HMGA1 is associated with unfavorable outcome and accelerates the aggressiveness of cholangiocarcinoma

Abstract

Introduction

Cholangiocarcinoma (CCA) is a highly metastatic bile duct cancer with the highest global incidence in Northeastern Thailand. Most patients are diagnosed at advanced stages, necessitating the identification of novel prognostic markers and therapeutic targets. High mobility group A1 (HMGA1) is a non-histone chromosomal protein that orchestrates the transcription of genes involved in tumor progression, and its overexpression has been implicated in multiple malignancies. Aims: This study aimed to investigate the clinical significance and oncogenic roles of HMGA1 in CCA progression.

Methods

HMGA1 expression was evaluated in a hamster CCA model and human CCA tissues using immunohistochemistry. The functional effects of HMGA1 on cell growth, migration, and invasion, along with the underlying molecular mechanisms were investigated in vitro using human CCA cell lines.

Results

HMGA1 upregulation was detected as an early event in the cholangiocarcinogenesis of a hamster model. In the human cohort (n = 81), high HMGA1 expression significantly correlated with histological type (p=0.014), metastatic stage (p=0.004) and shorter overall survival (p=0.024). In vitro, siRNA-mediated suppression of HMGA1 remarkably inhibited cell proliferation. While HMGA1 silencing increased cleaved caspase-3, it resulted in only a modest increase in the apoptotic cells. Instead, this caspase activation was primarily associated with a marked reduction in cell migration and invasion through the modulation of epithelial-mesenchymal transition (EMT) markers, and cytoskeletal remodeling in a cell line-specific manner. Conclusions: These retrospective and preclinical findings suggest that HMGA1 is a critical driver of CCA progression and a valuable prognostic indicator. HMGA1 promotes neoplastic transformation and aggressiveness of CCA. Targeting HMGA1 may serve as a potential therapeutic strategy to attenuate CCA progression, warranting further clinical validation.

Introduction

Cholangiocarcinoma (CCA), a bile duct cancer which is highly prevalent in Northeastern Thailand,^1,2 where the infection of Opisthorchis viverrini (Ov) is associated with this cancer.^3–5 CCA patients are usually diagnosed in advanced stages with metastasis and the surgery or chemotherapy are unsatisfied. ⁶ Therefore, understanding of the molecular mechanism of cancer development and metastasis of CCA is required for exploring an alternative molecular target for therapeutic strategies in CCA patients.

High mobility group A1 (HMGA1) is a non-histone chromatin-binding protein with three AT-hook domains that bind A/T-rich DNA. ⁷ It acts as a key regulator of chromatin structure and gene expression,^8–10 and is involved in processes such as development, differentiation, and tumorigenesis.^11,12 Overexpression of HMGA1 has been demonstrated in several cancers including breast,^7,13 colon,^14,15 liver, ¹⁶ lung, ¹⁷ and bile duct cancer.^18,19 High expression of HMGA1 was correlated with high tumor grade, metastatic progression, disease recurrence, poor prognosis and short survival of cancer patients.^8,9,20,21 Furthermore, silencing of HMGA1 expression significantly reduced the cell proliferation, migration and invasion of breast cancer cells. HMGA1 knockdown cells increased E-cadherin and decreased Snail and Vimentin expressions which indicated the transition from mesenchymal to epithelial phenotype. ¹³ The significances of HMGA1 have not yet been studied in Ov-associated Thai CCA. This information led us to select HMGA1 and uncover the functions of HMGA1 and investigate whether it could be a potential target for diagnosis or treatment of CCA.

In this study, the clinical impacts of HMGA1 were assessed in tissues of hamster CCA and Thai CCA patients by immunohistochemical staining. The oncogenic roles of HMGA1 in cell proliferation, apoptosis, migration and invasion were examined in KKU-100 and KKU-213A CCA cell lines.

Materials and methods

This study was a retrospective study of human and hamster CCA tissues combined with in vitro functional studies and molecular mechanisms analysis in CCA cell lines.

HMGA1 expression in human CCA from online databases

To explore the HMGA1 expression in human CCA from online databases, we retrieved the HMGA1 mRNA expression from the TGCA dataset on GEPIA website (https://gepia.cancer-pku.cn/). ²² The GEO dataset (GSE76297) was obtained by GEOquery package ²³ and normalized by lumi package ²⁴ in R program (Vienna, Austria).

