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Purpose: The purpose of this study was to characterize insulin-like growth factor-1 receptor (IGF1R) protein expression, mRNA expression, and gene copy number in surgically resected non-small-cell lung cancers (NSCLC) in relation to epidermal growth factor receptor (EGFR) protein expression, patient characteristics, and prognosis., Patients and Methods: One hundred eighty-nine patients with NSCLC who underwent curative pulmonary resection were studied (median follow-up, 5.3 years). IGF1R protein expression was evaluated by immunohistochemistry (IHC) with two anti-IGF1R antibodies (n = 179). EGFR protein expression was assessed with PharmDx kit. IGF1R gene expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 114 corresponding fresh-frozen samples. IGF1R gene copy number was assessed by fluorescent in situ hybridization using customized probes (n = 181)., Results: IGF1R IHC score was higher in squamous cell carcinomas versus other histologies (P < .001) and associated with stage (P = .03) but not survival (P = .46). IGF1R and EGFR protein expression showed significant correlation (r = 0.30; P < .001). IGF1R gene expression by qRT-PCR was higher in squamous cell versus other histologies (P = .006) and did not associate with other clinical features nor survival (P = .73). Employing criteria previously established for EGFR copy number, patients with IGF1R amplification/high polysomy (n = 48; 27%) had 3-year survival of 58%, patients with low polysomy (n = 87; 48%) had 3-year survival of 47% and patients with trisomy/disomy (n = 46; 25%) had 3-year survival of 35%, respectively (P = .024). Prognostic value of high IGF1R gene copy number was confirmed in multivariate analysis., Conclusion: IGF1R protein expression is higher in squamous cell versus other histologies and correlates with EGFR expression. IGF1R protein and gene expression does not associate with survival, whereas high IGF1R gene copy number harbors positive prognostic value.

Background: Epidermal growth factor receptor (EGFR) inhibitors are effective in a subset of patients with non-small-cell lung cancer (NSCLC). We previously showed that E-cadherin expression associates with gefitinib activity. Here, we correlated the expressions of ErbB-3 and E-cadherin in NSCLC tumors and cell lines, their effect on response to gefitinib, and induction of both by the histone deacetylase (HDAC) inhibitors vorinostat and SNDX-275., Methods: Real-time RT-PCR was carried out on RNA isolated from 91 fresh-frozen NSCLC samples and from 21 NSCLC lines. Protein expression was evaluated with western blot and flow cytometry. Apoptosis was assessed using vibrant apoptosis assay., Results: Expressions of E-cadherin and ErbB-3 correlated significantly in primary tumors (r = 0.38, P < 0.001) and in cell lines (r = 0.88, P < 0.001). Cotransfection of ErbB-3 and E-cadherin in a gefitinib-resistant cell line showed enhanced apoptotic response to gefitinib. vorinostat and SNDX-275 induced ErbB-3 and E-cadherin in gefitinib-resistant cell lines. When gefitinib-resistant lines were treated with vorinostat and gefitinib, synergistic effects were detected in four of the five lines tested., Conclusion: ErbB-3 and E-cadherin are coexpressed and induced by HDAC inhibitors. For tumors with low ErbB-3 and E-cadherin expressions, the combination of HDAC and EGFR-tyrosine kinase inhibitors increased expression of both genes and produced more than additive apoptotic effect.

Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance.

Purpose: Epidermal growth factor receptor (EGFR) mRNA expression and EGFR gene dosage by quantitative PCR in tumor samples obtained from patients with gefitinib-treated non-small cell lung cancer were analyzed in order to determine the association with treatment outcome, clinical, and biological features [EGFR copy number by fluorescent in situ hybridization (FISH), EGFR tyrosine kinase mutations, and EGFR protein expression]., Experimental Design: EGFR mRNA expression was measured by real-time quantitative reverse transcription-PCR in 64 patients, and EGFR gene dosage was analyzed by real-time quantitative PCR in 82 patients from paraffin-embedded specimens., Results: EGFR mRNA expression was higher in responders to gefitinib as compared with nonresponders (P = 0.012). Patients with high EGFR mRNA expression (>5.01) had 43% response probability, whereas patients with low EGFR mRNA expression had 8% response probability (P = 0.006). Patients with high EGFR mRNA expression had longer median progression-free (5.3 versus 2.8 months, P = 0.028) but not overall survival (13.8 versus 10.9 months, P = 0.87). EGFR mRNA expression was higher in FISH-positive patients (P = 0.001) and in patients with positive EGFR immunostaining (P < 0.001) but not in patients with EGFR mutations (P = 0.19). EGFR gene dosage did not predict response (P = 0.54), progression-free (P = 0.73), or overall survival (P = 0.89). EGFR gene dosage was not associated with FISH positivity (P = 0.15), relative mRNA expression (P = 0.27), EGFR mutation status (P = 0.39), and EGFR protein expression (P = 0.35)., Conclusion: EGFR mRNA expression is a predictive biomarker for response to gefitinib and to progression-free survival after gefitinib treatment. EGFR gene dosage is neither predictive for response nor progression-free nor overall survival.

