluorescent microscope (Lecia DMI 3000B, Leica Microsystems, Germany) by counting cells at the bottom of the chamber.
Further clinical studies using large BC cohorts to assess the prognostic and clinicopathological significance of HMGB3 at the transcriptomic and proteomic levels were performed.
Clinical studies
Study cohorts
In this study, three well characterised BC cohorts were used.
Molecular taxonomy of breast cancer international consortium (METABRIC) cohort
At the transcriptomic level, the METABRIC cohort was used to evaluate the expression of HMGB3. This study enrolled a total of 1980 patients with primary operable invasive BC, and information regarding the validated clinicopathological and transcriptomic data was readily available [23]. The Illumina Totalprep RNA amplification kit (Ambion, Warrington, UK) was used to generate biotin-labelled cRNA from total RNA, which was then hybridised on Illumina Human HT-12 v3 platforms to evaluate mRNA expression.
The cancer genome atlas (TCGA) cohort
mRNA expression of HMGB3 was also evaluated using the TCGA BC cohort (n=854). The cohort was accessed for RNA-SeqV2-derived mRNA expression. De-identified clinical information for the patients was also accessed, with certain clinicopathological features and outcomes from cBioPortal [24].
Nottingham BC cohort
The expression of HMGB3 at the protein level was assessed using tissue samples from the well-characterised Nottingham invasive BC cohort. A total of 1647 cases were valid for evaluating HMGB3 protein expression from patients who had previously undergone surgery at Nottingham City Hospital. For each patient, a robust clinicopathological profile and outcome data were readily available. These profiles include the patient’s age at the time of diagnosis, tumour size and grade, lymph node stage, LVI status, and the Nottingham Prognostic Index (NPI). This cohort has ER, PR, and HER2 data [25-28]. The patients’ profiles included also data on BC-specific survival (BCSS) and time to distant metastasis (TTDM). BCSS is defined as the time in months, from the time when the patients underwent surgery to when they died from BC. TTDM is referred to the time in months, from when the patients underwent surgery to when the first distant metastasis occurred. Patient management was uniform and based on tumour features as determined by NPI and hormone receptor status. Patients with an NPI score ≤3.4, representing the excellent prognostic group, underwent no adjuvant therapy, while those with an NPI>3.4 who were ER positive were offered tamoxifen (with or without goserelin [Zoladex] in premenopausal patients). Patients who were ER-negative and fit enough to receive chemotherapy, received cyclophosphamide, methotrexate, and 5-flurouracil (CMF). In this study, no patients received neoadjuvant therapy or Herceptin.
The associations between HMGB3 and the available epithelial-mesenchymal transition (EMT)-related markers, such as E-cadherin, N-cadherin, P-cadherin, TGFβ1, and TWIST2 [29,30], were investigated.
HMGB3 protein expression
For immunohistochemistry (IHC) staining of the primary rabbit polyclonal anti-HMGB3 antibody (HPA062583, Sigma-Aldrich, UK), HMGB3 protein expression was determined using the Nottingham BC cohort. Tissue microarrays (TMAs) of the study cohort were prepared using a TMA Grand Master® [31]. Antigen retrieval was performed according to the manufacturer’s guidelines (citrate buffer, pH 6, at 1000 W for 20 minutes using microwave energy). The expression of the HMGB3 antibody was assessed by staining the TMAs with a Novolink polymer detection systems kit (Code: RE7280-K, Leica, Biosystems, UK). This involved incubating 4 µm sections with HMGB3 antibody (dilution 1:500) for 60 minutes in Leica antibody diluent (RE AR9352, Leica, Biosystems, UK). As a positive control, ovarian tissue was used, whereas normal kidney tissue was used as a negative control (Figure 1A and 1B). Staining for immunoreactivity was quantified using a modified histochemical score (H-score) based on semi-quantitative scoring. The scoring was done for the entire field, and the nuclear staining intensity was classified as follows: score 0= negative, score 1= weak staining, score 2= moderate staining, and score 3= strong staining. The percentage of each group was calculated (0-100%). The H-score, which ranges from 0 to 300, was calculated by multiplying the intensity of staining by the percentage of staining [32]. Two observers scored the TMAs, and the interclass correlation coefficient (ICC) test was used to assess the concordance of HMGB3.
