Purpose: Deregulation of STAT3 activation is a hallmark of many cancer cells, and the underlying mechanisms are subject to intense investigation. We examined the extent of PIAS3 expression in mesothelioma cells and human tumor samples and determined the functional effects of PIAS3 expression on STAT3 signaling.

Experimental design: We evaluated the expression of PIAS3 in mesothelioma tumors from patients and correlated the expression levels with the course of the disease. We also measured the effects of enhanced PIAS3 activity on STAT3 signaling, cellular growth, and viability in cultured mesothelioma cells.

Results: Gene expression databases revealed that mesotheliomas have the lowest levels of PIAS3 transcripts among solid tumors. PIAS3 expression in human mesothelioma tumors is significantly correlated with overall survival intervals (P = 0.058). The high expression of PIAS3 is predictive of a favorable prognosis and decreases the probability of death within one year after diagnosis by 44%. PIAS3 expression is functionally linked to STAT3 activation in mesothelioma cell lines. STAT3 downregulation with siRNA or enhanced expression of PIAS3 both inhibited mesothelioma cell growth and induced apoptosis. Mesothelioma cells are sensitive to curcumin and respond by the induction of PIAS3. Corroborative evidence has been obtained from STAT3 inhibition experiments. Exposure of the cells to a peptide derived from the PIAS3 protein that interferes with STAT3 function resulted in apoptosis induction and the inhibition of cell growth.

Conclusion: These results suggest that PIAS3 protein expression impacts survival in patients with mesothelioma and that PIAS3 activation could become a therapeutic strategy. Clin Cancer Res; 20(19); 5124–32. ©2014 AACR.

This article is featured in Highlights of This Issue, p. 4975

Translational Relevance

The biologic principle of negative feedback regulation is being exploited by tumor cells. Mesothelioma cells suppress PIAS3, a negative regulator of STAT3 activity. Strategies for the reactivation of PIAS3 could result in STAT3 inhibition and the growth arrest of tumor cells.

The JAK–STAT signaling pathway provides an essential cellular communication route from the plasma membrane to the nucleus. It is initiated by the interaction of extracellular ligands with cellular surface receptors and results in alterations of transcription patterns. Cytokines and growth factors act as ligands and play central roles in the regulation of cell growth, differentiation, and metabolism in many different cell types and organs (1). STAT proteins, especially STAT3, are not only important in the development and function of normal cells, but can assume oncogenic properties when the extent and duration of their activation are disturbed (2). Inappropriate STAT3 activation results in the prevention of apoptosis and the promotion of tumor cell proliferation (3, 4). STAT3 is constitutively phosphorylated and thereby activated in many forms of cancer. The inhibition of STAT3 activity often results in apoptosis and cell death (5, 6). STAT3 serves as a central integration point for multiple oncogenic signaling pathways to regulate genes involved in cell-cycle control, apoptosis, angiogenesis, tumor invasion, and metastasis. This makes it an attractive target for cancer therapies.

PIAS3 is part of a small gene family with four members (7, 8). PIAS3 was initially found to interact specifically with phosphorylated STAT3, and not STAT1, in IL6-activated murine myeloblast M1 cells and to decrease STAT3 DNA-binding capacity and transcriptional activity (9). We (10, 11) and others (12) have demonstrated that overexpression of PIAS3 can inhibit STAT3 transcriptional activity and promote growth inhibition in vitro. If PIAS3 counteracts transactivation by STAT3, it would be conceivable that PIAS3 expression could be decreased in malignant tissues with high STAT3 activation. This hypothesis has been confirmed. PIAS3 expression is absent in glioblastoma multiforme tumor tissue when compared with the adjacent normal brain tissue (13). The expression of PIAS3 is inversely proportional to that of activated, phospho-STAT3 (pSTAT3) expression. We have examined expression of PIAS3 in 44 resected non–small cell lung cancer (NSCLC) specimens and found that 89% of adenocarcinomas stained positive for PIAS3 and only 38% of squamous cell carcinomas showed evidence of staining (14). In squamous cell carcinomas in which PIAS3 could be detected, only low expression levels were found. Intense PIAS3 expression in adenocarcinomas was associated with the absence of pSTAT3.

These studies suggest that PIAS3 may represent a useful target to devise therapeutic strategies against cancer cells harboring activated STAT3. Here, we show that mesothelioma cell lines exhibit high STAT3 activity and use three experimental approaches to manipulate PIAS3 levels to demonstrate the principle. PIAS3 is able to inhibit STAT3 activity and cell growth in mesothelioma cells.

