Introduction
Hepatocellular carcinoma (HCC) is one of the most common and highly malignant neoplastic diseases, with a global morbidity ranking sixth among all tumors and a mortality rate ranking third among tumor-related deaths [
1]. HCC is a highly heterogeneous disease with multiple risk factors and etiology, including chronic hepatitis B or hepatitis C virus (HBV/HCV) infection, excessive alcohol consumption, aflatoxin exposure, and diabetes or obesity-related metabolic syndrome [
2]. The underlying disease and difficulty in early diagnosis lead to poor prognosis and high mortality in HCC patients. Due to the serious lack of insight into the molecular machine of development and progression of hepatocellular carcinoma, we lack effective late-stage treatment options. Since sorafenib been approved as a systemic drug for HCC by FDA, lenvatinib, regorafenib, and cabozantinib have also been approved for the treatment of advanced HCC patients [
3‐
5]. But it still shows unfavorable efficacy [
6]. Therefore, it is urgent to further understand the mechanism of HCC progression so as to identify new drug targets for treatment.
The Src homology and collagen (SHC) family is one of the most studied adaptor protein families consisting of four members, SHC1, SHC2, SHC3, and SHC4. SHC1 is the most ubiquitous member of the family but limits to neurogenic areas in mature nervous system [
7]. In contrast, SHC2 and SHC3 are expressed almost exclusively in neurons of the central and peripheral nervous systems [
8]. SHC4 is also found in the adult brain, followed by skin and muscle [
9‐
11]. It has been demonstrated that abnormal expression of SHC1 and SHC3 plays a role in malignant transformation, including transformation leading to HCC disease [
12‐
15]. However, reports on SHC4 have been limited to studies of melanoma and glioma. High expression of SHC4 promoted the migration and invasion of melanoma cells and glioma cells [
16,
17]. The function and regulatory mechanism of SHC4 in HCC are still unknown.
MAPK signaling pathways have been shown to play an important role in tumor progression that SHC1 and SHC3 mediated [
18]. Involved in initiation of ERK pathway activation, JAK-STAT pathways intersects with MAPK pathways in multiple links. The activation of STAT requires MAPK auxiliary role as well. In HCC, aberrant activation of the JAK/STAT pathway promotes tumor growth, angiogenesis, invasion, and metastasis [
19‐
21]. STAT3 is generally accepted as a bona fide oncogene in promoting HCC development. In our current study, we found increased SHC4 expression in HCC, leading to poor prognosis. We further confirmed the function of SHC4 in cell growth, migration and invasion. The significant role of STAT3 pathway in tumor progression is also involving, which may provide a new idea for the treatment of HCC.
Materials and methods
Clinical samples
The HCC tumor tissues and adjacent normal tissues of 138 patients were collected from resected specimens at Tongji Hospital of Huazhong University of Science and Technology between 2012 and 2016. Tissue microarray plates containing 105 HCC cases were constructed from paraffin-embedded HCC tissues. The diagnosis was based on the pathological examination. HCC staging was defined according to the criteria of the seventh edition of AJCC (American Joint Committee on Cancer) TNM classification. All patients received a standardized follow-up protocol, and the median follow-up was 25 months (range 0.5–65 months) [
22]. Written informed consent for data analysis was obtained from all patients before operation.
Plasmids and reagents and antibodies
SHC4 cDNA was kindly provided by Han’s Lab and cloned into pLenti-CMV-GFP vector to generate SHC4 expression plasmid which was confirmed by sequencing. The target sequences in the pLKO.1-SHC4 shRNA vector against human SHC4 were 5′-GCCTAGCATTTCTCAGTGTTT-3′, 5′-GAATGGCCCAAGACGTCATAA-3′ and 5′-ATGTTGCCTACGTAGCTAAAG-3′. pLenti-CMV-GFP and PLKO.1 were purchased from Addgene. Smartpool siRNA against human STAT3 was from RiboBio. The STAT3 siRNA target sequence was 5′-UCUACUUGGCUCCCAACUU dTdT-3′. Stattic was from MedChemExpress. Lipofectamine 2000 was from Invitrogen. Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM reduced serum media and fetal bovine serum (FBS) were from Gibco. Polybrene and puromycin were from Sigma. Primary antibodies used in this study include: SHC4 (Abcam, ab174908, 1:1000), GAPDH (Aksomics, KC-5G4, 1:10,000), c-Myc (Cell Signaling Technology, #5605, 1:1000), Cyclin D1 (Cell Signaling Technology, #2978, 1:1000), p21 (Cell Signaling Technology, #2947, 1:1000), E-cadherin (BD Biosciences, 610182, 1:1000), Occludin (Cell Signaling Technology, #5446, 1:1000), Slug (Cell Signaling Technology, #9585, 1:1000), STAT3 (Cell Signaling Technology, #12640, 1:1000) and p-STAT3 (Cell Signaling Technology, #52075, 1:1000).