Archival Ov-induced CCA hamster tissues

Archival paraffin-embedded sections of Ov-induced CCA in hamster were the same samples as previous study. ²⁵ Sixty Syrian golden hamster were divided into 4 groups; untreated control group, Ov-infected group, N-nitrosodimethysamine (NDMA)-treated group and Ov-NDMA treated group. Liver tissues of five hamsters each group were harvested at 1, 3, and 6 months. All protocols were performed in accordance with the ARRIVE guidelines (Supplementary File S1) and approved by The Ethics Committee for Animal Research, Khon Kaen University (AEMDKKU 001/2558).

Human CCA tissues

Eighty-one paraffin embedded tissues from CCA and 21 Hepatocellular carcinoma (HCC) patients were obtained from Cholangiocarcinoma Research Institute (CARI), Khon Kaen University, Thailand between 1998 to 2011. Patients were included if they met the following criteria: (1) the histopathological diagnosis of CCA confirmed by pathologists; (2) samples were from hepatectomy; (3) clinical data were available; and (4) patients had no history of other cancers. Cases with radiotherapy or chemotherapy prior to surgical intervention and perioperative deaths were excluded from this study. For survival analysis, patients were followed up continuously from the time of surgery until death. Written informed consent was obtained from all patients prior to tissue collection. The study protocols were adhered to the STROBE checklist (Supplementary File S2) and received ethical approval from The Khon Kaen University Ethics Committee for Human Research following the Declaration of Helsinki (HE641574).

CCA cell lines

Two CCA cell lines, including KKU-100 (JCRB1568) and KKU-213A (JCRB1557) were obtained from the JCRB cell bank (Osaka, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako Pure Chemical Industries, Japan) supplemented with 10% fetal bovine serum and a 1% antibiotic-antimycotic (Gibco, Grand Island, NY). Cells were cultured at 37 °C in a 5% CO₂ humidified incubator.

Immunohistochemistry (IHC)

The IHC was performed using a standard protocol. Briefly, the tissue slides were deparaffinized in xylene and rehydrated by submerging in stepwise decreasing concentration of ethanol. Antigen retrieval was performed by autoclaving in Tris-EDTA buffer pH 9.0 for 3 minutes. Endogenous peroxidase was neutralized using 0.3% H₂O₂ in methanol for 30 minutes, followed by the blockage of non-specific binding with 1% skim milk in phosphate buffer saline (PBS) for 1 hour. The tissue slides were incubated with 1:200 rabbit anti-HMGA1 antibody (#129153, Abcam, UK) at 4 ˚C for overnight, followed by the peroxidase-conjugated Envision^TM secondary antibody (Dako, Denmark) at room temperature. The peroxidase activity was developed using diaminobenzidine tetrahydroxychloride solution (Sigma, St. Louis, MO). The HMGA1 expression was evaluated using H-score. ²⁶ H-score = [[1 × (% of 1+ cells)] + [2 × (% of 2+ cells)] + [3 × (% of 3+ cells)]]. The scoring was evaluated independently by the pathologists who were blinded to the patients’ clinical data, survival outcomes and the experimental groups of the hamster models.

Transient knockdown of HMGA1 mRNA by siRNA

CCA cell lines (1.5×10⁵ cells) were seeded into a 35 mm culture dish and transfected with 100 pmole of siHMGA1#1 (sense stand 5′-GUGCCAACACCUAAGAGACCUdTdT-3′ and antisense stand 5′-AGGUCUCUUAGGUGUUGGCACdTdT-3′) or siHMGA1#2 (sense stand 5′-GCAGGAAAAGGACGGCACUdTdT-3′ and antisense stand 5′-AGUGCCGUCCUUUUCCUGCdTdT-3′) ²⁷ or a scramble control (sense stand 5′ UUCUCCGAACGUGUCACGUdTdT 3′ and antisense stand 5′ ACGUGACACGUUCGGAGAAdTdT 3′) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Cells were cultured for 24-96 h and harvested for further analysis.