Cell culture and transient transfection

Human pulmonary epithelial cell line A549 and mesothelioma cell lines H2052, H2452, 211H, and H28 were purchased from ATCC and maintained in DMEM/Ham's F-12 medium supplemented with 10% (v/v) FBS (Hyclone, ThermoFisher Scientific) in a 5% CO2 humidified incubator at 37°C. Cells were transfected with either pCMV5 (empty vector) or pCMV5-mouse Pias3 using TransIT2020 (Mirus Bio LLC). After 5 hours, media were replaced with DMEM/F12 media containing FBS (10%). After 24 hours of incubation, cells were collected for further analysis.

STAT3 siRNA transfection

Downregulation of STAT3 by siRNA was achieved as described previously by Dabir and colleagues (15).

Immunoblotting and antibodies

Whole-cell lysates were prepared in RIPA buffer, as described previously (16). Antibodies used in Western blots were obtained from either Cell Signaling Technology (PIAS3 #4641, pSTAT3 #9145, Caspase 3 #9961, PARP #9542), or Sigma (β-actin #A5441).

Cell proliferation analysis

Cell growth and viability were assessed in manually counted cells by either Trypan blue dye exclusion or the MTS assay, as described previously (10).

Purification and cell treatment of peptide rPP-C8

Protein purification of the peptide rPP-C8 and cell treatment was performed as described previously (17). Briefly, 2,000 cells were seeded in 96-well plates, and the next day, medium was removed, 0.5 or 2.0 μmol/L of the peptide (or PBS as solvent control) was diluted in 100 μL medium and added to the cells.

Immunohistochemistry

The tissue microarray (TMA) slides were obtained from the National Mesothelioma Virtual Bank (NMVB; http://mesotissue.org/). The slides were deparaffinized with xylene rinses and then transferred through two changes of 100% ethanol. Endogenous peroxidase activity was blocked by 30-minute incubation in a 2.5% hydrogen peroxide/methanol buffer. Antigen retrieval was performed by boiling the slides in a pressure cooker filled with a sodium citrate buffer (pH 6.0). After antigen retrieval, the slides were blocked using Background Sniper (Biocare #BS966M) for 20 minutes. The tissues were incubated with 1:400 dilution of rabbit anti-human PIAS3 antibody (Cell Signaling Technology) overnight at 4°C. Bound antibody was detected using an anti-rabbit MACH4 horseradish peroxidase–labeled polymer secondary antibody from Biocare (#MRH534L) for 30 minutes. The slides were rinsed in a TBS series and visualized with a 10-minute incubation of liquid 3,3′-diaminobenzidine in buffered substrate in the dark. Finally, the slides were counterstained with hematoxylin for 30 minutes and mounted with Biomount. The intensity of immunostaining for PIAS3 was scored visually and stratified into 3 staining groups: 0 = no nuclear staining, 1+ = minimal staining, and 2+ = moderate-to-strong staining in >50% of tumor nuclei. At least two different cores were analyzed by immunohistochemistry (IHC) for approximately 70% of the tumors, approximately 50% had ≥ 3 cores analyzed per tumor. The multiple scores from cores of a single tumor specimen were averaged to yield the final score.

Statistical analysis

Overall patient survival (OS) was measured from the date of diagnosis to the date of death. The difference of OS between groups was examined by log-rank test. The effect of PIAS3 as a continuous measurement was further evaluated using the Cox model (18) after controlling for tumor grade (low, intermediate, or high). Histologic type (epithelioid, sarcomatoid, biphasic) was not included in the Cox analysis due to a lack of some histotypes. The large majority of patients had advanced stage disease (stages III/IV) and therefore staging was also not included in the Cox model used. The difference of PIAS3 expression between two groups was examined using a t test. All tests are two-sided and a P ≤ 0.05 was considered statistically significant.

PIAS3 mRNA expression is low in cancer tissues

We assessed global gene expression patterns for PIAS3 expression across all types of cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) database (http://www.broadinstitute.org/ccle/home; ref. 19). When cancer cell lines derived from solid tumors were compared, very low levels of PIAS3 were found in mesotheliomas (Fig. 1A). When we specifically compared PIAS3 expression between NSCLC and mesothelioma cells, we found a significant difference in PIAS3 mRNA expression (Fig. 1B; P < 0.0007). On the basis of these observations, we further searched the Oncomine database (https://www.oncomine.org/resource/login.html) for analysis of PIAS3 expression in tumor tissues. The study by Gordon and colleagues (20) compared PIAS3 expression in thoracic cancers. The results demonstrated that mesothelioma showed significantly lower expression of PIAS3 (P < 0.001) compared with adenocarcinoma (Fig. 1C).

Figure 1.

Mesothelioma shows low PIAS3 expression in public datasets. A, global gene expression pattern for PIAS3 across all types of cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) database. B, PIAS3 expression in NSCLC and mesothelioma cells, showing a significant difference in PIAS3 mRNA expression in the CCLE database. C, Whisker plots showing the Log2 median-centered intensity of PIAS3 expression in Gordon lung. Compared with adenocarcinoma, PIAS3 expression in mesothelioma tissue samples is significantly lower.