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, produce 9% to 27% response rates in NSCLC patients. E-Cadherin, a calcium-dependent adhesion molecule, plays an important role in NSCLC prognosis and progression, and interacts with EGFR. The zinc finger transcriptional repressor, ZEB1, inhibits E-cadherin expression by recruiting histone deacetylases (HDAC). We identified a significant correlation between sensitivity to gefitinib and expression of E-cadherin, and ZEB1, suggesting their predictive value for responsiveness to EGFR-tyrosine kinase inhibitors. E-Cadherin transfection into a gefitinib-resistant line increased its sensitivity to gefitinib. Pretreating resistant cell lines with the HDAC inhibitor, MS-275, induced E-cadherin along with EGFR and led to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive NSCLC cell lines including those harboring EGFR mutations. Thus, combined HDAC inhibitor and gefitinib treatment represents a novel pharmacologic strategy for overcoming resistance to EGFR inhibitors in patients with lung cancer.

To better understand the development of ventral mesencephalic dopamine neurons, we performed subtractive hybridization screens to find ventral mesencephalic genes expressed at rat embryonic day 10 when these neurons begin to differentiate. The most commonly identified genes in these screens were members of the Bex (Brain expressed X-linked) gene family, rat Bex1 (Rex3), and a novel gene, rat Bex4. After identifying these genes, we then sought to characterize the Bex gene family. Two additional novel Bex genes (human Bex5 and mouse Bex6) were discovered through genomic databases. Bex5 is present in humans and monkeys, but not rodents, while Bex6 exists in mice, but not humans. Bex4 and Bex5 are localized to the X chromosome, are expressed in brain, and are similar in sequence. Bex4 and Bex5 are 54% and 56% identical to human Bex3 (pHGR74, NADE). Mouse Bex6 is on chromosome 16 and is 67% identical to mouse Bex4. Human Bex gene expression was studied with tissue expression arrays probed with specific oligonucleotides. Human Bex1 and Bex2 have similar expression patterns in the central nervous system with high levels in pituitary, cerebellum, and temporal lobe, and Bex1 is widely expressed outside of the central nervous system with high expression in the liver. Human Bex4 is highly expressed in heart, skeletal muscle, and liver, while Bex3 and Bex5 are more widely expressed. The subcellular localization of the Bex proteins varies from nuclear (rat Bex1) to cytoplasmic (rat Bex3, human Bex5, and mouse Bex6) and to both nuclear and cytoplasmic (rat Bex2 and rat Bex4). Rat Bex3, rat Bex4, human Bex5, and mouse Bex6 are degraded by the proteasome, while rat Bex1 or Bex2 are not. Rat Bex3 protein can likely bind transition metals through a histidine-rich domain. Because this gene family was originally named Bex and because these genes are unified by sequence similarity and gene structure, we believe the Bex nomenclature should prevail over nomenclature based on function (NADE) that has not been extended to the other Bex genes. We conclude that the Bex gene family members are highly homologous but differ in their expression patterns, subcellular localization, and degradation by the proteasome.

Purpose: Exisulind (sulindac sulfone, FGN-1, Aptosyn) is a sulindac metabolite that induces apoptosis via inhibition of cyclic GMP-phosphodiesterase. This agent demonstrated tumor growth inhibition in rodent models of colon, breast, prostate, and lung carcinogenesis. In an orthotopic model of human non-small-cell lung cancer, the combination of exisulind and docetaxel prolonged survival in athymic nude rats, forming the basis of this phase I combination study., Experimental Design: This study evaluated the toxicity and pharmacokinetics of combining exisulind (150-250 mg) given orally twice daily and docetaxel (30-36 mg/m2) administered intravenously on days 1, 8, and 15 of a 4-week cycle., Results: Twenty patients with a range of advanced solid tumors (median age, 59 years; age range, 35-77 years; median performance status, 1) received a total of 70 courses. Observed adverse events were mild to moderate, and there was no dose-limiting toxicity at any level. Grade 3 gastrointestinal toxicities were present in 10 of the 70 cycles (10%) and included nausea, vomiting, dyspepsia, and elevated alkaline phosphatase. Neutropenia was present in four cycles in patients treated with a docetaxel dose of 36 mg/m2. Pharmacokinetic analysis did not demonstrate a clear effect of exisulind on docetaxel pharmacokinetics and vice versa. Relationships were evident between the plasma concentration of exisulind and the development of grade 2 or greater toxicities. One third of patients maintained stable disease for 3 to 12 cycles, but no objective responses were observed., Conclusions: The combination of docetaxel (36 mg/m2, weekly) and exisulind (500 mg/d) was reasonably well tolerated, and it is undergoing phase II testing in patients with non-small-cell lung cancer.

