Cathelicidin LL-37 promotes EMT, migration and metastasis of hepatocellular carcinoma cells in vitro and mouse model

ABSTRACT The effect of cathelicidin hCAP18/LL-37 in hepatocellular carcinoma (HCC) metastasis remains unclear. Here, we confirmed that LL-37 expression enhanced endothelial-mesenchymal transition (EMT), migration and invasion in HCC cells. And the HER2/EGFR-MAPK/ERK signal participated in the process above. More frequent lung metastases were observed in an LL-37-overexpressing hematogenous metastasis model. Interestingly, 1,25(OH)2D3 together with si-LL-37 significantly enhanced 1,25(OH)2D3-induced inhibition of migration and invasion in PLC/PRF-5 cells, and also enhanced reversion of the EMT process. Therefore, LL-37 is involved in HCC metastases, and may act as an important factor to attenuate the inhibitory activity of 1,25(OH)2D3 on HCC metastasis. Targeting hCAP18/LL-37 may offer a potential strategy to improve the anticancer activity of 1,25(OH)2D3 in HCC therapy.


Introduction
Hepatocellular carcinoma (HCC) has the third highest mortality rate among cancer patients, resulting in 830,000 deaths annually [1]. Due to unsatisfactory progress in developing effective strategies for early diagnosis and treatments, 70% to 80% of patients have already reached an advanced stage at the time of diagnosis [2]. Metastasis is one of the main reasons for the high recurrence rate and poor prognosis of HCC. Although development of intrahepatic metastases is the most common reason why patients with advanced stage HCC die of liver failure resulting from advanced intrahepatic lesions, extrahepatic metastasis (EHM) via hematogenous spread, lymphatic dissemination or direct invasion is another important cause of death in HCC patients [3]. HCC patients with EHM usually have a poor prognosis with an expected median survival time of 6 months [4]. The lung is considered the most likely organ for HCC metastatic colonization, accounting for 47% of all EHM, followed by lymph nodes, bones and adrenal glands [3,5]. To date, systemic therapy for advanced HCC includes molecular targeted therapy, immune checkpoint inhibitors or a combination of both [6]. Based on the results of recent clinical trials, it appears that a single drug may not be sufficient for the treatment of HCC.
Combination therapy now represents a major research direction for the systemic treatment of advanced HCC [7]. It thus will be necessary to further our understanding of the mechanisms underlying HCC metastases in order to develop new therapeutic strategies against advanced HCC in the future.
Our previous research demonstrated that hCAP18/ LL-37 has a promotional effect on HCC cell proliferation and tumor growth both in vitro and in vivo [19]. Furthermore, hCAP18/LL-37 expression can be significantly induced by 1,25(OH) 2 D 3 in HCC cells and in xenograft tumor tissue, which in turn suppresses the antitumor growth activity of 1,25(OH) 2 D 3 in HCC xenograft tumors. In addition, 1,25(OH) 2 D 3 inhibits the migration, invasion or metastasis of several cancers including colon cancer [20], ovarian cancer [21] and prostate cancer [22] in vitro or in vivo. Moreover, 1,25(OH) 2 D 3 or its analog prevents lung and bone metastasis, and prolonged animal survival time was also reported in a breast cancer mouse model [23,24]. Provvisiero et al. (2019) showed that 1,25(OH) 2 D 3 prolonged everolimus-induced transition to the mesenchymal phenotype by restoring the epithelial phenotype in everolimus-resistant HCC cells in vitro, suggesting that 1,25(OH) 2 D 3 may be involved in the endothelialmesenchymal transition (EMT) of HCC cells [25]. However, the role of 1,25(OH) 2 D 3 on HCC metastasis, and the effect of hCAP18/LL-37 expression on the antimetastatic effects of 1,25(OH) 2 D 3 remains unknown.
EMT is a transformation process mandatory for the local and distant progression of many malignant tumors including HCC [26]. Usually, epithelial cells lose their characteristic marker E-cadherin and gain mesenchymal markers (such as N-cadherin and Vimentin) during the EMT process [27]. During the process, a number of transcription factors (such as Snail, Slug and Twist) are involved in the EMT of HCC, and their presence is associated with a poor prognosis [28]. Similar to most cancers, HCC tumor invasion and metastasis depend to a large extent on the proteolytic activity of a large number of matrix metalloproteinases (MMPs) which affect cell-to-cell and cellmatrix communication [29]. MMP levels and the activation of EMT-related transcription factors are controlled by several signaling pathways, which participate in migration, invasion and metastasis. Among these pathways, the mitogen-activated protein kinase (MAPK) pathway plays an indispensable role in the development of EMT in a variety of cancer cells and is closely related to the malignant behavior of tumors [30,31]. Our previous study showed that the MAPK pathway was significantly enriched in LL-37overexpressing HCC cells [19]. However, whether MAPK pathway is involved in migration and invasion of HCC cells in the anticancer activity of LL-37 remained unclear.
In the present study, we analyzed the effects of LL-37 expression on EMT, migration and metastasis in cultured HCC cells and in HCC xenograft and hematogenous metastasis mouse models. We also assessed the effect of LL-37 expression on 1,25(OH) 2 D 3 -mediated regulation of EMT, migration and invasion by HCC cells. Our findings revealed the role of hCAP18/LL-37 in migration and metastases of HCC cells, and may also help to develop an effective anticancer strategy for 1,25(OH) 2 D 3 in HCC treatment.