Figure 1.

Mesothelioma shows low PIAS3 expression in public datasets. A, global gene expression pattern for PIAS3 across all types of cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE) database. B, PIAS3 expression in NSCLC and mesothelioma cells, showing a significant difference in PIAS3 mRNA expression in the CCLE database. C, Whisker plots showing the Log2 median-centered intensity of PIAS3 expression in Gordon lung. Compared with adenocarcinoma, PIAS3 expression in mesothelioma tissue samples is significantly lower.

Close modal

PIAS3 expression predicts survival intervals of patients with mesothelioma

We also investigated PIAS3 expression at the protein level. TMAs were obtained from the NMVB and PIAS3 expression analysis was performed by IHC. Representative pictures of PIAS3 staining and scoring for epithelioid cores are shown in Fig. 2A. PIAS3 protein expression observed in the mesothelioma TMA showed mostly nuclear localization. The mean score for PIAS3 expression was 1.46 ± 0.65; and the median was 1.71. Thus, about half of the tissue cores demonstrated low or negative PIAS3 staining. These results demonstrated that PIAS3 expression in a subset of mesothelioma tumors was consistent with the mRNA expression results.

Figure 2.

Mesothelioma tumors demonstrate reduced PIAS3 expression. A, IHC of mesothelioma tissue microarray: Representative pictures of PIAS3 staining and scoring. B, Kaplan–Meier estimation of OS by PIAS3 level (epithelioid histology only, from Cox model).

Figure 2.

Mesothelioma tumors demonstrate reduced PIAS3 expression. A, IHC of mesothelioma tissue microarray: Representative pictures of PIAS3 staining and scoring. B, Kaplan–Meier estimation of OS by PIAS3 level (epithelioid histology only, from Cox model).

Close modal

To determine whether PIAS3 expression levels are correlated with the survival intervals of patients upon diagnosis of the disease, we analyzed PIAS3 scoring of 39 epithelioid tumors with available patient survival information. We did not analyze other patients with mesothelioma with biphasic, sarcomatoid, and mixed histologies due to their low frequency. Kaplan–Meier survival analysis (21) showed that after controlling for the effects of tumor grade, PIAS3 expression significantly predicted OS intervals (P = 0.058). Mortality in patients with high PIAS3 expression was about 44% lower 12 months after diagnosis (Fig. 2B).

STAT3 is activated in mesothelioma cell lines

PIAS3 protein expression was also measured in four mesothelioma cell lines (H2052, H2452, 211H, and H28) by Western blotting and compared with the expression levels in A549 lung cancer cells (Fig. 3A), a representative NSCLC cell line (10, 11, 14, 15). Because PIAS3 is thought to be an endogenous inhibitor of STAT3 activity, we examined whether any decreased PIAS3 expression in mesothelioma cells was accompanied by increased p-STAT3 expression. The results in Fig. 3A demonstrate an inverse correlation between PIAS3 and p-STAT3 expression; the three mesothelioma cells with high p-STAT3 levels (H2052, H2452, 211H) all have little detectable PIAS3 expression. The mesothelioma cell with high PIAS3 expression, H28, demonstrated no detectable p-STAT3. PIAS3 expression did not correlate with total STAT3 expression. Reprobing the blot with anti-β-actin shows that variability in protein loading could not account for the observed differences in p-STAT3 expression.

Figure 3.

Mesothelioma cells are dependent on high STAT3 activity for growth. A, A549, H2052, H2452, 211H, and H28 cells were grown under basal conditions and protein lysates immunoblotted for PIAS3, p-STAT3 (active), total STAT3, and β-actin. B, H2052 and H2452 cells were treated with STAT3 siRNA, mismatch siRNA (mock), or untreated (control). Protein lysates were immunoblotted for total STAT3, cleaved caspase-3, PARP, and β-actin. C, knockdown of STAT3 in mesothelioma cells promotes apoptosis (MTS assay) and (D) cell growth inhibition.

Figure 3.

Mesothelioma cells are dependent on high STAT3 activity for growth. A, A549, H2052, H2452, 211H, and H28 cells were grown under basal conditions and protein lysates immunoblotted for PIAS3, p-STAT3 (active), total STAT3, and β-actin. B, H2052 and H2452 cells were treated with STAT3 siRNA, mismatch siRNA (mock), or untreated (control). Protein lysates were immunoblotted for total STAT3, cleaved caspase-3, PARP, and β-actin. C, knockdown of STAT3 in mesothelioma cells promotes apoptosis (MTS assay) and (D) cell growth inhibition.

