Upregulation of limb-bud and heart (LBH) drives liver cancer progression by interacting with the oncoprotein Mortalin

Abstract

Purpose

Hepatocellular carcinoma (HCC), the major subtype of primary liver cancer, is one of the most prevalent and lethal malignancies globally. Limb-bud and heart (LBH), a highly conserved transcriptional cofactor, regulates embryonic development and is implicated in various human diseases, including cancer. However, whether and how LBH relates to liver cancer progression remains elusive.

Methods

The expression patterns and prognostic value of LBH in HCC were evaluated using data from The Cancer Genome Atlas (TCGA) database. The role of LBH during HCC progression was characterized through in vitro cellular experiments, as well as subcutaneous xenograft and hydrodynamic tail vein injection (HTVi)-induced spontaneous HCC mouse models. Transcriptome sequencing (RNA-seq), quantitative real-time PCR (qPCR), the Cistrome Data database, and chromatin immunoprecipitation sequencing (ChIP-seq) were utilized to explore the signaling pathways governing LBH gene transcription. Mass spectrometry was performed to identify Mortalin as a binding partner of LBH, which was further verified by co-immunoprecipitation (co-IP) and immunofluorescence (IF) assays. Finally, in vitro rescue experiments were conducted to clarify the role of Mortalin in mediating the oncogenic effects of LBH in HCC.

Results

LBH is significantly upregulated in HCC tissues and functions as an oncogenic driver. The aberrant upregulation of LBH in HCC is attributable to transcriptional regulation by the TGF-β/Smad signaling pathway. LBH notably promotes tumor growth both in vitro and in mouse models. Furthermore, LBH physically interacts with the oncoprotein Mortalin, and the two proteins colocalize in the cytoplasm of HCC cells. Functionally, Mortalin is essential for LBH-mediated oncogenic promotion in HCC.

Conclusions

Collectively, these findings demonstrate that the TGF-β/LBH/Mortalin axis plays a crucial role in HCC progression, highlighting a potential therapeutic target for the treatment of liver cancer.

Introduction

Hepatocellular carcinoma is one of the most common malignancies and a leading cause of cancer-related mortality worldwide, posing a significant challenge to global healthcare [1, 2]. The rapid advancement of medical technology including the continuous refinement of diagnostic techniques, the gradual optimization of treatment protocols, and the development and application of targeted therapies and immunotherapies, have improved the clinical diagnosis and intervention of liver cancer to some extent [3–5]. However, the five-year survival rate for liver cancer patients remains low, at approximately 21% [2]. Consequently, it is urgent to elaborate the complex molecular mechanisms underlying the occurrence and development of liver cancer and to discover novel molecular therapeutic targets.

Limb-bud and heart (LBH) is an evolutionarily conserved transcription cofactor that was initially found to regulate the development of the heart and limb in vertebrates [6, 7]. LBH consists of an N-terminal hydrophobic region, a nuclear localization signal that contains eight basic amino acid residues, and a C-terminal acidic glutamic acid-rich domain [6]. In addition to regulating embryonic development, LBH has been found to be involved in several human diseases, such as tissue fibrosis, arthritis, and cancer [8–11]. Although acting as a tumor suppressor in nasopharyngeal, prostate, and lung cancer [12–15], LBH is overexpressed in glioma, gastric and breast cancer, exerting tumor-promoting functions [16–20]. For instance, LBH was reported to be upregulated upon hypoxic condition in glioma, and promote cancer progression via VEGFA/ERK signaling [16]. Moreover, LBH also plays a pivotal role during mammary development and promotes the progression of basal type or three-negative breast cancer via transcriptional induction of the key epithelial stem cell transcription factor ΔNp63 or modulation of the transcriptional responses mediated by transforming growth factor beta (TGF-β) [11, 21]. However, the role and molecular mechanisms of LBH in liver cancer remain largely unexplored.

As a prototype of a large secreted peptide superfamily, TGF-β plays a crucial role in the normal liver and various liver diseases including hepatic fibrosis and liver cancer [22–25]. The functions and mechanisms of TGF-β in cancer have been intensively and extensively investigated. As a functionally dichotomous cytokine, TGF-β acts as a tumor inhibitor in normal epithelial cells and early stage tumor cells, but switches to a tumor promoter in late stages [24, 26]. TGF-β drives cancer progression by enhancing the proliferation, invasion and metastasis of tumor cells, in addition to remodeling the tumor microenvironment [25, 27]. Various strategies have been developed to target the TGF-β pathway in preclinical animal models and tested in clinical cancer patients [28, 29]. In consideration of the pivotal role of TGF-β in cancer, identification of its downstream oncogenic effectors would provide additional opportunities for cancer intervention.

Mortalin, also known as heat shock protein A9 (HSPA9), glucose-regulated protein 75 (GRP75) or MOT/mot-2, is a stress-inducible mitochondrial chaperone belonging to the heat shock protein 70 chaperone protein family, which consists of 13 members in human [30, 31]. In addition to being involved in protein quality control in the mitochondria and cellular homeostasis maintenance in responses to stresses, Mortalin can regulate a wide range of cellular functions including cell proliferation, growth, death and metabolism [30]. Moreover, Mortalin is upregulated in a line of cancers and promotes their occurrence or progression, such as breast cancer, melanoma, non-small cell lung cancer, gastric cancer and colorectal cancer, etc [32–37]. The tumor-promoting effects of Mortalin have been attributed to its capabilities to interact with some key protein regulators or pathways involved in cancer. For instance, Mortalin interacts with the wild-type tumor suppressor protein p53, sequestering it in the cytoplasm and consequently boosting the survival and proliferation of tumor cells [38–40]. Mortalin also promotes cancer cell epithelial-mesenchymal transition (EMT), invasion and metastasis through regulation of the ERK/MAPK, Wnt/β-catenin and/or JAK/STAT pathways [41–45]. Likewise, evidence also implicates that Mortalin is highly expressed in liver cancer tissues and promotes tumor cell EMT, proliferation and migration [38, 46–48]. However, the regulatory mechanisms of Mortalin in liver cancer are less understood.