Cell culture, SHC4 overexpression and knockdown
HL7701, HL-7702, HepG2, Huh7, Sk-Hep1, Bel7402, HLE, HLF and Alex cell lines were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). MHCC-97 H and HCC-LM3 cell lines were obtained from Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China. 293T cells were purchased from the American Type Culture Collection. Cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 4.5 g/L glucose and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). All cell lines have been tested for their authenticity. Transfection were performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacture’s instruction. For lentivirus production, pLKO.1 vector and pLKO.1-shSHC4 or pLenti-CMV-GFP vector and pLenti-CMV-GFP-SHC4 (6 µg), psPAX2 (4.5 µg), pMD2.G (1.5 µg) were purchased from Addgene, Inc. (Cambridge, MA, USA) and were co-transfected into 293T cells. The virus-containing supernatants were collected and filtered 48 h after transfection. Freshly made virus supernatants supplemented with 8 µg/mL polybrene (Sigma-Aldrich) were added to exponentially growing HepG2, Huh7 or HCC-LM3 cells. After 8 h, fresh medium was added. SHC4 stable overexpression or knockdown cells were achieved by 1-week puromycin (5 µg/mL, Ann Arbor, MI, USA) selection.
Immunohistochemical staining
Immunohistochemistry (IHC) of clinical tumor samples and tumor xenograft samples was performed using antibodies against SHC4, Ki67 and E-cadherin. Briefly, tissue sections were deparaffinized in xylene, rehydrated with ethanol and subjected to antigen retrieval in boiling citrate buffer for 15 min. After peroxide block, the section was incubated with primary antibody at 4 °C overnight. The section was then treated with secondary antibody (Dako, Denmark) for 1 h at room temperature. The peroxidase reaction was developed with diaminobenzidine (DAB, Dako, Denmark). Images were acquired using 3DHIESTECH scan system and software. Cell-based average integrated option density (IOD) for SHC4 expression in each sample was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics Inc, Bethesda, USA). The cutoff for the definition of high expression group or low expression group was the median value. Accordingly, samples were segregated into two groups for further analysis.
Immunofluorescence staining
Cells were grown on coverslips in a 24-well culture plate, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min and blocked with 5% bovine serum albumin for 1 h. Cells were then incubated with primary antibodies overnight, followed by incubation with Alexa Fluor-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) for 1 h. Finally, coverslips were incubated with DAPI (Sigma) for 5 min and visualized under an inverted fluorescent microscope.
Western blotting
Cells or tissues were lysed in RIPA buffer supplemented with 1% protease (Roche) and 1% phosphatase inhibitor cocktail (Sigma). The protein samples were quantified using BCA assay, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocked with 5% skim milk, the membranes were probed with the indicated antibodies and then exposed to horseradish peroxidase (HRP)-linked secondary antibodies. The enhanced ECL was used for signal detection and western blot images were collected using Bio-Rad GelDoc system.
Cell proliferation assay
HCC cells were seeded in 96-well microplate at the density of 1000 cells per well. After culture for 24, 48, 72, 96 and 120 h, the cells were treated with Cell Counting Kit-8 (CCK-8, Dojindo Laboratories) for 2 h at 37 °C. Then the optical density was measured at 450 nm using an enzyme-linked immunosorbent assay plate reader (Bio-Tek Elx 800, USA).
HCC cells were seeded in 6-well plate at the density of 800 cells per wells. After 14 days, the colonies were fixed in 4% paraformaldehyde and stained with 1% crystal violet. The plates were taken picture and the number of colonies larger than 100 μm in diameter were counted.
Transwell assay
Cell migration and invasion abilities were determined by Transwell assay as previous described [
23]. In brief, cells in 200 µL of DMEM were placed in to the upper chamber of transwell inserts (8 μm, Corning, USA) for migration assay or Matrigel-coated transwell inserts for invasion assay, and 650 µL of 10% FBS-containing DMEM in the absence or presence of Stattic was added to the lower chamber. After incubation for 24 or 48 h, cells on the upper surface of the inserts were removed with a cotton swab. The migrative or invasive cells were fixed and stained. Photographs of six random fields across three replicate wells were captured for quantification analysis.
Tumor xenograft model
BALB/c nude mice (4–6 weeks old, male) were purchased from HUAFUKANG (HUAFUKANG BIOSCIENCE CO. INC. Beijing, China). 2 × 106 of Huh7 cells stably transfected with vector or SHC4 were subcutaneously inoculated into the flanks of nude mice. The mice were sacrificed by cervical dislocation 3 weeks after injection, and tumors were excised and weighed. Tumor volume was estimated according to the formula: volume = length × width2/2. The excised tumors were either embedded in paraffin for IHC analysis or snap frozen in liquid nitrogen for protein extraction. For the Stattic treatment, Huh7-vec and Huh7-SHC4 (2 × 106/mouse) cells were subcutaneously implanted into the flanks of nude mice. When tumors grew to 3–5 mm in diameter, the mice were peritoneally treated with Stattic (50 mg/kg, three times per week for 4 weeks). Xenograft tumor samples were then collected for further analysis.
