Comparison of anti-cancer effects of novel protein disulphide isomerase (PDI) inhibitors in breast cancer cells characterized by high and low PDIA17 expression

Protein disulphide isomerases (PDIs) are highly expressed proteins present predominantly in the endoplasmic reticulum but also in the cytosol, nucleus, on the cell surface and extracellularly [1]. PDIs were initially identified as chaperone proteins and are involved in the reduction, oxidation and isomerization of disulphide bonds [2]. Various functions of PDIs have been proposed, including the regulation of cancer cell phenotype. Indeed, the overexpression of PDIs in highly metabolically active cancer cells maintains the proper folding of the proteins and prevents protein aggregation [3].

The best described PDI isoforms in cancer cells are PDIA1 (P4HB) and PDIA3 (ERP57) [4‐9]. Suppression of cancer growth by PDIA1 inhibition was proposed as a novel approach to target cancer. Some PDI inhibitors (e.g., quercetin-3 rutinoside and PACMA 31) demonstrate promising anti-cancer activity [10]. Recently, it was shown that extracellular PDIA1 plays an important role in regulating the adhesion of cancer cells to the endothelium and their transendothelial migration, whereas intracellular PDIA1 was involved in regulating cell cycle and caspase 3/7 activation [11]. Additionally, growing evidence supports the important role of PDIA17 (anterior gradient protein 2, AGR2) in oncoproteins, stimulating proliferation and promoting metastasis [4, 12]. Accordingly, among the PDI proteins covering over 20 isoforms, PDIA17 has also emerged as a novel prometastatic and proangiogenic protein and an attractive target for anti-cancer therapy [13, 14].

It is still inconclusive which PDI isoform should be targeted in anti-cancer treatment and whether targeting single rather than a formulation of ‘PDI-pan inhibition’ is a better strategy [15‐18]. It is also not clear whether the pattern of PDI isoform expression is heterogeneous or homogeneous in various cancer cells. Here, we address this issue by characterizing the PDI repertoire of multiple cancer cell lines to determine which PDI isoforms are the most abundant and that might represent an optimal target for anti-cancer therapy. Then, we studied the effects of inhibiting the most abundant isoforms, including PDIA1 and PDIA3 as well PDIA17 in cancer cells identified as high and low PDIA17 expressing breast cancer cells. As pharmacological tools to inhibit PDIA1, PDIA3 or PDIA17, we used recently developed aromatic N-sulphonamides of aziridine-2-carboxylic acid derivatives [19, 20].

In the present work, we characterized PDI isoform contents in various cancer cell lines and provided evidence that PDIA1 and PDIA3 represent major isoforms of multiple cancer cells, and the non-selective inhibition of PDIA1 and PDIA3 displayed significant anti-proliferative effects irrespective of whether or not PDIA17 was present. On the other hand, PDIA17 was highly expressed in the hormone-sensitive MCF-7 breast cancer cell line when compared to the triple-negative MDA-MB-231 cell line, and PDIA17 inhibition displayed additional anti-proliferative effects in the MCF-7 but not in the MDA-MB-231 breast cancer cell line. Interestingly, the anti-adhesive effects in the hormone-sensitive MCF-7 cells featured by higher levels of PDIs were clearly more pronounced when compared to triple-negative MDA-MB-231 cells, suggesting that PDIA1-, PDIA3- and PDIA17-based therapy may represent a novel strategy for personalized anti-metastatic therapy in cancers with high PDI expression.

In previous studies, the overexpression of the PDIs in comparison to normal tissue was demonstrated (e.g., using RNA-Seq methodology), and there is a general agreement on the important role of PDIs in cancer survival [29]. The data from gene expression datasets showed that the expression levels of individual PDI members vary between the types of cancer. Higher expression of PDIA1 was, for example, noted in prostate, ovarian and oesophageal cancer [29]. However, to the best of our knowledge, our study is the first to comprehensively characterize the content of various PDI isoforms in multiple cancer cell lines using a proteomic approach to determine which of these isoforms are the most abundant and might represent an optimal target for anti-cancer therapy.