Close modal

STAT3 siRNA induces apoptosis in mesothelioma cells

We established the functional significance of STAT3 activation in mesothelioma cells by measuring their survival and proliferation as a function of STAT3 transactivation. Downregulation experiments with STAT3 siRNA were carried out in H2052 and H2452 cell lines. Specific reduction in total STAT3 was demonstrated after STAT3 siRNA treatment, mismatch siRNA (mock) served as a negative control in H2052 and H2452 cells (Fig. 3B). The reduction in STAT3 levels was accompanied by the induction of apoptosis, as shown by the appearance of cleaved caspase-3 and PARP. Further experiments demonstrated that knockdown of STAT3 with siRNA resulted in substantial growth inhibition in both mesothelioma cell lines, as measured by the MTS (Fig. 3C) or by cell viability assays (Fig. 3D). Taken together, these results demonstrate that STAT3 activity is required for mesothelioma cell growth and survival.

PIAS3 overexpression inhibits cell growth and viability in mesothelioma cells

Most mesothelioma cells appear to be STAT3-dependent and we hypothesized that overexpression of PIAS3 would inhibit their growth. Indeed, transient overexpression of FLAG-tagged PIAS3 in H2052 and H2452 cells decreased p-STAT3 levels (Fig. 4A). Empty vector (EV)-transfected cells and untransfected cells served as controls (Fig. 4C). The decrease in activated STAT3 levels by PIAS3 overexpression also led to a decrease in cell growth, as measured by MTS (Fig. 4B) or cell viability assays (Fig. 4C). These results support our hypothesis that endogenous PIAS3 expression is downregulated in most mesothelioma cells and may thus contribute to the increased STAT3 activity that drives cell proliferation.

Figure 4.

PIAS3 overexpression inhibits STAT3 activation and cell growth in mesothelioma cells. A, H2052 and H2452 cells were transfected with FLAG-Pias3 (TXN), empty vector (EV), or untreated (C); 48 hours later, whole-cell extracts were prepared and immunoblotted for PIAS3, FLAG, pSTAT3 (active), and β-actin. B, PIAS3 overexpression results in apoptosis (MTS assay) and cell growth inhibition (C).

Figure 4.

PIAS3 overexpression inhibits STAT3 activation and cell growth in mesothelioma cells. A, H2052 and H2452 cells were transfected with FLAG-Pias3 (TXN), empty vector (EV), or untreated (C); 48 hours later, whole-cell extracts were prepared and immunoblotted for PIAS3, FLAG, pSTAT3 (active), and β-actin. B, PIAS3 overexpression results in apoptosis (MTS assay) and cell growth inhibition (C).

Close modal

Curcumin induces PIAS3 expression in mesothelioma cell lines

Enhancement of PIAS3 expression represents a potential therapeutic strategy for cancers like mesothelioma which depend on activated STAT3 for sustained growth. Thus, the discovery of a small molecule that increases endogenous PIAS3 levels in STAT3-dependent cancer cells could potentially become beneficial. Curcumin may be such a molecule, as it is a well-known inhibitor of STAT3 activity (22–24). Indeed, there is a time-dependent increase in PIAS3 levels in H2052 mesothelioma cells exposed to 1.0 μmol/L curcumin (Fig. 5A). This increase in PIAS3 was associated with a parallel decrease in p-STAT3 levels. No change in total STAT3 expression was observed, similar to β-actin. A dose-dependent increase in PIAS3 protein expression was observed upon curcumin treatment in all four mesothelioma cell lines (Fig. 5B). Our experiments also demonstrated that curcumin treatment decreased mesothelioma cell growth (Fig. 5C). These results suggest that curcumin may produce growth inhibition in mesothelioma cells through increased PIAS3 expression.

Figure 5.

Curcumin increases endogenous PIAS3 expression in mesothelioma cells. A, H2052 cells were incubated with 1 μmol/L curcumin for up to 24 hours and protein lysates immunoblotted for PIAS3, pSTAT3 (active), total STAT3, and β-actin. B, curcumin treatment at 0.1 or 1.0 μmol/L for 24 hours shows an increase in PIAS3 protein expression level in most of the mesothelioma cell lines. C, curcumin inhibits cell proliferation. H2052 and H2452 cells were incubated for 24 hours in the absence or presence of 10 μmol/L of curcumin and cell growth determined by DNA assay.

Figure 5.

Curcumin increases endogenous PIAS3 expression in mesothelioma cells. A, H2052 cells were incubated with 1 μmol/L curcumin for up to 24 hours and protein lysates immunoblotted for PIAS3, pSTAT3 (active), total STAT3, and β-actin. B, curcumin treatment at 0.1 or 1.0 μmol/L for 24 hours shows an increase in PIAS3 protein expression level in most of the mesothelioma cell lines. C, curcumin inhibits cell proliferation. H2052 and H2452 cells were incubated for 24 hours in the absence or presence of 10 μmol/L of curcumin and cell growth determined by DNA assay.

