SRSF2 is a serine/arginine-rich protein belonging to the family of SR proteins that are crucial regulators of constitutive and alternative pre-mRNA splicing. Although it is well known that phosphorylation inside RS domain controls activity of SR proteins, other post-translational modifications regulating SRSF2 functions have not been described to date. In this study, we provide the first evidence that the acetyltransferase Tip60 acetylates SRSF2 on its lysine 52 residue inside the RNA recognition motif, and promotes its proteasomal degradation. We also demonstrate that the deacetylase HDAC6 counters this acetylation and acts as a positive regulator of SRSF2 protein level. In addition, we show that Tip60 downregulates SRSF2 phosphorylation by inhibiting the nuclear translocation of both SRPK1 and SRPK2 kinases. Finally, we demonstrate that this acetylation/phosphorylation signalling network controls SRSF2 accumulation as well as caspase-8 pre-mRNA splicing in response to cisplatin and determines whether cells undergo apoptosis or G 2 /M cell cycle arrest. Taken together, these results unravel lysine acetylation as a crucial post-translational modification regulating SRSF2 protein level and activity in response to genotoxic stress.

The INK4a/ARF locus which is frequently inactivated in human tumours encodes two dierent tumour suppressive proteins, p16 INK4a and ARF. p16INK4a is a major component of the RB pathway. ARF is part of an ARF-mdm2-p53 network that exerts a negative control on hyperproliferative signals emanating from oncogenic stimuli. Among these is the transcription factor E2F1, a ®nal eector of the RB pathway, that induces ARF expression. Recent data suggest that ARF function is not restricted to the p53 pathway. However, ARF target(s) implicated in this p53-independent function remains to be identi®ed. We show that ARF is able to inhibit the proliferation of human cell lines independently of their p53 status. In this context, we demonstrate that ARF interacts physically with E2F1 and inhibits its transcriptional activity. Moreover, we show that mdm2 is required for the modulation of E2F1 activity by ARF. Beside the well-known p53 and mdm2 partners, these results identify E2F1 as a new ARF target. Thus, ARF can be viewed as a dual-acting tumour suppressor protein in both the p53 and RB pathways, further emphasizing its role in tumour surveillance. Oncogene (2001) 20, 1033 ± 1041.

The INK4b-ARF-INK4a locus encodes two members of the INK4 family of cyclin-dependent kinase inhibitors, p15 INK4b and p16 INK4a , and a completely unrelated protein called ARF. ARF is a nucleolar protein with unusual structure that exhibits tumor suppressive functions. There is growing evidence that ARF signaling is complex, and involves p53-dependent or -independent pathways aiming mainly at restraining abnormal cell growth and at maintaining genomic stability. As such, ARF is a critical component of tumor surveillance, and its expression is decreased in human tumors. In this review, we present the current knowledge on ARF regulation and major functions. The ARF status in human tumors is also briefly summarized.ARF (known as p14 ARF in human and p19 ARF in mouse) was originally identified as an alternative transcript of the INK4b-ARF-INK4a locus located on human chromosome 9p21. 1 This locus encodes two members of the INK4 family of cyclin-dependent kinase inhibitors, p15 INK4b and p16 INK4a , that regulate progression through the G1 phase of the cell cycle. The intercalation of an additional exon (called exon 1b) between INK4b and INK4a renders the simple tandem arrangement more complex, as its transcription by a distinct promoter produces a transcript that also incorporates exons 2 and 3 of INK4a. However, because exon 2 of ARF is translated in an alternative reading frame (ARF) to that used for INK4a, the ARF product is unrelated to the INK4a protein.As a consequence, p16 INK4a and ARF are not isoforms, do not share any amino acid homology and have distinct functions in the cells. Nevertheless, like p16 INKA4 , ARF exhibits tumor suppressive functions as demonstrated by the tumor susceptibility phenotype of ARF/INK4a-deficient mice (Table 1). Indeed, mice that are defective for any one of the genes have increased susceptibility to spontaneous or carcinogeninduced tumors, albeit to different degrees. 3,5 Arf-null mice develop tumors early in life whereas p16 INK4a -null mice do not show predisposition to spontaneous tumor within 17 months. 6 Furthermore, the phenotype resulting from disruption of both p16 INK4a and p19 ARF is comparable to that produced by disruption of p19 ARF alone. 2,5 Although these results suggest a predominant role of p19 ARF over p16 INK4a , each product acts in a nonredundant manner to significantly contribute to tumorigenesis in vivo, and the effects are exacerbated in animals that lack both genes. Studies of mouse embryo fibroblasts (MEFs) derived from the knockout strains also emphasize the independent role of ARF. In wild type MEFs, both p19 ARF and p16 INK4a accumulate significantly after passaging. However, spontaneous escape from senescence occurs through loss of the ARF-p53 axis rather than INK4a-Rb. In addition, ARF-null MEFs expressing functional p16 INK4a fail to undergo crisis after multiple passages in vitro and are efficiently transformed by oncogenic Ha-ras. 5 In contrast, p16 INK4a -null MEFs display a phenotype similar to wild-type MEFs and are not sensitive to Ha...

