Snehal Dabir

The Association and Nuclear Translocation of the PIAS3-STAT3 Complex Is Ligand and Time Dependent

The epidermal growth factor (EGF) receptor activation of downstream signal transducers and activators of transcription 3 (STAT3) plays a crucial role in the pathogenesis of lung cancer. STAT3 transcriptional activity can be negatively regulated by protein inhibitor of activated STAT3 (PIAS3). We investigated the time-dependent PIAS3 shuffling and binding to STAT3 in an EGF-dependent model in lung cancer by using confocal microscopy, immunoprecipitation, luciferase reporter assay, and protein analysis of segregated cellular components. We also explored the role of phosphorylation at Tyr705 of STAT3 in the formation and intracellular shuffling of the PIAS3-STAT3 complex. In a growth factor–free state, PIAS3 was localized to the cytoplasm and unbound to STAT3 in both H520 and A549 cells. On exposure to EGF, we observed STAT3 phosphorylation and rapid formation of the PIAS3-STAT3 complex. Within 5 minutes, there was a progressive translocation of the complex to the nucleus, and by 10 minutes, PIAS3 was uniquely localized to the nuclear compartment. After 30 minutes, PIAS3 returned to the cytoplasm. Using site-directed mutagenesis, we substituted Tyr705 of STAT3 with a phenylalanine. Despite EGF stimulation, we observed a significant decrease in PIAS3-STAT3 binding and a significant reduction in nuclear translocation of PIAS3. Furthermore, there was a significant reduction in the capacity of PIAS3 to reduce STAT3-mediated gene transcription. In wild-type STAT3 cells, increasing concentrations of PIAS3 resulted in a proportional decrease in STAT3 phosphorylation. These data suggest an important role for the negative regulatory effect of PIAS3 on STAT3 in EGF-driven tumors. (Mol Cancer Res 2009;7(11):1854–60)

Low PIAS3 Expression in Malignant Mesothelioma Is Associated with Increased STAT3 Activation and Poor Patient Survival

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.

Protein inhibitor of activated STAT3 expression in lung cancer

Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is an endogenous inhibitor of STAT3 transcriptional activity. We have previously demonstrated the concentration‐dependent negative regulatory effect of PIAS3 on STAT3 signaling and its capacity to decrease lung cancer proliferation and synergize with epidermal growth factor inhibition. We now investigate PIAS3 expression in both non‐small cell lung cancer (NSCLC) cell lines and human resected NSCLC specimens. We also investigated the mechanism by which some lung cancers have significantly decreased PIAS3 expression. Expression of PIAS3 is variable in lung cancer cells lines with 2 of 3 squamous cell carcinoma (SCC) cell lines having no or little PIAS3 protein expression. Similarly, the majority of human SCCs of the lung lack PIAS3 expression by immunohistochemistry; this despite the finding that SCCs have significantly higher levels of PIAS3 mRNA compared to adenocarcinomas. High PIAS3 expression generally correlates with decreased phosphorylated STAT3 in both SCC cell lines and human specimens compatible with the negative regulatory effect of this protein on STAT3 signaling. To investigate this variable expression of PIAS3 we first performed sequencing of the PIAS3 gene that demonstrated single nucleotide polymorphisms but no mutations. Exposure of lung cancer cells to 5‐azacytidine and trichostatin A results in a significant increase in PIAS3 mRNA and protein expression. However, methylation‐specific PCR demonstrates a lack of CpG island methylation in the promoter region of PIAS3. Exposure of cells to an agent blocking proteosomal degradation results in a significant increase in PIAS3. Our data thus shows that SCC of the lung commonly lacks PIAS3 protein expression and that post‐translational modifications may explain this finding in some cases. PIAS3 is a potential therapeutic molecule to target STAT3 pathway in lung cancer.

Cooperative interaction between protein inhibitor of activated signal transducer and activator of transcription‐3 with epidermal growth factor receptor blockade in lung cancer

Epidermal Growth Factor Receptor (EGFR) targeting in nonsmall cell lung cancer (NSCLC) is an established treatment modality; however, it only benefits a minority of patients. STAT3 (signal transducer and activator of transcription‐3) plays an important role in the oncogenic signal transduction pathway of NSCLC. Inhibition of STAT3 results in NSCLC growth inhibition and apoptosis. We have previously shown that combined inhibition of EGFR and STAT3 by small molecules resulted in improved therapeutic efficacy as compared with blocking EGFR alone. However, the STAT3 protein has a number of endogenous negative regulators including PIAS3 (Protein Inhibitor of Activated STAT3). In this study, we investigated for the first time the role of PIAS3 in modulating oncogenic EGFR‐STAT3 signaling pathway in lung cancer and the anti‐proliferative effect of using PIAS3 in conjunction with EGFR blockade in NSCLC. We demonstrate that PIAS3 is expressed in variable degrees in all NSCLC cells. EGF and IL‐6 stimulation resulted in the association of PIAS3 with STAT3. The PIAS3/STAT3 complex then bound the STAT3 DNA binding sequence resulting in STAT3 regulated gene expression. Over‐expression of PIAS3, using a PIAS3 expression construct, decreases STAT3 transcriptional activity. Furthermore, over‐expression of PIAS3 consistently decreased proliferation. EGFR blockade and PIAS3 over‐expression in combination had significantly greater anti‐proliferative effects as compared with either EGFR blockade or PIAS3 over‐expression alone. In conclusion, PIAS3 is expressed in NSCLC cell lines and its over‐expression decreased STAT3 transcriptional activity, decreased proliferation of NSCLC cells and when used in conjunction with EGFR inhibitors, increased the anti‐proliferative effects.