Plasmids construction and cell transfection
The LL-37 coding sequences (GenBank accession no. 820) was cloned from the cDNA of human L02 cells using Oligo dT23 primers including a pair of Bam HI and Xbal I restriction sites. Amplify the LL-37 coding sequence, and the eukaryotic expression vector pcDNA3.0-LL-37 was constructed and then transformed into DH5α E. coli. The orientation of the pcDNA3.0-LL-37 was verified by sequencing. The plasmid was transfected into HCC cells using the HiTrans TM LipoPlus reagent. Briefly, cells (3 × 10 5 cells/mL) were seeded into six-well plates and incubated for 12 h until they reached 80-90% confluence. 2 μg of pcDNA3.0, pcDNA/LL-37, si-control (scrambled control RNA), and si-LL-37 (5'-GTCCAGAGAATCAAGGATT-3') were added, respectively. After 24 h, to select for transfected cells, 800 µg/ ml G418 was administered for 3-5 days and until antibiotic-resistant colonies were observed. Selection of recombinant transfectants was performed for at least 30 days. Finally, the constructed stable PLC/PRF-5 LL−37 cells were identified by qRT-PCR and western blotting.

Western blot analysis
Total proteins were extracted using RIPA containing PMSF and phosphatase inhibitors (Beyotime, Shanghai, China). Protein concentration was determined with a BCA Kit (Vazyme, Nanjing, China). A total of 20 μg of protein was loaded per lane, separated by 8-15% SDS-PAGE and transferred to PVDF membranes (Millipore, Darmstadt, Germany). Subsequently, the membranes were blocked and incubated with primary antibodies (1:1000) overnight at 4°C and then were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The visualization of bands was detected using an ECL detection system (Tanon, GE, USA) and analyzed with Image J densitometry analysis software (NIH).

Wound healing assays
Wound healing assays were used to assess cell migration. PLC/PRF-5 and Huh7 cells (~2 × 10 5 ) were plated in 12-well plates. The monolayers were scratched with a 200 μl sterile pipette tip. The cells were washed with PBS to remove non-adherent cells. Subsequently, the cells were cultured in serum-free DMEM. The wound surface was observed under light microscope at 0 h, 48 h and 72 h. The width of the scratched gaps at 0, 48 h and 72 h was measured using Image J software. The wound closure rate was calculated using the following formula: Wound closure rate (%) = (Original width−Width after migration)/Original width×100. Each independent experiment was repeated four times.

Transwell assays
Transwell analysis was used to determine cell invasive capacity. Briefly, transwell chambers with 8-μm pore size (Corning, USA) were coated with 100 μl of 1:8 diluted Matrigel (BD Biosciences, USA) and incubated at 37°C for 4 h. The transfected cells (0.5-1 × 10 5 cells) were cultured in the upper chamber with DMEM supplemented with 1% FBS. Then, 500 μl of DMEM supplemented with 15% FBS were added to the lower chamber. After incubation for 24 h, the cells in the upper chamber were removed with a cotton swab. The cells in the lower chamber were fixed using 4% paraformaldehyde and stained with 0.1% crystal violet. After imaging the cells under a light microscope, the cells were eluted with 33% acetic acid and detected at 570 nm with a microplate reader to calculate the invasion rate. Each independent experiment was replicated at least four times.

