Jih-Hsiang Lee

Array comparative genomic hybridization-based characterization of genetic alterations in pulmonary neuroendocrine tumors

The goal of this study was to characterize and classify pulmonary neuroendocrine tumors based on array comparative genomic hybridization (aCGH). Using aCGH, we performed karyotype analysis of 33 small cell lung cancer (SCLC) tumors, 13 SCLC cell lines, 19 bronchial carcinoids, and 9 gastrointestinal carcinoids. In contrast to the relatively conserved karyotypes of carcinoid tumors, the karyotypes of SCLC tumors and cell lines were highly aberrant. High copy number (CN) gains were detected in SCLC tumors and cell lines in cytogenetic bands encoding JAK2, FGFR1, and MYC family members. In some of those samples, the CN of these genes exceeded 100, suggesting that they could represent driver alterations and potential drug targets in subgroups of SCLC patients. In SCLC tumors, as well as bronchial carcinoids and carcinoids of gastrointestinal origin, recurrent CN alterations were observed in 203 genes, including the RB1 gene and 59 microRNAs of which 51 locate in the DLK1-DIO3 domain. These findings suggest the existence of partially shared CN alterations in these tumor types. In contrast, CN alterations of the TP53 gene and the MYC family members were predominantly observed in SCLC. Furthermore, we demonstrated that the aCGH profile of SCLC cell lines highly resembles that of clinical SCLC specimens. Finally, by analyzing potential drug targets, we provide a genomics-based rationale for targeting the AKT-mTOR and apoptosis pathways in SCLC.

Capillary isoelectric-focusing immunoassays to study dynamic oncoprotein phosphorylation and drug response to targeted therapies in non-small cell lung cancer

Developing proteomic biomarkers is valuable for evaluating therapeutic effects of drugs and generating better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, lack of assay reproducibility, accuracy, and sensitivity. A novel capillary isoelectricfocusing (IEF) immunoassay system (NanoPro) was used to study the dynamic phosphorylation status of signaling molecules in non–small cell lung cancer (NSCLC) cells treated with EGFR tyrosine kinase and MEK inhibitors. NanoPro showed the same dynamic ERK phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation in NanoPro system enables multiple protein phosphorylation isoforms to be resolved and detected simultaneously. With NanoPro, we identified a specific on-target mitogen-activated protein/extracellular signal–regulated kinase (MEK) response pattern to MEK inhibitor PD325901, which was not detectable by Western blot analysis. We also revealed a MEK2 signal that may be associated with NSCLC cell sensitivity to the EGF receptor inhibitor erlotinib, and distinguished erlotinib-sensitive cells from intrinsic as well as acquired resistant cells to erlotinib. Moreover, NanoPro could differentiate human ERK1 isoforms from the mouse isoforms based on their isoelectric point differences and showed that erlotinib effectively inhibited ERK phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. With 8 μg of tumor aspirates, we precisely quantified the response of 18 signaling molecules to erlotinib and MEK1 inhibitor treatments in an NSCLC patient. NanoPros higher sensitivity, better resolution of protein phosphorylation status, and reduced tissue requirement warrant NanoPros investigation for future drug development and evaluation of drug effects of targeted therapies. Mol Cancer Ther; 12(11); 2601–13. ©2013 AACR.