Close modal

A recombinant PIAS3 peptide induces apoptosis and inhibits growth in mesothelioma cells

It has recently been shown that rPP-C8, a C-terminal recombinant peptide derived from PIAS3, has the ability to inhibit STAT3 activity and cell growth in glioblastoma cells (17). The peptide is able to penetrate the plasma membrane upon addition to the growth medium. We incubated this peptide with mesothelioma cells to determine its effect on STAT3 activity. Western blotting experiments showed a decrease in p-STAT3 levels in H2052 and 211H cells after a 4-hour peptide treatment (Fig. 6A). There was no effect on total STAT3 or PIAS3 expression levels. Furthermore, addition of rPP-C8 to the medium of mesothelioma cells induced apoptosis, as shown by increased PARP cleavage (Fig. 6B). We next measured the effect of rPP-C8 on mesothelioma cell proliferation. H2052 and 211H cells were treated with 0.2 or 2.0 μmol/L rPP-C8 peptide for 3 days. The mesothelioma cells demonstrated a dose-dependent decrease in cell viability upon addition of the peptide, compared with PBS-treated cells (Fig. 6C). This was confirmed by cell counting (Fig. 6D). Thus, the rPP-C8 peptide appears to be efficiently taken up by mesothelioma cells and acts as a potent STAT3 inhibitor in mesothelioma cell lines.

Figure 6.

A recombinant PIAS3 peptide inhibits STAT3 activation and cell growth in mesothelioma cells. A, protein lysates were prepared after 4-hour exposure of H2052 and 211H cell lines to rPP-C8 peptide (0.5 and 2.0 μmol/L). Western blotting shows decrease in pSTAT3 (Tyr 705) without affecting total STAT3 or PIAS3 expression. β-Actin is a loading control. B, H2052 and 211H cells exposed to 0.5 or 2.0 μmol/L rPP-C8 peptide for 24 hours results in PARP cleavage at the higher concentration. +C is a positive control lysate. C, addition of the peptide to mesothelioma cells induces apoptosis (MTS assay) and cell growth inhibition (D).

Figure 6.

A recombinant PIAS3 peptide inhibits STAT3 activation and cell growth in mesothelioma cells. A, protein lysates were prepared after 4-hour exposure of H2052 and 211H cell lines to rPP-C8 peptide (0.5 and 2.0 μmol/L). Western blotting shows decrease in pSTAT3 (Tyr 705) without affecting total STAT3 or PIAS3 expression. β-Actin is a loading control. B, H2052 and 211H cells exposed to 0.5 or 2.0 μmol/L rPP-C8 peptide for 24 hours results in PARP cleavage at the higher concentration. +C is a positive control lysate. C, addition of the peptide to mesothelioma cells induces apoptosis (MTS assay) and cell growth inhibition (D).

Close modal

Our study addressed the question of whether the PIAS3 protein can be exploited as a potential drug target. We demonstrated elevated levels of STAT3 activation in mesothelioma and that mesothelioma may be “addicted” to STAT3 activity using siRNA. A recent report also showed that the active forms of STAT3 are highly expressed in most cases of mesothelioma (25). As the PIAS3 protein is being downregulated by cancer cells, a therapeutic strategy could be based upon the enhancement of PIAS3 expression because of its role in inhibiting activated STAT3 and thereby inducing apoptosis. The number of cases of mesothelioma is increasing and current chemotherapy, radiotherapy, and surgery treatments are mainly palliative (26). Clinical studies using molecular-targeted approaches have been promising for NSCLC, like EGFR inhibition and angiogenic blockade, but have failed to improve patient outcome in patients with mesothelioma (27, 28). Thus, new therapeutic strategies targeting PIAS3 could become valuable for cancers with persistent STAT3 activity, including mesotheliomas.

Many current cancer therapies target cell signal initiation at the level of membrane receptors, such as VEGFR and EGFR. However, multiple receptor tyrosine kinases are often activated in cancer, including mesothelioma (29, 30), making kinase-specific inhibitors less effective or subject to the development of resistance. For example, two recent phase II studies have shown that EGFR is highly expressed in 75% to 97% of mesothelioma tumor samples yet the disease was refractive to the EGFR tyrosine kinase inhibitors gefitinib (31) or erlotinib (32). These results indicate that downstream effectors of tyrosine kinase receptor signaling such as STATs may represent a more attractive choice for drug targeting because they represent an integration point for multiple oncogenic signaling pathways. Unfortunately, specific and effective STAT3 inhibitors are still only in early development, although promising lead compounds have recently been reported (33).