Parkinson's disease symptoms can be improved by transplanting fetal dopamine cells into the putamen of parkinsonian patients. Because the supply of human donor tissue is limited and variable, an alternative and genetically modifiable non-human source of tissue would be valuable. We have generated cloned transgenic bovine embryos, 42% of which developed beyond 40 days. Dopamine cells collected from the ventral mesencephalon of the cloned fetuses 42 to 50 days post-conception survived transplantation into immunosuppressed parkinsonian rats and cells from cloned and wild-type embryos improved motor performance. Somatic cell cloning can efficiently produce transgenic animal tissue for treating parkinsonism.

The replacement of dopamine (DA) by DA neuron transplants in the treatment of advanced Parkinson disease (PD) is a rational approach. Because of limitations associated with fetal tissue transplants, a clone (1RB3AN27) of simian virus 40 large tumor antigen (LTa) gene-induced immortalized DA neurons were used in this study. These allogeneic immortalized dopamine neurons, when grafted into striata of normal rats, did not divide, did not form tumors, did not produce LTa, did not extend neurites to host neurons, and were not rejected, for as long as 13 months after transplantation. Grafted cells when recultured in vitro resumed cell proliferation and LTa production, suggesting the presence of a LTa gene-inhibiting factor in the brain. The grafting of undifferentiated and differentiated 1RB3AN27 cells or differentiated murine neuroblastoma (NBP2) cells into striata of 6-hydroxydopamine-lesioned rats (an animal model of PD) caused a time-dependent improvement in neurological deficits (reduction in the methamphetamine-induced turning rate). At 3 months after transplantation, 100% of the animals receiving differentiated 1RB3AN27 cells, 63% of the animals receiving undifferentiated 1RB3AN27 cells, 56% of the animals receiving differentiated NBP2 cells, and 0% of the sham-transplanted animals showed improvements in neurological deficits. At 6 months after transplantation, there was a progressive increase in spontaneous recovery in sham-transplanted animals. These results suggest that immortalized DA neurons should be further studied for their potential use in transplant therapy in advanced PD patients.

XIPOU 2, a member of the class III POU-domain family, is expressed initially at mid-blastula transition (MBT) and during gastrulation in the entire marginal zone mesoderm, including Spemann's Organizer (the Organizer). To identify potential targets of XIPOU 2, the interaction of XIPOU 2 with other genes co-expressed in the Organizer was examined by microinjecting XIPOU 2's mRNA into the lineage of cells that contributes to the Organizer, head mesenchyme and prechordal plate. XIPOU 2 suppresses the expression of a number of dorsal mesoderm-specific genes, including gsc, Xlim-1, Xotx2, noggin and chordin, but not Xnot. As a consequence of the suppression of dorsal mesoderm gene expression, bone morphogenetic factor-4 (Bmp-4), a potent inducer of ventral mesoderm, is activated in the Organizer. Gsc is a potential target of XIPOU 2. XIPOU 2 is capable of binding a class III POU protein binding site (CATTAAT) that is located within the gsc promoter, in the activin-inducible (distal) element. Furthermore, XIPOU 2 suppresses the activation of the gsc promoter by activin signaling. At the neurula and tailbud stages, dorsoanterior structures are affected: embryos displayed micropthalmia and the loss of the first branchial arch, as detected by the expression of pax-6, Xotx2 and en-2. By examining events downstream from the Wnt and chordin pathways, we determined that XIPOU 2, when overexpressed, acts specifically in the Organizer, downstream from GSK-3beta of the Wnt pathway and upstream from chordin. The interference in dorsalizing events caused by XIPOU 2 was rescued by chordin. Thus, in addition to its direct neuralizing ability, in a different context, XIPOU 2 has the potential to antagonize dorsalizing events in the Organizer.

XIPOU 2, a member of the class III POU domain family, is expressed initially in Spemann's organizer, and later, in discrete regions of the developing nervous system in Xenopus laevis. XIPOU 2 may act downstream from initial neural induction events, since it is activated by the neural inducer, noggin. To determine if XIPOU 2 participates in the early events of neurogenesis, synthetic mRNA was microinjected into specific blastomeres of the 32-cell stage embryo. Misexpression of XIPOU 2 in the epidermis causes a direct switch in cell fate from an epidermal to a neuronal phenotype. In the absence of mesoderm induction, XIPOU 2 has the ability to induce a neuronal phenotype in uncommitted ectoderm. These data demonstrate the potential of XIPOU 2 to act as a master regulator of neurogenesis.