MTT assay

Following a 24 h post-transfection, the HMGA1-knockdown and a scramble control cells were harvested and re-plated (1500 cells) into triplicated wells of a 96-well plate. Cell proliferation was determined at 24, 48, 72 and 96 h using MTT assay following the manufacturer’s instructions (Sigma, St. Louis, MO). After incubation, 0.04N HCl in isopropanol was added to dissolve the formazan crystals. The absorption was measured at 595 nm using a microplate reader (iMark; Bio-Rad Laboratories, Hercules, CA). The proliferation rate of each time points was compared to the starting time (0 h) which was set as 1. The results were the averages from three independent biological experiments. The differences between the experimental groups and the control group was determined using two-way ANOVA with Dunnett’s post-hoc test.

Apoptosis assay

CCA cell lines (1.5×10⁵ cells) were seeded into a 35 mm culture dish and treated with siHMGA1 or scramble control for 72 h. Cells were harvested, double stained with Pacific Blue™ Annexin V (BioLegend, San Diego, CA) and propidium iodide (PI) staining (Invitrogen, Carlsbad, CA) and analyzed using the BD FACSCanto™ II flow cytometer. Data analysis was performed using FlowJo™ software. The number of apoptotic cells was quantified as a percentage. The results (mean ± SD) were the averages from three independent biological experiments.

Cell migration and invasion assay

The migration assay was performed using an 8 µm pore size-transwell insert (Corning, Danvers, MA). After 24 h siRNA transfection, thirty thousand CCA cells in 200 µl of serum free DMEM were added into 2 replicated transwell inserts. After 16 h incubation, cells in the upper chamber were scraped off using a cotton swab. The inserts were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of migrated cells was photographed with 10X objective lens and counted. For invasion assay, the protocol was similar to migration assay but the transwell insert was coated with 0.4 mg/ml of Matrigel^TM (Corning, Danvers, MA) before adding the cells. The results (mean ± SD) were the averages from three independent biological experiments.

Western blotting

To verify HMGA1 knockdown efficiency, cells were harvested between 24 to 96 h after the siRNA transfection. To determine the apoptotic markers and EMT-related proteins, cells were harvested at 72 h (related to the apoptosis assay) and 48 h post-transfection (covering knockdown incubation and migration/invasion time), respectively. Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM NaF, 1 mM Na₃VO₄) containing protease inhibitor cocktail (Nacalai Tesque, Japan). Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Thermo Science, Rockford, IL). Protein lysate (10-40 µg) was separated by SDS-PAGE and blotted onto a PVDF membrane (GE Healthcare, Japan), which was probed with primary antibodies; HMGA1 (Abcam; 1:5000), EMT antibody sampler kit (#9782, Cell Signaling, Danvers, MA; 1:1000), Phospho-FAK-Tyr925 (#3284, Cell Signaling; 1:1000), total FAK (#12636-1-AP, Proteintech; 1:1000), apoptotic markers antibodies (Cell Signaling; 1:1000) including Caspase 8 (#4790), Caspase 9 (#9502), Cleaved caspase 3 (#9664), Caspase 3 (#9662), Cleaved PARP (#5625) and PARP (#9542). The membranes were further incubated with HRP-conjugated secondary antibodies (#7074 and #7076, Cell Signaling; 1:2000). Heat shock protein (HSP70, # ADI-SPA-815B; EnZo Life Sciences, Farmingdale, NY; 1:1000) or β-actin (#A5441, Sigma; 1:5000) were used as internal control. Protein bands were detected using Chemi-Lumi One Super reagents (Nacalai Tesque, Japan) and visualized with an ImageQuant Las4000 system (GE Healthcare, Japan). Band intensities were quantified using ImageJ software. The relative protein expression was normalized with internal control and assigned the sc control as 1. The results (mean ± SD) were the averages from three independent biological experiments.

Statistical analysis

Statistical analyses were performed using SPSS version 17 (SPSS, Inc., Chicago, IL) for clinical and survival data, and GraphPad Prism v9 (GraphPad Software, San Diego, CA, USA) for in vitro experimental data. Categorical variables, including sex, TNM stage, and metastasis, were analyzed using the Chi-square test or Fisher’s exact test. For survival analysis, the Kaplan-Meier method was used to estimate survival probabilities, and the log-rank test was applied to compare distributions. The proportional hazards (PH) assumption was verified by inspecting log-minus-log (LML) survival plots and incorporating time-dependent covariates into the Cox model; the assumption was found to be satisfied. Independent prognostic factors were identified using univariate and multivariate Cox proportional hazards regression models. Evaluated variables included sex, age, tumor size, tumor stage, TNM stage, histological type, and HMGA1 expression. Variables with statistical significance (p < 0.05) in univariate analysis were included in the multivariate model. Continuous data from three independent biological experiments are presented as mean ± standard deviation (SD). Comparisons among three or more groups were evaluated using one-way or two-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons against a single control. A p-value < 0.05 was considered statistically significant.

