Abstract #5515: The p53 and Ink4A independent tumor suppressor activity of ARF in melanoma
Authors: Prasun Mishra, Linan Ha, Jennifer Rieker, Curtis Henrich, Dorothy Bennett, Elena Sviderskaya, Glenn Merlino
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

Cutaneous malignant melanoma is an extremely aggressive and often fatal disease, accounting for 75% of all deaths associated with skin cancer due to its inherent drug-resistant nature. Activating mutations in either NRAS or BRAF are common in melanoma, representing a critical driving force. Unlike other cancers, mutation/deletion of TP53 is relatively rare in melanoma, whereas expression of p14ARF, the positive regulator of p53, is often lost along with p16INK4A at the CDKN2A locus. We have recently demonstrated that Arf acts as a regulator of mouse melanocyte senescence, independent of p53 activity (Ha et al. 2007, PNAS). In fact, re-expression of ARF in two human melanoma cell lines (A375P and 451Lu) was found to induce senescence. Here, we provide evidence for a prominent role for Arf in melanoma genesis and progression, independent of Ink4A or p53. Primary melanocytes obtained from genetically engineered mice lacking either Arf, Ink4A, or both were transfected with a vector containing activated NRAS ; p53 levels in melanocytes were stably knocked down in order to provide insights into p53-independent Arf function. Melanocytes deficient in either Ink4A-Arf or Arf alone were transformed upon NRAS activation, demonstrating enhanced proliferation, anchorage-independentgrowth in soft agar, loss of pigmentation and rapid development of melanoma in nude mice. Remarkably, NRAS expressing, Arf-deficient melanocytes could form metastatic lesions in the lung, liver, pancreas and heart. In contrast, inactivation of Ink4A and p53 in combination with NRAS activation was insufficient to transform melanocytes or significantly alter their phenotype. Using microarray analysis, with validation by Western blotting, we discovered that the majority of genes involved in melanin biosynthetic/metabolic process were differentially expressed in non-transformed ( NRAS ; Ink4A-/-) relative to transformed ( NRAS ; Ink4A-Arf-/-) melanocytes. Specifically, in the transformed melanocytes genes involved in melanin synthesis ( Tyrp1, Tyr, Dct, Mc1r, Mitf ) and chromatin modification ( Dnmt3a, Smarca4 ) were down-regulated, whereas genes involved in antigen processing and presentation (H2-Q6, H2-D1, H2-K1, H2-L) were up-regulated. Our data suggest that in the presence of activated NRAS, Arf-deficiency is sufficient to block melanocyte differentiation, promote transformation and induce metastasis. Since melanoma is resistant to conventional chemotherapy, a therapeutic approach designed to restore Arf-mediated differentiation and/or senescence in transformed melanocytes may be a better anti-melanoma strategy. The NRAS-expressing, Arf-deficient melanocytes are currently being used to screen for compounds in the hope of developing novel anti-melanoma therapy (funded by NCI contract N01-CO-12400). Our work suggests that Arf, Ink4A and p53 suppress tumorigenesis through distinct, lineage-dependent mechanisms.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5515.