Figure 1.
Nuclear expression of HMGB3 protein in invasive breast cancer. (A) Positive control of ovarian tissue stained by HMGB3, (B) Negative control of normal kidney stained by HMGB3, (C) Positive HMGB3 IHC expression and (D) Negative HMGB3 IHC expression. Magnification 10×. Scale bars =200 μm. Inset, magnification 20×. Scale bars =100 μm.
Statistical analysis
For statistical analysis, GraphPad Prism 3.02 software and SPSS version 24 (Chicago, IL, USA) were used. In vitro results were represented as the mean ± standard error of the mean (SEM) of three independent experiments performed in triplicate. The significant differences between the control and silencing HMGB3 were determined using a student’s t-test.
Using continuous data on HMGB3 mRNA and protein levels, the correlations with clinicopathological characteristics were investigated. One-way analysis of variance (ANOVA) with the post-hoc Tukey multiple comparison test (for parametric data) or Kruskal-Wallis test (for non-parametric distribution) was used to analyse differences between three or more groups. To compare two groups, the Student t-test (parametric data) or Mann-Whitney test (non-parametric distribution) were employed. To categorise the expression of HMGB3 mRNA, the median was used, with H-score of 50 was used for protein. The Spearman correlation test was used for correlation analysis. For univariate survival analysis, the Log-rank and Kaplan-Meier curve tests were utilised, whereas for multivariate survival analysis, the Cox Regression model was used. A p-value <0.05 was considered statistically significant.
The guidelines for reporting recommendations for tumour marker prognostic studies (REMARK) were followed in this study [33] (Supplementary Table 1).
Results
Pre-clinical studies
Impacts of HMGB3 silencing in BC cells
The expression of the HMGB3 protein was determined in four BC cell lines, including HER2-enriched (SK-BR-3), luminal (MCF-7 and ZR-75-1), and triple-negative (MDA-MB-231) cells (Supplementary Figure 1A). The highest levels were seen in SK-BR-3, MDA-MB-231 and MCF-7, and the lowest ones in ZR-75-1 which was excluded from the subsequent experiments (Supplementary Figure 1B).
When HMGB3 knockdown expression was compared to β-actin expression in MCF-7, SK-BR-3, and MDA-MB-231 at days 3, 5 and 7 post-transfection, complete reduction of HMGB3 protein expression was seen (Supplementary Figure 1C-N).
Down regulation of HMGB3 resulted in a considerable decrease in BC proliferation rate, which was demonstrated in MCF-7 (P=0.0120 at T24h, P=0.0247 at T48h and P=0.0160 at T72h), SK-BR-3 (P=0.0027 at T24h, P<0.0001 at T48h and P<0.0001 at T72h), and MDA-MB-231 (P=0.0014 at T24h, P=0.0092 at T48h and P=0.0060 at T72h), as compared to the control (Figure 2A-C).
Figure 2.
The effect of HMGB3 knockdown (KD) by siRNA on cell proliferation in (A) MCF-7, (B) SK-BR-3 and (C) MD-MB-231 cells. (A-C) Cell proliferation was significantly reduced after KD in BC cell lines as detected by the MTS assay. HMGB3 siRNA transfection reduced the ability of BC cell lines to colonise in (D) MCF-7, (E) SK-BR-3 and (F) MD-MB-231 as detected by the colony formation assay. Results are presented as mean ± standard error of the mean (SEM) of three independent experiments. The p-values are * ≤0.05, ** ≤0.01, *** ≤0.001 and **** ≤0.0001.
