The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a.

BACKGROUND: CDKN2A/p16INK4a is frequently altered in human cancers and it is the most important melanoma susceptibility gene identified to date. p16INK4a inhibits pRb phosphorylation and induces cell cycle arrest, which is considered its main tumour suppressor function. Nevertheless, additional activities may contribute to the tumour suppressor role of p16INK4a and could help explain its specific association with melanoma predisposition. To identify such functions we conducted a yeast-two-hybrid screen for novel p16INK4a binding partners. RESULTS: We now report that p16INK4a interacts with the chromatin remodelling factor BRG1. We investigated the cooperative roles of p16INK4a and BRG1 using a panel of cell lines and a melanoma cell model with inducible p16INK4a expression and BRG1 silencing. We found evidence that BRG1 is not required for p16INK4a-induced cell cycle inhibition and propose that the p16INK4a-BRG1 complex regulates BRG1 chromatin remodelling activity. Importantly, we found frequent loss of BRG1 expression in primary and metastatic melanomas, implicating this novel p16INK4a binding partner as an important tumour suppressor in melanoma. CONCLUSION: This data adds to the increasing evidence implicating the SWI/SNF chromatin remodelling complex in tumour development and the association of p16INK4a with chromatin remodelling highlights potentially new functions that may be important in melanoma predisposition and chemoresistance.

Amino terminal hydrophobic import signals target the p14(ARF) tumor suppressor to the mitochondria.

The p14(ARF) tumor suppressor is frequently targeted for inactivation in many human cancers and in individuals predisposed to cutaneous melanoma. The functions of p14(ARF) are closely linked with its subcellular distribution. Nucleolar p14(ARF) dampens ribosome biosynthesis and nucleoplasmic forms of p14(ARF) activate the p53 pathway and induce cell cycle arrest. p14(ARF) can also be recruited to mitochondria where it interacts with many mitochondrial proteins, including Bcl-x(L) and p32 to induce cell death. It has been suggested that the movement of p14(ARF) to mitochondria requires its interaction with p32, but we now show that the ARF-p32 interaction is not necessary for the accumulation of p14(ARF) in mitochondria. Instead, highly hydrophobic domains within the amino-terminal half of p14(ARF) act as mitochondrial import sequences. We suggest that once this hydrophobic pocket is exposed, possibly in a stimulus-dependent manner, it accelerates the mitochondrial import of p14(ARF). This allows the interaction of p14(ARF) with mitochondrial proteins, including p32 and enables p53-independent cell death.

Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells.

Duox NADPH oxidases generate hydrogen peroxide at the air-liquid interface of the respiratory tract and at apical membranes of thyroid follicular cells. Inactivating mutations of Duox2 have been linked to congenital hypothyroidism, and epigenetic silencing of Duox is frequently observed in lung cancer. To study Duox regulation by maturation factors in detail, its association with these factors, differential use of subunits and localization was analyzed in a lung cancer cell line and undifferentiated or polarized lung epithelial cells. We show here that Duox proteins form functional heterodimers with their respective DuoxA subunits, in close analogy to the phagocyte NADPH oxidase. Characterization of novel DuoxA1 isoforms and mispaired Duox-DuoxA complexes revealed that heterodimerization is a prerequisite for reactive oxygen species production. Functional Duox1 and Duox2 localize to the leading edge of migrating cells, augmenting motility and wound healing. DuoxA subunits are responsible for targeting functional oxidases to distinct cellular compartments in lung epithelial cells, including Duox2 expression in ciliated cells in an ex vivo differentiated lung epithelium. As these locations probably define signaling specificity of Duox1 versus Duox2, these findings will facilitate monitoring Duox isoform expression in lung disease, a first step for early screening procedures and rational drug development.

Oncogene-induced senescence does not require the p16(INK4a) or p14ARF melanoma tumor suppressors.

Oncogene-induced senescence is considered to act as a potent barrier to cell transformation, and has been seen in vivo during the early stages of tumor development. Human nevus cells frequently express oncogenic N-RAS or B-RAF, and are thought to be permanently growth arrested. Many studies have suggested that the p16(INK4a) and, to a lesser extent, the p14ARF tumor suppressor proteins act as critical triggers of oncogene-induced senescence in nevi, and thus these proteins represent major inhibitors of progression to melanoma. There have also been reports, however, showing that p16(INK4a) and/or p14ARF is not sufficient to execute the oncogene-induced senescence program. In this study, we examined the impact of melanoma-associated N-RAS(Q61K) on melanocyte senescence and utilized RNA-interference vectors to directly assess the individual contribution of human p14ARF and p16(INK4a) genes to the N-RAS-induced senescence program. We formally show that cultured human melanocytes can initiate an effective oncogene-mediated senescence program in the absence of INK4a/ARF-encoded proteins. Our data are consistent with observations showing that senescent nevus cells do not always express p16(INK4a), and highlight the need to thoroughly explore INK4a/ARF-independent molecular pathways of senescence in human melanocytes.

Inhibitory action of NoxA1 on dual oxidase activity in airway cells.

Imbalance between pro- and antioxidant mechanisms in the lungs can compromise pulmonary functions, including blood oxygenation, host defense, and maintenance of an anti-inflammatory environment. Thus, tight regulatory control of reactive oxygen species is critical for proper lung function. Increasing evidence supports a role for the NADPH oxidase dual oxidase (Duox) as an important source for regulated H2O2 production in the respiratory tract epithelium. In this study Duox expression, function, and regulation were investigated in a fully differentiated, mucociliary airway epithelium model. Duox-mediated H2O2 generation was dependent on calcium flux, which was required for dissociation of the NADPH oxidase regulatory protein Noxa1 from plasma membrane-bound Duox. A functional Duox1-based oxidase was reconstituted in model cell lines to permit mutational analysis of Noxa1 and Duox1. Although the activation domain of Noxa1 was not required for Duox function, mutation of a proline-rich domain in the Duox C terminus, a potential interaction motif for the Noxa1 Src homology domain 3, caused up-regulation of basal and stimulated H2O2 production. Similarly, knockdown of Noxa1 in airway cells increased basal H2O2 generation. Our data indicate a novel, inhibitory function for Noxa1 in Duox regulation. This represents a new paradigm for control of NADPH oxidase activity, where second messenger-promoted conformational change of the Nox structure promotes oxidase activation by relieving constraint induced by regulatory components.