Real-time quantitative polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using TRIzol reagent (Vazyme, China) according to the manufacturer's protocols. RNA was quantified using a NanoDrop ND-1000 spectrometer. Then, RNA was reverse-transcribed into cDNA using HiScriptIII RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China). The relative expression of genes was examined using AceQ qPCR SYBR Green Master Mix (Vazyme, China). Gene expression was calculated using the 2 −ΔΔCt method. The primers used in this study are listed in Table 1.

Immunofluorescence (IF) staining
PLC/PRF-5 cells were cultured on cell slides for 24 h, followed by treatment with 1,25(OH) 2 D 3 (200 nM) for 24 h , then fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 15 min. Cells were incubated with goat anti-rabbit and antimouse conjugated antibodies at room temperature for 1 h in the dark, followed by counterstaining with DAPI for 30 min at room temperature. Fluorescence images were collected with a Ti-E-A1R confocal laser microscope (Nikon, Japan).

Hematogenous metastasis model by intravenous HCC cell injection
BALB/c nude mice (4-6) weeks old were housed in specific pathogen-free conditions, and were randomly divided into three groups (6 mice per group). Then PLC/PRF-5 cells (5 × 10 6 cells) or PLC/PRF-5 LL−37 cells (5 × 10 6 cells) were injected into mice of each group via the tail vein. The remaining six mice were injected with PBS as a control. Mouse body weights were measured every week. After 13 weeks, the mice were euthanatized and dissected. The lungs and livers were harvested at necropsy and fixed in 4% paraformaldehyde. The fixed lung and liver tissues were paraffin-embedded for hematoxylin/eosin (HE) staining and analyzed for the presence of metastasis.

Xenograft mouse model
The xenograft animal assays were carried out in two batches to assess (1) the effect of LL-37 expression on EMT, and (2) the effect of LL-37 on EMT regulated by 1,25(OH) 2 D 3 . The establishment of the HCC mouse xenograft model and the different treatments were described in our previous paper [19]. After 28 days, the mice were euthanatized and the tumors were removed for western blot analysis or for preparation of paraffin sections to detect changes in EMT markers.

HE staining
Tissues were fixed in 4% paraformaldehyde followed by paraffin embedding, and then cut into 7-μm-thick sections. Longitudinal slices were dewaxed and rehydrated, then stained with hematoxylin solution for 5 min. The sections were then stained for 5 min with 1% acid ethanol, rinsed with distilled water, and stained with eosin solution for 3 min. Finally, the sections were dehydrated with graded alcohol and cleared with xylene. Representative images were obtained using an Olympus IX51 fluorescence microscope (Nikon, Japan).

Immunohistochemical (IHC) staining
IHC was performed using an SABC-AP kit (BOSTER, USA). The sections were dewaxed and rehydrated, then pretreated with sodium citrate for antigen retrieval. The sections were rinsed with PBS three times and then 5% bovine serum albumin was used to block nonspecific staining at 37°C for 30 min, followed by the appropriate primary antibody incubated overnight at 4°C, secondary antibody incubated at room temperature for 30 min, and incubation with SABC at room temperature for 30 min. After three washes with PBS, DAB was used for color reaction and hematoxylin solution was used for nuclear counterstaining. The sections were dehydrated in gradient ethanol, made transparent with xylene and neutral adhesive sealing compound was applied. Representative images were obtained using an Olympus IX51 fluorescence microscope.

Statistical analysis
Values were expressed as means ± SEM from four to six independent experiments. Two-tailed Student's t-test and one-way ANOVA with Tukey's multiple comparison test were used to determine the significance of differences. For all cases, p < 0.05 was considered statistically significant. Statistical analysis was assessed using Statistical Package for the Social Sciences (SPSS/ PC 20.0, Chicago, USA).

LL-37 expression promoted migration and invasion of cultured HCC cells
To determine whether the expression of LL-37 affects HCC cell migration and invasion, overexpression and knockdown systems were first established. The LL-37 level was significantly increased or decreased after transfection with pcDNA3.0/LL-37 or si-LL-37 for 48 h in HCC cells, respectively ( Figure 1a). The wound healing assay showed that LL-37 overexpression significantly promoted migration of both PLC/PRF-5 and Huh7 cells (p < 0.01), whereas LL-37 knockdown significantly inhibited migration (p < 0.05, Figure 1b). Consistently, LL-37 overexpression also significantly promoted invasion of PLC/PRF-5 and Huh7 cells (p < 0.001), while the invasive ability of HCC cells after LL-37 knockdown was significantly impaired (p < 0.001, Figure 1c). Taken together, these data demonstrated that LL-37 could significantly promote migration and invasion of cultured HCC cells.

