An ATP-grasp superfamily enzyme induced under serum starvation and hypoxia as a potential biomarker for pancreatic cancer diagnosis

Pancreatic cancer is one of the most prognostically unfavorable cancers worldwide. On the other hand, favorable outcomes have been reported when treatment is initiated at an early stage, and there is great expectation for the development of early diagnostic technologies. Most solid tumors, including pancreatic cancer, contain areas of hypoxia where the dominant cells show increased resistance to both chemo- and radiotherapy. We hypothesized that tumor cells proliferate under the ischemic environment, which is generally unfavorable for cell survival, through specific gene expression. Genes exhibiting such expression changes may serve as candidates for early detection or therapeutic targeting. To identify such candidates, we cultured human pancreatic cancer cell lines under serum starvation and hypoxic conditions to mimic the ischemic tumor microenvironment. Genes synergistically induced by these combined conditions were identified by DNA microarray analysis. One of the candidates was an ATP-grasp superfamily enzyme, RIMKLA, which is involved in glutamine family amino acid metabolic process, and its inducible expression was presented by qRT-PCR for at least four pancreatic cancer cell lines. We raised antibodies against peptides corresponding to specific regions of the polypeptide and showed that the protein synthesis is also induced. In mouse xenograft models, RIMKLA expression was seen in hypoxic regions where HIF-1alpha was induced, and the product of this enzyme was quantitatively detected by mass spectrometry. Tumorigenicity of the gene knockout cells was significantly reduced compared to that of wild-type cells, and necrotic regions were observed within hypoxic areas even in small xenografts. Immunohistochemical staining and mRNA in situ hybridization performed on surgical specimens from pancreatic cancer patients demonstrated that this enzyme is specifically expressed in pancreatic cancer tissues. These observations support the potential application of this protein as a clinical biomarker for pancreatic cancer diagnosis. In addition to detecting and targeting the enzyme molecule in tissue samples, minimally invasive diagnostic methods could be developed based on detecting this relatively small product in peripheral blood. Since the genetic disruption of the enzyme impairs tumor formation, its enzymatic activity appears to play an important role in tumor cell survival. These findings support the potential development of molecular targeted therapies based on gene disruption or enzymatic activity inhibition.