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K. Krysan



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    P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      P1.04-063 - Exposure to IL-1β Leads to EMT via Distinct Mechanisms in Acute and Chronic Inflammation in NSCLC (ID 3034)

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

      • Abstract

      Background:
      Dysregulated inflammation is associated with the development and progression of lung cancer. Pulmonary diseases characterized by increased inflammation, including emphysema and pulmonary fibrosis, are strongly related to heightened risk of lung cancer. Moreover, lung cancer patients with increased levels of inflammatory mediators or inflammatory cells have poor outcomes. It has been shown that dysregulated inflammatory cytokines in the tumor microenvironment can promote cancer metastasis. However, the mechanisms of this effect in lung cancer have not been fully understood. Interleukin 1β (IL-1β), a key pro-inflammatory cytokine, is associated with tumor aggressiveness and poor patient outcomes in NSCLC. Herein, we report that treatment of IL-1β leads to epithelial-to-mesenchymal transition (EMT) in NSCLC cell lines. Delineation of the underlying molecular pathway(s) may potentiate novel therapeutic strategies.

      Methods:
      We treated NSCLC cell lines with IL-1β acutely (3 days) and chronically (21 days) in vitro and identified EMT mediators using RNA interference and chemical inhibitors. Histone modifications and DNA methylation were analyzed with chemical inhibitors, ChIPassays and methylation-specific PCR. We utilized transwell migration, cell proliferation and anchorage-independent cell growth assays to evaluate the functional phenotypes

      Results:
      We found that following acute IL-1β exposure (within 7 days), the activator protein 1 (AP-1) transcription factor components, including Fra-1 and c-jun, mediate EMT. AP-1 functions downstream of ERK1/2 and JNK signaling and resides upstream of the transcription factors Slug and Zeb2. Importantly, inhibition of slug, zeb2, fra-1 or ERK1/2 and JNK signaling by RNA interference or chemical inhibitor is sufficient to abolish IL-1β-induced E-cadherin repression. This occurs concomitantly with decreased cell migration and invasion. Surprisingly, following prolonged IL-1β exposure (21 days), cells do not revert back to the epithelial state despite inhibition of these acute EMT mediators. We also found that following withdrawal of IL-1β after twenty one-day exposure, the treated cells are able to maintain their mesenchymal phenotype for more than 30 days before reverting back to an epithelial phenotype. We refer to this prolonged but reversible EMT program that persists in the absence of the original inflammatory stimulus as EMT “memory.” Further studies showed that fra-1 is only required to establish but not to maintain EMT memory. Chemical inhibition of a variety of enzymes involved in histone modifications and DNA methylation indicates the repression of E-cadherin is mediated by different mechanisms depending on the duration of IL-1β exposure. H3K27Me3 and histone acetylation mediate E-cadherin repression during acute EMT but DNA methylation is responsible for the downregulation of E-cadherin in EMT memory. In fact, we have found increased CpG island methylation in the E-cadherin promoter region in EMT memory. In vitro functional studies further showed that EMT memory enables cancer cells to enhance their motility but gradually regain proliferative advantage.

      Conclusion:
      We conclude that lung cancer cells utilize distinct mechanisms for EMT in response to acute and chronic inflammation. We also demonstrate that dynamic alteration of histone modification and DNA methylation can lead to prolonged but reversible EMT, subsequently creating a time window for cancer cells to migrate to distant organs and eventually undergo mesenchymal-epithelial transition to form macro-metastases.

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    P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)

    • Event: WCLC 2015
    • Type: Poster
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      P3.04-063 - The Mutational Landscape of Pulmonary Premalignancy in the Context of Lung Adenocarcinoma (ID 1614)

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

      • Abstract

      Background:
      While genomic alterations in lung cancer are being actively investigated, the early mutational events that occur within the pulmonary field of cancerization that subsequently drive early carcinogenesis are poorly understood. As a result, the clinical importance of premalignant lesions remains enigmatic. Epithelial cells in the field of lung injury can give rise to distinct premalignant lesions that may bear unique genetic aberrations. A subset of these lesions may progress to invasive cancer, however the mutational landscape that may predict progression has not been determined. In the present study we performed whole exome DNA sequencing to measure the incidence of somatic DNA alterations in matched sets of primary tumor, premalignant lesions and adjacent normal lung tissues.

      Methods:
      FFPE tissue blocks from 41 patients were obtained from the UCLA Lung Cancer SPORE Tissue Repository. The following regions were dissected from distal airways utilizing Laser Capture Microdissection: a) normal airway epithelial cells (1-3 regions), b) premalignant atypical adenomatous hyperplasia (AAH, 2-4 regions), c) adenocarcinoma in situ (AIS, 1-3 regions) and, d) adenocarcinoma (ADC, 1-3 regions). DNA was extracted and sequencing libraries were constructed followed by exome capture. Sequencing was performed on an Illumina HiSeq2000 with a mean coverage of ~50x per base.

      Results:
      Data analysis included analyses for germline and somatic variants, loss of heterozygosity and copy number alternations. Within each case, position-specific missense and nonsense mutations were compared. Different cases were compared for the mutations at a gene-specific level. Mutations found only in AAH lesions were defined as premalignant, in ADC as malignant, and in both AAH and ADC as progression-associated mutations. The analysis demonstrated that AAH lesions from the same patient often have different mutational profiles. We identified novel recurring progression-associated mutations in 33 genes, most of which have not been previously described as key drivers for lung cancer. Interestingly, recurring mutations were found in genes involved in calcium signaling and extracellular matrix/receptor interaction. The data was compared to the TCGA and COSMIC databases. Among affected proteins, only 3% overlapped with the COSMIC and approximately 6% with the TCGA database. Interestingly, all of the mutations overlapping with the COSMIC, were found to be common mutations in AAH. Furthermore, pathways affected by the mutated genes were identified utilizing Gene Ontology and pathways from the KEGG, Biocarta or Reactome databases. The observation that few genes mutated in both AAH and ADC are known as key drivers, indicates that: a) progression-associated mutations might facilitate malignant transformation by mutated key driver(s), or b) a combination of two or more progression-associated mutations that are not oncogenic alone, might drive malignant transformation. These hypotheses will be further tested by mapping progression- and malignant-associated genes in the context of pathways.

      Conclusion:
      Our data indicate that premalignant lesions from the same patient may have different mutational profiles. This inter-lesion heterogeneity suggests that a progression-associated mutational landscape could be defined in longitudinal studies of pulmonary premalignancy. These results could help identify targets for the development of targeted chemopreventive strategies for lung cancer. Supported by EDRN (U01CA152751-AS).