In addition, a clonogenic experiments were performed to examine how HMGB3 knockdown affected cell growth and survival. The capacity of a single cell colony to survive after being transfected was much lower than the control in MCF-7, SK-BR-3, and MDA-MB-231 (all P<0.0001) (Figure 2D-F). The knockdown of HMGB3 resulted in a significantly larger unhealed wound in comparison to the negative controls in MCF-7, SK-BR-3, and MDA-MB-231 (P<0.0001, P=0.0040, and P=0.0002, respectively) (Figure 3A-C).
Figure 3.
The effect of HMGB3 knockdown (KD) by siRNA on cell migration in (A) MCF-7, (B) SK-BR-3 and (C) MD-MB-231 cells. The wound repair rate of HMGB3 KD and control cells was observed by measuring the width of the gap left unhealed at T0h, T24h and T48h. (A-C) Silencing HMGB3 significantly reduced the migration rate in BC cell lines as detected by the wound healing assay. Results are presented as mean ± standard error of the mean (SEM) of three independent experiments. The p-values are * ≤0.05, ** ≤0.01, *** ≤0.001 and **** ≤0.0001.
The role of HMGB3 in the interaction between BC cells and endothelial cells
There was a higher adherence to HUVECs in the HMGB3-untransfected cells than HMGB3-transfected cells in MCF-7 (P=0.0002), SK-BR-3 (P=0.0068), and MDA-MB-231 (P<0.0001); higher adherence to DLECs than HMGB3-transfected cells was observed in cell lines MCF-7 (P=0.0205), SK-BR-3 (P=0.0006), and MDA-MB-231 (P=0.0001) (Figure 4A-C).
Figure 4.
Representative photomicrographs of tumour cell adhesion across vascular and lymphatic endothelial cells (HUVECs and DLECs). (A) MCF-7, (B) SK-BR-3 and (C) MD-MB-231. (A-C) Silencing HMGB3 decreased the number of all BC cells that adhered with HUVECs and DLECs. Results are presented as mean ± standard error of the mean (SEM) of three independent experiments. The p-values are * ≤0.05, ** ≤0.01, *** ≤0.001 and **** ≤0.0001. Magnification 10×. Scale bars =200 μm.
Tumour cell transmigration through HUVECs was higher in the control than in the knockdown group as demonstrated in MCF-7 (P=0.0208), SK-BR-3 (P=0.0260), and MDA-MB-231 (P<0.0001); transmigration through DLECs was higher in the control than in the knockdown group in MCF-7 (P=0.0307), SK-BR-3 (P=0.0006), and MDA-MB-231 (P=0.0008) (Figure 5A and 5B).
Figure 5.
Representative photomicrographs of tumour cell (MCF-7, SK-BR-3 and MD-MB-231) transmigration across (A) HUVECs and (B) DLECs. (A, B) The number of tumour cells that transmigrated across HUVECs and DLECs was higher in the control group than in the KD group. Results are presented as mean ± standard error of the mean (SEM) of three independent experiments. The p-values are * ≤0.05, ** ≤0.01, *** ≤0.001 and **** ≤0.0001.
The role of HMGB3 in LVI and BC outcome in the clinical BC cohorts
HMGB3 mRNA expression
The METABRIC and TCGA cohorts were used to assess HMGB3 mRNA expression. High HMGB3 mRNA expression was observed in 901/1980 (46%) of the METABRIC BC cases, and in 427/854 (50%) of the TCGA cases. In both transcriptomic datasets, high expression of HMGB3 mRNA was significantly associated with LVI positivity, and high tumour grade (all P<0.0001). In the METABRIC cohort, high expression of HMGB3 mRNA was significantly correlated with larger tumour size (P=0.001), poor NPI (P<0.0001), and higher LN stage (P=0.048; Table 1).
Table 1.
