Nature. 2005;436:642. Nature. 2005;436: Emerging Concept of Oncogene-induced senescence  Oncogene induced senescence (OIS) was first observed following expression of an oncogenic form of RAS in normal human fibroblasts.  The demonstration that certain oncogenes can elicit a senescence response in vivo provided unambiguous evidence that OIS is an important mode of tumor suppression.

Critical requirement for mitogenic signal pathways in Ras-induced senescence  Paradoxically the Raf-MEK-ERK signaling cascade that provides cells with constitutive mitogenic signals also triggers Ras-induced cell cycle arrest through accumulation of p16 Ink4a, p19 Arf, p21 Waf1 and p53.  Perturbation of PI3K signalling through loss of PI3K or the use of PI3K inhibitors renders primary murine embryonic fibroblasts (MEFs) susceptible to premature senescence. J Biol Chem. 2000;275: Nature. 2005;436:

 Loss of p53 or its upstream regulator p19 Arf permits cellular transformation of H-Ras V12 - transduced primary MEFs, while re-establishment of p53 expression in tumors induces features of cellular senescence and tumor regression.  The functional significance of p53 in human fibroblast senescence is eclipsed by p16 Ink4a which governs the irreversibility of the phenotype but conditional activation of p53 in p53 deficient human tumor cells has been reported to promote irreversible cell cycle arrest with features of senescence. Methods Mol Biol. 2013;965: Regulation of p53 function and consequences for Ras- induced senescence

Cell. 2006;127: Cell. 2007;130:  The INK4a/ARF/INK4b locus encodes three genes within 35kb; ARF, p15 INK4b, and p16 INK4a. Members of the INK4 family of cyclin-dependent kinase inhibitors bind to and inactivate CDK4/6.  ARF inhibits MDM2, resulting in p53 stabilization. RD INK/ARF indicates a newly discovered origin of replication 5′ top15 INK4b that participates in the transcriptional silencing of theINK4a/ARF/INK4b locus. Senescence-promoting activity of p16INK4a and ARF

Genes Dev. 2012;26:  PTEN depletion abrogates BRAF V600E -induced senescence in human fibroblasts and melanocytes in melanocytic nevi (moles).  The PI3K pathway was often activated through either decreased PTEN or increased AKT3 expression in melanomas relative to their adjacent nevi. Abrogation of BRAF V600E -induced senescence by PI3K pathway activation contributes to melanomagenesis 4-OHT-treated Tyr::CreER; BRaf CA Acute in vivo PTEN depletion in BRAF V600E - expressing nevi drives tumor formation. a population of plump to elongated cells (red arrows) some of which are laden with melanin pigment, highlighting the characteristic melanocytic dendrites (black arrows).

Genes Dev. 2012;26: Trends Biochem Sci. 2014;39:  There is an optimal window of PTEN expression allowing BRAF V600E -expressing melanocytes to abrogate OIS bypass: Too high levels do not allow for proliferation, whereas too low levels are cytotoxic. Abrogation of BRAF V600E -induced senescence by PTEN depletion is shRNA dose-dependent.

Working Hypothesis What kind of changes in signal transduction are required for the development of Braf V600E -induced melanoma???

Tyrosinase::CreERT2 (TC) Braf v600E Cdkn2a flox mice 4-OHT Cdkn2a/Braf nevi were approximately three times larger and more numerous than nevi induced by Braf V600E alone melanocyte escape from hair follicles prior to nevus formation growth arrest The similarities in nevus formation and progression kinetics were striking and suggested that similar processes may be enforcing growth arrest in both the Cdkn2a/Braf and Trp53/Braf models.

28-day-old 90-day-old Cdkn2a/Braf mice Despite inactivation of p53, melanocytic nevi still exhibited a growth-arrest phenotype that was similar to the Cdkn2a/Braf mode and stochastically progressed to melanoma.