The major contribution of PDIA1 and PDIA3 to the total PDI profile of various cancer cells, their secretion and important role outside the cell agrees with growing evidence for the involvement of PDIA1 and PDIA3 in cancer progression [16, 30‐35].

Importantly, independent of this general rule, the profile of individual PDIs did vary to some extent between cancer types (e.g., prostate, breast cancer) and cell lines within the same type of cancer. For example, among lung cancers, the NCIH1299 cell line had very low expression of PDIA1, whereas in the NCIH358 cell line, this expression was 14 folds higher. In particular, in breast cancer cell lines, PDIA17 expression was high in the hormone-sensitive MCF-7 cell line but not detectable in the triple-negative MDA-MB-231 cell line. This was an important finding of our work in the context of the suggested role of PDIA17 in genomic integrity, proliferation, apoptosis, angiogenesis, adhesion, migration, stemness and inflammation [18, 36], indicating that PDIA17 is a promising target for anti-cancer treatment. In fact, the high expression of PDIA17 in the primary tumour of breast cancer patients was found to be a possible prognostic indicator of poor patient outcome [37]. Increased plasma concentration of PDIA17 was positively correlated with cancer progression [38]. In our hands, this protein was expressed only in a few studied cell lines, including PC-3, MCF-7, HT-29 and T47D, thus forming an anti-cancer target for selected cancers. Unexpectedly, higher PDIA17 expression was noted in the less-invasive breast cancer cell line [3, 39], suggesting the importance of hormonal sensitivity or involvement of other regulatory mechanisms [10, 40‐42]. Interestingly, in breast cancers, the rationale for anti-PDIA17 therapy was offered for oestrogen receptor (ER)-positive breast cancers (e.g., MCF-7) but not ER-negative breast cancers (e.g., MDA-MB-231) [43‐45].

In the context of the emerging literature on the role of PDIA17 in tumorigenesis, based on our proteomic analysis confirmed by Western blotting, we were able to clearly define MCF-7 as a breast cancer cell line with high PDIA17 expression, whereas MDA-MB-231 had very low PDIA17 expression, and we used these two cell lines for pharmacological studies. Importantly, the intercellular expression of PDIA1 and PDIA3 in these two cell lines displayed only modest differences in their relative intracellular abundance; therefore, these two lines were well suited to delineate the relative functional importance of PDIA1 and PDIA3 as compared with PDIA17. As pharmacological tools to inhibit PDIA1, PDIA3 or PDIA17, we used recently developed aromatic N-sulphonamides of aziridine-2-carboxylic acid derivatives [19, 20]. These compounds represent a novel group of potent and relatively selective PDIA1 inhibitors with additional activity towards PDIA3. Many of them show low or moderate cytotoxic activity in vitro towards a panel of cancer cell lines that was for numerous compounds more pronounced as compared with their cytotoxicity against normal cell lines [19, 20]. However, their anti-cancer effects have not been previously investigated in the context of heterogeneous contents of PDIs in various cancer cell lines.

Here, we demonstrated in the anti-proliferative assays that MCF-7 and MDA-MB-231 exhibited similar responses to the PDI inhibitors used, suggesting that targeting the most abundant PDI isoforms regardless of PDIA17 levels may be an effective strategy to inhibit the proliferation of cancer cells.