Statistical associations between HMGB3 mRNA expression and clinicopathological parameters in the METABRIC (n=1980) and TCGA (n=854) breast carcinoma datasets
| Parameters | HMGB3 mRNA (METABRIC) | HMGB3 mRNA (TCGA) | ||||
|---|---|---|---|---|---|---|
|
|
|
|||||
| Number (%) | Mean Rank | P value | Number (%) | Mean Rank | P value | |
| Patient Age (year) | ||||||
| ≤50 | 424 (21.4) | 7.58 | 0.695 | 231 (27) | 435.0 | 0.587 |
| >50 | 1556 (78.6) | 7.56 | 623 (73) | 424.1 | ||
| Lymphovascular Invasion (LVI) | ||||||
| Negative | 930 (59) | 7.46 | <0.0001 | 559 (65) | 403.1 | <0.0001 |
| Positive | 635 (41) | 7.65 | 295 (35) | 437.7 | ||
| Tumour Size | ||||||
| ≤2 cm | 622 (31.7) | 7.42 | 0.001 | 239 (28) | 411.8 | 0.245 |
| >2 cm | 1338 (68.3) | 7.62 | 615 (72) | 433.6 | ||
| Tumour Grade | ||||||
| I | 170 (9.0) | 7.09 | <0.0001 | 89 (11) | 267.2 | <0.0001 |
| II | 770 (40.6) | 7.43 | 375 (46) | 338.9 | ||
| III | 952 (50.3) | 7.77 | 352 (43) | 518.4 | ||
| Lymph node stage | Not available | |||||
| I (Negative nodes) | 1035 (52.5) | 7.51 | 0.048 | |||
| II (1-3 positive nodes) | 622 (31.5) | 7.63 | ||||
| III (>3 positive nodes) | 316 (16.0) | 7.62 | ||||
| Nottingham prognostic index (NPI) groups | Not available | |||||
| Good | 680 (34.3) | 7.35 | <0.0001 | |||
| Moderate | 1101 (55.6) | 7.67 | ||||
| Poor | 199 (10.1) | 7.78 | ||||
| Oestrogen Receptor (ER) | ||||||
| Negative | 474 (23.9) | 7.82 | <0.0001 | 185 (22) | 580.9 | <0.0001 |
| Positive | 1506 (76.1) | 7.49 | 639 (78) | 363.7 | ||
| Progesterone Receptor (PR) | ||||||
| Negative | 940 (47.4) | 7.65 | <0.0001 | 272 (33) | 510.4 | <0.0001 |
| Positive | 1040 (52.6) | 7.49 | 546 (67) | 359.2 | ||
| Human epidermal growth factor receptor 2 (HER2) | ||||||
| Negative | 1733 (87.5) | 7.48 | <0.0001 | 567 (81) | 339.9 | 0.004 |
| Positive | 247 (12.5) | 8.18 | 133 (19) | 395.6 | ||
| PAM50 Subtypes | Not available | |||||
| Luminal A | 718 (36.4) | 7.31 | <0.0001 | |||
| Luminal B | 488 (24.7) | 7.81 | ||||
| HER2+ enriched | 240 (12.1) | 8.15 | ||||
| Basal like | 329 (16.7) | 7.65 | ||||
| Normal like | 199 (10.1) | 7.04 | ||||
P values in bold are statistically significant.
In both METABRIC and TCGA datasets, ER-, PR-, and HER2+ tumours all demonstrated significantly high expression of HMGB3 mRNA (all P<0.0001) (Table 1). Analysis of the METABRIC cohort regarding the intrinsic (PAM50) subtypes showed that high HMGB3 mRNA expression was associated with HER2+, luminal B, basal-like, luminal A, and normal-like subtypes in descending order (all P<0.0001) (Table 1).
Survival analysis of HMGB3 mRNA showed that high expression was associated with shorter BCSS in both the METABRIC and TCGA cohorts (P<0.0001 and P=0.003, respectively) (Figure 6A and 6B).
Figure 6.
Kaplan-Meier survival plots showing the association between HMGB3 mRNA expression and breast cancer-specific survival (BCSS) in (A) whole cohort (METABRIC) and (B) whole cohort (TCGA). Associations are also shown in the Nottingham cohort between HMGB3 protein expression and (C) BCSS and (D) total time to distant metastasis (TTDM).