Results

HMGA1 is upregulated during CCA carcinogenesis in a hamster model

The protein expression of HMGA1 during hamster CCA carcinogenesis was evaluated using immunohistochemistry (IHC). HMGA1 was found to be predominantly localized within the nucleus. As shown in Figure 1(A), low HMGA1 expression was observed in normal bile ducts (NBD) across all treatment groups from months 1 to 6. However, HMGA1 staining intensity increased in hyperplastic and dysplastic (HP/DP) tissues in both the O. viverrini (Ov)-infected and NDMA-treated groups at 1, 3, and 6 months. In the Ov + NDMA-treated group, HP/DP lesions exhibited predominantly positive HMGA1 staining at all time points. Notably, the strongest HMGA1 expression was observed in developed CCA at 3 and 6 months. To further quantify these findings, HMGA1 expression was categorized into low and high groups based on a median H-score of 155. The proportion of cases with high HMGA1 expression increased progressively from NBD to HP/DP and peaked in CCA at both 3 and 6 months (Figure 1(B)).

Figure 1.

HMGA1 expression in hamster CCA tissues. (A) Representative IHC staining of HMGA1 in four groups of hamsters including untreated control, Ov-infection, NDMA treatment and Ov + NDMA at 1, 3, and 6 months. (B) H-score of HMGA1 in hamster CCA tissues (median = 155, a cut off value). Bar graph displays the percentage of low and high HMGA1 cases between NBD, HP/DP, and CCA.

HMGA1 overexpression correlates with metastasis and poor prognosis in CCA patients

To validate the clinical relevance of HMGA1, we first analyzed transcriptomic data from public databases. Analysis of the TCGA dataset via the GEPIA web tool revealed significantly higher HMGA1 mRNA levels in CCA tissues (n = 36) compared to normal tissues (n = 9, p < 0.05; Figure 2(A)). This finding was further supported by the GEO dataset (GSE76297), which showed a marked overexpression of HMGA1 mRNA in 91 CCA cases compared to 92 normal controls (p < 0.0001). We subsequently evaluated HMGA1 protein expression in 81 Ov-associated CCA tissues and 21 HCC cases using IHC. HMGA1 showed weak expression in normal bile ducts (NBD), whereas moderate to strong staining was observed in CCA tissues (Figure 2(B)). Elevated HMGA1 levels were also detected in HCC tissues compared to surrounding hepatocytes (Figure 2(C)). Quantitative analysis using H-scores revealed median values of 150 for NBD and 165 for CCA. In comparison, the median H-scores for hepatocytes and HCC were 10 and 90, respectively (p = 0.017). Notably, HMGA1 expression in CCA was significantly higher than in HCC samples (p < 0.0001; Figure 2(D)).

Figure 2.

Overexpression of HMGA1 in CCA tissues. (A) HMGA1 transcript level of CCA were obtained from GEPIA website. TPM is transcript per million, *p < 0.05. Another HMGA1 dataset was retrieved from GEO database (GSE76297). A dot plot of HMGA1 transcript level showed significant overexpression of HMGA1 in 91 human CCA cases compared to 92 normal cases (**** p < 0.0001). Representative IHC figures of HMGA1 in (B) human CCA tissues (n=81) and (C) human HCC cases (n=21) and their normal counterparts. (D) H-score of HMGA1 expression in bile ducts, CCA tissues, hepatocytes and HCC tissues. (E) Overall survival curves between low (blue line) and high HMGA1 (red line) expressing CCA patients.

CCA patients were divided into two groups based on the median H-score: low HMGA1 (<165) and high HMGA1 (≥165). As summarized in Table 1, high HMGA1 expression significantly correlated with metastasis stage (p = 0.004) and histological subtype (p = 0.014). Kaplan-Meier survival analysis (Figure 2(E)) demonstrated that patients with high HMGA1 expression had a significantly shorter median survival (167 days; 95% CI: 61–272 days) compared to those with low expression (302 days; 95% CI: 235–368 days; p = 0.024). Univariate Cox regression analysis identified metastasis (M) stage (p = 0.009), histological subtype (p = 0.004), and HMGA1 expression (p = 0.026) as significant prognostic factors. However, multivariate analysis revealed that only histological subtype remained an independent prognostic indicator (HR = 1.961; 95% CI: 1.080–3.563; p = 0.027; Table 2).