In this study, we found that the expression of LBH is upregulated in HCC tissues when compared to normal liver tissues. LBH promotes the malignant properties of HCC cells in vitro and accelerates HCC xenograft growth in mice. As an upstream signal, TGF-β could induce LBH expression transcriptionally through Smad2/3 in cancer cells, and the expression level of LBH positively correlated with those of the signature genes of the TGF-β pathway. Mass spectrometry and experimental data demonstrated that the oncoprotein Mortalin is not only a novel binding partner of LBH, but also mediated the oncogenic functions of LBH in liver cancer cells. These results shed light on the novel function and mechanism of LBH in liver cancer progression.

Results

LBH is upregulated in HCC and acts as a tumor promoter

To explore the potential functions of LBH in HCC, we firstly analyzed its expression in cancer by mining The Cancer Genome Atlas (TCGA) database. As a result, pan-cancer analysis revealed that the LBH expression level is upregulated in the majority of tumor tissues than their adjacent normal counterparts (Fig. 1A). In particular, the mRNA expression level of LBH in HCC tissues was significantly higher than that in normal liver tissues in the TCGA liver hepatocellular carcinoma (LIHC) database (Fig. 1B, C). Kaplan-Meier analyses showed that, based on TCGA, high expression level of LBH is slightly relevant to shorter overall survival (OS) and disease-free survival (DFS) rates in HCC patients (Fig. 1D). Next, we established HCCLM3 and SMMC-7721 stable cell lines that expressed LBH or the control lentivirus vector, and the expression efficacy of LBH was confirmed by qPCR and immunoblotting (Fig. 1E, F). Then HCCLM3 stable cells were inoculated subcutaneously into nude mice to generate a HCC xenograft model. The results displayed that LBH overexpression significantly promoted the growth of HCC xenografts in vivo, in terms of either tumor volume or weight (Fig. 1.G-I). In addition, tumors with ectopic LBH expression exhibited an enhanced level of cell proliferation as indicated by Ki67 staining (Fig. 1J). In line with this, in the Chinese Liver Cancer Atlas (CLCA) database [49], the LBH expression level is positively correlated with those of Ki67 and PCNA, two key molecules marking cell proliferation (Fig. 1K-J). These results suggest that the upregulation of LBH in HCC exerts a promoting effect on liver cancer growth.

Fig. 1.

LBH is associated with HCC progression and promotes HCC growth. (A) The LBH mRNA expression levels in different tumor samples and their normal tissue counterparts in pan-cancer based on the TCGA database. (B-C) Comparison of the mRNA expression level of LBH in normal human liver tissues with that in tumor tissues of HCC patients (B) or in different TNM stages. (C), in the TCGA-LIHC database. (D) Kaplan-Meier curve diagrams illustrating the overall survival (OS) and the disease-free survival (DFS) rates, respectively, in LBH-high or -low HCC patient groups from the TCGA-LIHC database. (E-F) Assessment of the stable expression of LBH and the control vector in HCCLM3 and SMMC-7721 cells by qPCR (E) and immunoblotting (F), respectively. (G) Representative images of xenograft tumors derived from nude mice that were subcutaneously inoculated with LBH- or vector-expressing HCCLM3 cells (n = 7/group). (H-I) Upon mouse sacrifice, the xenograft tumors were quantified in terms of volume (H) and weight (I), respectively. (J) Representative H&E and Ki67 IHC staining images of the above xenografts. Scale bar: 75 μm. (K-L) Correlation analyses of the mRNA expression levels between LBH and Ki67 or between LBH and PCNA in human HCC tissues from the CLCA database. The representation includes data and error bars, demonstrating the mean ± standard deviation (SD) from three independent trials. In all the qPCR experiments, gene expression levels were normalized to that of GAPDH. *p < 0.05, **p < 0.01 and ***p < 0.001 were calculated using two-tailed student t-test (A-C, E, H and I). p-values for (D) were calculated using log-rank, while pearson correlation tests were employed to assess gene expression correlations (K, L). ns, no statistical significance

LBH promotes the growth and invasion of HCC cells in vitro

To further verify the tumor-promoting functions of LBH in liver cancer, we studied how LBH affects the malignant properties of HCC cells in vitro. HCC cells stably carrying LBH-specific shRNAs or the control shRNA lentivirus vector were generated using HCCLM3 and HLE cells. Indeed, the expression of LBH could be significantly reduced by shRNAs (Fig. 2A-B). CCK8 cell-counting and Transwell assays demonstrated that depletion of LBH led to an obvious decrease in the capabilities of tumor cell proliferation (Fig. 2C-D), migration and invasion (Fig. 2G-J). On the contrary, LBH overexpression had the opposite effects on HCCLM3 and SMMC-7721 cells (Fig. 2E-F and K-N). These findings demonstrated that LBH exerts a tumor-promoting effect in liver cancer both in vivo and in vitro.