Statistical analysis
Statistical analyses were performed using SPSS software (version 21.0, IBM Corp, Armonk, NY, USA) or the GraphPad Prism software (version 6.01, GraphPad Software Inc., San Diego, CA). Values were expressed as the mean ± SD from at least three independent experiments. Quantitative variables were compared using Student’s t-test or Mann–Whitney-U test when applicable. Also, the one-way ANOVA followed by a Turkey post hoc test was performed for multigroup comparison. The Pearson’s χ2 or Fisher’s exact test was used to analyze qualitative variables. Comparisons between Kaplan–Meier curves were performed using the log-rank test. P < 0.05 was considered statistically significant.
Discussion
SHC4 was first identified and confirmed in melanoma in 2007. Ernesta Fagiani etc. found that SHC4 expression is essential for the growth of metastatic melanoma in vivo [
17]. Subsequent studies indicated that SHC4 is highly expressed in malignant gliomas and it can promote invasion in U87 glioma cells [
16]. SHC4 has also been reported to be expressed at high levels in adult brain tissue and at low levels in skeletal muscle [
10,
11]. Melanie et al. detected enhanced SHC4 expression in astrocytomas and it was again shown to promote monolayer cell healing [
25]. Furthermore, SHC4 widely expressed in the developing nervous system was further confirmed to be specifically expressed early during embryonic stem cell differentiation and embryonic development [
9,
26]. On the basis of the studies above mentioned, the important role of SHC4 in the development of several tumors can be seen. In our current study, we for the first time showed the expression of SHC4 in HCC tissues and cell lines, with its high expression being associated with aggressive clinicopathological characteristics and poor prognosis in HCC patients. In consistent with the studies on glioma and melanoma, our functional analysis showed that SHC4 enhanced cancer proliferation and invasion abilities at both the cellular and organismal levels.
The typical structural domain of the SHC family is (CH2)-PTB-CH1-SH2. Although SHC articulators have a conserved structure and common binding partners, the SHC adaptors and their isoforms can generate distinct downstream signals and make unique physiological contributions [
27]. The molecular mechanisms of SHC1-mediated signal transduction have been widely documented: SHC1 bind to the cell membrane receptor through PTB domain, promoting CH1 domain binding to Grb2 and solicits SOS to form SHC1-Grb2-SOS complex, and further activates Raf/MEK/MAPK signaling pathway [
13]. Along this context, Yun Liu and others found that SHC3 forms a complex with MVP, MEK, and ERK, which potentiates ERK activation, independent of the classic SHC1, Grb2, SOS, Ras, and Raf pathway [
15]. However, the mechanism of SHC4 action in cancer cells, especially in HCC, largely remains unknown.
SHC4 promotes migration and invasion of melanoma cells through activation of Ras-dependent or Ras-independent pathways [
17,
28]. SHC proteins are involving in the epidermal growth factor receptor (EGFR) internalization process and the increased expression level of SHC4 in glioma promoted the phosphorylation of EGFR specific sites [
16,
25,
29]. Recent studies have shown that SHC4 regulate EV secretion and promote tumorigenesis of prostate [
30]. During the process that embryonic stem cells differentiate into ectodermal stem cells, SHC4 knockout promoted the enrichment of CDX2-positive cells and result in the activation of MAPK-ERK1/2 [
26]. In our study, we explored the potential mechanisms of SHC4 in HCC by analyzing the transcription factor STAT3. Belongs to the signal transducer and activator of transcription (STAT) family, STAT3 is inactive in non-stimulated cells but is rapidly activated by various cytokines and growth factors, for instance, the receptor-associated Janus kinase (JAK) [
31,
32]. Activated STAT3 plays a role in most cancers, mediating the expression of various genes in response to cellular stimulation and playing an important role in cell growth and apoptosis [
33]. Notably, STAT3 has been widely recognized to play an important role in the progression and treatment of HCC [
34‐
37]. Nonetheless, the exact mechanism of STAT3 activation in HCC remains unclear. In this study, we confirmed that SHC4 overexpression can activate STAT3. Further research on cell lines showed that knockdown and inhibition of STAT3 attenuates the proliferation, migration and invasion of tumor cells caused by overexpression of SHC4. Experiments on mice had also been conducted to verify that SHC4 promoted tumor growth, and Stattic could reverse this effect. Therefore, STAT3 was essential for the effects of SHC4 on HCC cell proliferation, migration and invasion. STAT3 inhibitors may be a potential option for liver cancer treatment.
As is mentioned above, studies on how SHC1 and SHC3 activate downstream molecules are well established. Even though we have proposed a new viewpoint on HCC progression, deeper mechanisms how SHC4 activate STAT3 signaling is yet to be explored. Acting as an adaptor protein, SHC4 itself don’t have the activity of protein kinase. Therefore, it usually interacts with other proteins to regulate signal transduction, so our further study will focus on the molecular mechanisms underlying the SHC4 induced STAT3 signaling activation.
Conclusions
In conclusion, overexpression of SHC4 potentiates STAT3 activation and stimulates a repertoire of downstream tumorigenic responses, including proliferation, migration, invasion, and EMT, leading to aggressive clinicopathological characteristics and poor prognosis in HCC patients. These evidences support the feasibility of SHC4 as novel therapeutic target for HCC.
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