RET Mutation and Expression in Small-Cell Lung Cancer

Background: There is growing interest in defining the somatic mutations associated with small-cell lung cancer (SCLC). Unfortunately, a serious blockade to genomic analyses of this disease is a limited access to tumors because surgery is rarely performed. We used our clinical/pathologic database of SCLC patients to determine the availability of biopsy specimens that could be used for genomic studies and to identify tumors for initial oncogene analysis. Methods: DNA was extracted from six tumors, three primary and three metastatic, and analyzed by SEQUENOM platform technology. Results: Primary-resected tumor tissue represents less than 3% of all diagnostic specimens in this disease, highlighting the limited access to tissue sufficient for comprehensive genomic analyses. We identified an activating M918T RET somatic mutation in a metastatic SCLC tumor specimen. Bioinformatic search identified RET mutations in other SCLC studies. Stable overexpression of both mutant M918T and wild-type RET in two SCLC cell lines, H1048 and SW1271, activated ERK signaling, MYC expression, and increased cell proliferation, particularly by mutant RET. Stable cells became sensitized to the RET tyrosine kinase inhibitors, vandetanib and ponatinib. Further analysis of RET mRNA expression in SCLC revealed wide variability in both cells and tumors, and SCLC cells demonstrated significantly higher RET expression compared with adenocarcinoma lung cells. Conclusions: Our data suggest that a subpopulation of SCLC patients may derive benefit from tyrosine kinase inhibitors targeting RET. Coupled with the presence of RET fusion proteins in non-small-cell lung cancer, our data indicate an emerging role for RET in SCLC.

PIAS3 activates the intrinsic apoptotic pathway in non-small cell lung cancer cells independent of p53 status

Protein inhibitor of activated signal transducer and activator of transcription 3 (STAT3) (PIAS3) is an endogenous inhibitor of STAT3 that negatively regulates STAT3 transcriptional activity and cell growth and demonstrates limited expression in the majority of human squamous cell carcinomas of the lung. In this study, we sought to determine whether PIAS3 inhibits cell growth in non‐small cell lung cancer cell lines by inducing apoptosis. Our results demonstrate that overexpression of PIAS3 promotes mitochondrial depolarization, leading to cytochrome c release, caspase 9 and 3 activation and poly (ADP‐ribose) polymerase cleavage. This intrinsic pathway activation was associated with decreased Bcl‐xL expression and increased Noxa expression and was independent of p53 status. Furthermore, PIAS3 inhibition of STAT3 activity was also p53 independent. Microarray experiments were performed to discover STAT3‐independent mediators of PIAS3‐induced apoptosis by comparing the apoptotic gene expression signature induced by PIAS3 overexpression with that induced by STAT3 siRNA. The results showed that a subset of apoptotic genes was uniquely expressed only after PIAS3 expression. Thus, PIAS3 may represent a promising lung cancer therapeutic target because of its p53‐independent efficacy and its potential to synergize with Bcl‐2 targeted inhibitors.

CD30 Is a Potential Therapeutic Target in Malignant Mesothelioma

CD30 is a cytokine receptor belonging to the TNF superfamily (TNFRSF8) that acts as a regulator of apoptosis. The presence of CD30 antigen is important in the diagnosis of Hodgkin disease and anaplastic large cell lymphoma. There have been sporadic reports of CD30 expression in nonlymphoid tumors, including malignant mesothelioma. Given the remarkable success of brentuximab vedotin, an antibody–drug conjugate directed against CD30 antigen, in lymphoid malignancies, we undertook a study to examine the incidence of CD30 in mesothelioma and to investigate the ability to target CD30 antigen in mesothelioma. Mesothelioma tumor specimens (N = 83) were examined for CD30 expression by IHC. Positive CD30 expression was noted in 13 mesothelioma specimens, primarily those of epithelial histology. There was no significant correlation of CD30 positivity with tumor grade, stage, or survival. Examination of four mesothelioma cell lines (H28, H2052, H2452, and 211H) for CD30 expression by both FACS analysis and confocal microscopy showed that CD30 antigen localized to the cell membrane. Brentuximab vedotin treatment of cultured mesothelioma cells produced a dose-dependent decrease in cell growth and viability at clinically relevant concentrations. Our studies validate the presence of CD30 antigen in a subgroup of epithelial-type mesothelioma tumors and indicate that selected mesothelioma patients may derive benefit from brentuximab vedotin treatment. Mol Cancer Ther; 14(3); 740–6. ©2015 AACR.