Role of CYB5A in Pancreatic Cancer Prognosis and Autophagy Modulation

BACKGROUND Loss of 18q22.3 is a prognostic marker in pancreatic ductal adenocarcinoma (PDAC). This study investigated genes encoded by this cytoband. METHODS We studied mRNA/protein expression in radically resected (n = 130) and metastatic patients (n = 50). The role of CYB5A was tested in 11 PDAC cell lines and five primary cultures through retrovirus-mediated upregulation and small interfering RNA using wound-healing, invasion, annexin-V, electron microscopy, and autophagic assays, as well as autophagy genes and kinases arrays. CYB5A+ orthotopic models (n = 6 mice/group) were monitored by Firefly and Gaussia-luciferase bioluminescence, magnetic resonance imaging, and high-frequency ultrasound. Data were analyzed by t test, Fisher exact-test, log-rank test and Cox proportional hazards models. All statistical tests were two-sided. RESULTS Both resected and metastatic patients with low mRNA or protein expression of CYB5A had statistically significantly shorter survival (eg, median = 16.7 months, 95% confidence interval [CI] = 13.5 to 19.9; vs median = 24.8 months, 95% CI = 12.8 to 36.9; P = .02, two-sided log-rank test; n = 82 radically resected PDACs), and multivariable analyses confirmed prognostic relevance. Moreover, we characterized a novel function to CYB5A, autophagy induction, concomitant with reduced proliferation and migration/invasion of PDAC cells. Network analysis of proautophagic pathways suggested CYB5A interaction with TRAF6, which was confirmed by TRAF6 downregulation after CYB5A reconstitution (-69% in SU.86.86-CYB5A+; P = .005, two-sided t test). CYB5A silencing had opposite effects, restoring TRAF6 expression and wound healing. In vivo studies showed that CYB5A induced autophagy while inhibiting tumor growth/metastasis and increasing survival (median = 57 days, 95% CI = 52 to 61; vs median = 44 days, 95% CI = 21 to 57; P = .03, two-sided log-rank test). CONCLUSIONS These results define CYB5A as a novel prognostic factor for PDAC that exerts its tumor-suppressor function through autophagy induction and TRAF6 modulation.

Characterization of Fibroblast Growth Factor Receptor 1 in Small-Cell Lung Cancer

Introduction: There remains a significant therapeutic need for small-cell lung cancer (SCLC). We and others have reported high frequency of copy number gains in cytogenetic bands encoding fibroblast growth factor receptor 1 (FGFR1) in SCLC tumors and cell lines. Methods: Thirteen SCLC cell lines and 68 SCLC patient tumor samples were studied for FGFR1 amplification. Growth inhibition assays were performed using PD173074, a pan-FGFR inhibitor to determine the correlation between FGFR1 expression and drug sensitivity. Results: We did not detect FGFR1 mutations in SCLC cell lines. Focal amplification of FGFR1 gene was found in five tumor samples (7%), with high-level focal amplification in only one tumor sample (1%). Amplification owing to polysomy of chromosome 8, where FGFR1 locates, was observed in 22 tumor samples (32%). There was no correlation between FGFR1 gene copy number and messenger RNA expression or protein expression in SCLC cells. FGFR inhibitor sensitivity correlated with FGFR1 copy number determined by real-time polymerase chain reaction assay (r= −0.79; p = 0.01). Conclusion: FGFR1 gene mutations and focal amplification are rare in SCLC, but polysomy of chromosome 8 is relatively common. FGFR1 copy number gain predicts sensitivity to FGFR inhibition, and FGFR expression correlates inversely with chemosensitivity.

Loss of 18q22.3 Involving the Carboxypeptidase of Glutamate-like Gene Is Associated with Poor Prognosis in Resected Pancreatic Cancer

Purposes: Pancreatic cancer is the fourth leading cause of cancer-related death, and studies on the clinical relevance of its genomic imbalances are warranted. Experimental Design: Recurrent copy number alterations of cytobands and genes were analyzed by array comparative genomic hybridization (aCGH) in 44 resected pancreatic cancer specimens. Prognostic markers identified by aCGH were validated by PCR gene copy number assay in an independent validation cohort of 61 resected pancreatic cancers. The functions of gene identified were evaluated by proliferation, cell cycle, and migration assays in pancreatic cancer cells. Results: We showed recurrent copy number gains and losses in the first cohort. Loss of 18q22.3 was significantly associated with short-term overall survival in the first cohort (P = 0.019). This cytoband includes the carboxypeptidase of glutamate-like (CPGL) gene. CPGL gene deletion was associated with shorter overall survival in the validation cohort (P = 0.003). CPGL deletion and mutations of TP53 or Kras seem to be independent events. A Cox model analysis of the two cohorts combined showed that loss of 18q22.3/deletion of the CPGL gene was an independent poor prognostic factor for overall survival (HR = 2.72, P = 0.0007). Reconstitution of CPGL or its splicing variant CPGL-B into CPGL-negative pancreatic cancer cells attenuated cell growth, migration, and induced G1 accumulation. Conclusion: Loss of 18q22.3/deletion of the CPGL gene is a poor prognostic marker in resected pancreatic cancer, and functional studies suggest the CPGL gene as growth suppressor gene in pancreatic cancer. Clin Cancer Res; 18(2); 524–33. ©2011 AACR.

