Therapeutic utility of targeting protein synthetic machinery in melanoma
Open Access
- Author:
- Kardos, Gregory Robert
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 13, 2014
- Committee Members:
- Gavin Peter Robertson, Dissertation Advisor/Co-Advisor
Jin Ming Yang, Committee Member
Gregory Steven Yochum, Committee Member
Scot R Kimball, Special Member - Keywords:
- melanoma
RPL13
ALDH18A1
ribosome
proline
nanoliposome
protein synthesis
P5CS
GCN2
eIF2alpha - Abstract:
- Malignant melanoma is a cancer with few treatment options and thus, a very poor prognosis. Targeted therapies directed at commonly over-activated signaling pathways in melanoma have limited success due to development of drug resistance. The mitogen-activated protein (MAP) kinase and PI3K/AKT pathways are commonly over-activated in melanoma. Both pathways regulate protein synthetic machinery through RNA polymerase I transcription and mRNA translation initiation. Resistance to mutant V600EBRAF targeted therapies can be mediated through the mRNA translation initiation complex eIF4F, revealing that the therapeutic efficacy of MAPK targeting in melanoma is mediated though protein synthesis. Thus, common drivers of melanoma progression regulate protein synthesis to promote melanoma development. Since protein synthesis deregulation is a critical facilitator of melanoma development, regulation of protein synthesis is a potential therapeutic approach to impair melanoma growth. The aim of this dissertation is to identify novel therapeutic targets for the treatment of malignant melanoma. The protein synthetic machinery is an attractive target to therapeutically investigate. Ribosome biogenesis is one component of this machinery, which is required for proper ribosome formation. Disruption of ribosome biogenesis can impair ribosome production and activate a stress response mediated by p53. Chapter 2 discusses how targeting ribosomal proteins such as RPL13 impaired melanoma growth by decreasing protein synthesis and stabilizing p53. Another approach to impair protein synthesis is by disruption of mRNA translation initiation. Translation initiation requires the eukaryotic initiation factor eIF2. Stresses such as amino acid insufficiency cause the phosphorylation and inactivation of the α subunit of eIF2 by GCN2. Chapter 3 investigates how proline biosynthesis disruption by targeting ALDH18A1 with siRNA to deplete P5CS protein activated GCN2, impaired protein synthesis, and decreased melanoma tumor growth. Neither RPL13 nor P5CS are currently druggable targets. A major limitation of targeted therapies is the inability to effectively and specifically disrupt many targets. RNA interference (RNAi) by the delivery of short interfering RNA (siRNA) in liposomes offers a potential solution to these limitations. Chapter 4 examines the utility of targeting RPL13 or ALDH18A1 via siRNA-loaded nanoliposomes for the therapeutic treatment of melanoma. The protein synthetic machinery is a key mediator of melanoma development. Ribosome biogenesis is one component capable of modulating protein synthesis through ribosome production. In chapter 2, the role of large subunit ribosomal proteins (RPLs) in melanoma was dissected to determine the therapeutic potential of targeting RPLs. Based on the consequences of siRNA-mediated knockdown, two groups of RPLs were identified and categorized with respect to their effects on melanoma cell viability and protein synthesis. Targeting the first group negligibly affected cell viability and protein synthesis while targeting the second group significantly decreased cell viability and protein synthesis. A subset of this second group was capable of stabilizing p53 following protein knockdown. Targeting RPL13, a representative of this group, increased p53 stability mediated by the inhibition of MDM2 by RPL5 and RPL11. RPL13 knockdown caused p53-dependent cell cycle arrest, decreased protein synthesis, and impaired melanoma tumor development. Thus, certain ribosomal proteins can be therapeutically targeted for the treatment of melanoma. Regulation of mRNA translation initiation can also modulate protein synthesis. The ternary complex of met-tRNA, eIF2, and GTP is necessary for mRNA translation initiation to proceed. Regulation of this complex through eIF2 controls protein synthesis. Amino acid insufficiency can activate the GCN2 kinase to inactivate eIF2. Aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) encodes pyrroline-5-carboxylate synthase (P5CS), an enzyme necessary for proline biosynthesis. Chapter 3 studies the effect of impairing proline biosynthesis on melanoma development. siRNA targeting of ALDH18A1 decreased intracellular proline levels by 66 to 85% and decreased melanoma cell growth rate by 56 to 96% without affecting apoptosis, autophagy, or cell cycle arrest. Melanoma tumor growth was inhibited by 45 to 99% upon ALDH18A1 inhibition. Mechanistically, ALDH18A1 knockdown proline-dependently activated the GCN2 pathway and impaired protein synthesis. Collectively, these data suggest that the protein synthetic machinery can be impaired by proline biosynthesis disruption, providing a novel therapeutic target for melanoma treatment. Chapters 2 and 3 discuss novel therapeutic targets capable of disrupting the protein synthetic machinery to impair melanoma growth, however neither target is currently druggable. To overcome this limitation, chapter 4 investigates the use of siRNA-encapsulated liposomes to target RPL13 or ALDH18A1 in melanoma. Transfection of melanoma cells with liposome-polycation-DNA (LPD) complexes carrying siRNA targeting RPL13 or ALDH18A1 knocked down target protein levels and inhibited melanoma cell viability by up to 50%. Melanoma xenograft mice treated with siRNA-LPD complexes decreased melanoma tumor growth by 40 to 55%. These results suggest that RPL13 and ALDH18A1 are effective targets for melanoma treatment however better approaches are needed to improve upon the limitations of targeted therapies.