Table 1.

HMGA1 expression and clinico-pathological findings of 81 CCA patients.

Clinical characteristics	No. of patients	HMGA1 expression
		Low <165	High ≥165	p-value
Age (years)	​	​	​	0.095
< 56	40	16	24	​
≥ 56	41	24	17	​
Sex	​	​	​	0.116
Male	54	30	24	​
Female	27	10	17	​
Histological type	​	​	​	0.014
Non-papillary	55	22	33	​
Papillary	26	18	8	​
T stage	​	​	​	0.693
T1	6	4	2	​
T2	10	4	6	​
T3	33	15	18	​
T4	32	17	15	​
N stage (n=71)	​	​	​	0.076
N0	38	23	15	​
N1	33	13	20	​
M stage (n=74)	​	​	​	0.004
M0	64	38	26	​
M1	10	1	9	​
Tumor stage	​	​	​	0.585
I-III	30	16	14	​
IVA-B	51	24	27	​
Tumor size (cm)	​	​	​	0.904
< 7	37	18	19	​
≥ 7	44	22	22	​

Table 2.

Univariate and multivariate Cox regression analyses of prognostic indicators of survival in CCA patients.

Variables		No. of patients	HR	95%CI	p-value
Univariate analysis
Age (years)	< 56	40	1	​	​
	≥ 56	41	1.081	(0.695-1.682)	0.729
Sex	Male	54	1	​	​
	Female	27	1.056	(0.661-1.689)	0.819
Tumor size (cm)	<7	37	1	​	​
	≥7	44	1.197	(0.764-1.873)	0.432
Tumor stage	I-III	30	1	​	​
	IVA-B	51	1.097	(0.696-1.729)	0.689
T stage	T1	6	1	​	​
	T2	10	0.596	(0.247-1.440)	0.250
	T3	33	1.266	(0.612-2.617)	0.525
	T4	32	1.299	(0.781-2.160)	0.313
N stage (n=71)	N0	38	1	​	​
	N1	33	1.297	(0.797-2.110)	0.296
M stage (n=74)	M0	64	1	​	​
	M1	10	2.497	(1.255-4.965)	0.009
Histological type	Papillary	26	1	​	​
	Non-papillary	55	2.126	(1.269-3.562)	0.004
HMGA1 expression	< 165	41	1	​	​
	≥ 165	40	1.665	(1.063-2.609)	0.026
Multivariate analysis
M stage (n=74)	M0	64	1	​	​
	M1	10	1.657	(0.796-3.451)	0.177
Histological type	Papillary	26	1	​	​
	Non-papillary	55	1.961	(1.080-3.563)	0.027
HMGA1 expression	< 165	41	1	​	​
	≥ 165	40	1.408	(0.845-2.346)	0.190

HMGA1 silencing inhibits cell proliferation, induces apoptosis and possibly affects cytoskeleton remodeling in CCA cells

To investigate the functional role of HMGA1 in CCA, HMGA1 mRNA expression was suppressed using two specific siRNAs (siHMGA1-1 and siHMGA1-2) in KKU-100 and KKU-213A cell lines, both derived from primary tumors of Ov-associated CCA patients. Effective knockdown was achieved, with HMGA1 protein levels reduced by 20–40% in KKU-100 (48–96 h) and 20–90% in KKU-213A (24–96 h) compared to scrambled (sc) controls (Figure 3(A) and Supplementary File S3). MTT assays revealed that HMGA1 knockdown significantly suppressed cell proliferation in both cell lines over a 96-hour period (Figure 3(B)). In KKU-100 cells, while the sc control showed a 5-fold increase in growth by 96 h, siHMGA1-transfected cells exhibited marked inhibition starting at 48 h (1.2–2.1 fold, p < 0.0001) and persisting through 72 h (1.5–1.6 fold, p < 0.0001) and 96 h (2.5–2.9 fold, p < 0.0001). Similarly, in KKU-213A cells which showed a 10-fold growth in the control group, HMGA1 depletion significantly reduced proliferation rates at 48 h (1.9–3.0 fold, p < 0.0001), 72 h (3.1–4.1 fold, p < 0.0001), and 96 h (4.0–5.5 fold, p < 0.0001). Furthermore, apoptosis assays demonstrated an increase in apoptotic cells in HMGA1-silenced KKU-100 cells (13.1–19.2%) compared to the sc control (7.5%; si-1, p = 0.017 and si-2, p = 0.012). A modest increase in apoptotic cells was also observed in KKU-213A cells (Figure 3(C)). Western blotting analysis showed elevated levels of cleaved caspase-8, cleaved caspase-9, cleaved PARP, and cleaved caspase-3 in both cell lines following HMGA1 knockdown. To explore whether caspase-3 affect to cytoskeletal remodeling in HMGA1 knockdown cells. Western blotting of the phosphorylation of Focal Adhesion Kinase (pFAK) at the Tyr925 (Y925) and total FAK were investigated. The results showed that pFAK (Y925) levels decreased in the HMGA1 knockdown KKU-100 cell line (both si-1 and si-2) and in the si-1-treated KKU-213A cell line, whereas total FAK expression remained constant as displayed in Figure 3(D)–3(E) and Supplementary File S4.