HMGB3 protein expression
HMGB3 protein expression ranged from negative to strong in the nucleus of invasive BC cells (Figure 1C and 1D). High HMGB3 protein expression was observed in 787/1647 (47.8%) invasive BC cases. There was a high degree of concordance between the TMAs scored by both scorers in HMGB3 immunoscoring (ICC=0.8, P<0.0001).
Similar to the transcriptomics results, high HMGB3 protein expression was significantly associated with aggressive tumour features, including LVI positivity, younger age, higher tumour grade, poor NPI, high mitotic count (all P<0.0001), high pleomorphism (P=0.005), high tubular formation (P=0.001), and higher LN stage (P=0.049) (Table 2). The correlation of HMGB3 protein expression with IHC subtypes was comparable to the mRNA findings, with HMGB3 protein expression being highest in HER2+ tumours, followed by TN tumours. Within the ER+/HER2- group, the high-proliferation class had considerably higher HMGB3 expression than the low-proliferation class (P<0.0001) (Table 2). At the protein level, there was a significant association between the high expression of HMGB3 and shorter BCSS and TTDM (all P<0.0001) (Figure 6C and 6D). In all cohorts, multivariate Cox regression analysis showed that higher HMGB3 expression predicted poor BCSS independent of the tumour size and grade, LN stage, and LVI (HR 1.4; 95% CI=1.1-1.7; P=0.004 for METABRIC cohort, HR 1.7; 95% CI=1.1-2.8; P=0.028 for TCGA cohort, and HR 1.4; 95% CI=1.1-1.7; P=0.003 for Nottingham cohort) (Table 3).
Table 2.
Statistical associations between HMGB3 protein expression and the clinicopathological factors in the Nottingham breast cancer (BC) cohort (n=1647)
| Parameters | HMGB3 protein | ||
|---|---|---|---|
|
| |||
| Number % | Mean Rank | P value | |
| Patient Age (year) | |||
| ≤50 | 574 (35) | 882.3 | <0.0001 |
| >50 | 1070 (65) | 790.4 | |
| Lymphovascular Invasion (LVI) | <0.0001 | ||
| Negative | 1096 (67) | 790.1 | |
| Positive | 541 (33) | 877.6 | |
| Tumour Size | 0.668 | ||
| ≤2 cm | 914 (56) | 815.5 | |
| >2 cm | 725 (44) | 825.6 | |
| Tumour Grade | <0.0001 | ||
| I | 206 (13) | 697.9 | |
| II | 584 (52) | 779.4 | |
| III | 854 (35) | 882.0 | |
| Mitosis Scores | <0.0001 | ||
| I | 622 (38.3) | 715.66 | |
| II | 312 (19.2) | 838.60 | |
| III | 686 (42.5) | 883.71 | |
| Pleomorphism Scores | 0.005 | ||
| I | 21 (1.3) | 674.48 | |
| II | 465 (28.7) | 759.01 | |
| III | 1135 (70) | 834.83 | |
| Tubular formation | 0.001 | ||
| I | 83 (5.1) | 619.11 | |
| II | 472 (29.1) | 814.69 | |
| III | 1066 (65.8) | 824.31 | |
| Lymph Node Stage | 0.049 | ||
| I (Negative nodes) | 975 (60) | 799.5 | |
| II (1-3 positive nodes) | 500 (31) | 843.9 | |
| III (>3 positive nodes) | 167 (9) | 882.7 | |
| Nottingham Prognostic Index (NPI) groups | <0.0001 | ||
| Good | 462 (28) | 733.2 | |
| Moderate | 880 (54) | 853.1 | |
| Poor | 295 (18) | 851.8 | |
| Oestrogen Receptor (ER) | 0.001 | ||
| Negative | 386 (23) | 895.1 | |
| Positive | 1262 (77) | 802.9 | |
| Progesterone Receptor (PR) | 0.108 | ||
| Negative | 673 (41) | 834.7 | |
| Positive | 951 (59) | 796.9 | |
| HER2 status | <0.0001 | ||
| Negative | 1402 (86) | 84.5 | |
| Positive | 225 (14) | 997.9 | |
| Triple Negative phenotype | 0.185 | ||
| No | 1359 (83) | 811.1 | |
| Yes | 276 (17) | 852.2 | |
| Immunohistochemistry Subtypes | <0.0001 | ||
| ER+/HER2- Low Proliferation | 528 (34) | 655.6 | |
| ER+/HER2- High Proliferation | 522 (33.7) | 802.2 | |
| Triple Negative | 275 (17.7) | 809.2 | |
| HER2+ | 225 (14.6) | 953.5 | |
P values in bold are statistically significant.
