†These authors contributed equally.
Academic Editor: Graham Pawelec
Background: p62 is a multi-domain protein and participates in a variety
of cellular biological activities. p62 is also related to tumor malignancy.
However, the underlying molecular mechanism of p62 regulating the progression of
hepatocellular carcinoma (HCC) remains unclear. Methods: The expression
levels of p62 in HCC tissues and adjacent non-tumor tissues were confirmed using
the TCGA dataset and immunohistochemistry. Stable p62-overexpressing HepG2 cells
and p62-knockdown MHCC97H cells were established with lentiviral vectors. Cell
proliferation, migration, and invasion assays were carried out to investigate the
role of p62 in HCC cells and HCC-derived exosomes. The relationship between p62
and
Hepatocellular carcinoma (HCC) is a neoplastic disease with high morbidity and mortality all over the world, especially in Asia and Africa [1]. Although there are many therapeutic options for HCC, long-term survival prognosis remains poor [2]. The metastatic property of tumor is one of the main causes of death in patients with HCC, so it is necessary to learn more about the mechanism of HCC metastasis [3].
As a classic autophagy receptor, protein p62 is encoded by the gene SQSTM1 [4]. It is also a multi-domain protein that selectively interacts with different signaling mediators to regulate selective autophagy, metabolic reprogramming, and antioxidant reactions [5]. The function of p62 in different cancers remains controversial, with evidence suggesting that p62 is promoted in many kinds of cancer including colorectal cancer, prostate cancer, breast cancer, and others [6, 7, 8, 9, 10]. High p62 expression is relevant to aggressive clinical features, highlighting the potential of p62 as an important target for anticancer therapy [11, 12]. However, the detailed mechanisms of p62-mediated cancer invasion and metastasis have not been fully elucidated.
Exosome is a kind of extracellular vesicle with a size range of 30–150 nm and secreted by most species [13]. It can carry proteins, lipids, and various RNAs for cell-to-cell transport [14, 15]. Tumor cells may cooperate with other cells by releasing exosomes and transferring oncogenic molecules to the recipient cells [16]. In recent years, increasing evidence has indicated that exosome may participate in the process of cancer invasion and metastasis, indicated that it can be used as a biomarker for the diagnosis of cancers [17, 18, 19]. However, the role of exosome in the metastasis of cancer and the mechanisms related to the regulation of selective sorting of exosomal cargos remain largely unknown.
The purpose of this study was to elucidate the molecular mechanism and function
of p62 in HCC, and the results ultimately showed that p62 was significantly
upregulated in HCC tissues and metastatic HCC cell lines. Overexpressing p62 can
significantly promote the oncogenic behavior including cell growth, mobility, and
invasiveness of HCC by regulating the localization of
The data of gene expression, together with the corresponding clinical data of hepatocellular carcinoma and normal liver tissue samples, were downloaded from the TCGA database (http://ualcan.path.uab.edu/analysis.html).
The HepG2 cell line was obtained from the American Type Culture Collection
(ATCC, USA). SNU182, SNU387, Hep3B, and Li-7 were provided by Stem Cell Bank,
Chinese Academy of Sciences (China). Huh7 cell line was purchased from the
Japanese Collection of Research Bioresources (Japan). MHCC97H cell line was
provided by CELLCOOK Biological Technology (China). All HCC cell lines were
cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented
with 10% fetal bovine serum (FBS, Gibco, USA) at 37
The HCC cells and isolated exosomes were harvested and lysed in RIPA buffer
(Beyotime, China) supplemented with a protease-inhibitor cocktail (SIGMA, USA).
BCA assay was used to measure the protein concentration. Equal amounts of lysates
were loaded and separated on 9% SDS-PAGE gels. Then, the proteins were
electrotransferred to polyvinylidene difluoride membranes (PVDF), blocked in 5%
skim milk at 37
TRIZOL Reagent (SIGMA, USA) was used for the RNA extraction in accordance with
the manufacturer’s instruction. Reverse transcription was conducted using Prime
Script RT Master Mix (TOYOBO, Japan) in accordance with the standard protocol.
