Targeted ablation of gonadotropin dependent endocrine tumors in transgenic mice through their luteinizing hormone receptor (LHR)

Gonadal somatic cell and adrenocortical endocrine tumors are rare. The incidence of adrenocortical carcinomas is only 1-2/1000000 a year. However, they are aggressive, especially in adulthood and currently surgery is the only curative treatment. Cytotoxic agents are in use in advanced cancers, but side effects and multidrug resistance are often problems. Thus there is a need for novel curative treatment methods. In contrast, ovarian granulosa cell tumors and testicular Leydig cell tumors are usually benign, especially at a younger age. The aim of the present thesis was to study a novel targeted treatment method through luteinizing hormone/chorionic gonadotropin receptor (LHCGR) in a transgenic mouse tumor model. The cytotoxic agent was lytic peptide Hecate-CGbeta conjugate where 23 amino acid Hecate, a synthetic form of honeybee venom melittin, was conjugated to 15 amino acid fragment of human chorionic gonadotropin β subunit. Lytic peptides are known to act only on negatively charged cells, such as bacteria and cancer cells and hereby, due to hCGbeta fragment, the conjugate is able to bind directly to LHCGR bearing cancer cells, saving the healthy ones. The experiments were carried out in inhibin-alpha-Simian Virus 40-T-antigen transgenic mice that are known to express LHCGR-bearing gonadal tumors, namely Leydig and granulosa cell tumors by 100% penetrance. If the mice are gonadectomized prepubertally they form adrenocortical tumors instead. Transgenic and wild type mice were treated for three consecutive weeks with control vehicle, Hecate or Hecate-CGbeta conjugate. GnRH antagonist or estradiol was given to a group of mice with or without Hecate-CGbeta conjugate to analyze the additive role of gonadotropin blockage in adrenocortical tumor treatment efficacy. Hecate-CGbeta conjugate was able to diminish the gonadal and adrenal tumor size effectively in males. No treatment related side effects were found. Gonadotropin blockage through GnRH antagonist was the best treatment in female adrenal tumors. The mode of cell death by Hecate-CGbeta conjugate was proven to be through necrosis. LHCGR and GATA-4 were co-expressed in tumors, where the treatment down-regulated their expression simultaneously, suggesting their possible use as tumor markers. In conclusion, the present thesis showed that Hecate-CGbeta conjugate targets its action selectively through LHCGR and selectively kills the LHCGR bearing tumor cells. It works both in gonadal somatic and in ectopic LHCGR bearing adrenal tumors. These results establish a more general principle that receptors expressed ectopically in malignant cells can be exploited in targeted cytotoxic therapies without affecting the normal healthy cells.

Vesicular Trafficking in Osteoclasts

Osteoclasts are multinucleated bone-degrading cells that undergo large changes in their polarisation and vesicular trafficking during the bone resorption cycle. Rab proteins are small GTPases that offer both temporal and spatial regulation to the transport between membranous organelles. Previously the presence and function of only few of the currently known 60 Rab proteins in osteoclasts have been reported. In this study, the expression of 26 Rab genes in bone-resorbing osteoclasts was demonstrated with gene-specific primer pairs. The further analysis of three Rab genes during human osteoclast differentiation revealed that Rab13 gene is highly induced during osteoclastogenesis. The presence of Rab13 protein in the secretory vesicles directed towards the ruffled border and in the endocytotic or transcytotic pathways in resorbing osteoclasts was excluded. The localisation of Rab13 suggests that that it is associated with a previously unknown vesicle population travelling between the trans-Golgi network and the basolateral membrane in bone resorbing osteoclasts. Rab proteins convey their functions by binding to specific effector proteins. We found a novel Rab13 interaction with endospanins-1 and -2 that are yet poorly characterised small transmembrane proteins. The Rab13 subfamily member Rab8 also bound to endospanins, while Rab10 and unrelated Rabs did not. Rab13 and endospanin-2 co-localised in perinuclear vesicles in transfected cells, demonstrating the interaction also in vivo. The inhibition of Rab13 did not interfere with the localisation of endospanin-2 nor did it affect the cell surface expression of growth hormone receptor, as has been previously described for endospanins. The physiological role of this novel protein-protein interaction thus remains to be clarified. The analysis of the transcytotic route in bone resorbing osteoclasts revealed that multiple vesicle populations arise from the ruffled border and transport the bone degradation products for exocytosis. These vesicles are directed to the functional secretory domain that is encircled by an actin-based molecular barrier. Furthermore, the transcytotic vesicles contain abundant Helix pomatia lectin binding sites and represent lipid raft concentrates. Finally, autophagosomal compartments may also be involved in the transcytosis.