Table 3.
Multivariate Cox regression for predictors of breast cancer-specific survival (BCSS) and HMGB3 mRNA expression in the METABRIC and TCGA cohorts and protein expression in the Nottingham BC cohort
| METABRIC Cohort | ||||
|
| ||||
| Parameters | Hazard ratio (HR) | 95% confidence interval (CI) | Significance P value | |
|
| ||||
| Lower | Upper | |||
|
| ||||
| HMGB3 mRNA Expression | 1.391 | 1.113 | 1.739 | 0.004 |
| Tumour Size | 1.474 | 1.126 | 1.928 | 0.005 |
| Lymph Nodal Stage | 1.969 | 1.538 | 2.520 | <0.0001 |
| Tumour Grade | 1.456 | 1.192 | 1.779 | <0.0001 |
| Lymphovascular Invasion (LVI) | 1.469 | 1.159 | 1.862 | 0.001 |
|
| ||||
| TCGA Cohort | ||||
|
| ||||
| Parameters | Hazard ratio (HR) | 95% confidence interval (CI) | Significance P value | |
|
| ||||
| Lower | Upper | |||
|
| ||||
| HMGB3 mRNA Expression | 1.728 | 1.061 | 2.816 | 0.028 |
| Tumour Size | 1.523 | 0.873 | 2.656 | 0.138 |
| Lymph Nodal | 1.196 | 0.712 | 2.010 | 0.498 |
| Tumour Grade | 1.092 | 0.761 | 1.567 | 0.634 |
| Lymphovascular Invasion (LVI) | 1.878 | 1.149 | 3.070 | 0.012 |
|
| ||||
| Nottingham BC Cohort | ||||
|
| ||||
| Parameters | Hazard ratio (HR) | 95% confidence interval (CI) | Significance P value | |
|
| ||||
| Lower | Upper | |||
|
| ||||
| HMGB3 Protein Expression | 1.350 | 1.104 | 1.650 | 0.003 |
| Tumour Size | 1.383 | 1.116 | 1.714 | 0.003 |
| Lymph Nodal Stage | 1.708 | 1.478 | 1.973 | <0.0001 |
| Tumour Grade | 1.649 | 1.375 | 1.977 | <0.0001 |
| Lymphovascular Invasion (LVI) | 1.620 | 1.307 | 2.009 | <0.0001 |
P values in bold are statistically significant.
A positive linear correlation between HMGB3 protein and mRNA expression was observed in the Nottingham subset (n=288) of the METABRIC cohort based on the Spearman’s rank correlation coefficient (r=0.2, P=0.016).
HMGB3 expression and LVI related biomarkers
To further assess the role of HMGB3 in BC and its relationships with other genes involved in various LVI processes, the METABRIC and TCGA datasets were analysed for correlations between HMGB3 mRNA expression and other genes related to invasion, EMT and adhesion. A significant weak to moderate positive linear correlation was observed between HMGB3 and N-cadherin, P-cadherin, GSK3B, TWIST1, TWIST2, ZEB1, ZEB2, NFKB1, and CTNNB1, while the correlation was negative with E-cadherin (all P<0.05). Moreover, there was a weak to moderate positive linear correlation between HMGB3 and the expression of various MMPs, including MMP1, MMP7, MMP9, MMP12, MMP15, and MMP25 (all P<0.05) (Table 4).