Fig. 2.

LBH promotes the growth and invasion of HCC cells in vitro. (A) qPCR assessment of LBH expression levels in HCCLM3 and HLE cells that stably express LBH-targeting shRNAs or the control vector. (B) HCCLM3 and SMMC-7721 cells stably overexpressing LBH-3×Flag were infected with LBH-targeting shRNA virus, and then immunoblotting assays were performed with anti-Flag or anti-TUBULIN antibodies. (C-D) HCC cells stably expressing LBH-targeting shRNAs or its control vector were subjected to CCK8 cell viability assays at the indicated time points. (E-F) The growth of LBH- or the vector-expressing HCC cells was detected similarly as above. (G-J) The migration and invasion of HCCLM3 (G) or SMMC-7721 (I) cells that stably express LBH shRNAs were assessed via Transwell assays, and the results were quantified (H and J). Scale bar: 100 μm. (K-N) Transwell assays were similarly carried out with HCC cells that stably express LBH or the vector. Scale bar: 100 μm. One-way ANOVA was applied for comparisons among three groups, followed by Dunnett’s post hoc test (A, C, D, H, and J). For two-group comparisons, a two-tailed Student’s t-test was used (E, F, L, and N). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant

LBH is transcriptionally upregulated by TGF-β in liver cancer cells

In consideration that LBH is highly expressed in HCC tissues (Fig. 1), we wondered how LBH expression is controlled in HCC cells. To this end, we referred to our transcriptome sequencing (RNA-seq) data from three HCC cell lines (MHCC97H, MHCC97L and HCCLM3) that were treated with or without TGF-β1 [50]. Intriguingly, TGF-β1 administration induced an upregulation of the mRNA level of LBH, along with some well-known TGF-β target genes like Smad7, PAI-1, CYR61, JUNB, CXXC5, TGFB1, amongst others [23, 50, 51]. To consolidate the regulation of LBH expression by TGF-β, three HCC cell lines, i.e. HCCLM3, HLE and SMMC-7721, were treated with TGF-β1 for different time periods. Not surprisingly, the mRNA expression levels of LBH and PAI-1 (serving as a positive control) were elevated in response to TGF-β in a time-dependent manner (Fig. 3C-E). In accordance with this, administration with SB431542, a small chemical inhibitor of the kinase activity of TGF-β type I receptor, resulted in a decrease of LBH expression (Fig. 3F-H). Furthermore, TGF-β-elicited upregulation of LBH was dramatically reduced upon knockdown of Smad2 or Smad3 (Fig. 3I-J). Subsequently, analysis of the Cistrome Data database revealed potential binding sites of Smad2/3 on the promoter region of LBH gene (Fig. 3K), and chromatin immunoprecipitation (ChIP)-qPCR assays approved that Smad2/3 could bind to the LBH gene promoter (+ 241/+500) upon TGF‑β signaling activation (Fig. 3L). These results demonstrated that TGF-β is an upstream signal that drives LBH expression in liver cancer cells through the Smad2/3 pathway.

Fig. 3.

LBH is transcriptionally upregulated by TGF-β in liver cancer cells. (A) Heatmap displaying the common 474 DEGs whose expressions were altered upon treatment with 100 pM of TGF-β1 for 4 h in three different HCC cell lines, i.e. MHCC97H, MHCC97L and HCCLM3. (B) Heatmap classification displaying some typical upregulated genes in response to TGF-β1 administration in HCC cell lines. (C-H) After being treated with 100 pM of TGF-β1 (C-E) or 10 µM of SB431542 (F-H) for the indicated periods, HCCLM3, HLE and SMMC-7721 cells were harvested for RNA purification and gene expression analyses through qPCR. (I-J) HCCLM3, HLE cells transfected with a control siRNA (NS) or those targeting Smad2/3 were treated with 100 pM of TGF-β1 for 6 h. Then the expression levels of Smad2, Smad3 and LBH were analyzed by qPCR. (K) Gene track view of Smad2/3, H3K4me3, and H3K27ac at the LBH promoter from the Cistrome Data database (top), and schematic diagram of ChIP primers designed within and near the LBH promoter region (bottom). HCCLM3 cells were treated with 2.5 ng/ml TGF-β for 24 h. ChIP assays were then performed to examine the binding of Smad2/3 to the LBH promoter region, and the enriched DNA fragments were quantitatively analyzed by qPCR. qPCR experiments described above were analyzed by one-way ANOVA followed by Tukey’s or Dunnett’s post hoc test. Statistical significance: *p < 0.05, **p < 0.01 and ***p < 0.001; ns, no significance. Data were shown as mean ± SD

Depletion of LBH ameliorates TGF-β-mediated protumor activities in HCC

Given that LBH is a downstream transcriptional target gene of TGF-β in liver cancer cells, we reasoned that LBH might mediate the functions of TGF-β. To test this, HCCLM3 and HLE cells that stably express LBH-specific shRNAs or the control vector were treated with TGF-β, followed by CCK8 cell-counting and Transwell assays. As a result, TGF-β treatment led to enhanced proliferation (Fig. 4A-B), migration and invasion (Fig. 4C-F) of liver cancer cells, which were greatly attenuated upon LBH knockdown (Fig. 4A-F). In support of this functional relationship between TGF-β and LBH, mining of the CLCA database showed that the expression of LBH was positively correlated with those of genes encoding TGF-β ligands and receptors, including TGFB1, TGFB2, TGFB3, TGFBR1 and TGFBR2 (Fig. 4G-K). Moreover, HCC patients with high expression levels of both TGFB1 and LBH had an apparent shorter OS rate than patients with TGFB1- and LBH-low expression levels in the TCGA-LIHC database (Fig. 4L). Together, the above results suggest that LBH acts as a critical mediator of the oncogenic functions of TGF-β in liver cancer.

