ADENYLOSUCCINATE SYNTHETASE 1: A NOVEL TARGET OF DELETION IN LUNG ADENOCARCINOMA WITH IMPLICATIONS FOR METABOLIC STRESS TOLERANCE

Open Access
Author:
Miller, Joshua Christopher
Graduate Program:
Pharmacology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 02, 2010
Committee Members:
  • Dr Christopher Herzog, Dissertation Advisor
  • Chris Herzog, Committee Chair
  • Kristin Ann Eckert, Committee Member
  • Jong Kak Yun, Committee Member
  • John Peter Richie Jr., Committee Member
  • Melvin Lee Billingsley, Committee Member
Keywords:
  • lung cancer
  • lung adenocarcinoma
  • metabolic reprogramming
  • adenylosuccinate synthetase
  • , novel deletion
Abstract:
Tobacco smoke consists of numerous carcinogens whose effect on lung tumor development includes the induction of mutations in key metabolic genes as well as the induction of chromosome instability (CIN). Consequently, carcinogen-induced mouse lung adenocarcinomas (LAC) display many more recurrent site- and chromosome-specific changes in DNA copy number compared with non-induced LAC. Here we identified the Adenylosuccinate synthetase 1 (Adss1) gene located on distal chromosome 12q as a focus of biallelic or homozygous deletion (HD) in lung adenocarcinoma (LAC). HDs of Adss1 were detected at a high rate in both mouse LAC cell lines and primary LAC tumors induced in mice. Losses of Adss1 not meeting the quantitative threshold of HD were also detected at a high frequency. Adss1 losses were found to be significantly associated with a more extensive CIN phenotype in the primary mouse tumors indicating that this change may be more relevant to carcinogen induced tumors. A similar frequency of ADSS1 deletion was observed in human LAC cell lines, suggesting relevance in human lung cancer. These results suggest ADSS1 inactivation as a novel somatic alteration in lung carcinogenesis, and suggest that its selective deletion in LAC may be triggered by CIN. In human lung tumors quantitative PCR detected bi-allelic or homozygous deletion of ADSS1 at a high frequency in LAC, however only one Squamous cell carcinoma (SCC) tumor exhibited biallelic loss and this was not isolated to ADSS1 specifically. ADSS1 loss not reaching the level of HD was detected at a much higher frequency than HD in both LAC and lung SCC, including the human cell lines. In all cases, for both HD and for deletions not reaching the threshold of HD, ADSS1 loss occurred at a higher frequency than was observed in either flanking gene, Inverted-Feron 2 (INF2) and SIVA1. The ADSS1 isozyme plays a key role in the inter-conversion of inosine 5´-monophophate (IMP) and adenosine 5´-monophophate (AMP), which is tightly linked with the production of cellular energy in the form of adenosine 5´-triphophate (ATP). Purine salvage pathway disruption is also reported in a number of cancers, primarily by deletion or mutation of the gene 5'-methylthioadenosine phosphorylase (MTAP). It was observed here that ADSS1 and MTAP are deleted in a mutually exclusive fashion and HD of one gene was detected in the majority of the cell lines examined. Knockdown of the remaining pathway in several cell lines resulted in a significant reduction in cell viability. Due to the potential impact of these pathways on cellular energy levels, analysis of the nucleotide pools was undertaken in stable knockdowns for ADSS1. HPLC showed an increase in adenylate levels in both cell lines examined as well as to the ratio of ATP to AMP in the H522 LAC cell line. Cellular energy status can affect both rates of proliferation and response to metabolic stress. While no increase was observed in growth rates from this change in cellular energy levels, succinate dehydrogenase activity, an indicator of cellular metabolism, was increased in knockdowns under hypoxia. Metabolic remodeling is often associated with changes in metabolic stress tolerance and low ADSS1 protein expressing cell lines were shown to be resistant to inhibition of glycolysis. Inhibition of glycolysis by 2-deoxyglucose (2-DG) in a panel of human non-small cell lung cancer (NSCLC) cell lines showed that levels of ADSS1 protein expressed relate to sensitivity to metabolic stress. Restoration of ADSS1 expression to an ADSS1 deficient immortalized human bronchial epithelial cell line was shown to sensitize it to both inhibition of glycolysis by 2-DG and to inhibition of oxidative phosphorylation by rotenone which functions by inhibiting electron transport. This indicates a role in ADSS1 loss for protecting tumor cells during times of metabolic stress. Results from these studies demonstrate the frequent inactivation of ADSS1 by deletion in both mouse and human LAC, which suggests that this novel and irreversible change in cellular metabolism is selected in and therefore advantageous for LAC tumorigenesis. Our results suggest that ADSS1 loss increases AMP availability for energy production and promotes survival under energy stress characteristic of early tumorigenesis. As the underlying metabolic changes of cancer are better understood, these pathways are becoming increasingly relevant for therapeutic development.