Here, we report for the first time the clinical significance of PIAS3 expression on overall patient survival. In this regard, our study has provided preliminary data that PIAS3 expression may be useful as a prognostic factor in mesothelioma. Previous reports have used immunohistochemistry to suggest an important role for PIAS3 expression in subsets of gastrointestinal carcinomas, glioblastoma cell tumors, and colorectal carcinoma (13, 34, 35). This further emphasizes the potential role of PIAS3-targeted therapy in malignancies beyond mesothelioma.

Curcumin is regarded as the most active constituent derived from the spice turmeric (22). Turmeric has a long history in ancient Asian medicine and curcumin has been extensively studied in modern science as an anticancer agent both in vitro and in vivo. Here, we show that curcumin treatment can increase PIAS3 levels and thereby decrease STAT3 phosphorylation and cell viability in mesothelioma cells. In support of our findings, curcumin has been shown to increase PIAS3 levels and decrease STAT3 phosphorylation and cell viability in ovarian and endometrial cancer cells in vitro (24). These results suggest that curcumin may represent a lead compound to explore the potential of increasing endogenous PIAS3 levels as a therapeutic strategy against STAT3-dependent cancers. Although limited bioavailability of curcumin has hindered its clinical use (23), recent efforts to produce more pathway-specific analogs of curcumin, coupled with methods to increase its bioavailability, make this avenue more attractive for the treatment of mesothelioma (36, 37).

STAT3 inhibition can also be affected by peptides (5, 17, 38). Our study indicates that the uptake of a STAT3-inhibiting peptide into mesothelioma cells is effective and induces cell growth inhibition and apoptosis. There is an increase in the number of peptide drugs in preclinical and clinical development. Peptide degradation by proteolysis can be prevented by chemical modifications such as incorporation of d-amino acids or cyclization (39). Because of the tremendous advancement in the large-scale synthesis of peptides it will be possible to make peptide-based anticancer drugs more affordable to patients (40). Thus, peptides are poised to make a huge impact in the near future in the area of cancer treatment and diagnosis.

In conclusion, here we present PIAS3 as a potential prognostic marker that would be beneficial in the management of mesothelioma due to the variable biologic behavior of these tumors despite similar tumor stage, grade, and clinical presentations. Moreover, we present evidence that downregulation of STAT3 or induction of PIAS3 protein expression by curcumin or using a recombinant PIAS3 peptide in vitro inhibit cell growth and induce apoptosis. Because the three-dimensional crystal structure of PIAS3 has recently been solved (http://www.ebi.ac.uk/pdbe-srv/view/entry/4mvt/summary.html), this should hasten work on developing effective PIAS3-directed therapeutics using small-molecule drug screens or finding structural analogs of the PIAS3 protein.

No potential conflicts of interest were disclosed.

Conception and design: S. Dabir, G. Wildey, A. Dowlati

Development of methodology: S. Dabir, A. Dowlati

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Dabir, A. Kluge, A.M. Kresak, M. Yang, B. Groner, A. Dowlati

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Dabir, P. Fu, G. Wildey, A. Dowlati

Writing, review, and/or revision of the manuscript: S. Dabir, A.M. Kresak, B. Groner, G. Wildey, A. Dowlati

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Dabir, A.M. Kresak, G. Wildey, A. Dowlati

Study supervision: S. Dabir, G. Wildey, A. Dowlati

The authors are thankful to the Mesothelioma Research Bank [CDC NIOSH 1-U19-OH009077-01 National Mesothelioma Virtual Bank for Translational Research] for providing them with the tissue microarrays of mesothelioma.