LL-37 promoted EMT of HCC cells in vitro
In order to determine the effect of LL-37 on EMT of HCC cells, the levels of mesenchymal markers (N-cadherin and Vimentin), epithelial marker (E-cadherin) and EMTrelated transcription factors (Snail and Slug) were detected. After overexpression of LL-37 in PLC/PRF-5 or Huh7 cells for 48 h, western blot assays showed that the levels of N-cadherin, Vimentin and Slug were obviously increased, while E-cadherin levels decreased significantly (Figure 2a). After knockdown of LL-37 by si-LL-37, the levels of N-cadherin, Vimentin and Slug were significantly downregulated, while E-cadherin was significantly up-regulated ( Figure 2b). qRT-PCR analysis further found that the mRNA levels of N-cadherin, Vimentin and Slug were significantly up-regulated in LL-37-overexpressing HCC cells (Figure 2c). On the contrary, the mRNA level of E-cadherin was significantly down-regulated by si-LL-37 treatment of HCC cells (Figure 2d). However, no significant change was observed in the Snail level either by overexpression or knockdown of LL-37. These results indicated that LL-37 promoted EMT in PLC/PRF-5 and Huh7 cells.

HER2/EGFR-MAPK/ERK signaling participated in LL-37-induced EMT, migration and invasion
To explore the role of the MAPK pathway on EMT, migration and invasion induced by LL-37 in HCC cells, two inhibitors were used in the following study. Results showed that LL-37 overexpression significantly increased the MMP14, MMP9 and p-ERK1/2 level in both PLC/PRF-5 and Huh7 cells (Figure 3a

Upregulation of LL-37 promoted EMT in mouse model
In order to assess the effect of LL-37 expression on EMT in vivo, we established PLC/PRF-5 xenografted mice

LL-37 overexpression promoted lung metastasis through a hematogenous pathway
Hematogenous metastasis is one of the main modes of HCC metastasis. To further investigate the effect of LL-37 on the hematogenous metastasis of HCC cells, a model of hematogenous metastasis in nude mice was established. The experimental mice were divided into two groups, which then received PLC/PRF-5 LL−37 or PLC/PRF-5 cells injected via the tail vein. After 13 weeks, though no significant difference in body weight between the two groups was observed (Figure 5a), anatomic studies revealed microscopically visible metastatic nodules on the lung surface. The number of lung metastatic nodules in PLC/PRF-5 LL−37 mice was significantly higher than in PLC/PRF-5 control mice (p < 0.01, Figure 5b). HE staining showed that LL-37-overexpressing cells (PLC/PRF-5 LL−37 ) had stronger lung metastatic ability than control PLC/PRF-5 cells (Figure 5c). High levels of LL-37 and p-EGFR were confirmed in lung metastatic nodules (Figure 5d). These results indicated that LL-37 over-expression enhanced the metastatic ability of HCC cells through the hematogenous approach.

Silencing LL-37 enhanced the inhibition of EMT, migration and invasion induced by 1,25(OH) 2 D 3 in vitro
Our previous studies indicated that 1,25(OH) 2 D 3 significantly induced the expression of hCAP18/LL-37 in both HCC cells and xenograft tumor tissue [19].

