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.

Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays.

BACKGROUND: The EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] gene is known to harbor genomic alterations in advanced lung cancer involving gene amplification and kinase mutations that predict the clinical response to EGFR-targeted inhibitors. Methods for detecting such molecular changes in lung cancer tumors are desirable. METHODS: We used a nanofluidic digital PCR array platform and 16 cell lines and 20 samples of genomic DNA from resected tumors (stages I-III) to quantify the relative numbers of copies of the EGFR gene and to detect mutated EGFR alleles in lung cancer. We assessed the relative number of EGFR gene copies by calculating the ratio of the number of EGFR molecules (measured with a 6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit) (measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each panel. To assay for the EGFR L858R (exon 21) mutation and exon 19 in-frame deletions, we used the ARMS and Scorpion technologies in a DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array. RESULTS: The digital array detected and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations (0.02%-9.26% abundance) that were present in formalin-fixed, paraffin-embedded samples of early-stage resectable lung tumors without an associated increase in gene copy number. Our results also demonstrated the presence of intratumor molecular heterogeneity for the clinically relevant EGFR mutated alleles in these early-stage lung tumors. CONCLUSIONS: The digital PCR array platform allows characterization and quantification of oncogenes, such as EGFR, at the single-molecule level. Use of this nanofluidics platform may provide deeper insight into the specific roles of clinically relevant kinase mutations during different stages of lung tumor progression and may be useful in predicting the clinical response to EGFR-targeted inhibitors.

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.

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.

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.

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.

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/ß-catenin signaling and functions such as cell death and proliferation. A model is proposed by which PIAS3 effects EGR1 regulated pathways.

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.

SCH66336, inhibitor of protein farnesylation, blocks signal transducer and activators of transcription 3 signaling in lung cancer and interacts with a small molecule inhibitor of epidermal growth factor receptor/human epidermal growth factor receptor 2.

Signal transducer and activators of transcription 3 (STAT3) is an important transcription factor that is essential for lung cancer cell survival. STAT3 is activated by diverse upstream receptor and nonreceptor tyrosine kinases, and blockade of STAT3 results in tumor growth inhibition. Therefore, a search for STAT3 inhibitors is under way. We demonstrate that SCH66336, at 4 mumol/l, completely blocks STAT3 phosphorlyation in a variety of nonsmall cell lung carcinoma (NSCLC) cell lines, whereas the effect on AKT and extracellular signal-regulated kinase activation is variable. Furthermore, SCH66336 has antiproliferative effects on NSCLC cells. When NSCLC cells are exposed sequentially to SCH66336 and a small molecule dual tyrosine kinase inhibitor of epidermal growth factor receptor and human epidermal growth factor receptor 2, synergistic activity is observed with an increase in the fraction of cells undergoing apoptosis. Concurrent exposure to both agents is, however, associated with antagonism and decreased apoptosis. We conclude that blockade of STAT3 phosphorylation might be one of the mechanisms by which SCH66336 exerts its antitumor activity, and that this can be synergistic in vitro when administered sequentially with epidermal growth factor receptor inhibitors.

Speech sound disorder influenced by a locus in 15q14 region.

Despite a growing body of evidence indicating that speech sound disorder (SSD) has an underlying genetic etiology, researchers have not yet identified specific genes predisposing to this condition. The speech and language deficits associated with SSD are shared with several other disorders, including dyslexia, autism, Prader-Willi Syndrome (PWS), and Angelman's Syndrome (AS), raising the possibility of gene sharing. Furthermore, we previously demonstrated that dyslexia and SSD share genetic susceptibility loci. The present study assesses the hypothesis that SSD also shares susceptibility loci with autism and PWS. To test this hypothesis, we examined linkage between SSD phenotypes and microsatellite markers on the chromosome 15q14-21 region, which has been associated with autism, PWS/AS, and dyslexia. Using SSD as the phenotype, we replicated linkage to the 15q14 region (P=0.004). Further modeling revealed that this locus influenced oral-motor function, articulation and phonological memory, and that linkage at D15S118 was potentially influenced by a parent-of-origin effect (LOD score increase from 0.97 to 2.17, P=0.0633). These results suggest shared genetic determinants in this chromosomal region for SSD, autism, and PWS/AS.

Pleiotropic effects of a chromosome 3 locus on speech-sound disorder and reading.

Speech-sound disorder (SSD) is a complex behavioral disorder characterized by speech-sound production errors associated with deficits in articulation, phonological processes, and cognitive linguistic processes. SSD is prevalent in childhood and is comorbid with disorders of language, spelling, and reading disability, or dyslexia. Previous research suggests that developmental problems in domains associated with speech and language acquisition place a child at risk for dyslexia. Recent genetic studies have identified several candidate regions for dyslexia, including one on chromosome 3 segregating in a large Finnish pedigree. To explore common genetic influences on SSD and reading, we examined linkage for several quantitative traits to markers in the pericentrometric region of chromosome 3 in 77 families ascertained through a child with SSD. The quantitative scores measured several processes underlying speech-sound production, including phonological memory, phonological representation, articulation, receptive and expressive vocabulary, and reading decoding and comprehension skills. Model-free linkage analysis was followed by identification of sib pairs with linkage and construction of core shared haplotypes. In our multipoint analyses, measures of phonological memory demonstrated the strongest linkage (marker D3S2465, P=5.6 x 10(-5), and marker D3S3716, P=6.8 x 10(-4)). Tests for single-word decoding also demonstrated linkage (real word reading: marker D3S2465, P=.004; nonsense word reading: marker D3S1595, P=.005). The minimum shared haplotype in sib pairs with similar trait values spans 4.9 cM and is bounded by markers D3S3049 and D3S3045. Our results suggest that domains common to SSD and dyslexia are pleiotropically influenced by a putative quantitative trait locus on chromosome 3.