Table 4.
Correlations of HMGB3 expression with mRNA and protein expression of epithelial-mesenchymal transition (EMT) and matrix metalloproteinase- (MMP-) related genes
| Gene names | METABRIC cohort | TCGA cohort | Nottingham cohort | |||
|---|---|---|---|---|---|---|
|
|
|
|
||||
| Correlation value | P value | Correlation value | P value | Correlation value | P value | |
| EMT related genes | ||||||
| E-cadherin | -0.252 | <0.0001 | -0.197 | <0.0001 | -0.128 | 0.001 |
| N-cadherin | 0.118 | <0.0001 | 0.169 | <0.0001 | 0.099 | 0.023 |
| P-cadherin | 0.064 | 0.005 | 0.196 | <0.0001 | 0.125 | 0.002 |
| TGFβ1 | 0.001 | 0.947 | 0.216 | <0.0001 | 0.052 | 0.827 |
| TWIST1 | 0.149 | <0.0001 | 0.149 | <0.0001 | Not available | |
| TWIST2 | 0.282 | <0.0001 | 0.2173 | <0.0001 | 0.062 | 0.186 |
| ZEB1 | 0.202 | <0.0001 | 0.347 | <0.0001 | Not available | |
| ZEB2 | 0.231 | <0.0001 | 0.197 | <0.0001 | ||
| NFKB1 | 0.114 | <0.0001 | 0.150 | <0.0001 | ||
| GSK3B | 0.286 | <0.0001 | 0.121 | <0.0001 | ||
| CTNNB1 | 0.138 | <0.0001 | 0.074 | 0.032 | ||
| MMPs related genes | ||||||
| MMP1 | 0.188 | <0.0001 | 0.382 | <0.0001 | Not available | |
| MMP7 | 0.056 | 0.013 | 0.104 | 0.002 | ||
| MMP9 | 0.133 | <0.0001 | 0.184 | <0.0001 | ||
| MMP11 | 0.138 | <0.0001 | 0.016 | 0.640 | ||
| MMP12 | 0.137 | <0.0001 | 0.273 | <0.0001 | ||
| MMP15 | 0.315 | <0.0001 | 0.214 | <0.0001 | ||
| MMP20 | 0.023 | 0.299 | 0.031 | 0.367 | ||
| MMP25 | 0.051 | 0.023 | 0.069 | 0.043 | ||
P values in bold are statistically significant.
The correlation between HMGB3 and EMT-related markers, such as E-cadherin, N-cadherin, and P-cadherin was further studied at the protein level using the Nottingham BC cohort. There was a significant weak negative correlation between HMGB3 and E-cadherin (P=0.001), while the correlation was positive with N-cadherin (P=0.023), and P-cadherin (P=0.002) (Table 4).
Disscussion
HMGB3 is a multifunctional protein that performs a variety of functions in many cellular compartments, and has been identified as a critical regulator of tumour development [34]. Recently, aberrant HMGB3 was identified as a pro-carcinogen, promoting tumour development, proliferation, invasion, and metastasis in a variety of tumour types, including gastric [35], lung [36], colorectal [12], and urinary bladder tumours [20]. Although HMGB3 has previously been associated with cancer cell proliferation and migration [12], no studies have been performed to date to investigate the potential role of HMGB3 in the development of LVI.
HMGB3 was determined as a key gene associated with LVI positivity through stringent bioinformatics analysis [16]. Similarly, subsequent research employing bioinformatic and co-expression analyses revealed a link between HMGB3 and the development of human cancers, including gastric cancer [8]. This study aimed to evaluate the in vitro mechanistic role of HMGB3 in BC cell lines with emphasis on the role in LVI development, and to investigate the clinicopathological significance of HMGB3 at the transcriptomic and proteomic levels using large BC cohorts with long-term clinical follow-up.