SYBR Premix EX Taq TMII (TOYOBO, Japan) was used for RT-PCR. The differences in
relative expression levels between groups were analyzed using the
2
We analyzed samples from 81 HCC patients who underwent hepatectomy at Sun Yat-sen Memorial Hospital, Sun Yat-sen University (Guangzhou, China), between January 2018 and November 2019. The ethical approval was approved by the Ethics Committee of Sun Yat-sen Memorial Hospital [approval number: 2017(107)], and informed consent was obtained from each patient. Immunohistochemistry analysis was used to detect the expression of p62 in paraffin embedded adjacent non-tumor tissues and HCC tissues. The protocol was performed according to previously described [26].
The EGFP:T2A:Puro-U6
CCK-8 assay was used to detect the cell proliferation. Cells were seeded in
96-well plates at a density of 5
A scratch wound was created using a 1 mm wide sterile pipette tip after cells
reached 80–90% confluence in 6-well plate. The detached cells were removed by
washing with PBS three times, and then serum-free culture medium was added into
each well. A microscope was used to capture the images at 0, 24 and 48 h at 100
A Transwell Chamber (Corning, USA) was used for cell migration and invasion
assays. For the migration assay, 700
Cells were grown on glass dishes for 24 h, and then treated with 0.3% Triton X-100 for 15 min after being fixed in 4% paraformaldehyde for 15 min at room temperature. After that, 10% goat serum in PBS was used to block the cells for 60 min at room temperature. After the incubation with primary antibodies and Alexa-conjugated secondary antibody (Cell Signaling Technology, USA), the cells were stained with DAPI for 5 min and visualized on a confocal microscope (Olympus LV3000, Japan).
Cells were seeded in 15ml vesicle-depleted medium on 10 cm dishes for 2–3 days.
The culture media were harvested and centrifuged at 300 g for 10 min and then
2000 g for 20 min. After centrifugation at 10,000 g for 30 min at 4
The animal experiments were carried out with approval by the Ethics Committee of
Sun Yat-sen University. Male athymic nude mice (BALB/c-nu/nu, 6 weeks old) were
obtained from the Animal Center of Sun Yat-sen University. HepG2-Control and
HepG2-p62-OE xenograft tumors were established by subcutaneous injection of cells
(5
All statistical analyses were performed using SPSS version 20.0 (IBM, Chicago,
Illinois, USA) and GraphPad Prism 7 (GraphPad Software, San Diego, California,
USA). Statistical differences were evaluated using a student’s t-test
and the data were presented as the mean
The TCGA dataset showed that the level of p62 in HCC tissues (n = 371) was much higher than that in normal liver tissues (n = 50) (Fig. 1A). The analysis of the survival curve of the TCGA dataset showed that the survival time of HCC patients with high expression of p62 (n = 91) was significantly shorter than that of patients with low expression of p62 (n = 274), suggesting that the level of p62 was a risk factor for poor prognosis of patients with HCC (Fig. 1B). Moreover, the expression of p62 was upregulated with the decrease of tumor differentiation of HCC according to the TCGA dataset (Fig. 1C). Similar results were obtained in the clinical tissue samples collected specifically for this study. IHC analysis showed that the expression of p62 in HCC tissues was higher than that in adjacent non-tumor tissues (Fig. 1D,E). To screen suitable HCC cell lines for the follow-up experiments, the expression of p62 was explored in 7 HCC cell lines, including HepG2, SNU182, SNU387, Huh7, MHCC97H, Hep3B, and Li-7. p62 was upregulated in Huh7, MHCC97H, and Li-7, while it was silenced in HepG2, SNU182, SNU387, and Hep3B, according to the results of western blotting and RT-PCR (Fig. 1F,G).