Fig. 4.

Downregulation of LBH ameliorates TGF-β-mediated protumor activity in vitro. (A-B) HCCLM3 (A) or HLE (B) cells stably carrying LBH-targeting shRNAs or its control vector were treated with or without 100 pM of TGF-β1, followed by cell growth assay by CCK8 cell-counting. (C-F) After being treated with or without 200 pM of TGF-β1 in 2% FBS-containing DMEM, HCCLM3 (C, D) or HLE (E, F) cells expressing LBH-targeting shRNAs or its control vector were subjected to Transwell migration and matrigel invasion assays, respectively. The results were quantified (D, F). (G-K) The expression of LBH positively correlates with those of TGFB1 (G), TGFB2 (H), TGFB3 (I), TGFBR1 (J) and TGFBR2 (K), respectively, in the CLC Atlas. (L) The Kaplan-Meier curve of the OS rates of HCC patients associated with TGFB1/LBH-high or -low expression levels from the TCGA-LIHC database. For the above CCK-8 and transwell assays, results were analyzed by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons (A, B, D and F). Statistical significance: *p < 0.05, **p < 0.01 and ***p < 0.001; ns, no significance. Data were shown as mean ± SD. Pearson correlation tests were employed to assess gene expression level correlations (G-K). p-values for (L) were calculated using log-rank

LBH interacts with Mortalin

The next issue is the underlying molecular mechanism by which LBH boosts HCC progression. To address this, HEK293-FT cells overexpressing GST-fused LBH or the control GST-expressing vector were subjected to a GST pulldown assay followed by mass spectrometry (MS) analysis (Fig. 5A). Among the potential interacting partners of LBH is Mortalin (also named HSPA9) (Fig. 5A), which functions as a mitochondrial chaperone, or localizes in the cytosol or nucleus to regulate cellular homeostasis [30, 31]. Of interest, Mortalin has been reported to be highly expressed in a wide range of cancers including liver cancer, exerting an oncogenic role by promoting tumor cell proliferation, EMT and invasion [30]. Immunofluorescence (IF) analysis indicated that stably expressed LBH resided in both the cytoplasm and the nucleus in HCC cells, while Mortalin is mainly distributed in the cytoplasm, where the two proteins colocalize with each other (Fig. 5B). Then, GST-pulldown assays were performed to show that overexpressed LBH and Mortalin could form a protein complex in HEK293-FT or different HCC cells (Fig. 5C-F). Moreover, co-IP experiments verified that stably expressed LBH-3×Flag associated with endogenous Mortalin (Fig. 5G-I). These results demonstrated that Mortalin is a novel binding partner of LBH in the cytoplasm.

Fig. 5.

LBH interacts with Mortalin. (A) HEK293-FT cells expressing GST-LBH or the control GST-expressing vector were subjected to GST pulldown assays using glutathione-Sepharose beads, followed by coomassie brilliant blue staining of LBH overexpressing cells (left) and mass spectrometry (MS) analysis. Mortalin (HSPA9) was identified as a potential interacting protein by MS (right) (B) HCCLM3 or SMMC-7721 cells expressing LBH-3×Flag were subjected to immunofluorescence assays. Scale bar = 20 μm (C-F) HEK293-FT (C), HCCLM3 (D), HLE (E) or SMMC-7721 (F) cells transfected with plasmids encoding GST-LBH and Mortalin-3×Flag were subjected to GST pulldown assays using glutathione-Sepharose beads, followed by immunoblotting assays with anti-Flag and anti-GST antibodies. The protein expression levels in the input were examined by indicated antibodies. (G-I) HCC cells expressing LBH-3×Flag were subjected to co-IP assays with anti-Flag antibody or the control IgG, followed by immunoblotting assays

LBH boosts the malignant properties of liver cancer cells through Mortalin

Before examining the role of Mortalin in liver cancer, we firstly explored its expression in the TCGA database, which may have updated and expanded data from cancer patients than previously reported. As a result, the expression of Mortalin is upregulated in the majority of cancer types (Fig. 6A). Importantly, the expression level of Mortalin is much higher in the human HCC tissues than the normal liver tissues (Fig. 6B), and high Mortalin expression did predict a worse progression-free interval (PFI) in patients in the TCGA-LIHC (Fig. 6C). Through high-pressure hydrodynamic tail vein injection (HTVi), c-Myc (20 µg) and Δ90-β-catenin were expressed in mouse hepatocytes, driving spontaneous HCC development in male C57BL/6 mice. Exploiting this approach, expression of either LBH or Mortalin was capable of promoting HCC growth in mice (Fig. 6D). Analyses of the tumor samples revealed that LBH and Mortalin enhanced tumor cell proliferation and tumor angiogenesis, as indicated by elevated levels of Ki67 and CD34, respectively (Fig. 6E). To further explore the funcitonal link between LBH and Mortain in HCC, we first approved the efficacy of siRNAs targeting the Mortalin 5’ UTR region (Fig. 6F). Indeed, knockdown of Mortalin alleviated LBH-mediated migration and invasion of HCC cells, while ectopic expression of Mortalin further reinforced the effects (Fig. 6G-K). Moreover, restoration of Mortalin expression when siRNA was transfected rescued the phenotypes (Fig. 6G-K). Collectively, these results indicate that Mortalin plays a critical role in the tumor-promoting function of LBH-mediated HCC progression.

