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K. Shilo



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    P2.01 - Poster Session 2 - Cancer Biology (ID 145)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.01-004 - Type of P53 mutation influences oncogenic potential and spectrum of associated K-ras mutations in lung specific transgenic mice. (ID 1972)

      09:30 - 09:30  |  Author(s): K. Shilo

      • Abstract

      Background
      p53 mutations have been categorized as type I (contact) and type II (conformational) mutations. Differential effects of type I vs. type II p53 mutations in spontaneous lung tumor formation, and their relationship with secondary genetic alterations have not been previously reported. .

      Methods
      We evaluated the potential of two common type I (273H) and type II (175H) mutations under the transcriptional control of the human surfactant protein C (SP-C) promoter to induce lung tumors in transgenic mice. Necropsies of 138 non-transgenic, 207 SP-C-p53-273H and 171 SPC-p53-175H transgenic mice in progressive age cohorts were performed.

      Results
      Ninety-one tumors, all adenocarcinomas, were observed; 8 (5.8%), 37 (17.9%) and 46 (26.9%) in non-transgenics, p53-273H and p53-175H, respectively (non-transgenic vs. 273H, p=0.010; non-transgenic vs. 175H, p= 0.0003; logistic regression). Type II p53 mutants had an earlier onset of tumors; 23 of 98 p53-175H mice developed tumors before the age of 13 months, compared to 7 of 108 p53-273H mice (p=0.012, logistic regression). K-ras mutations occurred in a substantial proportion (21 of 50, 42%) of murine lung tumors sequenced. For both the non-transgenic and the p53-273H transgenics, tumor K-ras codon 12-13 mutations occurred after 13 months with a peak incidence at 16–18 months. However, for the p53-175H transgenics K-ras codon 12-13 mutations were observed as early as six months, with a peak incidence between the ages of 10-12 months. Codons 12-13 were the predominant location in p53-175H transgenics (6 of 7), whereas codon 61 (6 of 10) was more common in p53-273H transgenics.

      Conclusion
      The observation of accelerated tumor onset, early appearance and high frequency of K-ras codon 12-13 mutations in type II p53-175H mice confirms the enhanced oncogenic function of conformational p53 mutations, and the gains in early genetic instability for tumors containing these mutations compared to contact mutations. These data would suggest that not only the presence, but also the type of p53 mutations in human lung cancer should be considered when evaluating prognosis and developing treatment strategies for this malignancy. These mice develop a single-lung tumor that is easy to follow with CT imaging. Thus, these animal models provide a framework for evaluation of the effects of these mutations on response to standard and novel anticancer treatment interventions.

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    P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.02-016 - PRMT5, a novel epigenetic target in lung cancers (ID 2886)

      13:45 - 14:02  |  Author(s): K. Shilo

      • Abstract

      Background
      PRMT5 is an arginine methyltransferase that regulates cellular events by methylation of Arginine residues on histone and non-histone proteins. PRMT5 cooperates with chromatin remodelers and co-repressors to induce epigenetic silencing, and is overexpressed in several human cancers. It also has impact on cell growth and transformation pathways by modulation of E2F1, p53, EGFR and CRAF. We investigated the role of PRMT5 in lung cancer.

      Methods
      The expression pattern of PRMT5 using immunohistochemistry from resection specimens obtained with an IRB approved protocol. Immortalized lung cancer cells (A549, H719, H520 and H1299) were obtained from ATCC and normal bronchial airway cells (HPAEpiC, HBEpiC) were obtained from ScienCell. Western immunoblot and cell cycle analysis by flow cytometry was performed using standard techniques. A novel and specific inhibitor of PRMT5 developed by colleagues at OSU was applied to in vitro culture systems.