Identification of STAT3-independent regulatory effects for protein inhibitor of activated STAT3 by binding to novel transcription factors

Protein Inhibitor of Activated Signal Transducer and Activators of Transcription 3 (PIAS3) is a molecule that regulates STAT3 and has antiproliferative properties. Glioblastoma and squamous cell lung cancer lack PIAS3 expression. To test the hypothesis that PIAS3 transcriptional effects are STAT3-independent, we developed models for STAT3 knockdown and PIAS3 over-expression. PIAS3 expression results in a distinct transcriptional profile that does not occur with STAT3 knockdown. We identify novel transcription factor binding partners for PIAS3 including ETS, EGR1, NR1I2, and GATA1. PIAS3 binds to these factors and regulates their transcriptional effects resulting in alterations in canonical pathways including Wnt/β-catenin signaling and functions such as cell death and proliferation. A model is proposed by which PIAS3 effects EGR1 regulated pathways.

Abstract 847: PIAS3 induction of apoptosis is p53-independent and has STAT3-independent mediators.

Protein inhibitor of activated STAT3 (PIAS3) is an endogenous inhibitor of STAT3 that negatively regulates STAT3 transcriptional activity and cell growth and demonstrates limited expression in the majority of human squamous cell carcinomas of the lung. In the present study we sought to determine if PIAS3 inhibits cell growth in non-small cell lung cancer (NSCLC) cell lines by induction of apoptosis and further determine the dependence of PIAS3 activity on p53 status by using both wild-type and p53-null cells. Our results demonstrate that over-expression of PIAS3 promotes caspase 3 activation and PARP cleavage. Furthermore, the expression of pro-survival family members BclXL and Bcl-2 is decreased. These effects were observed after both transient and regulated expression of exogenous PIAS3 and were independent of p53 status. PIAS3 inhibition of STAT3 luciferase activity was also p53 independent. Microarray experiments were performed to further investigate the STAT3-dependence of PIAS3-induced apoptosis by comparing its gene expression signature with that of STAT3 siRNA treated cells. IPA pathway analysis showed that a number of genes related to cell death were significantly increased by PIAS3. Furthermore, a subset of apoptotic genes, including CIDEC and DAPK2, were uniquely expressed only after PIAS3 expression and not after STAT3 siRNA. Thus, PIAS3 may represent a promising lung cancer therapeutic target because of its p53-independent efficacy as well as its potential to synergize with direct STAT3 inhibitors. Citation Format: Snehal Dabir, Yu Liu, Gary Wildey, Afshin Dowlati. PIAS3 induction of apoptosis is p53-independent and has STAT3-independent mediators. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 847. doi:10.1158/1538-7445.AM2013-847

Abstract 2249: Induction of PIAS3 by curcumin as a novel treatment strategy in mesothelioma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Curcumin, a natural drug derived from the spice turmeric, has been extensively studied as an anti-cancer agent. The mechanism of action behind this promising bioactivity of curcumin, previously unclear, is now beginning to emerge. Curcumin has been shown to inhibit STAT3 phosphorylation and activity in melanoma cells, ovarian and endometrial cancer cells and small-cell lung cancer cells. In many cancers constitutively activated STAT3 (Signal Transducers and Activators of Transcription 3) is essential for cell proliferation. In this study we demonstrate that four mesothelioma cell lines demonstrate high levels of active STAT3. The addition of curcumin can suppress mesothelioma cell growth and proliferation, demonstrating the STAT3-dependence of these cells. Interestingly, the inhibitory effect of curcumin on STAT3 appears to be mediated through an upregulation of an endogenous inhibitor of STAT3 called PIAS3 (protein inhibitor of activated STAT3). Thus, the significance of our results is that curcumin not only targets an effector of mesothelioma cell proliferation, STAT3, but does this by increasing the expression of its endogenous inhibitor, PIAS3. This result prompted ongoing studies to determine if PIAS3 levels are deficient in mesothelioma tumor specimens obtained from the National Virtual Mesothelioma bank. Taken together, our data support the idea that mesothelioma is a STAT3-driven cancer caused by a lack of PIAS3 expression and that strategies to increase endogenous PIAS3 expression will have tumor suppressor effects. The ability of curcumin to increase PIAS3 expression will give us a jump start on pursuing this strategy because it represents a bioactive lead compound with many available structural analogs. Citation Format: Snehal Dabir, Gary Wildey, Afshin Dowlati. Induction of PIAS3 by curcumin as a novel treatment strategy in mesothelioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2249. doi:10.1158/1538-7445.AM2013-2249