Fig. 6.

LBH promotes the proliferation and invasion capabilities of HCC cells through Mortalin. (A) The Mortalin expression levels in pancancer tissues and their normal counterparts in the TCGA database. (B) The expression of Mortalin in HCC tissues and normal liver tissues of human patients from the TCGA-LIHC database. (C) Kaplan-Meier diagrams illustrating the progression-free interval (PFI) in different Mortalin expression groups from the TCGA-LIHC database. (D) Images of the gross morphology and tumor weight of in situ HCC induced by HTVi-mediated expression of c-Myc and Δ90-β-catenin (n = 4/group). (E) Analyses of the above HTVi-induced HCC tumor tissues by H&E and IHC (Ki67, CD34) stainings. (F) HCCLM3 and SMMC-7721 cells transfected with siRNAs specifically targeting the 5’UTR region of Mortalin or a non-specific control one (NC) were lysed for immunoblotting assay. (G-I) HCCLM3 cells stably expressing LBH-3×Flag, Mortalin-3×Flag or the control lentivector were transfected with siRNAs. Then, these cells were subjected to Western blot (G), or transwell assays followed by cell number quantification (H-I). Scale bar: 100 μm. (J-K) The migration and invasion capabilities of SMMC-7721 cells were similarly carried out as above. Scale bar: 100 μm. Two-group comparisons were analyzed using a two-tailed Student’s t-test (B). p-values for (C) was calculated using log-rank. The liver-to-body weight ratio in HCC mouse models was assessed by one-way ANOVA followed by Dunnett’s post hoc test (D). Transwell assays were subjected to one-way ANOVA with Tukey’s post hoc test for multiple comparisons (I and K). Statistical significance: *p < 0.05, **p < 0.01 and ***p < 0.001

Discussion

As a developmental transcription cofactor, LBH has been connected with several human diseases, including congenital heart disease, heart and liver fibrosis, inflammatory arthritis, Alzheimer’s disease, and cancer [7, 8, 10, 52, 53]. It seems like that the expression and function of LBH differ in distinct cancer types. LBH has been shown to be highly expressed in glioma, gastric and breast cancer, and acts as a tumor promoter [16, 19, 20, 54]. Conversely, LBH is expressed at low to moderate levels in nasopharyngeal carcinoma, prostate cancer and lung cancer, where it functions as a tumor suppressor. This differential expression and functional duality of LBH across distinct cancer types may beattributable to tissue-specific epigenetic landscapes, distinct upstream and downstream signaling pathways, or heterogeneous cytokine milieus within the tumor microenvironment. However, whether and how LBH is involved in liver cancer progression is less understood. In this study, we found that LBH is upregulated in human HCC tissues in comparison to the normal liver tissues, and exerts a tumor-promoting role as revealed by in vitro cellular assays and in vivo mouse xenograft and primary HCC models (Figs. 1, 2 and 6D-E).

The expression of LBH is controlled by some cancer-related signaling pathways. For instance, it has been well documented that LBH is a direct target gene of the Wnt/β-catenin pathway in mammary epithelial cells and basal-like or triple-negative breast cancer cells, leading to expansion of mammary stem cells and cancer progression [20, 21]. LBH is transcriptionally upregulated by the hypoxic condition through the transcription factor HIF-1, thereby mediating the tumor-promoting effects of hypoxia in glioma [17]. Moreover, another study suggested that the high expression level of LBH could be attributed to circular RNA- and miRNA-mediated regulation in glioma stem cells [55]. Here, we revealed that the elevated expression of LBH in liver cancer is under the control of the TGF-β/Smad pathway. This was supported not only by our transcriptome sequencing data and experimental evidence in HCC cells, but also by the facts that the expression of LBH positively correlates with those of TGF-β ligand- and receptor-encoding genes (Fig. 3). Mounting evidence has established that TGF-β plays a critical tumor-promoting role in various cancers by regulating tumor cell malignant properties and the remodeling of tumor microenvironment including the immune microenvironment [23, 24, 27, 28, 56]. Thereafter, different strategies and drugs have been developed to target the TGF-β pathway [28, 29]. Although promising in preclinical cellular or mouse models, these drugs are unsuccessful in clinical trials due to the bidirectional functions of TGF-β in cancer and the nonnegligible side effects when administration of these drugs [24, 28, 56]. Our results that LBH acts as a functional mediator of TGF-β in promoting HCC progression provide an alternative opportunity to target the downstream oncogenic arm of TGF-β. In support of our observations, TGF-β has also been evidenced to regulate the expression of LBH in breast cancer, cardiomyocytes and heart fibroblasts, suggesting that the TGF-β/LBH axis could play a wider range of biological functions [8, 11, 57]. It is notable that although LBH upregulation in HCC is driven by TGF-β signaling, the correlation between LBH expression and patient survival outcomes in the TCGA database is relatively weak. This observation may be explained by the high heterogeneity of HCC, the biphasic functional properties of the TGF-β pathway, and the complex regulatory network governing LBH expression.