Discussion
Accumulating studies have revealed that hCAP18/LL-37 plays a role in the promotion of tumor growth in several types of tumors through direct stimulation of malignant cells, initiation of angiogenesis or suppressing immunity in the tumor microenvironment [8,9]. However, only a few studies have reported its promotion of migration and metastasis in breast cancer [12,32], ovarian cancer cells [33] and melanoma [34].
Previously, we showed that hCAP18/LL-37 promotes the growth of HCC through the ERK/EGFR-PI3K/Akt signaling pathways [19]. Our current study further reveals the promotional effect of hCAP18/LL-37 on HCC metastasis via the ERK/EGFR-MAPK/ERK pathway both in vitro and in vivo. Currently, because only very limited and poorly effective therapeutic options exist for HCC, this is still one of the tumors with the highest metastatic capacity and greatest risk for recurrence. EMT plays an important role in HCC metastasis and several biomarkers have been identified during this process. Here, both in cultured HCC cells and HCC xenograft tumors, LL-37 expression significantly decreased the E-cadherin level, a key biomarker for epithelial cells, and obviously increased the levels of N-cadherin and Vimentin, both biomarkers for mesenchymal cells. By losing E-cadherin-mediated cell adhesion and acquiring mesenchymal properties, carcinomatous cells acquire mobility and invasiveness, and are thus able to penetrate the surrounding stroma [35]. Although Snail is important EMT-related transcription factors in HCC, its expression was not affected by LL-37. However, the Slug level was significantly increased by LL-37 expression in HCC cells and xenograft tumors. Slug participates in EMT during cancer metastasis by binding to the promoter of downstream target genes like E-cadherin, thereby promoting the function of EMT [36]. Thus, the decreased E-cadherin level may partly result from reduced Slug induced by LL-37 in HCC cells. This implies that Slug plays an important role in LL-37-induced EMT during HCC progression.
MMPs are key factors conferring invasive and metastatic traits on malignant tumor cells by enabling their infiltration and migration in the process of EMT [29]. EMT and migration depend on increased release and activation of MMPs, as well as their cell membrane expression. Although several MMPs have been reported in HCC, secreted MMP9 is considered to be one of the most important MMPs and its functions have been well-characterized in HCC [37]. We found that LL-37 overexpression significantly increased the MMP9 level, while knockdown of LL-37 significantly decreased MMP9 levels in both HCC cells and xenograft tumors, suggesting an obvious promotional effect of LL-37 on MMP9 expression. More interestingly, membrane-type MMP14 was also significantly induced by LL-37 expression. MMP-14 has a central role among the MMPs and acts in cancer metastasis by degrading the ECM, increasing the secretion of pro-MMP2, pro-MMP9 and pro-MMP13, while cleaving membrane-anchored growth factors and cytokines [38,39]. A report showed that the increased expression of MMP14 was correlated with high rates of portal vein invasion, intrahepatic metastasis and recurrence in HCC [40].
For almost two decades, it has been known that hCAP18/LL-37 can activate EGFR signaling in a variety of cells, but the mechanisms involved are poorly understood [41]. Our previous study showed that hCAP18/LL-37 can increase HB-EGF release from membrane-anchored pro-HB-EGF and activate EGFR/HER2 in HCC cells [19]. Research further revealed that heterotrimeric G proteins regulated MMP14 directly, resulting in HB-EGF release and EGFR transactivation [42]. MMP14 activity mediated proteolytic processing to activate HB-EGF, stimulating the EGFR signaling pathway to increase proliferation and promote tumor growth [43]. One proposed model is that hCAP18/LL-37-induced G protein coupled receptors activation stimulates MMPs (such as MMP14), which subsequently cleave HB-EGF. Interestingly, Co-IP assay and MS confirmed the interaction between hCAP18 and MMP14 in PLC/PRF-5 cells ( Figure S1). We speculate that hCAP18/LL-37 not only increases the expression level of MMP14, but may also be involved in the regulation of MMP14 activity to promote cell migration and invasion. Further study is needed to clarify the interactions among hCAP18/LL-37 and MMP14, which may reveal the effect of hCAP18/LL-37 on the activity of MMP14 in HCC.