Figure 3.

Suppression of HMGA1 expression inhibited cell proliferation and induced apoptosis in CCA. (A) KKU-100 and KKU-213A CCA cell lines were treated with scramble (sc) or siHMGA1-1 (si-1), -2 (si-2) for 24, 48, 72 and 96 h. The proliferation rate of each time points was compared to the starting time (0 h) which was set as 1. The results were the averages from three separated biological experiments. Western blotting showed that both siHMGA1 silencers effectively decreased HMGA1 expression (B) Knockdown of HMGA1 reduced cell proliferation of CCA cell lines when compared to sc control. (C) Suppression of HMGA1 induced apoptotic cell death in CCA cell lines. Annexin V-PI apoptosis assay displayed an increase % of apoptotic cells in siHMGA1 treated cells. (D) Representative blots of the apoptotic-related proteins (cleaved and total forms of caspase 3, 8, 9, PARP) and the pFAK(Y925) and total FAK. Band intensities were quantified using ImageJ software. Quantification of protein expression was normalized with internal control and assigned the sc control as 1. (E) Bar graph illustrates the fold-change ratio between the cleaved or phosphorylated forms and their total proteins. The results (mean ± SD) were the averages from three independent biological experiments. *p<0.05, ** p < 0.01, **** p < 0.0001.

HMGA1 knockdown inhibits CCA cell migration and invasion by suppressing EMT

Knockdown of HMGA1 mRNA expression using specific siRNAs (siHMGA1-1 and siHMGA1-2) significantly reduced the migratory capacity of KKU-100 cells by 70–90% (si-1, p = 0.0011; si-2, p = 0.0247) and KKU-213A cells by 31–94% (si-1, p = 0.0003; si-2, p = 0.0045), as shown in Figure 4(A). Similar inhibitory effects were observed in invasion assays (Figure 4(B)). To investigate whether these processes were regulated by epithelial-mesenchymal transition (EMT), the expression of EMT-related markers was analyzed via Western blotting. In both HMGA1-silenced KKU-100 and KKU-213A cells, the epithelial markers E-cadherin and ZO-1 were upregulated compared to the sc control. Conversely, the expression levels of mesenchymal markers, including Snail, β-catenin, and matrix metalloproteinase-2 (MMP-2), were downregulated. (Figures 4(C)-4(D) and Supplementary File S5).

Figure 4.

Downregulation of HMGA1 expression diminished migration and invasion of CCA. Suppression of HMGA1 reduced (A) migration and (B) invasion abilities in CCA cell lines. The numbers of migrated and invaded cells were compared to sc control which was set as 100%. (C) The expression of EMT markers including E-cadherin, Snail, β-catenin, MMP-2 and ZO-1 were determined using Western blotting. Band intensities were quantified using ImageJ software. Quantification of protein expression was normalized with HSP70 as an internal control (ZO-1 was normalized with β-actin) and assigned the sc control as 1. (D) Bar graph shows the relative fold change of protein expression. The results (mean ± SD) were the averages from three independent biological experiments. *p<0.05, ** p < 0.01, *** p < 0.001.

