p62 is elevated in ICC tissues and associated with lymph-node metastasis and poor prognosis of ICC patients
To identify the potential role of p62 in ICC, we compared the protein expression of p62 in 12 ICC tissue samples with adjacent nontumor tissues (Fig. 1A). A significant increase in p62 expression was observed in the tumor tissues compared with adjacent tissues (P=0.0018). Among above tumor samples, 7 cases increased over two-fold, and 5 cases exhibited over one-fold increase (Fig. 1B). In addition, the results showed higher expression of protein p62 in tumor samples with lymph-node metastasis. (P=0.0260) (Fig. 1C).
We further validated our hypothesis using TMA to evaluate the correlation between p62 and prognosis in 140 ICC patients who underwent curative liver resection. Notably, the p62 protein level in cytoplasm was higher in tumor tissues, especially in ICC with lymph-node metastasis (Fig. 1D). From the data quantified, only nine para-tumor samples had a IHC score of 3 or 4 points, while most of the tumor tissues had a score of more than 3 points, especially in ICC with lymph-node metastasis (Fig. 1E). Then, we divided the whole study cohort into p62 high expression (the IHC score >4 points; n=41) and p62 low expression (the IHC score ≤4 points; n=99) subgroup, and analyzed the relationship between the level of p62 expression and clinicopathological characteristics of ICC patients. The results indicated that older patients had higher expression of p62. However, there was no significant correlation between p62 expression and clinical characteristics such as gender, liver cirrhosis, tumor number, α-fetoprotein level, tumor capsule, tumor differentiation and vascular invasion (Supplemental Table 1). Moreover, high expression of p62 was significantly associated with lymph-node metastasis (P=0.0346) (Fig. 1E). The heterogeneity of the degree of p62 expression in different status of lymph-node metastasis promoted us to examine whether p62 expression was associated with ICC patients’ survival. Kaplan–Meier survival analysis revealed that ICC patients with high levels of p62 had a significantly shorter overall survival and higher cumulative recurrence rate than those with low expression of p62 (P=0.0082 and P=0.0034, respectively) (Fig. 1F-G).
These observations indicated that high p62 expression in ICC not only contributes to lymph-node metastasis, but also influences patients’ survival after liver resection.
Inhibition of p62 impaired metastasis potential of ICC cells in vitro
To further evaluate the cellular functions, especially metastasis potential, of p62 in vitro, we successfully constructed ICC cells with stably knockdown of p62 by shRNA, of which inhibitory efficiency was evaluated by western blotting (Fig. 2A). We investigated the role of p62 expression in cell proliferation and found that inhibition of p62 expression in ICC cells resulted in the decreased proliferation rate (Fig. 2B). Cellular migration is a characteristic of metastatic tumors and the first step of invasion. To assess whether p62 down-regulation in ICC cells affects cell migration, we performed transwell migration and wound-healing assays. Microscopic observation at 24 hours revealed a significant delay in the wound closure of p62-knockdown ICC cells compared with the control groups (Fig. 2E). As shown in Fig. 2C, silencing p62 expression was associated with a 33.8% and 47.9% reduction in migrated cells through transwell chamber in the HCCC-9810-shp62 and QBC939-shp62 cells compared with control cells, respectively. In addition, matrigel invasion assay was used to validate the invasive potentials of ICC cell lines. The cells with p62 knockdown showed a significant reduction in their ability to migrate through matrigel compared with control groups (Fig. 2D). Collectively, these in vitro assays suggested that knockdown of p62 expression attenuated proliferation, migration and invasion potentials of ICC cells.
p62 inhibition impairs tumor growth and metastasis in vivo
Next, we constructed a subcutaneous xenograft model using QBC939-shp62 and QBC939-shCtrl cells. Two weeks after inoculation, all mice successfully formed palpable tumors. Compared with controls, mice injected with QBC939-shCtrl cells showed significantly faster tumor growth which reflected by increased tumor size (Fig. 3A). After inoculation for 40 days, tumor volume of QBC939-shCtrl xenografts was 686.4 ± 333.2 mm3, which was signiଁcantly larger than that derived from QBC939-shp62 group (207.3±93.6mm3, P=0.0147, Fig. 3B). Furthermore, metastatic analysis in vivo found that the down-regulation of p62 reduced lung metastasis capacity in QBC939-shp62 group mice. In contrast, the lung metastasis rate was 80% (4/5) in the QBC939-shCtrl group with more metastatic lung nodules (Fig. 3C-D). These results indicated that reduction of p62 expression could signiଁcantly inhibited tumor growth and progression of ICC in vivo.
p62 promoted tumor progression of ICC cells through induction of EMT
It has been reported that tumor cells mainly undergo epithelial-to-mesenchymal transitions to acquire the migratory and invasive properties. Therefore, we performed western blotting and immunofluorescence assays to determine the expression of EMT-related markers in ICC cells with different level of p62. The WB assay demonstrated a diminished expression of Snail and Vimentin, and increased expression levels of E-Cadherin in ICC cells after interference of p62 expression (Fig. 4A). qRT-PCR results also further confirmed that E-Cadherin mRNA was upregulated in ICC cells with p62 knockdown whereas Vimentin mRNA expression was significantly inhibited (Fig. 4B-C). Consistently, immunofluorescence assay also showed that down-regulation of p62 in ICC cells resulted in the increased expression of epithelial marker E-Cadherin and the decreased expression of mesenchymal marker Vimentin (Fig. 4D). We further used the IHC to investigate the relationship between expression of p62 and EMT-related markers in the 140 ICC tissues. Tumor samples expressing high p62 tended to have up-regulation of Vimentin and Snail while down-regulation of E-Cadherin (Fig. 4E). Taken together, these data implicated that the higher expression of p62 significantly may promote ICC tumor progression by inducing EMT process.
Inhibition of p62 compromises mitochondrial function and mitophagy in ICC cells
The mutual interplay between mitochondrial dysfunction and EMT in tumors has been recently highlighted, which is believed to be associated with progression to a metastatic and drug-resistant phenotype. We investigated the functionality of mitochondria in ICC cells after inhibition of p62 expression. Measuring the mitochondrial OCR which quantify mitochondrial function more precisely demonstrated that basal and maximal respiratory capacity were significantly reduced in the p62-knockdown ICC cells compared with control cells (Fig. 5A-C).
As is showed in the results above, inhibition of p62 caused mitochondria dysfunction. We hypothesized that p62 may be required for maintenance of mitochondrial respiration through mitophagy which could degrade dysfunctional mitochondrial. We then use the iron chelator deferiprone (DFP) triggered PINK1/Parkin-independent mitophagy and assessed early stage of mitophagy by visualizing colocalization of mitochondrial marker TOMM20 with the autophagosomal marker LC3. p62-knockdown ICC cells were found to have significant reduction of LC3 recruitment to mitochondria after DFP treatment (Fig. 5D). Together, these results suggested that inhibition of p62 impaired autophagic clearance of dysfunctional mitochondria, and disrupt mitochondrial homeostasis which may be associated with compromised metastatic potential of ICC.