Background
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The HCC population in Asia and Africa accounts for the majority of all cases [
1]. HCC has the characteristics of slow onset, high aggressiveness and rapid growth [
2]. Despite improvements in comprehensive treatment regimen and considerable progress made in understanding its epidemiology, etiology, biology, diagnosis and treatment, the long-term prognosis of patients with HCC remains poor. Approximately 90% of cancer death are caused by metastasis, a complicated process involving tumor cell motility, intravasation, circulation, extravasation and growth in new tissues and organs [
3]. The increased motility and invasive properties of metastatic tumor cells are reminiscent of events that occur during epithelial-mesenchymal transition (EMT). EMT is a process in which epithelial cells lose their cell polarity and cell-cell adhesion, and then gain a mesenchymal phenotype with migratory and invasive properties. EMT has been proposed as a vital mechanism for epithelial cancer cells to acquire a malignant phenotype, especially invasion and metastasis [
4]. EMT activation is usually initiated and controlled by cellular signals that respond to extracellular cues, such as TGF-β, Wnt, Notch, and Hedgehog signaling pathways. However, the mechanism by which tumors induce EMT to facilitate invasion and metastasis remains largely unknown.
The calcium signal is a key mechanism well suited to the rapid translation of signals from the tumor microenvironment into cellular responses. In addition to its important roles in the growth of the primary tumor, calcium signaling is also important in the context of cancer cell migration and invasion [
5]. Calcium-dependent migration and invasion has been observed in prostate and breast cancer cells [
6,
7]. A number of well-known molecular players in cellular Ca
2+ homeostasis, such as the constituents of store-operated Ca
2+ entry and calcium release-activated calcium channel proteins, have been implicated in the process of cancer cell metastasis and migration.
As the “power factory” of the cell, mitochondria have also been generally considered to regulate the intracellular calcium homeostasis. Mitochondrial calcium uptake is believed to be essential in regulating cellular signaling events, energy status, reactive oxygen species (ROS) production and survival [
8]. The uptake of Ca
2+ by mitochondria depends on mitochondrial Ca
2+ uptake channel (mitochondrial calcium uniporter, MCU) and its regulatory subunits, such as MICU1 (mitochondrial calcium uptake 1) and MCUR1 (mitochondrial calcium uniporter regulator 1) [
9‐
11]. Previous studies have demonstrated that MCU complex and its regulatory proteins are frequently dysregulated in several types of cancer, including breast, prostate, ovarian and colorectal cancer [
7,
12‐
14]. Moreover, the expression aberration of these proteins facilitates proliferation, migration, invasion and apoptotic resistance of cancer cells and is frequently associated with poor prognosis of cancer patients [
15].
In our previous study, we have found that MCU expression is significantly elevated in HCC cells and MCU-dependent mitochondrial Ca
2+ uptake promotes ROS production, cell metastasis and poor prognosis of HCC patients [
15]. Moreover, we have also found that MCUR1-mediated mitochondrial calcium signaling promotes HCC cell survival via ROS signaling [
16]. These data suggest that MCUR1 may be involved in the metastasis and invasion of HCC. However, this hypothesis remains to be tested and the underlying mechanisms need further investigation.
In the present study, we systematically investigated the effects of MCUR1 on HCC metastasis and the underlying mechanisms. We have demonstrated that MCUR1-mediated mitochondrial calcium signaling triggers ROS generation and subsequent Notch signaling pathway, which contributes to the EMT of HCC cells and poor prognosis of patients. Our data provide novel evidence supporting MCUR1 as a potential promising therapeutic target for the treatment of HCC patients.
Methods
Antibodies and reagents
Antibodies used in this study were listed in Additional file
1: Table S1. The mitoROS scavenger MitoTEMPO, Nrf2 activator OPZ, Notch1 inhibitor DAPT, H
2O
2, histamine were purchased from Sigma-Aldrich (St Louis, MO, USA).
Cell culture and tissue
Human HCC cell lines BEL7402 and MHCC97L were from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCC cell lines were authenticated using short tandem repeat DNA testing by the FMMU Center for DNA Typing in 2018. And all cell lines were routinely cultured. Human HCC tissue samples without metastasis (n = 74) or with metastasis (n = 63) from surgical HCC patients were obtained in Xijing Hospital affiliated with Fourth Military Medical University, Xi’an, China, with signed informed consents. All tissues were assessed by H&E staining to select suitable regions for further examination. The latest follow-up date was January 2016 and the median follow-up duration was 28.5 months (ranging from 2.4 to 85.6 months).
Knockdown and forced expression for the target genes
A small hairpin RNA (shRNA) specifically targeting the human MCUR1 mRNA sequence (5′-AAGGACAUCGUCUACAAAGAU-3′) was cloned into the vector of pSilencer™ 3.1-H1 puro (Ambion). For overexpression of MCUR1, the cDNA of MCUR1 was cloned into the vector of pcDNA™3.1(+) (Invitrogen). All siRNAs were synthesized by GenePharma (Shanghai, China) and the sequences were provided in Additional file
1: Table S2. The cDNA of the Ca
2+ binding protein parvalbumin (PV) was cloned into pAc1GFP1-Mito vector using primers listed in Additional file
1: Table S2 to establish PV-Mito-green fluorescent protein and PV-Mito construct. The pAd-Easy adenovirus system was used for generation of recombinant adenoviruses carrying mitochondria-targeted PV (Ad-PV-Mito).