Metastasis is the most lethal aspect of cancer, due to the challenges in treating the metastasis and spread of cancer to key organs. Among them, hematogenous metastasis is usually the main cause of death related to HCC, and the most common sites of hematogenous metastases are lung (in up to 60% of patients who have metastatic disease) and bone (in up to 40% of patients) [44,45]. The intravenous injection (tail vein injection) method is frequently used to generate lung metastasis models [46]. Metastatic colonization of distant tissues is a key process in tumor metastasis [47]. In this study, using a hematogenous metastasis model, we found more lung metastatic nodules in PLC/PRF-5 LL−37injected mice than in PLC/PRF-5 control mice, suggesting stronger lung colonization ability induced by LL-37 overexpression. Subcutaneous tumor models are also widely used in pre-clinical cancer metastasis research. An early study observed a significant increase in metastases in a xenograft tumor model using hCAP18overexpressing breast cancer MJ1105 cells [12]. In our HCC xenograft tumor model, we observed that LL-37 expression significantly promoted the EMT process. More importantly, knockdown of LL-37 significant inhibited EMT in xenografted mice. Therefore, cathelicidin LL-37 is involved in HCC metastases by EMT.
Several pathways have been implicated in the progression of EMT in HCC, such as the Wnt/β-catenin, c-Met/HGF/Snail, Notch-1/NF-κB, TGF-β/SMAD and basic fibroblast growth factor-related signaling pathways [48]. Here we found that LL-37 promoted EMT, migration and invasion of HCC cells via MAPK/ERK signaling. Actually, the MAPK/ERK signaling pathway plays an important role in tumor invasion and metastasis [49]. ERK1/2 activation has also been linked to TGF-β-induced EMT and cell invasiveness [31]. Other signaling pathways which promote migration and invasion induced by LL-37 have been reported in other malignant tumors, such as the NF-κB pathway in melanoma cells [15] and the MAPK/ERK pathway in breast cancer and prostate cancer cells [13,17]. A study reported that LL-37 enhanced invasion, metastasis and tumorigenesis through FPR2 and P2X7 in pancreatic cancer stem cells [14]. Receptor tyrosine kinases are key factors lying upstream of the MAPK pathway. Our previous data revealed that the receptor tyrosine kinases HER2/EGFR were targets for LL-37 in HCC cells. Here, inhibiting the phosphorylation of HER2/ EGFR by neratinib significantly inhibited LL-37induced EMT, migration and invasion. Therefore, the HER2/EGFR-MAPK/ERK pathway participates in LL-37-induced EMT and migration of HCC cells. Actually, several pathways are related to the effect of LL-37 overexpression in HCC cells, including the PI3K/Akt, MAPK and JAK/STAT pathways [19]. However, whether these signalling pathways are also involved in the migration and invasion of HCC cells were not examined in our current study, further study is needed.
Although hCAP18/LL-37 was down-regulated in human HCC cells and HCC tumors [19], results from our si-LL-37 experiment confirmed that low LL-37 levels may be sufficient to promote the EMT, migration and invasion of HCC cells. Moreover, under special circumstances such as 1,25(OH) 2 D 3 treatment for cancer, microbial infections and UVB ultraviolet light, the LL-37 level will be significantly increased [50][51][52]. Here we found that 1,25(OH) 2 D 3 significantly induced VDR expression and nuclear transport, as well as hCAP18/ LL-37 expression. The mesenchymal-like phenotype could revert to an epithelial-like phenotype in HCC cells caused by 1,25(OH) 2 D 3 resulting from the increased E-cadherin level, which is consistent with previous evidence [25]. Our study further revealed that 1,25(OH) 2 D 3 significantly decreased N-cadherin, Vimentin, MMP9 and MMP14, in conjunction with inhibition of migration and invasion of HCC cells.
More interestingly, 1,25(OH) 2 D 3 treatment together with knockdown of LL-37 further enhanced 1,25(OH) 2 D 3 -induced inhibition of migration and invasion by HCC cells, along with enhanced reversion of the EMT process in HCC cells. As mentioned above, LL-37 promoted EMT, migration, invasion and metastasis in HCC cells. Although the detailed mechanism by which 1,25(OH) 2 D 3 inhibits HCC metastasis is unclear, here for the first time, we revealed that hCAP18/LL-37 may be an important factor which suppresses the therapeutic benefit of 1,25(OH) 2 D 3 in HCC tumors by promoting metastasis, which further supports the hypothesis that hCAP18/LL-37 may be an important target which can improve the anticancer activity of 1,25(OH) 2 D 3 in HCC therapy.

Conclusions
In conclusion, for the first time to our knowledge, our study shows that LL-37 promotes EMT, migration and lung metastasis of HCC cells in vitro and in vivo. The HER2/EGFR-MAPK/ERK signaling pathway mediates LL-37-induced EMT, migration and invasion (Figure 7c). We believe that in addition to promoting tumor growth, LL-37 can also play an important role in HCC tumor progression by promoting HCC metastasis. Additionally, LL-37 may be an important factor interfering with 1,25(OH) 2 D 3 inhibition of HCC cell metastasis. Therefore, 1,25(OH) 2 D 3 treatment combined with silencing hCAP18/LL-37 expression may be a potential strategy to increase the anticancer activity of 1,25(OH) 2 D 3 in treating HCC progression.