The conducted pre-clinical experiments showed that silencing HMGB3 suppressed cell proliferation and growth. Tumour cells must proliferate and evade apoptosis to penetrate surrounding tissue and develop metastatic cascades; proliferation continues until tumour cells invade the vascular or lymphatic channels. In vitro functional assays showed that silencing HMGB3 in BC cell lines reduced migration. This finding supports a study that evaluated the expression of β-catenin, a major WNT pathway protein, to determine that HMGB3 stimulated colorectal cancer migration via the WNT/beta-catenin pathway. HMGB3 can increase the expression of β-catenin, and the downstream genes c-Myc and MMP7 [12].
The malignant features and mechanism of action of HMGB3 in LVI remain unknown. Importantly, in this study, HMGB3 knockdown reduced adhesion and transmigration across endothelial cell lines in MCF-7, SK-BR-3, and MDA-MB-231; the positive associations between EMT- and MMP-related markers support these results. Although the association between HMGB3 and these biomarkers ranged from weak to moderate correlation, it was statistically significant which indicates that these markers are contributing to the same oncogenic pathway in the context of LVI process. N-cadherin is associated with EMT, which is required for cell invasion and intravasation into the bloodstream and for extracellular matrix (ECM) destruction induced by protease synthesis. Increased N-cadherin expression thus results in the loss of the connection between the epithelium of BC cells and other epithelial cells, leading to invasion into the stroma [37]. Additionally, P-cadherin, a critical protein that may activate integrin molecules, enables cancer cells to adhere to the ECM and initiates invasion [38]. The microenvironment of tumours exhibiting LVI is closely associated with the expression of MMPs, notably MMP9 and MMP1. The expressions of these MMPs are involved in the intravasation and metastasis of BC cells [39]. This finding also corroborates a previous study reporting that, by limiting the activity of MMP9, downregulation of HMGB3 expression decreased the invasion of gastric cancer cells [22]. Although the in vitro studies revealed that overexpression of HMGB3 aided migration, adhesion and transmigration via endothelial cell lines, triggering the LVI process in the MCF-7, SK-BR-3, and MDA-MB-231 cell lines, more mechanistic investigations into how HMGB3 triggers LVI and in vivo animal experiments are warranted.
This study evaluated the clinicopathological and prognostic significance of HMGB3 expressions using large, and swell-annotated BC cohorts. High expression of HMGB3 at the mRNA and protein levels was associated with aggressive BC features, including LVI positivity, higher tumour grade, poor NPI, ER/PR negativity, and HER2 positivity. High HMGB3 expression at the mRNA and protein levels was significantly associated with poor BCSS and TTDM, and this association was independent of other prognostic factors, which is consistent with previous studies [15,36]. A weak correlation between the protein and mRNA expressions was observed which could be explained by the rate at which mRNA is translated into protein, which is often referred to as ‘translational efficiency’. Translational efficiency has a significant influence on both mRNA and protein levels [40]. The subjectivity of the H-score method in the interpretation of expression in IHC staining sections is another potential reason [41], in addition to the use of whole tissue, including different cell types, in the METABRIC cases.
In conclusion, our findings suggest that HMGB3 has an oncogenic role in BC, and that it is involved in the pathogenesis of LVI. This study demonstrated the impact of HMGB3 silencing on several processes of tumour development related to LVI, including migration, adhesion and transmigration. Although the findings of this study suggest that HMGB3 is a critical gene in LVI, especially in light of its impact on lymphatic invasion, and is a precursor for the metastatic cascade, more research is required to further substantiate these findings.
Acknowledgements
Abrar I Aljohani is supported and funded by Taif University, Kingdom of Saudi Arabia. The authors are part of the PathLAKE digital pathology consortium. These new Centres are supported by a £50m investment from the Data to Early Diagnosis and Precision Medicine strand of the government’s Industrial Strategy Challenge Fund, managed and delivered by UK Research and Innovation (UKRI).
Informed consent was obtained from all individuals prior to surgery to use their tissue materials in research.
Disclosure of conflict of interest
None.
Supporting Information
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