In turn, the anti-adhesive properties of the tested PDIA1 and PDIA3 inhibitors were more pronounced in the MCF-7 cell line, a result that might be attributed to higher expression of PDIA1 on the surface membrane of the cells in comparison to the MDA-MB-231 cell line because extracellular PDIA1 seemed more abundant in the cell line. The role of PDIA1 in cancer cell adhesion was in accordance with our recent work demonstrating that extracellular PDIA1 plays an important role in regulating the adhesion of cancer cells to matrix proteins, endothelium and their transendothelial migration [11]. Similar extracellular mechanisms of action could be attributed to PDIA3. Indeed, PDIA3 was also present extracellularly in both MCF-7 and MDA-MB-231 cell lines. The compound C-3399, which inhibited PDIA1 and PDIA3 rather unselectively, with some preferences to the inhibition of PDIA3 was the most potent inhibitor of adhesion of MDA-MB-231 cells to collagen, underscoring the therapeutic value of non-selective inhibition of PDIA3 and PDIA1 in the regulation of cancer cell adhesion to the cellular matrix. The relative importance of PDIA3 vs PDIA1 in this effects remains to be delineated in further studies. Finally, we demonstrated that C-3353 inhibition of PDIA17 was quite potent in inhibiting the adhesion of MCF-7 to collagen. Altogether, our results show that targeting PDIA1 and PDIA3 as well as PDIA17 might afford effective anti-cancer effects, particularly in highly malignant cancer cell types by targeting cancer cell adhesion. Such a mechanism of action involving extracellular disulphide exchange and most likely integrin activation might afford important anti-metastatic effects independent of cytostatic or anti-proliferative potential of PDI inhibition [11]. Furthermore, it seems that effects of the tested PDI inhibitors cannot be ascribed to apoptosis. The studies of Stojak et al. [11] showed that the PDI inhibition was not accompanied by increased caspase 3/7 activity in MDA-MD-231 cand MCF-7 cell lines. This conclusion was confirmed by using the two strategies of PDI inhibition–PDIA1 and PDIA3 silencing and bepristat treatment. Additionally, the authors did not observe changes in mitochondrial respiration and only moderate changes in cell cycle. An increase in caspase 3/7 activity was noted in the studies of the effects of other azaridine-based PDI inhibitors in colon cancer, but only with the highest concentration used (data not published). In contrast, in reports using cell permeable PDI-inhibitors e.g. BAP2 and 35G8 the activation of apoptosis and autophagy were reported [46] the effects most likely linked to ER stress and the unfolded protein response (UPR) [3]. Accordingly, we claim that PDI inhibitors that do not penetrate across the cell membrane would target extracellular PDIs and their action is not necessarily associated with caspase inhibition but affords the inhibition of cancer cell adhesion to the cellular matrix or to endothelial cells. Indeed, membrane-impermeant thiol blocker pCMBS, inhibited cancer cell adhesion to collagen type I and fibronectin [11] supporting the notion that extracellular PDIs is a target for anti-adhesive effects of PDIA1 inhibitors.

Noteworthy, the majority of available inhibitors of PDIs are non-selective sulfhydryl-reactive compounds that mostly target intracellular PDIs. For example, the irreversible PDIA1 inhibitor—PACMA31—efficiently suppressed ovarian tumour growth, suggesting non-selective intracellular PDI inhibition as a major target for anti-cancer PACMA 31 action [10, 47]. Similarly, a small molecule intracellular inhibitor of PDI activated the apoptosis signalling pathway and reduced the malignancy of colorectal cancer [48]. CCF642 caused acute ER stress in multiple myeloma cells accompanied by apoptosis in addition to a robust ER stress response [49]. E64FC26 induced the induction of oxidative stress in multiple myeloma [50]. All these mechanisms involved intracellular targets, and the compounds used were cell-permeable and unselective PDI inhibitors. The development of effective and selective cell-impermeable PDI inhibitors may pave the way for further studies targeting cancer cell adhesion and metastasis by targeting extracellular PDIs.

In conclusion, PDIA1 and PDIA3 represent the most abundant isoforms in multiple cancer cell lines and are also bound to membranes and released extracellularly providing the rationale of the development of anti-PDIA1 and anti-PDIA3 therapy targeted to extracellular PDIs. PDIA17 expression appears to be cell specific and related to cancer cell malignancy, and its inhibition could present additional benefits in cancer cells expressing PDIA17. In particular, it seems that PDIA17 targeting might be of importance in some ER-positive cancers. Targeting extracellular PDIA1-, PDIA- and PDI17-dependent regulation of cancer cell adhesion may represent a novel, effective, personalized anti-adhesive and anti-metastatic therapy in cancers with high PDI expression. This notion agrees with an increasing interest in the molecular mechanisms of action of extracellular PDIs [51]. Further studies are required to explore the extracellular PDIs as anti-cancer targets.