p62 is highly expressed in HCC tissues and indicates
poor prognosis in human HCC. (A) The expression of p62 in HCC based on sample
types from TCGA dataset (p
The high expression of p62 in clinical HCC tissues indicated that p62 may plays a potential tumor-promoting role in the occurrence and development of HCC. To further evaluate the role of p62 in HCC, stable p62-overexpressing HepG2 and p62-knockdown MHCC97H cell lines were established (Fig. 2A,C). The plasmids used for knockdown experiment have been verified by western blotting (Supplementary Fig. 1). The overexpression of p62 in HepG2 cells significantly promoted cell proliferation compared with control HepG2 cells as determined by CCK-8 assays, while knockdown of p62 inhibited cell proliferation of MHCC97H cells (Fig. 2B,D). Then, the abilities of migration and invasion in p62-overexpressing HepG2 cells and p62-knockdown MHCC97H cells were further evaluated. Wound healing assays showed that migration ability was significantly enhanced in p62-overexpressing HepG2 cells and weakened in p62-knockdown MHCC97H cells (Fig. 2E,F). In addition, transwell assays showed that the migration and invasion abilities were significantly promoted after overexpressing p62 in HepG2 cells, while knockdown of p62 inhibited the migration and invasion abilities of MHCC97H cells (Fig. 2G,H).
p62 promotes cell proliferation, migration, and invasion
abilities in HCC cells. (A) HepG2 was stably transduced with a
p62-overexpression lentiviral vector (p62-OE), and the control group was
transduced with the corresponding lentiviral carrying an empty vector (Control).
(B) Overexpression of p62 significantly promoted cell proliferation in HepG2,
according to CCK-8 assays, compared to that of the control groups (***p
As a multi-domain protein, p62 is widely involved in a variety of cellular life activities [5]. In our previous related studies, mass spectrometry was used to find the Vps4A-related protein [27]. Surprisingly, the mass spectrometry showed that p62 interacted with Vps4A (Supplementary Table 3). So, this study explored whether p62 affected the exosome pathway. Exosomes were isolated from culture medium of both control and p62-overexpressing HepG2 cells by precipitation reagent. The markers of exosomes including HSP90, Alix, Tsg101, and CD63 were examined by western blotting (Fig. 3A). Then, Nanoparticle Tracking Analysis (NTA) and Transmission Electron Microscopy (TEM) were used to identify the morphology and concentration of the exosomes (Fig. 3B,C). The results showed that overexpression of p62 had no effect on the size but increased the number of the exosomes released into the culture medium, according to the concentration of NTA.
p62 increases exosome secretion but does not affect the size of
exosomes. (A) Immunoblotting showed the typical exosomal markers (HSP90, Alix,
Tsg101, and CD63) in isolated exosomes. (B) Exosomes isolated from the culture
medium of HepG2 control cells and p62-overexpressing cells by precipitation
reagent were identified by Transmission Electron Microscopy. (C) Nanoparticle
Tracking Analysis showed that the majority of isolated exosomes were about 130
nm, and the exosome concentration in the p62-overexpressing HepG2 group (5.07
To investigate the bioactive effect of the exosomes derived from p62-overexpressing HepG2 cells, cell growth, wound healing, as well as transwell assays were performed. The recipient HepG2 cells were incubated with N-Rh-PE labeled exosomes for 24 h and observed under a confocal microscope to confirm that the recipient cells could absorb exosomes (Fig. 4A). HepG2 cells were incubated with different groups of exosomes, and it was found that exosomes from both control (CTexo) and p62-overexpressing HepG2 cells (p62exo) could promote the proliferation of the recipient cells. The promoting effect of exosomes in the p62-overexpressing group was stronger than that in the control group (Fig. 4B). Similar results were also obtained in wound healing and transwell assays. Exosomes derived from p62-overexpressing HepG2 cells could significantly promote the migration and invasion of the recipient cells (Fig. 4C,D). These results indicated that overexpression of p62 can promote the oncogenic behavior of HCC cells in vitro via exosomes.