Discussion

To our knowledge, the expression of HMGA1 in Ov-associated CCA is revealed for the first time in this study. The significance of HMGA1 was examined in the CCA carcinogenesis in a hamster model. Upregulation of HMGA1 was detected in hyperplasia/dysplasia (precancerous lesions) as early as one month following Ov infection and was retained throughout the carcinogenesis process (3-6 months). This observation represents a finding of significant translational value. Because human CCA is asymptomatic in its initial stages and is frequently diagnosed at an advanced stage, the identification of early molecular drivers is clinically required. The early detection of HMGA1 suggests its potential utility as a novel predictive biomarker for the early detection of precancerous biliary lesions. Furthermore, from a therapeutic perspective, this finding implies that HMGA1 maybe an active driver of early malignant transformation. Consequently, targeting HMGA1 or its downstream signaling pathways in the early stage of disease progression could serve as a promising strategy for early intervention or chemoprevention, particularly in Ov-endemic regions.

HMGA1 was also overexpressed in human CCA tissues. High level of HMGA1 expression was significantly correlated with shorter survival, histological type and metastasis of CCA patients. In agreement with the previous report, overexpression of HMGA1 was correlated with lymph node metastasis and recurrence in non Ov-associated CCA patients. ²⁸ Compare to the previous studies on HMGA1 in CCA,^28,29 the novel findings from our study demonstrated for the first time that HMGA1 was detected in early event of CCA carcinogenesis in hamster model; HMGA1 was associated with poor clinical outcomes of Ov-related CCA patients. Our study was in line with other cancer studies which revealed overexpression of HMGA1 was associated with tumor progression in many different types of human cancers.^16,18,30 Moreover, the HMGA1 expression level in CCA cases were substantially higher than HCC, which was similar to the earlier publication. ¹⁸ This result provided preclinical evidence suggesting that HMGA1 could be explored as a candidate differential diagnostic biomarker between CCA and HCC in future clinical studies.

The functions of HMGA1 in CCA progression has been explored in the recent study. Knockdown of HMGA1 mRNA expression using siRNA inhibited the proliferation, migration and invasion in CCA cell lines. These data were corresponded with the previous reports in a number of cancers including hepatocellular carcinoma, ¹⁶ cervical cancer, ²⁰ breast cancer, ¹³ colorectal cancer ³¹ and non Ov-associated CCA.^28,29,32,33 The recent study revealed that knockdown of HMGA1 inhibited cell proliferation of CCA cell lines via apoptosis pathway which in agreement with several publications.^7,34–36 While HMGA1 depletion significantly suppressed CCA cell proliferation, our flow cytometry data revealed a modest increase in the apoptotic cell population. Interestingly, this was accompanied by a marked elevation in cleaved caspase-3, with minor alterations in cleaved caspase-8 and -9. This evidence suggests, cleaved caspase-3 is not solely an executioner of apoptosis but also actively participates in non-apoptotic processes, particularly cytoskeletal remodeling. High levels of active caspase-3 can cleave key cytoskeletal and focal adhesion proteins, thereby impairing the cell’s migratory capacity. This hypothesis aligns with our in vitro findings, where HMGA1 knockdown substantially inhibited CCA cell migration and invasion. Thus, the activation of caspase-3 observed here likely contributes to the disruption of cytoskeletal dynamics, leading to impaired motility rather than full execution of apoptosis. Upon HMGA1 knockdown, pFAK (Y925) decreased in both si-1 and si-2 treated KKU-100 cells. However, in the KKU-213A cells, a decrease was observed only in si-1 treated cells. Total FAK levels remained constant across both cell lines. Tyr925 is primarily regulated by Src kinase during focal adhesion turnover and cell disassembly, rather than serving as the direct autophosphorylation initiator site (which occurs at Tyr397). This finding suggests that HMGA1 may affect cytoskeletal remodeling in a cell line-specific response and operate via a different branch of the FAK pathway that highlights the heterogeneity and dynamic variability in CCA. This suggests that while the HMGA1/caspase-3 axis significantly suppresses cell migration and invasion in both CCA cell lines, the downstream of cytoskeletal remodeling may vary due to genetic backgrounds, aggressiveness of the cells and cellular heterogeneity. In KKU-213A cell line, compensatory upstream signals (such as Src or integrin redundancies) may sustain pFAK levels. Alternatively, active caspase-3 might bypass FAK to preferentially cleave alternative structural or focal adhesion substrates (such as Gelsolin, Rock1, or alpha-actinin) to achieve motility inhibition. Due to the complexity of cytoskeletal remodelling and focal adhesion dynamics, investigating these processes in order to prove the deeper molecular insight of HMGA1/caspase-3/cytoskeletal remodelling axis are of interest for future study. Furthermore, we investigated whether alternative programmed cell death pathways were activated; however, our evaluation of autophagy markers revealed no significant differences following HMGA1 depletion (Supplementary File S6), suggesting that autophagy is not the primary compensatory mechanism in this context.

