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P. Dospoy



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    MS 26 - Genomic Alterations and Drug Targets in Small Cell Lung Cancer (ID 44)

    • Event: WCLC 2015
    • Type: Mini Symposium
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MS26.03 - Targeting ASCL1 in Neuroendocrine Lung Cancers via a MAPK-Regulated Double-Negative Feedback Loop (ID 1965)

      14:50 - 15:05  |  Author(s): P. Dospoy

      • Abstract
      • Presentation

      Abstract:
      ASCL1 is a lineage-specific transcription factor responsible during development for the formation of pulmonary neuroendocrine cells. ASCL1 is highly expressed in the majority of neuroendocrine lung tumors including small cell lung cancer (SCLC) and non-small cell lung cancer with neuroendocrine features (NSCLC-NE). Others have shown that SCLC survival depends on continued ASCL1 expression while we showed that ASCL1 is also required for the survival of NSCLC-NEs; that ASCL1 down-stream targets predict for poor survival in NSCLC patients; and that BCL2 is a therapeutically actionable ASCL1 target gene (PNAS 2014;111(41):14788-93). Thus, we are trying to target ASCL1 and its “druggable” downstream genes by developing ASCL1 based ChIP-Seq datasets in SCLC and NSCLC-NE tumors. We have now discovered a way to reliably regulate ASCL1 protein expression through “upstream” targeting. Phorbol 12-myristate 13-acetate (PMA) is an agonist of the MAPK pathway via specific activation of Protein Kinase C. Treatment of ASCL1(+) HCC1833 cells for 24 hours with nM quantities of PMA resulted in a robust down-regulation of ASCL1 mRNA and protein. Tumor cell death was apparent and apoptosis confirmed via induction of cleaved PARP. ASCL1 down-regulation was associated with activation of the MAPK pathway, measured by increased protein levels of phosphorylated ERK (pERK), and decreased ASCL1 mRNA expression was found to be at least partly due to mRNA degradation. These data indicate that activation of the MAPK pathway in high-grade neuroendocrine tumors has potential for therapeutic intervention and also provides a reason for the previously unexplained low levels of MAPK activation (pERK) in SCLC. Unexpectedly, we also found that siRNA mediated knockdown of ASCL1 resulted in activation of the MAPK pathway. In addition, pERK was significantly induced with ASCL1 knockdown even when we also knocked down MEK1 (MEK1 knockdown by itself completely eliminated pERK expression). The MAPK pathway depends on active phosphorylation/dephosphorylation and this is regulated in part by dual-specificity phosphatases (DUSPs). Using our ASCL1 ChIP-Seq data, we identified a conserved ASCL1 binding site in the promoter region of DUSP6. DUSP6 mRNA was found to be dramatically elevated in ASCL1(+) lines HCC1833 and H889, while by contrast there was little or no DUSP6 expression in ASCL1(-) SCLC lines H82 and H526, and knockdown of ASCL1 resulted in a decrease of DUSP6 protein suggesting transcriptional regulation. This led us to try a DUSP6 allosteric inhibitor (E/Z-BCI, Sigma-Aldrich) which induced pERK, decreased ASCL1 protein expression, and inhibited soft agar colony forming ability of H889 SCLC cells. In conclusion: Our data indicate that the MAPK pathway regulates ASCL1 expression, where activation of pERK signaling is correlated with decreased ASCL1 mRNA and protein. In addition, ASCL1 in turn, actively down-regulates the MAPK pathway. Our hypothesis is that high-grade neuroendocrine lung cancers down-regulate the MAPK pathway in order to maintain ASCL1 expression, which promotes cell survival and maintenance of the neuroendocrine lineage. This points to a double-negative feedback loop involving the MAPK pathway, ASCL1, and at least one DUSP. Targeting components of the MAPK pathway regulating ASCL1 expression is thus a new therapeutic avenue for high-grade neuroendocrine lung cancers. (Lung Cancer SPORE P50CA70907, NIH 1F30CA168264, CPRIT).

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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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      P3.04-134 - Cancer-Specific Production of N-Acetylaspartate via NAT8L Overexpression in Non-Small Cell Lung Cancer and its Potential as a Biomarker (ID 3570)

      09:30 - 09:30  |  Author(s): P. Dospoy

      • Abstract

      Background:
      Lung cancer is the leading cause of cancer death worldwide, leading to 1.6 million deaths every year. The majority of lung cancer cases are diagnosed in late stages, and early-stage detection and treatment are now known to reduce mortality rates, as recently reported for non-invasive screening with low-dose CT (LDCT) scan. Currently, LDCT screening is recommended only for the high-risk population of smokers over 55 years of age. This limitation is due to high false positive rates (96.4%) as well as risks of radiation exposure in LDCT. For better screening methods, recent studies have attempted to use diverse biological fluid samples from patients for finding new lung cancer biomarkers. Unlike diagnostic biomarkers that are required to have high sensitivity for clinical application, screening biomarkers must have high specificity (i.e. low false positive rates) in order to avoid a large number of people without lung cancer from undergoing invasive or costly procedures for confirmation. Among recent studies on new lung cancer biomarkers, only one small-scale study identified a panel of blood microRNAs with cancer-specificity higher than 99%.

      Methods:
      In order to expedite the discovery of candidates for cancer-specific metabolites in lung cancer, we exploited a unique system of a non-small cell lung cancer (NSCLC) cell line and a line of immortalized bronchial epithelial cells derived from the same patient, HCC4017 and HBEC30KT, for the initial discovery. After molecular characterization, we validated the selected candidate’s cancer specificity in additional NSCLC cell lines and NSCLC tumors. The mechanistic basis of this cancer specificity was further investigated with NSCLC cell lines, and its clinical potential as a circulating biomarker of lung cancer was evaluated with selected blood samples from lung cancer patients.

      Results:
      Among several metabolites with significant cancer/normal differences, we identified a unique metabolic compound, N-acetylaspartate (NAA) in cancer cells ¾ undetectable in normal lung epithelium. NAA’s cancer-specific detection was validated in additional cancer and control lung cells as well as selected NSCLC patient tumors and control tissues. NAA’s cancer-specificity was further supported in our analysis of NAA synthetase (gene symbol: NAT8L) gene expression levels in The Cancer Genome Atlas: elevated NAT8L expression in approximately 40% of adenocarcinoma and squamous cell carcinoma cases (N=577), with minimal expression in all non-malignant lung tissues (N=74). We then showed that NAT8L is functionally involved in NAA production of NSCLC cells through siRNA-mediated suppression of NAT8L, which caused selective reduction of intracellular and secreted NAA. Our cell culture experiments also indicated that NAA biosynthesis in NSCLC cells depends on glutamine availability. For preliminary evaluation of NAA’s clinical potential as a circulating biomarker, we developed a sensitive NAA blood assay and found that NAA blood levels were elevated in approximately 40% of NSCLC patients (N=13) in comparison with age-matched healthy controls (N=21) among individuals aged 55 years or younger.

      Conclusion:
      Taken together, these results indicate that NAA is produced specifically in NSCLC tumors through NAT8L overexpression and its extracellular secretion can be detected in blood.