POMC Overexpression Stimulates MITF/HIF-1α Survival Pathway in B16-F10 Melanoma Cells

• Main Authors: Yu-Fen Kuo, 郭餘芬
• Other Authors: Ming-Hong Tai
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/a6q458

碩士 === 國立中山大學 === 生物科學系研究所 === 96 === Melanoma is a cancer of the pigment producing cells, melanocytes, and is the most serious type of skin cancer. Cancer is a condition in which one type of cell grows without limit in a disorganized fashion, disrupting and replacing normal tissues and their functions. Normal melanocytes reside in the outer layer of the skin and produce a brown pigment called melanin, which is responsible for skin color. Melanoma occurs when melanocytes become cancerous, grow, and invade other tissues. Pro-opiomelanocortin (POMC) is a precursor polypeptide of 241 amino acids and the prohormone of various neuropeptide, including corticotropin (ACTH), α-melanocyte-stimulating hormone (α-MSH), and β-endorphin (β-EP). Recently, we demonstrated that systemic POMC overexpression potently suppresses the growth and metastasis of B16-F10 melanoma in vitro and in vivo. However, despite potent inhibition of tumor proliferation and angiogenesis, B16-F10 melanoma still managed to survive after POMC gene therapy. The underlying survival mechanism of B16-F10 melanoma remains unclear. Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix transcription factor that plays a key role not only in melanin synthesis, but also in melanocyte development and survival. Besides, MITF binds to the hypoxia-inducible factor-1α (HIF-1α) promoter to stimulate its transcriptional activity. In this study, we investigate the influence of POMC gene delivery on the pro-survival MITF/HIF-1α pathway in B16-F10 melanoma cells. Quantitative RT-PCR and western blot analysis revealed that POMC gene delivery increased the MITF mRNA and protein level in B16-F10 melanoma cells. Besides, POMC gene delivery significantly enhanced the HIF-1α-driven luciferase activities in melanoma cells. By transfection and puromycin selection, we generated and characterized a MITF-knockdown B16-F10 melanoma cells (MITF KD) stably expressing short hairpin RNA against MITF. The growth, invasion, and colonies formation of MITF-KD were similar to those of vector control. However, implantation of MITF-KD cells led to melanoma with significantly reduced tumor size compared with those in mice implanted with vector control cells. Histological analysis revealed a significant reduction of CD31-positive blood vessels in implantation of MITF-KD cells-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, POMC-mediated upregulation of MITF and HIF-1 α was significantly attenuated in MITF KD-B16-F10 cells. Acetylsalicylic acid (aspirin; ASA) is widely used as an analgesic/antipyretic drug. ASA exhibits a wide range of biological effects, including preventative effects against heart attack, stroke, and the development of some types of cancer. In our study, we found ASA enhanced cell proliferation. However, in invasion test, ASA had no effect on cell migration. POMC gene delivery elevated the mRNA and protein level of hemeoxygenase-1 (HO-1), a downstream effector of HIF-1α pathway and an enzyme catalyzing the converting reaction of heme to carbon monoxide, ion and biliverdine. Inhibition of HO-1 activities augmented the inhibitory effect of POMC gene delivery on proliferation, migration and anchorage-independent growth of B16-F10 melanoma cells. These studies indicated that activation of MITF/HIF-1α/HO-1 indeed contributes to melanoma survival after POMC gene delivery.