ER is not only high in calcium-dependent molecular chaperones such as GRP78 for helping stabilize protein-folding intermediates, also has an oxidative environment for the formation of disulphide bonds with the aid of PDI and for the proper folding of proteins [23, 24]. Under non-stressed conditions, GRP78 combines with three UPR sensors (PERK, ATF6 and IRE1) keeping them inactive. Once ER stress occurs, GRP78 preferentially binds to the unfolded proteins, dissociates from the sensors, resulting in activation of the UPR transducers [6, 25]. The outcome of it involves in protein folding and transporting, as well as increasing unfolded protein clearance through pathways such as ERassociated degradation (ERAD) and autophagy. Upon unresolved stress, cells enter apoptosis [8].
PDI family is a part of the thioredoxin (TRX) superfamily, mainly act as catalysts for the formatting and rearranging S-S bonds, also can function as chaperones involved in ERAD [26], playing an important role in the maintenance and regulation of proteostasis. P4HB, also known as PDIA1 (usually referred as PDI), as the archetype PDI protein, the chaperone activity of who is independent of catalytic activity, while the former is vital for many client proteins [27]. There is sufficient evidence supporting that PDIA1 high expressed in a wide variety of cancers tissues [9–11, 24] and cancer cells [28]. And the upregulated PDIA1 is closely correlated with cancer metastasis and invasiveness [27].
GRP78 is encoded by HSPA5, as the most abundant protein of heat shock protein-70 (Hsp70) family, mainly exits in ER lumen, corrects the folding and assembly, as well as prevents the transport of misfolded proteins or protein subunits [29, 30]. GRP78 works as a molecular chaperone in cells, its high expression is crucial for proliferation, invasion, metastasis of many cancers, proves to be correlated with tumor resistance, recurrence, and a low OS [17, 18, 31–33]. With the help of GRP78, tumor cells can organize the stimulation of processes including macroautophagy, combat reactive oxygen species (ROS), and activate pro-survival signaling pathways [29]. Thus, GRP78 will be a useful prognostic biomarker and a promising target for cancer.
P4HB and GRP78 are both ER chaperone proteins involved in UPR, and represent for poor prognosis of various cancers from the above mentioned. However, rare research pays attention to the correlation of them, especially in cancer. An early study shows that BiP and PDI perform synergistically in vitro in the oxidative folding of antibodies, BiP bind to the unfolded polypeptide chains to keep them in a conformation where cysteine residues are accessible for PDI [34]. Different from P4HB negatively correlated with GRP78 in hepatocellular cancer in vitro [19], we confirmed that P4HB and GRP78 showed a positive correlation in protein (from our cohort) and mRNA (from the databases) levels, with disease progress, the two proteins might more closely related since the correlation coefficients were significantly elevated in tumor size ≥ 5 cm, TNM Ⅲ/Ⅳ stage and positive vessel invasion subgroups. Together with single or combined high expression of P4HB and GRP78 represented for poor prognosis in GC. The specific mechanisms need further exploration.
A lot of studies have shown in a wide range of primary tumors, there exists a sustained and high-level activation of all three branches within UPR [35]. From bioinformatics analyses, we also found P4HB and GRP78 might participate in UPR, since they positively correlated with all typical genes within UPR, as well as correlation coefficients of most upstream genes were higher than downstream genes. UPR may act as a key driver in resistance to chemotherapy [36, 37]. A new encouraging research finds that activation of the PERK branch with UPR is required for colon cancer cells surviving from treatment of 5-fluorouracil, the usage of PERK inhibitor synergizes with 5-FU could suppress the growth of colon cancer cells in vivo [38]. It may explain in fluorouracil-based postoperative adjuvant chemotherapy subgroup, GC patients with a high co-expression of P4HB and GRP78 had a shorter OS of our cohort. When stratified by TNM stage, only patients in TNM Ⅲ/Ⅳ stage had a significant shorter OS, one reason might be that in early stage GC, the positive effects from chemotherapy could cancel out the negative effects from UPR, while in advanced GC, the negative effects probably exceeded.
Our nomogram integrated co-expression of P4HB and GRP78 with other prognostic variables, was a simple visualized tool for predicting OS after surgery of patients with OS. Comparing to TNM stage, the discrimination ability and clinical usefulness of the nomogram was much better through AUROCs and DCA curves of 3-year and 5-year, may be great valuable for clinic.
There still remain several limitations in our study. Firstly, this is a retrospective study of small sample group of GC patients, exists the statistical limitations. Secondly, further researches are required to confirm the correlation and regulation mechanisms of P4HB and GRP78 expression of GC in vitro and in vivo. Finally, it needs an independent external cohort to verify our research findings.
In conclusion, we revealed that P4HB expression is positively correlated with GRP78 expression, the correlationship may closer in advanced GC; P4HB and GRP78 may involve in UPR of GC from bioinformatics analyses; high co-expression of P4HB and GRP78 predicts a shorter OS with fluorouracil-based postoperative adjuvant chemotherapy in the advanced stage; co-expression of P4HB and GRP78 can independently predict unfavorable OS for GC patients; the prognostic nomogram based on stepwise Cox regression model is much better than TNM stage in the discrimination ability and clinical usefulness.