A nanofluidic immunoassay system to develop proteomic responsive biomarkers in non-small cell lung cancer.

27 Background: Molecular targeted therapy is widely used to treat non-small cell lung cancer (NSCLC). However, errors in predicting response to targeted therapies are 20-30%, based on sequencing or FISH, and patient specimen are at times not sufficient for conventional protein technologies. Developing clinically feasible proteomic biomarkers may be valuable to identify patients who may benefit from targeted therapy. METHODS NanoPro technology (ProteinSimple), is a capillary-based isoelectric-focusing (IEF) immunoassay system, which detects and quantifies multiple protein phosphorylation isoforms that are not readily assessed by traditional immunoassays. The platform only uses nanograms of protein for analysis. RESULTS We studied dynamic phosphoprotein activities in NSCLC cells (PC9, H827, H4006, H2122 and H322) treated with EGFR or/and MEK inhibitors. In MEK inhibitor (PD325901) treated cells, NanoPro showed that the drug efficiently inhibited Erk phosphorylation, and also revealed a complex MEK response profile which was not detectable by Western blotting, demonstrating its on-target effect. NanoPro also identified a MEK2 signature that associated with erlotinib sensitivity and distinguished erlotinib sensitive from resistant cells. This MEK2 signature was further confirmed in acquired resistant cells to erlotinib (H827R, H4006R). In a H827 xenograft study, NanoPro was able to detect and distinguish human Erk1 isoform from mouse Erk1 based on their pI difference, and clearly demonstrated that erlotinib effectively inhibited Erk phosphorylation in human xenograft cancer cells but not in surrounding mouse stromal cells. We further demonstrated that Nanopro could monitor erlotinib and AZD2644 response in a KrasG12C NSCLC patient (NCT01229150) by profiling 18 signaling molecules with as little as 4ug tumor material. CONCLUSIONS NanoPro provides a valuable platform to monitor signaling response to targeted therapy. Nanopro assesses protein phosphorylation both qualitatively and quantitatively using small sample size, thus creates new opportunities for pharmacodynamic biomarker assessment in clinical studies.