The work at the GSH was supported by the LOEWE program “Oncogenic Signaling” of the State of Hessen. The Georg-Speyer-Haus is funded jointly by the German Federal Ministry of Health (BMG) and the Ministry of Higher Education, Research and the Arts of the State of Hessen (HMWK).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Groner
B
. 
Molecular and cellular endocrinology: special issue on cytokine signaling
.
Mol Cell Endocrinol
2014
;
382
:
551
.
2.
Groner
B
. 
Determinants of the extent and duration of STAT3 signaling
.
JAKSTAT
2012
;
1
:
211
5
.
3.
Turkson
J
,
Jove
R
. 
STAT proteins: novel molecular targets for cancer drug discovery
.
Oncogene
2000
;
19
:
6613
26
.
4.
Bromberg
J
. 
Stat proteins and oncogenesis
.
J Clin Invest
2002
;
109
:
1139
42
.
5.
Borghouts
C
,
Delis
N
,
Brill
B
,
Weiss
A
,
Mack
L
,
Lucks
P
, et al
A membrane penetrating peptide aptamer inhibits STAT3 function and suppresses the growth of STAT3 addicted tumor cells
.
JAKSTAT
2012
;
1
:
44
54
.
6.
Turkson
J
. 
STAT proteins as novel targets for cancer drug discovery
.
Expert Opin Ther Targets
2004
;
8
:
409
22
.
7.
Sharrocks
AD
. 
PIAS proteins and transcriptional regulation-more than just SUMO E3 ligases?
Genes Dev
2006
;
20
:
754
8
.
8.
Shuai
K
. 
Regulation of cytokine signaling pathways by PIAS proteins
.
Cell Res
2006
;
16
:
196
202
.
9.
Chung
CD
,
Liao
J
,
Liu
B
,
Rao
X
,
Jay
P
,
Berta
P
, et al
Specific inhibition of Stat3 signal transduction by PIAS3
.
Science
1997
;
278
:
1803
5
.
10.
Dabir
S
,
Kluge
A
,
Dowlati
A
. 
The association and nuclear translocation of the PIAS3-STAT3 complex is ligand and time dependent
.
Mol Cancer Res
2009
;
7
:
1854
60
.
11.
Kluge
A
,
Dabir
S
,
Kern
J
,
Nethery
D
,
Halmos
B
,
Ma
P
, et al
Cooperative interaction between protein inhibitor of activated signal transducer and activator of transcription-3 with epidermal growth factor receptor blockade in lung cancer
.
Int J Cancer
2009
;
125
:
1728
34
.
12.
Ogata
Y
,
Osaki
T
,
Naka
T
,
Iwahori
K
,
Furukawa
M
,
Nagatomo
I
, et al
Overexpression of PIAS3 suppresses cell growth and restores the drug sensitivity of human lung cancer cells in association with PI3-K/Akt inactivation
.
Neoplasia
2006
;
8
:
817
25
.
13.
Brantley
EC
,
Nabors
LB
,
Gillespie
GY
,
Choi
YH
,
Palmer
CA
,
Harrison
K
, et al
Loss of protein inhibitors of activated STAT-3 expression in glioblastoma multiforme tumors: implications for STAT-3 activation and gene expression
.
Clin Cancer Res
2008
;
14
:
4694
704
.
14.
Kluge
A
,
Dabir
S
,
Vlassenbroeck
I
,
Eisenberg
R
,
Dowlati
A
. 
Protein inhibitor of activated STAT3 expression in lung cancer
.
Mol Oncol
2011
;
5
:
256
64
.
15.
Dabir
S
,
Kluge
A
,
Aziz
MA
,
Houghton
JA
,
Dowlati
A
. 
Identification of STAT3-independent regulatory effects for protein inhibitor of activated STAT3 by binding to novel transcription factors
.
Cancer Biol Ther
2011
;
12
:
139
51
.
16.
Unkila
M
,
McColl
KS
,
Thomenius
MJ
,
Heiskanen
K
,
Distelhorst
CW
. 
Unreliability of the cytochrome c-enhanced green fluorescent fusion protein as a marker of cytochrome c release in cells that overexpress Bcl-2
.
J Biol Chem
2001
;
276
:
39132
7
.
17.
Borghouts
C
,
Tittmann
H
,
Delis
N
,
Kirchenbauer
M
,
Brill
B
,
Groner
B
. 
The intracellular delivery of a recombinant peptide derived from the acidic domain of PIAS3 inhibits STAT3 transactivation and induces tumor cell death
.
Mol Cancer Res
2010
;
8
:
539
53
.
18.
Cox
DR
. 
Regression Models and Life-Tables
.
J R Stat Soc Ser B-Stat Methodol
1972
;
34
:
187
220
.
19.
Barretina
J
,
Caponigro
G
,
Stransky
N
,
Venkatesan
K
,
Margolin
AA
,
Kim
S
, et al
The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity
.
Nature
2012
;
483
:
603
7
.
20.
Gordon
GJ
,
Jensen
RV
,
Hsiao
LL
,
Gullans
SR
,
Blumenstock
JE
,
Ramaswamy
S
, et al
Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma
.
Cancer Res
2002
;
62
:
4963
7
.
21.
Kaplan
EL
,
Meier
P
. 
Nonparametric Estimation from Incomplete Observations
.
J Am Stat Assoc
1958
;
53
:
457
81
.
22.
Sharma
RA
,
Gescher
AJ
,
Steward
WP
. 
Curcumin: the story so far
.
Eur J Cancer
2005
;
41
:
1955
68
.
23.
Kunnumakkara
AB
,
Anand
P
,
Aggarwal
BB
. 
Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins
.
Cancer Lett
2008
;
269
:
199
225
.
24.
Saydmohammed
M
,
Joseph
D
,
Syed
V
. 
Curcumin suppresses constitutive activation of STAT-3 by up-regulating protein inhibitor of activated STAT-3 (PIAS-3) in ovarian and endometrial cancer cells
.
J Cell Biochem
2010
;
110
:
447
56
.
25.
Achcar Rde
O
,
Cagle
PT
,
Jagirdar
J
. 
Expression of activated and latent signal transducer and activator of transcription 3 in 303 non-small cell lung carcinomas and 44 malignant mesotheliomas: possible role for chemotherapeutic intervention
.
Arch Pathol Lab Med
2007
;
131
:
1350
60
.
26.
Robinson
BW
,
Lake
RA
. 
Advances in malignant mesothelioma
.
N Engl J Med
2005
;
353
:
1591
603
.
27.
Jakobsen
JN
,
Sorensen
JB
. 
Review on clinical trials of targeted treatments in malignant mesothelioma
.
Cancer Chemother Pharmacol
2011
;
68
:
1
15
.
28.
Pasello
G
,
Favaretto
A
. 
Molecular targets in malignant pleural mesothelioma treatment
.
Curr Drug Targets
2009
;
10
:
1235
44
.
29.
Menges
CW
,
Chen
Y
,
Mossman
BT
,
Chernoff
J
,
Yeung
AT
,
Testa
JR
. 
A phosphotyrosine proteomic screen identifies multiple tyrosine kinase signaling pathways aberrantly activated in malignant mesothelioma
.
Genes Cancer
2010
;
1
:
493
505
.
30.
Ou
WB
,
Hubert
C
,
Corson
JM
,
Bueno
R
,
Flynn
DL
,
Sugarbaker
DJ
, et al
Targeted inhibition of multiple receptor tyrosine kinases in mesothelioma
.
Neoplasia
2011
;
13
:
12
22
.
31.
Govindan
R
,
Kratzke
RA
,
Herndon
JE
 II
,
Niehans
GA
,
Vollmer
R
,
Watson
D
, et al
Gefitinib in patients with malignant mesothelioma: a phase II study by the Cancer and Leukemia Group B
.
Clin Cancer Res
2005
;
11
:
2300
4
.
32.
Garland
LL
,
Rankin
C
,
Gandara
DR
,
Rivkin
SE
,
Scott
KM
,
Nagle
RB
, et al
Phase II study of erlotinib in patients with malignant pleural mesothelioma: a Southwest Oncology Group Study
.
J Clin Oncol
2007
;
25
:
2406
13
.
33.
Zhang
X
,
Yue
P
,
Page
BDG
,
Li
T
,
Zhao
W
,
Namanja
AT
, et al
Orally bioavailable small-molecule inhibitor of transcription factor Stat3 regresses human breast and lung cancer xenografts
.
Proc Natl Acad Sci U S A
2012
;
109
:
9623
8
.
34.
Li
H
,
Gao
H
,
Bijukchhe
SM
,
Wang
Y
,
Li
T
. 
PIAS3 may represent a potential biomarker for diagnosis and therapeutic of human colorectal cancer
.
Med Hypotheses
2013
;
81
:
1151
4
.
35.
Liu
LM
,
Yan
MG
,
Yang
DH
,
Sun
WW
,
Zhang
JX
. 
The expression of protein inhibitor of activated signal transducers and activators of transcription 3 in the evolutionary process of gastric cancer
.
Eur J Intern Med
2011
;
22
:
e31
5
.
36.
Anand
P
,
Kunnumakkara
AB
,
Newman
RA
,
Aggarwal
BB
. 
Bioavailability of curcumin: problems and promises
.
Mol Pharm
2007
;
4
:
807
18
.
37.
Rocks
N
,
Bekaert
S
,
Coia
I
,
Paulissen
G
,
Gueders
M
,
Evrard
B
, et al
Curcumin-cyclodextrin complexes potentiate gemcitabine effects in an orthotopic mouse model of lung cancer
.
Br J Cancer
2012
;
107
:
1083
92
.
38.
Borghouts
C
,
Kunz
C
,
Delis
N
,
Groner
B
. 
Monomeric recombinant peptide aptamers are required for efficient intracellular uptake and target inhibition
.
Mol Cancer Res
2008
;
6
:
267
81
.
39.
Li
L
,
Zhao
YL
,
Wang
H
,
Li
YJ
,
Xu
X
,
Liu
Q
. 
Base-catalyzed bicyclization of dialkyl glutaconates with cinnamoylacetamides: a synthetic strategy for isoquinolinedione derivatives
.
Chem Commun
2014
;
50
:
6458
60
.
40.
Groner
B
,
Weiss
A
. 
Targeting survivin in cancer: novel drug development approaches
.
Biodrugs
2014
;
28
:
27
39
.