Immunofluorescence staining assay
Cells grown in 15-mm coverglass-bottom dish (NEST, Wuxi, China) were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) with 0.2% Triton. Cells were then blocked for 1 h with 2% bovine serum albumin followed by incubation with primary antibody overnight at 4 °C and then with fluorophore-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA). Cells were examined with a confocal laser scanning microscope FV1000 (Olympus).
Western blot analysis
Protein expression levels in HCC cells were examined by Western blot as our previous description [
17] . The antibodies and their dilutions were listed in the Additional file
1: Table S1.
Scratch wound healing assay
Cells were seeded in six-well plates and cultured to almost total confluence in 24 h, and then a wound was scratched in each well using a 10-μl pipette tip. Cells were washed with PBS for several times and then incubated in culturing medium without serum. Wound closure was monitored at 0 and 48 h on a Nikon Eclipse TS100 microscope. The distance of cell migration at 0 and 48 h after scratching was evaluated. Migration rate (%) was calculated as (Migrated distance at indicated time - initial distance) / (initial distance) × 100%.
Transwell assays
Cell migration and invasion ability was assessed using transwell assay. For cell invasion assays, transwell chambers were coated with Matrigel. The upper chamber was seeded with 3 × 104 cells suspended in 500 μl of serum-free medium, while the lower chamber was filled with 20% FBS medium. After 24 h incubation, the cells in the upper chamber were carefully removed with a cotton swab. The chamber was then immersed in 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 5 min. The invasive cells attaching to the lower surface of the membrane were counted under microscopy. For cell migration assays, the procedure was similar to that for the invasion assays without Matrigel coating.
For in vivo metastasis assay, five-week-old and weighing 18-20 g each nude mice (BALB/c) were randomly divided into subgroups (five mice per group). Cells (2.0 × 106 for each mouse) were resuspended in 50 μL PBS and orthotopically injected into the left hepatic lobe of nude mice (male). Eight weeks later, mice were sacrificed under anesthesia. The liver and lung were resected and fixed with 4% paraformaldehyde. Serial 5-μm sections were stained with H.E. for histopathological examination. Metastasis lesions from 10 random high-power fields were counted. Housing and all other procedures were performed according to protocols approved by the Animal Experimentation Ethics Committee of the Fourth Military Medical University. All animals received human care and study protocols were complied with the institution’s guidelines.
Immunohistochemical (IHC) staining
Four-μm-thick tissue sections were cut from human HCC tissue blocks and xenograft tumor nodes. IHC was performed as previously described [
17]. The intensity and extent of immunostaining were evaluated for all samples under double-blinded conditions as previously described [
17].
Measurement of mitochondrial Ca2+
For measurement of basal mitochondrial Ca2+, cells were transiently transfected with the plasmid carrying mitochondrial matrix-targeted inverse pericam, which was defined as mitopericam (kindly provided by Dr. Atsushi Miyawaki from RIKEN Brain Science Institute, Japan). Then cells were examined with a confocal laser scanning microscope FV1000 (Olympus, Tokyo, Japan). For measurement of dynamic mitochondrial Ca2+, histamine (10 μM) was added after 30 s of baseline recording, 380 and 490 nm excitation filters were used together with a 540 nm emission filter and images were recorded every 3 s.
Detection of reactive oxygen species
Fluorescence probe DCFH-DA (Beyotime, Beijing, China) was used to detect cellular reactive oxygen species (ROS). The fluorescence intensity was assessed by flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The fluorescence probe mitoSOX (Invitrogen) was used to detect mitochondrial reactive oxygen species (mitoROS) under microscopy (FluoView 1000). Images were captured and analyzed by ImagePro image analysis software (Media Cybernetics, Silver Spring, MD, USA).
Statistical analysis
Experiments were repeated three times, where appropriate. Data represent mean ± SD. SPSS 17.0 software (SPSS, Chicago, IL) was used for all statistical analyses and P < 0.05 was considered significant. Unpaired t tests were used for comparisons between 2 groups where appropriate. Correlations between measured variables were tested by Spearman’s rank correlation analyses.
Discussion
Previous studies have demonstrated the important role of MCUR1 in HCC cell survival. Similar with previous reports, our study further confirmed the clinical significance of MCUR1 in EMT and HCC metastasis. More importantly, we provided the first evidence indicating high MCUR1 expression in HCC tissues with metastasis and significant association between MCUR1 expression and tumor progression in HCC patients. Moreover, the in vitro and in vivo experiments also first demonstrated that MCUR1-induced mitochondrial Ca
2+ uptake activated the ROS/Nrf2/Notch signaling and thus facilitated the epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma (Fig.
6g). Thus, MCUR1 has a great potential to be used as a prognosis marker or therapy target in clinical management of HCC patients.
