Hepatocellular carcinoma (HCC) is the fifth-leading cause of cancer-related death worldwide [1]. Curative resection is the primary therapeutic modality for HCC, except in advanced cases, but the prognosis of individual patients varies and several molecular markers of the prognosis of HCC have been identified [2]. Unfortunately, few molecular markers for HCC are targetable by drugs and there is a pressing need to identify new molecular candidates to improve therapeutic strategies and predict the prognosis in the era of targeted anti-cancer therapy.

Protein disulfide isomerase (PDI) is a 57-kDa dithiol: disulfide oxidoreductase and molecular chaperone. It is one of the most abundant soluble proteins in the endoplasmic reticulum (ER) and plays a key role in maintaining cellular homeostasis by mediating oxidative protein folding [3]. PDI expression is upregulated in some cancer types and involved in aggressive phenotypes of breast [4], brain [5], ovary [6], and prostate [7] cancers. Increasing evidence from functional studies indicates that PDI has an important role in tumor survival and cancer progression [3]. In human HCC cell lines, PDI was expressed in a hypoxia-inducible manner [8]. However, the expression of PDI in human HCC tissues and its prognostic role in patients with HCC has not been determined.

This study investigated whether PDI is expressed in human HCC tissues and evaluated the effect of PDI expression on the time to progression (TTP) and overall survival (OS) after surgical resection in patients with HCC. We found that PDI expression was upregulated in HCC samples and that high PDI expression was an independent predictor of a poor prognosis.

This study found that the expression of PDI was increased in HCC compared to nontumor tissue and that increased expression was associated with the aggressive clinicopathological features of HCC. Moreover, upregulated PDI expression in HCC tissues was an independent risk factor for a poor OS and shorter TTP in patients with HCC. This is the first report that the level of PDI expression adversely affects the OS and progression of HCC patients who have undergone surgical resection.

One can argue that the increased PDI level is a byproduct of cancer, such as the cellular environment, disease progression status, or degree of tumor differentiation. However, recent studies indicate that PDI is important for tumor survival and progression, emphasizing its role as a tumor-promoting factor [5,11,12,13]. PDI expression seems to be associated with protection of cancer cells from proteotoxic stress and hypoxic damage. Hypoxia can induce the accumulation of unfolded or misfolded proteins and lead to ER-stress-induced cell death [14]. Altered ER function results in ER stress and subsequent activation of the unfolded protein response (UPR), an important ER stress response that protects the cell by removing unfolded or misfolded proteins [15]. PDI proteins are key molecules in the UPR. Many ER-stress-response chaperones have PDI activity or PDI-like domains; therefore, blocking this activity might increase ER stress and enhance apoptosis [16].

Locally recurrent HCC nodules that survive hypoxic insults after transarterial chemoembolization sometimes have significantly shorter doubling times than those in neighboring nodules [17]. Moreover, HCC sometimes undergoes an infiltrating rather than a mass-forming growth pattern; these advanced infiltrative HCCs seldom show hypervascularity, grow more rapidly, and have a poorer prognosis than the mass-forming hypervascular types. These findings suggest that hypoxia activates the signals that allow HCC cells to survive and proliferate [18] and that PDI contributes to the survival and progression of these cancer cells. This explains the correlation between PDI expression and an aggressive cancer phenotype.

The expression of PDI family proteins, an important arm of the UPR, correlates with cancer invasion, metastasis, and drug resistance in various tumor types, including melanomas and gliomas [5,11]. UPR, a prosurvival pathway that is activated during hypoxia, which is a hallmark of solid tumors [19], is also an important mediator of metastasis in human cervical cancer [20]. UPR promoted the polarization of tumor-infiltrating myeloid cells toward an inflammatory/immunosuppressive phenotype, leading to T-cell dysfunction and, ultimately, to the evasion of tumor cells from immunosurveillance [21]. Therefore, we postulate that intratumoral hypoxia induces PDI expression as a component of the UPR, which leads to tumor evasion from immunosurveillance via T-cell dysfunction. The escape from immunosurveillance contributes to cancer recurrence or metastasis [22].

In addition, PDI was reported to be associated with resistance to the growth-inhibitory effects of transforming growth factor-β1 (TGF-β1), which frequently occurs in cancer cells. TGF-β1 downregulates PDI microRNA in TGF-β1-sensitive cells, but not in cells insensitive to the growth inhibitory effects of TGF-β1 [12]. Moreover, PDI protects cancer cells from apoptosis. In melanoma, the inhibition of PDI activity using bacitracin enhanced apoptosis triggered by fenretinide or velcade [13]. Although PDI expression was reported to be increased in the stressful microenvironment of human HCC cell lines and in the serum of HCC patients [23,24], its functional implications in HCC have not been elucidated fully.

Previously, we observed that PDI was expressed in a human HCC cell line under hypoxic conditions. This suggests that PDI participates in the UPR to compensate for the ER stress that occurs under innate intratumoral hypoxic conditions [8]. Indeed, the expression of PDI was increased in HCC compared to nontumor tissue, and the high PDI expression in HCC tissue adversely affected the clinical outcome of HCC patients following surgical resection in our series.

The development of robust PDI activity assays has led to the recent discovery of a series of novel PDI inhibitors, such as the irreversible inhibitors PACMA 31, 16F16, and RB-11-ca, and the reversible inhibitors juniferdin and rutin [3]. Tumor cells require higher levels of PDI to cope with marked ER stress and a global increase in protein synthesis to sustain rapid proliferation. Increased protein synthesis leads to an abundance of misfolded proteins in the ER that must be refolded by PDI. As such, tumor cells are more vulnerable to PDI inhibition than are normal cells. Therefore, PDI is an important target in the treatment of cancers.

Together, these findings suggest that PDI expression is upregulated in HCC, tissues and is associated with the aggressive histological features of HCC. More importantly, upregulated PDI expression in patients with HCC is predictive of poor OS after surgical resection via frequent recurrence. Therefore, PDI can serve as a novel independent prognostic factor and may be a useful therapeutic target in patients with HCC.