Although having been shown to regulate gene expression or cancer-related signaling pathways in certain cancer contexts, the molecular mechanisms underlying the actions of LBH are quite limited. We carried out a GST-pulldown assay followed by mass spectrometry (MS) analysis, identifying Mortalin/HSPA9, a member of the HSP70 family chaperone proteins, as a crucial interacting partner of LBH (Fig. 5). Our results also showed that the aberrant expression of Mortalin is linked to a worse clinical outcome in HCC patients (Fig. 6). In addition, depletion of Mortalin greatly alleviated the oncogenic functions of LBH (Fig. 6), suggesting that LBH and Mortalin could interact with each other both physically and functionally in HCC. As a matter of fact, Mortalin is elevated across various cancers and associated with poor prognosis in different types of cancers [32, 44, 58, 59]. Mortalin plays important tumor-promoting effects in multiple cancers by interacting with and inactivating the tumor suppressor p53 protein, thereby promoting cancer cell survival, proliferation, invasion and metastasis [40]. Mortalin may also exert its oncogenic role by regulating different signaling pathways, such as the ERK/MAPK, Wnt/β-catenin and JAK/STAT pathways [30, 31, 43, 48]. Intriguingly, Mortalin was suggested to serve as a biomarker for the early recurrence of HCC and promote the migration and invasion of liver cancer cells through the RECK/STAT3 pathway [46–48]. Our study has established a pivotal connection between Mortalin and LBH during HCC progression. It is notable that LBH resides not only in the nucleus but also in the cytoplasm, where it colocalizes with Mortalin (Fig. 5B). Our observations are supported by some other studies indicative of a cytoplasmic function of LBH [12, 13]. Although the present study demonstrates that Mortalin plays an essential role in LBH-mediated tumorigenic functions in liver cancer through their protein-protein interaction, further in-depth investigations are warranted to explore whether this interaction modulates the function of Mortalin as a chaperone protein and its role in regulating mitochondrial activity.

In summary, this study elucidated a pivotal role of the TGF-β/LBH/Mortalin axis in liver cancer progression, thus providing a promising target for cancer intervention.

Materials and methods

Cell culture

Human embryonic kidney epithelial cell lines HEK293-FT and HEK293T, and human HCC cell lines HCCLM3 and SMMC7721 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Corning Inc.) cultured with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (P/S, Solarbio, China). HLE cells were cultured in RPMI-1640 medium (Solarbio, China) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). All cell lines were maintained at 37℃ in a humidified, 5% CO₂ incubator. All cell lines utilized in this study have been authenticated via Short Tandem Repeat (STR) genotyping.

Plasmids, siRNAs and reagents

Mammalian expression constructs of LBH and Mortalin, fused with GST- or 3×Flag tag, were generated based on the pEGB2, pSEB-3×Flag and pT3-EF1α-MCS vectors, respectively. The DNA sequence encoding LBH-3×Flag was cloned into the pCDH-CMV-MCS-EF1-copGFP-Puro lentivirus vector. LBH-specific shRNAs (seed sequences: shRNA#1, 5’-GCGAAGAGACAGCGAAAGAA-3’ and shRNA#2, 5’-GATGAGCAAGATAACTGCGAA-3’) were constructed based on the pLKO.1-TRC vector. All primers were synthesized by Sangon Biotech (Hunan, China), and all constructs were validated by sequencing.

The siRNA sequences in this study were as follows: Smad2, 5’-GTCCCATGAAAAGACTTAA-3’; Smad3, 5’-GAGGCTGTCTACCAGTTGA-3’; Mortalin siRNA #1, 5’-GGCUCAUGCUUCCCAUAUUTT-3’, #2, 5’-GGGAAACUUAGCUAUUUAUTT-3’ and #3, 5’-GGAGGUAGGAUGAAUUGAUTT-3’. All siRNAs were synthesized by RiboBio (China) and transfected with the si-Tran™ siRNA Transfection reagent (OriGene).

Recombinant human TGF-β1 protein was obtained from R&D Systems Inc., and the small molecule inhibitor SB431542 was purchased from MedChemExpress (MCE, USA).

Lentivirus production and stable cell line establishment

A three-plasmid system was used for lentivirus production. HEK293-FT cells were plated in 60 mm dishes and allowed to reach approximately 80–90% confluence the next day. HEK293-FT cells were starved with 4 mL of opti-MEM (Gibco, USA) for 1–2 h. Then, two package plasmids psPAX2 and pMD2.G, along with the lentivirus plasmids (pCDH-CMV-MCS-EF1-copGFP-Puro for gene overexpression and pLKO.1-TRC for knockdown), were transfected using Lipofectamine 3000 (Invitrogen). At 12 h post-transfection, the cell culture medium was changed to DMEM with 5% FBS. The cell supernatants containing virus particles were collected at 48–72 h, centrifuged at 500×g for 5 min to remove cell debris, filtered with a 0.45 μm membrane filter, and stored in a -80℃ refrigerator. HCC cells were infected with lentivirus supernatants that included 8 µg/ml of polybrene. 1 µg/mL of puromycin (Sigma-Aldrich, St. Louis, MO) were added to select the stable cells.

