Background: Insulin is a hormone released by the pancreatic beta cells in response to elevated levels of nutrients in the blood. Insulin triggers the uptake of glucose, fatty acids and amino acids into the liver, adipose tissue and muscles. Genes regulating insulin signalling are thus of vital importance for metabolic homeostasis and for preventing the development of diabetes. This thesis aims to identify new modifiers of insulin signalling, while carrying out functional studies of a homolog to human arsenite translocating ATPase, ASNA1. ASNA1 activates the insulin signalling pathway and promotes insulin secretion in mammalian cell lines and in Caenorhabditis elegans. A second aim is to better understand how ASNA1 and its interactors regulate sensitivity to the chemotherapeutic drug, cisplatin. Results: Regulators of insulin/IGF signalling (IIS) in C. elegans were identified based on the Larval arrest arrest aspect of the asna-1 depletion phenotype. Sixty-five genes were selected by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest upon inactivation of the genes . mrps-2, mrps-10, mrpl-43 encoding mitochondrial ribosomal protein subunits, and enpl-1 encoding an ER chaperone, GRP94 homolog were identified as the genes which when inactivated caused larval arrest without any associated feeding defects. IIS was weaker and insulin secretion was defective in these knockdown animals. ENPL-1 and ASNA-1 proteins interacted with one another both ex vivo and in vitro. ASNA-1 protein and mRNA level swere greatly reduced in enpl-1 mutants and enpl-1(-);asna-1(-) double-mutant worms displayed synthetic lethality. Overexpression of the insulins INS-4 and DAF-28 caused partial rescue of the germline phenotype of enpl-1 mutants, indicating that the phenotype of enpl-1 mutants was due at least in part to insufficient insulin levels. Studies of enpl-1 mutants also helped to understand the role of asna-1 in cisplatin sensitivity. The unfolded protein response (UPR) was induced in asna-1 and enpl-1 knockdown animals. enpl-1 mutants displayed higher sensitivity to cisplatin, when compared to asna-1 mutants and this correlated to higher UPR in enpl-1 knockdown animals. Pharmacological induction of the UPR in intrinsically cisplatin resistant wildtype worms also resulted in increased cisplatin sensitivity. This suggests that manipulation of ENPL-1 levels or of the UPR could enhance the anti-tumoral effects of cisplatin based cancer therapy. With a yeast two hybridscreen 27 putative physical interactors of ASNA-1 were identified. Amongst these candidate swas smn-1, which encodes survival of motor neuron protein homolog. RNAi knockdown of smn-1 caused a larval arrest phenotype similar to asna-1 depleted animals and smn-1 positively regulated IIS, like asna-1. Defects in IIS may be at the level of insulin release because neuropeptide secretion was impaired upon smn-1 knockdown. Further in vitro binding studies showed that SMN-1 and ASNA-1 interacted and inactivation of smn-1 in asna-1 mutants resulted in decreased viability. This implies that SMN-1 is another modifier of ASNA-1 and also a new component in IIS. Conclusion: With a directed RNAi screen and a yeast two hybrid screen several interactors of ASNA-1 that are also IIS modifiers were identified. ENPL-1 and SMN-1 are both involved in insulin release. We also found that induction of the UPR in enpl-1 and asna-1 mutants is a possible mechanism for increased sensitivity to cisplatin.

Platinum based chemotherapy is widely used to treat cancer. Cisplatin (diamminedichloroplatinum) combination treatments provide cure for metastatic testicular cancer and prolong survival for patients suffering from ovarian, head and neck, bladder and non small cell lung cancer. Tumors that initially respond to treatment may eventually acquire resistance, resulting in treatment failure. Cisplatin resistant cells are crossresistant to arsenite and antimonite and these metalloids are exported from bacteria by the ars-operon. In this thesis, we describe the human ArsA homolog, ASNA1, as a protein involved in a novel resistance mechanism to cisplatin, arsenite and antimonite. ASNA1 was downregulated by antisense and siRNA techniques in human melanoma and ovarian carcinoma cell lines. These cells displayed increased sensitivity to arsenite and the platinum based drugs cisplatin, carboplatin and oxaliplatin. In both melanoma and ovarian carcinoma, cisplatin resistant cells overexpressed ASNA1. Blockage of ASNA1 resulted in increased apoptosis and retarded growth, complicating further characterization of ASNA1 in human cell lines. ASNA1 also promotes insulin signaling and mediates membrane insertion of tail-anchored proteins. To explore different aspects of ASNA1 function with respect to cisplatin resistance, we used the model organism C. elegans. In the nematode C. elegans, asna-1 (rnai) treated larvae were hypersensitive to cisplatin, arsenite and antimonite. Adult asna-1 mutant worms were cisplatin sensitive and this hypersensitivity was seen even when apoptosis was blocked. Expression of human ASNA1 rescued the cisplatin hypersensitivity in asna-1 mutants, showing conservation of function. Transgene expression of mutated forms of asna-1 separated the cisplatin hypersensitivity phenotype from the insulin signaling phenotype of asna-1 mutants. Three ASNA-1 residues, His164, Cys285 and Cys288 were identified as essential for ASNA-1 promoted cisplatin resistance but not for insulin signaling. Finally, studies of the C. elegans germline revealed increased numbers of apoptotic cells in asna-1 mutants. In conclusion, C. elegans is a suitable model organism to identify and characterize cisplatin response mechanisms. A targeted therapy against ASNA1 could sensitize cisplatin resistant cells and improve outcome for cancer patients.