Several studies demonstrated the oncogenic role of HMGA1 in tumor formation in vivo experiments. For instance, the establishment of a xenograft model using CCA cell lines with HMGA1 knockdown showed that HMGA1 promoted xenograft tumor growth of CCA^29,32 and metastatic lesions. ³³ This information represented an early stage of translational research. Further validation in vivo models e.g., patient-derived xenografts (PDX) is required. Evaluating the safety, efficacy, and potential off-target effects of HMGA1-targeted interventions is essential before these experimental concepts can be translated into therapeutic applications for CCA patients.

Epithelial mesenchymal transition (EMT) is a critical step in cancer metastasis. This process involves in an alteration of cell-cell adhesion, obtaining migration/invasion properties and becoming mesenchymal. Loss of epithelial marker (e.g., E-cadherin) and increase of mesenchymal markers (e.g., Snail, β-catenin, MMP) are observed during metastasis stage. Overexpression of HMGA1 promotes EMT by coordinately downregulating the epithelial markers and upregulating the mesenchymal markers. ³² Knockdown of HMGA1 demonstrated decreasing of migration and was concordant with increasing of E-cadherin and decreasing of N-cadherin and Snail in lung cancer cells. ¹⁷ Our results showed that knockdown of HMGA1 increased E-cadherin, decreased Snail, β-catenin and MMP-2 in both CCA cell lines. These data corresponded with earlier studies in breast cancer, ¹³ non Ov-associated CCA^29,32,33 and uterine cancer. ³⁷ Compare to the previous studies of HMGA1 in CCA, our study is the first report showing knockdown of HMGA1 altered the EMT-related molecules including ZO-1, β-catenin and MMP-2 that those previous studies did not explore. These in vitro findings suggested that silencing HMGA1 reversed EMT process to MET, providing evidence that HMGA1 is likely involved in driving the metastatic potential of CCA.

Limitations

There are limitations in this study. First, a sample size calculation was not performed for the human CCA cohort. The sample size was determined based on the availability of archived paraffin-embedded tissues that met our inclusion criteria during the study period. Consequently, this study may have limited statistical power to detect subtler associations between HMGA1 expression and certain clinical parameters, and there is a potential risk of overestimating the effect sizes. While the current sample size was sufficient to observe significant correlations regarding metastasis and survival, these findings should be interpreted with caution. Second, this study was retrospective nature in a single-center which could lead to selection bias. Future large-scale, prospective, and multi-center studies with statistically justified sample sizes are warranted to validate our clinical findings. Third, targeting HMGA1 using knockdown strategy or specific inhibitor are required for further development and take long time before the translation application.

Conclusion

Our results demonstrate for the first time that HMGA1 upregulation is an early event in cholangiocarcinogenesis, as shown in a hamster model. In human clinical samples, high HMGA1 expression significantly correlates with metastasis and shorter survival in CCA patients. In vitro, HMGA1 suppression significantly inhibits cell proliferation and modestly induces apoptosis alongside caspase-3 activation. This molecular change possibly alters to the cytoskeletal remodeling which leads to a substantial reduction in migration and invasion through the modulation of EMT-related markers in CCA cell lines. Taken together, these preclinical and retrospective findings highlight the significant prognostic value and roles of HMGA1 in CCA. This evidence supports the hypothesis that targeting HMGA1 could serve as a novel therapeutic strategy to attenuate CCA progression, pending future clinical validation.

ORCID iDs

Ubon Cha’on https://orcid.org/0000-0002-9909-8671

Charupong Saengboonmee https://orcid.org/0000-0003-1476-1129

Kanlayanee Sawanyawisuth https://orcid.org/0000-0003-2260-6372

Consent to participate

Written informed consent was obtained from all patients prior to tissue collection.

Data Availability Statement

Data are available from the corresponding author upon reasonable request.*