Reverse transcription (RT) and quantitative real-time PCR (qPCR)

Total RNAs were extracted using Trizol (Life Technologies) according to the manufacturer’s protocol. The quality and integrity of the total RNAs were assessed using a NanoDrop spectrophotometer to determine the A260/A280 ratio (ranging from 1.8 to 2.0) and A260/A230 ratio, as well as by agarose gel electrophoresis to verify RNA integrity. Then 1 µg RNA was reverse-transcribed using the PrimeScript RT Reagent Kit (Takara). qPCR was performed in triplicate using the Power SYBR-Green PCR SuperMix (Mei5 Biotechnology, Co., Ltd., Beijing, China) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc. USA). Gene expression was normalized to GAPDH or Actin as internal reference genes. Overall, the results obtained using the two reference genes were consistent in the liver cancer cell lines used in this study.

The comparative threshold cycle (Ct) method was used to determine the fold change (2^−ΔΔCt) in mRNA levels of various genes. The mRNA levels were normalized to the reference gene GAPDH. Primers for human gene qPCR are listed in Table 1.

Table 1.

qPCR primers for human genes

Gene	Sequence
LBH	Forward	5’-GCCCCGACTATCTGAGATCG-3’
	Reverse	5’-GCGGTCAAAATCTGACGGGT-3’
PAI-1	Forward	5’-ACCGCAACGTGGTTTTCTCA-3’
	Reverse	5’-AATCCCATAGCTGCTTGAAT-3’
Smad2	Forward	5’-CGTCCATCTTGCCATTCACG-3’
	Reverse	5’-CTCAAGCTCATCTAATCGTCCTG-3’
Smad3	Forward	5’-GCGTGCGGCTCTACTACATC-3’
	Reverse	5’-GCACATTCGGGTCAACTGGTA-3’
GAPDH	Forward	5’-ACCACAGTCCATGCCATCAC-3’
	Reverse	5’-TCCACCACCCTGTTGCTGTA-3’

Immunoblotting, immunoprecipitation, immunofluorescence, and mass spectrometry

Immunoblotting and immunoprecipitation were similarly performed as previously described [50]. For immunofluorescence, cells were grown on chamber slides (BD Falcon, MA), washed with cold 1×PBS, fixed with 4.0% paraformaldehyde for 10 min at room temperature, and permeabilized with 0.5% Triton X-100 for 20 min. After blocking with 0.5% of BSA (Solarbio, China) for 1 h, samples were incubated with the indicated primary antibody at 4 °C overnight. After washing, samples were incubated with a secondary antibody for 1 h at room temperature in the dark. The nuclei were counterstained with DAPI (Sigma). Images were obtained with using a confocal ZEISS LSM 800 microscope.

For mass spectrometry, HEK293-FT cells expressing GST-LBH or the empty vector pEBG2 were lysed for GST pulldown using glutathione-Sepharose beads. SDS-PAGE gels were minimally stained with Coomassie brilliant blue, cut into small pieces, digested with sequencing-grade modified trypsin (Promega, USA), and then incubated at 37℃ overnight. Prior to mass spectrometry analysis, the peptide preparations were desalted using StageTip and reconstituted with 0.1% formic acid (FA). The protein complexes were identified on a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer instrument (Thermo Fisher) coupled with an Easy-nLC 1200 system. The resulting raw data were analysed using MaxQuant (version 2.0.3.0) and were searched against the reviewed Human Universal Protein Resource (UniProt) sequence database. The pulldown-mass spectrometry results were transferred into a FileMaker-based relational database generated in-house, where protein identification numbers (protein GIs) were converted to GeneID identifiers according to the NCBI ‘gene accession’. Proteins with a fold change ≥ 2.0 and a p-value < 0.05 were considered as candidate interacting proteins.

Antibodies used in this study were as follows: anti-Flag (700002) antibody was bought from ZEN-BIO (China). Anti-GST (66031-1-Ig); anti-TUBULIN (14887-1-AP), anti-Mortalin (HS201-01), CoraLite488-conjugated Goat Anti-Mouse IgG(H + L) (RGAM002), and Cy3–conjugated Goat Anti-Rabbit IgG(H + L) (RGAM002) antibodies were bought from Proteintech (China); ProteinFind^®Goat anti-Mouse IgG (H + L) HRP conjugate (HS101-01) and ProteinFind^®Goat anti-Rabbit IgG (H + L) HRP conjugate (SA00013-1) antibodies were from TransGen Biotech (China).

Chromatin immunoprecipitation (ChIP)

HCCLM3 cells were cross-linked with 1% paraformaldehyde at 37°C for 10 minutes, followed by termination of the cross-linking reaction with 125 mM glycine at room temperature for 5 minutes. The cells were then washed twice with cold PBS and lysed in ChIP lysis buffer for 10 minutes. Ultrasonic fragmentation was performed to generate DNA fragments of 100–800 bp. After centrifugation at 15,000×g for 20 minutes at 4°C, the supernatant was collected for chromatin immunoprecipitation. Subsequently, protein A agarose beads blocked with bovine serum albumin were pre-washed, and then Smad2/3 antibody or rabbit-derived IgG was added to the samples and incubated overnight at 4°C. The beads were washed, and the bound DNA was eluted with elution buffer. Decross-linking was carried out at 65°C overnight. Finally, the extracted DNA was analyzed by qPCR. The ChIP-PCR primers are as follows: pair #1, -918 to-659 bp from the transcription start site (TSS) of the LBH gene, forward 5’-ACTCCTCGGAGAAGACGTGG-3’ and reverse 5’-CAAGGATGTGGTGTCGTCCT-3’; pair #2, -484 to -234 bp from LBH TSS, forward 5’-GCCAGTGGATGGGAAGGAG-’ and reverse 5’-CGGGACATGACAGCTCGG-3’; pair #3, + 241 to + 500 bp from the TSS, forward 5’-CCGTGCAGGAGTCAACGTCA-3’ and reverse 5’-CGCCAACCAATACAAACCCT-3’; primer #ns, forward 5’-CCGTGCAGGAGTCAACGTCA-3’ and reverse 5’-AGGCACCCAGAGGTGATTTT-3’.