      Results
      The expression of PRMT5 was analyzed in 9 lung cancer resection specimens (3 adenocarcinoma, 3 squamous, 2 small cell and 1 large cell neuroendocrine cancer). All 9 showed diffuse cytoplasmic and variable nuclear PRMT5 expression. PRMT5 was also seen in reactive type 2 pneumocytes and respiratory epithelium adjacent to the tumors but not in alveolar parenchyma, fibroblasts or endothelial cells. Using Western immunoblot, PRMT5 is highly expressed in A549, H719, H1299 and H520 cells compared with normal cells such as HPAEpiC and HBEpiC. We knockdowned the expression of PRMT5 by lentiviral shRNAs and identified several clones with effective PRMT5 inhibition. Inhibition of PRMT5 was associated with slow cell growth. The cell proliferation decreased 58.4% and 62.3% in H1299 and A549 knockdown cells respectively. Interestingly, while H4R3 methylation was decreased with PRMT5 knockdown in A549 it was not in H1299 cells. We further analyzed the role of PRMT5 by using a specific inhibitor developed by researchers at OSU, CPD5. One of the PRMT5 specific marks of histone H4R3 methylation was inhibited and significant cell cycle changes were observed in A549 and H1299 cells treated for 24 hr and 48 hr. At 24 hours, the percentage of cells in G0/G1 was 57.1% (control) compared with 66.4% in CPD5 treated A549 cells, and 52.5% (control) compared with 70.9% in CPD5 treated H1299 cells. The expression of p21 was increased while cyclin E1 was decreased in A549 cells treated with CPD5. In contrast, the expression of cell cycle related proteins were not found in PRMT5 knock-down cells. Immunoprecipitation and other protein interaction techniques are in process to identify new PRMT5 targets.

      Conclusion
      In summary, PRMT5 is highly expressed in lung cancer cells compared to normal lung. A novel PRMT5 inhibitor and shRNAs can inhibit cell proliferation. Although PRMT5 inhibitor could induce cell death by cell cycle regulation and apoptosis, different pathways may be involved in PRMT5 knock-down cells and speaks to the complexity regulating mechanisms in different histological lung cancer patients. The differences in H4R3 methylation suggest that epigenetic repression may be dominant in some cancers, but that non-epigenetic mechanisms may be relevant in others. Further exploring of PRMT5 targets are ongoing.

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    P3.24 - Poster Session 3 - Supportive Care (ID 160)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Supportive Care
    • Presentations: 1
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      P3.24-026 - Solitary Fibrous Tumor of the Pleura: Presentation and management (ID 1703)

      09:30 - 09:30  |  Author(s): K. Shilo

      • Abstract

      Background
      Solitary fibrous tumor of the pleura (SFT) is an infrequent diagnosis and presents with a spectrum of radiographic and clinical findings. SFT can follow indolent or agressive clinical courses. This report describes clinical, radiologic and pathologic findings from four patients with this diagnosis. Surgical management options and the role of surveillance are reviewed.

      Methods
      The medical records and pathology reports of these patients were abstracted. The pathology slides were reviewed and interpreted by a single pathologist.

      Results
      Each patient had unique presentation and clinical course. Two patients did not have cardiopulmonary symptoms; two had progressive dyspnea, cardiac arrythmia, or pleuritic pain. Surgical approaches included robotic-assisted surgery (2), limited thoracotomy (1), and posterolateral thoracotomy with median sternotomy (1). All patients did well in the short term and there were no mortalities. All tumors showed typical spindle cell morphology with hemagiopericytoma like vasculature. Two patients showed significant necrosis. One patient developed bilateral pulmonary and subcutaneous metastasis one year after R0 resection; this tumor showed increased mitotic activity and necrosis on original pathology. FIGURE 1: CT chest of a large fibrous tumor of the pleura Figure 1 FIGURE 2: CT Chest of pulmonary metastasis Figure 2

      Conclusion
      Our experience with management of solitary fibrous tumor of the pleura emphasizes the variety of radiographic and clinical presentations for this entity. In our series, one patient had disease progression despite original pathology interpretation indicating typical morphology. Interestingly, this patient had the greatest number of mitotic figures in the tumor. Surgical resection remains the standard of care. Because these tumors have malignant potential, ongoing radiographic surveillance is appropriate management in patients with this diagnosis.