S100 nevus cells melanoma cells Cdkn2a/Braf mice

reverse tetracycline-activated transactivator rtTA2s-M2 (Nature. 2011;469: ) To enable detailed study of melanocyte biology in vivo, a mouse model was established in which expression of the reverse tetracycline-activated transactivator rtTA2s-M2, characterized by minimal leakiness and background, was regulated by the melanocyte- specific dopachrome tautomerase (Dct) gene promoter. iDct-GFP mice exhibited an inducible GFP profile from embryonic through adult stages consistent with known Dct expression patterns. (Nature. 2011;469: ) melanocyte escape from HF Cdkn2a depletion caused increased number of nevi and induced growth-arrested conditions. Day17 Day50

GSEA revealed that mTOR signaling was the most differentially regulated pathway between pre-arrest and growth-arrested melanocytes, with it being downregulated in growth-arrested cells. Both the activation of IGF1R/mTOR and increased protein synthesis were associated with progression of nevi to melanoma.

PI3K/Akt and mTOR survival signals were transiently inactivated at the growth-arrested nevi and again reactivated in melanoma. P, pregrowth arrest nevi G, growth arrested nevi M, melanoma

Cdkn2a/Braf nevi showed strong nuclear staining of cyclin kinase inhibitors (CKIs), such as p21 and p27, which were often lost or mis-localized to the cytoplasm in tumors Pre-arrest Growth-arrested Melanoma Cdkn2a -/- Cyclin kinase inhibitors gradually decrease in the transition from pre-arrest nevi to melanoma via growth-arrested nevi.

 The serine–threonine liver kinase B1 (LKB1) is inactivated in Peutz–Jeghers syndrome and a large percentage of sporadic non-small cell lung carcinomas and cervical carcinomas.  The LKB1–AMPK pathway serves as a metabolic checkpoint in the cell, arresting cell growth in conditions of low intracellular ATP levels, such as in low nutrient conditions. LKB1 regulates Akt-mediated survival independently of PI3K. Nature Reviews Cancer 2009;9:

Lkb1/Braf mice Braf mice Inactivation of Lkb1 during Braf-induced proliferation abrogated detectable growth arrest.  Rather than forming discrete growth-arrested lesions, melanocytic proliferations in Lkb1/Braf mice grew to confluence.  In contrast to Lkb1/NRAS mice, Lkb1/Braf mice did not develop frank melanomas. Nevus 9-month-old Lkb1/Braf mouse dorsal skin after removal of hair P: confluent areas of melanocytes in the dermis

As compared with the nevi of Cdkn2a/Braf mice, those of Lkb1/Braf show higher PI3K/Akt and mTOR signal activities.

p-Akt (p-S473) ; mTORC2 activity Cdkn2a/Braf tumors showed avid 2-NBDG uptake but growth- arrested nevi did not. In Lkb1/Braf lesions, 2-NBDG uptake was also not detectable. In striking contrast to Lkb1/Braf lesions, 2-NBDG was taken up diffusely in the palpable masses in Cdkn2a/Lkb1/Braf mice. Lkb1/Braf lesions, which have bypassed OIS, are not frankly malignant, suggesting additional factors are required for full progression to malignancy such as Cdkn2a depletion.

Cdkn2a/Lkb1/Braf mice die soon after the distant metastasis. liver spleen

Activation of the mTORC2/Akt pathway is associated with full progression to malignancy. mTOR activity Activation of Akt via activation of mTORC2 is critical for inducing a fully malignant phenotype characterized by diffuse 2-NBDG uptake and rapid, infiltrative growth.

Treatment of melanoma cell lines with miR-99/100 mimics resulted in down-regulation of IGF1R in addition to the inhibition of proliferation.

Take Home Message how melanocytes escape OIS at the very earliest stages of transformation in Braf-induced melanoma. Activation of mTORC1 and mTORC2 is required to bypass Braf-induced OIS and identifies a miRNA family that regulates mTOR in this setting. This article represent significant progress in understanding how melanocytes escape OIS at the very earliest stages of transformation in Braf-induced melanoma. The combination of Braf activation and Cdkn2a loss was sufficient to prime progression of nevi to melanoma.