Abstract 5521: Application of an automated capillary isoelectric-focusing immunoassay to study dynamic oncoprotein phosphorylation in non-small cell lung cancer and for evaluating drug response to targeted therapies.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Molecular targeted therapy is widely used to treat non-small cell lung cancer (NSCLC) and genomic analysis has been commonly used to identify patients who may benefit from targeted therapies. Nevertheless, the accuracy of predicting responses to targeted therapies is only 50-80% based on sequencing or FISH. Developing proteomic biomarkers is valuable for evaluating therapeutic effects and better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, low throughput or/and lack of assay reproducibility, accuracy and sensitivity. In this study, a novel capillary isoelectricfocusing (IEF) immunoassay system (ProteinSimple, Santa Clara, CA) was used to study the dynamic phosphorylation status of signaling molecules in tyrosine kinase or/and MEK inhibitor treated NSCLC cells. NanoPro showed the same dynamic Erk phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation enables multiple protein phosphorylation isoforms to be resolved and detected using pan-reactive antibodies. In PD325901 MEK inhibitor treated cells, a specific MEK response pattern to drug treatment was revealed by NanoPro. This pattern demonstrated on-target effect of the drug, which was not detectable by Western blotting. NanoPro also identified a MEK2 response signature that may be associated with NSCLC cell sensitivity to the tyrosine kinase inhibitor, erlotinib, and distinguished erlotinib-sensitive from erlotinib-resistant cells. This MEK2 signature was further confirmed in cells with acquired resistance to erlotinib when compared to respective parental cells. In an HCC827 xenograft study, Western blotting was not able to differentiate human cancer cell signals from mouse stromal cell interference. In contrast, NanoPro detected and distinguished human Erk1 isoforms from the mouse isoforms based on their pI differences and demonstrated that erlotinib effectively inhibited Erk phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. We further used NanoPro to monitor responses of protein phosphorylations to erlotinib and AZD6244 (a MEK1/2 inhibitor) in a NSCLC patient by profiling 18 signaling molecules with as little as 8 μg tumor material. Signals from baseline and inhibitor treated samples were precisely quantified and compared. In conclusion, NanoPro can assess protein phosphorylation both qualitatively and quantitatively to provide more information about the activation status of proteins, which is not readily assessed by traditional Western blotting. Its higher sensitivity, better reproducibility and reduced tissue requirement are appealing for drug development and evaluation of drug effects in targeted therapies. Citation Format: Jinqiu Chen, Jih-Hsiang Lee, Michelle Herrmann, Kang-Seo Park, Madeleine Heldman, Paul K. Goldsmith, Yisong Wang, Giuseppe Giaccone. Application of an automated capillary isoelectric-focusing immunoassay to study dynamic oncoprotein phosphorylation in non-small cell lung cancer and for evaluating drug response to targeted therapies. [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 5521. doi:10.1158/1538-7445.AM2013-5521

Abstract 5466: The role of fibroblast growth factor receptor 1 (FGFR1) in small cell lung cancer (SCLC).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Although molecularly-targeted treatment options for solid tumors have advanced in recent years, there remains a significant therapeutic need for SCLC, which constitutes 12% of all lung cancers and carries a poor prognosis. FGFR1, a transmembrane tyrosine kinase receptor encoded by the FGFR1 gene in chromosome 8p12 is involved in cell cycle, differentiation, survival, apoptosis, and angiogenesis. In SCLC, FGF/FGFR pathway is known to induce proliferation, disrupt apoptosis and mediate chemo-resistance. Pharmacologic FGFR inhibition in SCLC reduces proliferation and increases apoptosis both in vitro and in vivo. We and others have reported high copy number gains (CNG) in cytogenetic bands encoding FGFR1 in SCLC suggesting that FGFR1 may be driver mutation and a potential therapeutic target in SCLC. In this study we further characterize FGFR1 as a therapeutic target in SCLC. Methods: Thirteen SCLC cell lines, 16 SCLC patient samples, and 14 SCLC tumors from commercially available tissue microarray (TMA) were screened for copy number alterations using array comparative genomic hybridization (aCGH) and focal FGFR1 amplifications using fluorescence in situ hybridization (FISH). In the cell lines, FGFR1 exons were sequenced from genomic DNA, mRNA expression evaluated using real-time polymerase chain reaction and protein expression determined semi-quantitatively using western blot. Growth inhibition assays were performed using PD173074, a selective FGFR inhibitor and standard cytotoxic agents (cisplatin and etoposide) to determine the correlation between FGFR1 expression and drug sensitivity. Results: CNG was detected in 10/16 (63%) patient samples and in 5/14 (36%) of tumors from TMA (range 4-9 copies per cell). 5/13 (38%) of cell lines demonstrated CNG, but we found no FGFR1 mutations or focal amplifications in SCLC cell lines. FGFR1 mRNA expression did not correlate with CNG and the FGFR1 protein expression was unrelated to the mRNA expression. FGFR1 CNG predicted sensitivity to [PD173074][1] with cell lines carrying higher copy numbers being more sensitive. No such correlation was detected between FGFR1 mRNA expression and [PD173074][1] sensitivity. Higher FGFR1 mRNA expression was associated with etoposide resistance. Conclusion: FGFR1 protein expression in SCLC cell lines is unrelated to copy number alterations and mRNA expression. CNG of 8p12 region which encodes FGFR1 predicts sensitivity to PD173074, a selective FGFR inhibitor. Citation Format: Anish Thomas, Jih-Hsiang Lee, Yisong Wang, Ziedulla Abdullaev, Lola Saidkhodjaeva, Svetlana D. Pack, Giuseppe Giaccone. The role of fibroblast growth factor receptor 1 (FGFR1) in small cell lung cancer (SCLC). [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 5466. doi:10.1158/1538-7445.AM2013-5466 [1]: /lookup/external-ref?link_type=GEN&access_num=PD173074&atom=%2Fcanres%2F73%2F8_Supplement%2F5466.atom

