טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLehvy Adrian Israel
SubjectZinc Transporter Mutations and Expression in Cancer and
Transient Neonatal Zinc Deficiency
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf
Full Thesis textFull thesis text - English Version


Abstract

Zinc is vital for the proper functioning of ~3,200 proteins. Consequently, impaired zinc homeostasis is associated with human diseases, including transient neonatal zinc deficiency (TNZD) and cancer. Zinc transporters termed ZnTs and ZIPs, as well as metallothioneins which are non-selective metal chelators, maintain zinc homeostasis. ZIPs increase cytosolic zinc levels by importing it from the lumen of organelles or from the extracellular milieu, while ZnTs lower cytosolic zinc levels by compartmentalizing it within organelles or exporting it to the extracellular milieu. To explore zinc homeostasis in cancer, we used bioinformatics analysis to compare the relative zinc transporter mutation rates in cancer genomes to a healthy population, using the COSMIC and gnoMAD database, respectively. We discovered that ZnT1, ZnT10 and ZIP10 have the most significant increase in LoF mutations in cancer. Moreover, ZnT1, the ubiquitously expressed, sole exporter of zinc, harbored the most significant increase in predicted deleterious missense mutations. Furthermore, five cancer types displayed significantly increased levels of ZnT1 mRNA, according to the GEPIA database. Remarkably, in these five cancer types, ZnT1 gene expression levels negatively correlated with patient survival rates. We therefore propose that ZnT1 mRNA levels may serve as a prognostic factor predicting cancer patient survival. We next examined the impact of ZnT1 mutations or overexpression on zinc homeostasis. Using novel zinc transport assays, we showed that overexpression of representative ZnT1 mutants impairs zinc export function, while overexpression of the WT ZnT1 causes a significant decrease in intracellular zinc levels. These results show that qualitative and quantitative alterations in ZnT1, observed in tumor specimens, impair zinc homeostasis on a mechanistic level. Since impaired zinc homeostasis was previously implicated in pathways that support cancer initiation and/or cancer progression, we propose that ZnT1 is a potential cancer marker gene and drug target. Loss of function (LoF) mutations in ZnT2 result in impaired zinc secretion into breast milk, consequently causing TNZD in exclusively breastfed infants. Low levels of 2 serum zinc in infants cause a systemic array of symptoms, which also affect development, and can be fatal if untreated. Dietary zinc supplementation is the recommended treatment for infants with TNZD. Unfortunately, clinical diagnosis of TNZD often occurs after the onset of severe symptoms. To date, the frequency of TNZD in the general population was unknown. We therefore first studied ZnT2 mutations in the ExAC database which contains the exome sequences of 60,706 healthy individuals. Since TNZD-causing mutations are haploinsufficient, deleterious mutations in one allele are sufficient to cause TNZD or mild zinc deficiency in the nursing infant. A total of 115 missense ZnT2 mutations are harbored at a frequency of 1/182 individuals. Following bioinformatics analyses and zinc transport assays of ZnT2 mutants, we calculated that the frequency of TNZD-causative mutations in the general population is 1/2,334 individuals. We showed that the frequency of TNZD is significantly higher than previously expected and, considering that exclusive breast-feeding rates are predicted to increase by 50% by 2025 according to the WHO, the timely diagnosis and preventative treatment of TNZD patients is of paramount importance.