Exosomes derived from p62-overexpressing HepG2 cells promote the
proliferation, migration, and invasion of recipient cells. (A) The recipient
HepG2 cells were incubated with N-Rh-PE labeled exosomes for 24 h and observed
under a confocal microscope (Red, N-Rh-PE, Exosomes; Blue, DAPI, Nucleus). (B)
Cell proliferation (CCK-8) assays were performed using HepG2 cells cocultured
with exosomes derived from HepG2 control cells and p62-overexpressing cells
(*p
In previous studies, we found that Vps4A functions as a tumor suppressor to
inhibit EMT by regulating the localization and exosome release of
p62 interacts with
To confirm that p62 can regulate the localization of
p62 regulates the expression and localization
of
The experiments of subcutaneous exnograft tumor formation in nude mice were
carried out to explore the function of p62 in HCC in vivo. The tumor
volumes of nude mice were significantly increased in the mice injected with
p62-overexpressing HepG2 cells compared with those injected with control HepG2
cells (Fig. 7A,B), concomitant with significantly greater tumor weights at the
end of the animal experiments (Fig. 7C). Western blotting and IHC assays were
used to detect the expression level of relevant proteins in exnograft tumor
tissues. The results of western blotting and IHC showed that the expression level
of p62 was higher in exnograft tumors injected with p62-overexpressing HepG2
cells than those injected with control HepG2 cells while the expression level of
p62 promotes tumor growth of HCC and down-regulates the
expression of
As a key protein of autophagy, p62 is indispensable in the growth and development of organisms [4]. Moreover, because p62 has multiple domains, it can participate in many signal pathways, affecting not only cell selective autophagy, but also metabolic reprogramming, antioxidation and so on [5]. In recent years, SQSTM1/p62 has been reported in many kinds of tumors as an oncogene, and its importance has been given increasing attention [6, 7, 8, 9, 10]. However, the mechanism of p62 in promoting cancer invasion and metastasis remains unclear.
Based on the TCGA dataset and clinical samples obtained from the center, the data confirmed that p62 was highly expressed in HCC tissues and highly metastatic HCC cell lines. Further experiments showed that by overexpressing p62 in HepG2 cells, the abilities of cell growth, mobility, and invasiveness were promoted, while the opposite results we got in p62-knockdown MHCC97H cells. This effect of regulating cancer biological behaviors was supported by previous reports [29, 30]. In a previous related study on Vps4A, a key protein of endosomal sorting complex required for transport (ESCRT), an interaction was observed between Vps4A and p62 in mass spectrometry (Supplementary Table 3). Thus, the current study boldly speculated that p62 may affect the exosome pathway to regulate the secretion of cytoplasmic materials [27]. The overexpression of p62 can increase the concentration but not change the size of exosomes in HepG2 cells according to the results of NTA and TEM. The recipient HepG2 cells incubated with exosomes derived from both control and p62-overexpressing HepG2 cells showed enhanced cell proliferation, migration, and invasion abilities. Previous related studies also supported these results [26]. In the current study, the exosomes derived from p62-overexpressing HepG2 cells had a stronger promoting effect than in the control group, suggesting that p62 could strengthen the oncogenic behavior of HCC cells via exosomes. However, the mechanism of how p62 finely regulates the exosomes pathway needs further research.
In previous related studies,
The expression of exosomal
We also established the HCC xenograft tumors in nude mice by using the
p62-overexpressing HepG2 cells and the control HepG2 cells. The tumor growth
curve and the histogram of tumor weight showed that overexpression of p62 in
HepG2 aggravates xenograft tumor growth. Meanwhile, the expression level of p62
and
At the same time, the level of EMT-related markers including Vimentin, Twist1,
and Snail were increased after overexpressing p62, while the expression of
E-cadherin was down-regulated (Supplementary Fig. 2). As we know that,
Taken together, this study demonstrated that p62 functions as an oncogene to
promote cell proliferation, migration, and invasion abilities in HCC cell lines
by regulating the cell structure and secretion of exosomes. p62 down-regulate the
expression of
HCC, hepatocellular carcinoma; EMT, epithelial-mesenchymal transition; N-Rh-PE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis; ESCRT, endosomal sorting complex required for transport.
WY, JM and PL designed the research study. WY, JW, ZZ and WZ performed the research. WY, LL, AL, and JX analyzed the data. All authors contributed to the manuscript writing and revision. All authors read and approved the final manuscript.
All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). All enrolled patients signed informed consent, and the study was approved by the ethics committee of the Sun Yat-sen Memorial Hospital, Sun Yat-sen University [approval number: 2017(107)].
Not applicable.
This research was funded by the National Natural Science Foundation of China (grant numbers 81672420, 81702406, 81872860, 81401987), and the Guangdong Science and Technology Department (2020B1212060018).
The authors declare no conflict of interest.