Abstract 1143: Unraveling the role of CYB5A in pancreatic ductal adenocarcinoma (PDAC): correlation with clinical outcome and functional characterization in the modulation of autophagy and oncogenic phenotypes.

Loss of 18q22.3 was reported to be a prognostic factor in PDAC. This study aimed at evaluating whether the genes of this cytoband were associated with PDAC outcome and provides mechanistic insights on their role in the suppression of oncogenic properties. The expression of FBXO15, C18orf55, CYB5A, CPGL and CPGL-B was evaluated in cells (11 PDAC cell lines, 5 primary cultures, and a normal pancreatic ductal cell line) and in 48 stage IIB PDAC specimens, isolated by laser-microdissection after surgical resection. Low CYB5A expression correlated with 18q22.3 deletion and shorter survival (16.3 vs. 29.5 months, P=0.01). Association with outcome was validated in a tissue-microarray of a second cohort radically resected patients (N=100): patients with CYB5A protein expression levels below median value had significantly shorter OS, and multivariate analysis confirmed CYB5A prognostic relevance (increased risk of death, 2.0, P=0.02). CYB5A expression in primary cultures correlated with expression in their respective tissues, and CYB5A retrovirus-mediated up-regulation of both PDAC-2 primary cells and Su86.86 cells suppressed proliferation and migration/invasion, while enhancing apoptosis and autophagy induction. Marked accumulation of autophagic vacuoles was detected by electron microscopy, while immunofluorescence revealed CYB5A colocalization with LC-3. Activation of pro-autophagic pathways was corroborated by PCR arrays showing a significant up-regulation of several ATG-genes, accompanied by down-regulation of BCL-2 and MAPK14. The phosphorylation status of MAPK14 was also inhibited, together with phospho-EGFR and the pro-invasive kinases phospho-Src/STAT6, as revealed by 144-kinase peptide substrate arrays. Network analysis suggested that this down-regulation was caused by CYB5A interaction with the NF-κB activator TRAF6, whose expression was significantly reduced after reconstitution of CYB5A in a genetic and histopathologic characterized patient-derived orthotopic mouse model. Furthermore, CYB5A upregulation increased survival, while decreasing primary tumor dimension and metastatic spread, as longitudinally monitored with Firefly- and Gaussia-luciferase. We thus identified CYB5A as a novel prognostic factor that modulates autophagy and oncogenic phenotypes, holding a potential as a novel therapeutic approach in the clinical management of PDAC patients. Citation Format: Elisa Giovannetti, Qiuyan Wang, Amir Avan, Niccola Funel, Elena Glavani, Tonny Lagerweij, Davide Chiasserini, Jih-Hsiang Lee, Viola Caretti, Matilde Masini, Ugo Boggi, Yisong Wang, Enrico Vasile, Godefridus J. Peters, Thomas Wurdinger, Giuseppe Giaccone. Unraveling the role of CYB5A in pancreatic ductal adenocarcinoma (PDAC): correlation with clinical outcome and functional characterization in the modulation of autophagy and oncogenic phenotypes. [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 1143. doi:10.1158/1538-7445.AM2013-1143