Cell growth, migration and invasion assays

For cell proliferation assay, 3,000 cancer cells were seeded per well in 96-well plates, and treated with or without 200 pM of TGF-β1 for distinct time periods. Then, cell growth was assessed using a Cell Counting Kit-8 (life-ilab, China) following the manufacturer’s instructions, and detected on a multifunctional hyphenated microplate reader (Molecular Devices, INC., USA). Transwell cell migration and matrigel invasion assays were carried out as described previously [50]. Migrated or invasive cells were counted using ImageJ. All the above experiments were designed in duplicates.

Animal model, immunohistochemistry (IHC), hematoxylin and eosin (H&E) staining

Animal experiments in this study were conducted according to guidelines approved by the Ethics Committee of the Institutional Animal Care and the Committees of Nanchang University. Male BALB/c nude mice (5 weeks old, 18–20 g) and C57BL/6 mice (8 weeks old, 20–25 g) were purchased from GemPharmatech (Nanjing, China). For nude mouse xenograft experiments, 1 × 10⁶ HCCLM3 cells that stably overexpressed LBH or the control lentivirus vector were inoculated into the armpit regions of the male BALB/c nude mice (n = 7 mice/group). All mice were sacrificed three weeks after tumor cell transplantation, and tumors were collected. Tumor size was measured by a vernier caliper, and tumor volume was calculated following the formula: volume = length×width²/2.

Hydrodynamic tail vein injection (HTVi) was used to establish mouse spontaneous HCC models. Plasmids encoding c-Myc (20 µg), Δ90-β-catenin (20 µg) and SB100 × transposition enzyme (4.5 µg), along with the pT3-EF1α plasmids expressing LBH-3×Flag or Mortalin-3×Flag were mixed with 2 ml of sterile 0.9% NaCl solution. Then, the mixed injection agents were administered to 8-week-old C57BL/6 mice (n = 4 mice/group) via the lateral caudal vein within a time frame of 4–6 s. When the mice had a distended abdomen, euthanasia was performed, and the mouse and their livers were weighed.

Tumor tissue samples were fixed in 4% formaldehyde and embedded in paraffin, and cut into 5 μm sections. For IHC staining, sections were deparaffinized and rehydrated, and then antigens were recovered in 10 mM citrate buffer (pH 6.0) for 10 min in a microwave oven. H₂O₂ (3%) was used to block endogenous peroxidase, followed by PBS wash. After being blocked in 5% goat serum at room temperature for 10 min, tissue sections were incubated with primary antibodies for Ki67 (GB121141-50, 1:300, Servicebio, Wuhan, China) and CD34 (GB111693-100, 1:500, Servicebio, Wuhan, China) for 12 to 16 h at 4 °C. Then, samples were incubated with the second antibody, followed by a diaminobenzidine (substrate of peroxidase) revelation and counterstaining with Mayer’s hematoxylin. For H&E staining, sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin. Slices were analyzed under a microscope.

Statistical analyses

All experiments were performed at least in replicates. Data are presented as the mean ± standard deviation (SD). Statistical significance was evaluated using a two-tailed Student’s t-test for comparisons between two groups. For multi-group comparisons, one-way ANOVA was applied, followed by Tukey’s post hoc test for multiple comparison correction or Dunnett’s test for comparisons relative to a defined control group. All statistical analyses were performed using GraphPad Prism 10.0 software, and p < 0.05 was considered statistically significant. pvalues for the survival rates of HCC patients were calculated using log-rank, while pearson correlation tests were employed to assess gene expression correlations.

Author contributions

XP (Xingqi Peng) and ZL conducted the bioinformatics analysis, performed the majority of the experiments, analyzed the results, prepared the figures, and wrote the manuscript. CL and YM assisted with data analysis and conducted several experiments. YL and WL contributed to the completion of some functional experiments. CZ and JY assisted with most of the plasmid construction. WW, ZL and YZ provided critical technical supports for the study. XY and LC conceived and designed the study, revised the manuscript, and approved the final version.

Funding

This work was supported by grants from the National Natural Science Foundation of China (NSFC, 32370765), the International Science and Technology Cooperation Project of Jiangxi Province (20244BDF60013), the Natural Science Foundation of Jiangxi Province (20224ACB206032 and S20257359), the Jiangxi Provincial Key Research and Development Program (20203BBGL73196), the Jiangxi Provincial Youth Science Fund (GJJ210218), and the Cultivation Fund Project of School of Basic Medical Sciences, Nanchang University.

Data availability

The RNA-seq raw data and processed DEGs have been documented in the NCBI GEO database with accession No. GSE168515. All other data are included within thearticle. The materials and methods in this study are available from the correspondingauthors upon reasonable request.

Declarations

Ethical approval

The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of Nanchang University (Ethics Approval No. NCULAE-20250912003). All procedures were conducted in strict accordance with the relevant institutional guidelines and regulations.

Consent for publication

All authors have read and approved the final manuscript and consent to its publication.

Competing interests

The authors declare no competing interests.