EMT is closely involved in a series of pathological processes of HCC progression, including invasion, metastasis and chemoresistance. High expression of mesenchymal markers often indicates worse prognosis in HCC patients. EMT is regulated by several key transcriptional factors including Snail (zinc finger proteins Snail and Slug), Zeb (zinc finger and homeodomain proteins Zeb1 and Zeb2) and Twist (Twist1, Twist2) [
19]. In the present study, we for the first time demonstrated that MCUR1 expression was significantly associated with the EMT of HCC cells both in vitro and in vivo. Moreover, MCUR1 knockdown and overexpression caused the expression change of Snail instead of Slug, suggesting that MCUR1 may promote the EMT of HCC cells through regulating Snail. Simultaneously, we found that MCUR1 expression was significantly associated with the metastasis of HCC, and MCUR1 expression level significantly affected the invasion in vitro and metastasis in vivo of HCC cells. These data suggest that MCUR1 expression may contribute to the metastasis of HCC by promoting EMT.
Ca
2+ is the most abundant second messenger in human cells and has a substantial diversity of roles in fundamental cellular physiology, including gene expression, cell cycle control, cell motility, autophagy and apoptosis. Disruption of normal Ca
2+ signaling contributes to the development of malignant phenotypes. There has been an increasing awareness that tumorigenic pathways are associated with altered expression level or abnormal activation of Ca
2+ channels or transporters. Previous studies have demonstrated that abnormal Ca
2+ signaling is involved in the proliferation, adhesion, migration, invasion and EMT of cancer cells [
20]. Intracellular Ca
2+ homeostasis is largely regulated by mitochondria mainly through MCU complex and its regulators, such as MCUR1. Previous studies have demonstrated that the dysregulation of mitochondrial Ca
2+ uptake-associated proteins contributes to the survival, proliferation, metastasis and chemoresistance of cancer cells. MCUR1 is a critical component of MCU complex which is required for mitochondrial Ca
2+ uptake and maintenance of normal cellular bioenergetics. MCUR1 silencing resulted in a dramatic reduction in the [Ca
2+]mito (− ~ 85% in Rhod-2 fluorescence compared to the control) without modifying the cytosolic Ca
2+ content [
21]. However, its biological roles in cancer development remain largely unclear. Our previous studies have demonstrated that MCUR1 expression is frequently upregulated in HCC and MCUR1-mediated Ca
2+ signaling promotes HCC cell survival. In this study, our findings that chelating mitochondrial Ca
2+ by PV-Mito significantly suppressed MCUR1 overexpression-induced EMT and invasion of HCC cells further indicate that mitochondrial Ca
2+ signaling mediates MCUR1-induced HCC metastasis.
Mitochondria are an essential source of reactive oxygen species in most mammalian cells. [Ca
2+]
m has been reported to play an important role in the generation of ROS. Our previous studies have demonstrated that the overexpression of either MCU or MCRU1 promotes the production of ROS and oxidative status in HCC cells. ROS play a key role in many intracellular signaling pathways which contributes to the tumorigenesis. Nrf2 as the major effector of ROS in human cells regulates the expression of more than 100 genes, including Notch1 [
18]. Upon oxidative stress, Nrf2 is detached from its inhibitor Keap-1 and translocated to the nucleus to promote the transcription of target genes by binding to promoter regions [
22,
23]. Currently, growing evidences suggest that constitutive upregulation of Nrf2 is linked to cancer development, progression and resistance to radiotherapy. Moreover, Nrf2 promotes the invasion of cancer cells and contributes to poor prognosis of patients. In this study, we found that similar like H
2O
2, overexpression of MCUR1 had a remarkable effect to induce the Nrf2 nuclear translocation and Notch1 activation, which can be abolished by ROS scavenger. Moreover, Notch1 activation can be suppressed by either Nrf2 silencing or H
2O
2 scavenging, suggesting that ROS/Nrf2/Notch1 axis may act as the downstream signaling of MCUR1-induced Ca
2+ homeostasis remodeling.
Notch signaling is a critical regulator in embryo development through the modulation of cell–cell communication [
24]. Increasing evidences indicate that Notch signaling plays important roles in cancer development and progression [
25,
26]. Elevated Notch1 expression has been observed in a series of malignancies including HCC and is commonly associated with aggressive cancer phenotypes. Notch signaling has been shown to promote EMT of cancer cells [
27,
28]. Previous studies have demonstrated that the inhibition of Notch signaling by DAPT leads to the decrease of EMT in HCC cell lines, whereas activation of Notch signaling by Jagged1 promotes the increase of EMT [
29‐
31]. In this study, we found that MCUR1 overexpression promoted the activation of Notch1 signaling, which can be inhibited by Ca
2+ chelating, ROS scavenging, or Nrf2 silencing. Moreover, MCUR1-induced EMT as well as Snail transcription can be suppressed by Nrf2 silencing or Notch1 inhibitor DAPT. These findings suggest that MCUR1 facilitated HCC EMT, invasion and migration mainly through Nrf2/Notch1 signaling activation.