Purpose: Epigenetic modifications of histone have crucial roles in the control of gene activity, nuclear architecture, and genomic stability. In this respect, they may contribute to the development and progression of cancer. We investigated whether epigenetic changes of histone H4 are involved in lung carcinogenesis. Experimental Design: Epigenetic modifications of histone H4 were studied by immunohistochemistry in normal lung and 157 lung carcinoma using antibodies specifically recognizing the acetylated (Ac) lysines 5 (K5), K8, K12, K16, and trimethylated (me3) K20 residues of histone H4. Western blotting was used to validate the immunohistochemistry results. H4K20me3 was also studied in 17 preneoplastic lesions. Expression of the Suv4-20h1/2 trimethyltransferases was analyzed by quantitative reverse transcription-PCR in a subset of tumor samples. Results: As compared with normal lung, cancer cells displayed an aberrant pattern of histone H4 modifications with hyperacetylation of H4K5/H4K8, hypoacetylation of H4K12/H4K16, and loss of H4K20 trimethylation. Alteration of H4K20 trimethylation was frequent in squamous cell carcinoma (67%) and was observed in early precursors lesions in which the level of H4K20me3 staining strongly decreased with disease progression. In adenocarcinoma, the down-regulation of H4K20me3 was less frequent (28%) but allowed the identification of a subgroup of stage I adenocarcinoma patients with reduced survival (P = 0.007). Loss of H4K20 trimethylation was associated with decreased expression of Suv4-20h2, a specific H4K20 trimethyltransferase involved in telomere length maintenance. Conclusions: Our findings indicate an important role of histone H4 modifications in bronchial carcinogenesis and highlight H4K20me3 as a candidate biomarker for early detection of and therapeutic approaches to lung cancer.Lung cancer is the leading cause of death from cancer among males in Europe and both men and women in the United States. Non -small cell lung cancer (NSCLC) accounts for almost 80% of such deaths (1 -3). In spite of diagnosis and treatment improvements, the 10-year overall lung cancer survival is less than 10%, claiming more deaths than breast, colon, and prostate cancers all together. Early diagnosis allowing surgical resection with a 60% to 80% 5-year survival is done in only less than 25% of the patients. Therefore, the identification of the molecular biomarkers of clonal selection in lung cancer as well as the comprehension of the mechanisms of their participation in the lung carcinogenesis process are required to establish the prognostic factors of progression of preneoplastic and invasive tumors toward earlier diagnosis, and to define more efficient therapies with lower side effects.Lung cancer is the end result of a process that associates multifocal morphologic transformation and multistep accumulation of molecular abnormalities (4). The clonal selection leading to proliferation and invasion is achieved by disruption of the cell growth controls, with a tumor progression k...

Until recently, the ability of ARF (human p14(ARF), murine p19(ARF)) tumour-suppressor protein, encoded by the INK4A/ARF locus, to inhibit cell growth in response to various stimuli was related to its ability to stabilize p53 through the so-called ARF/MDM2/p53 pathway. However, recent data have demonstrated that ARF is not implicated in this unique p53-dependent pathway. By use of transient and stable expression, we show here that human p14(ARF) inhibits the growth of human tumoral cells lacking functional p53 by inducing a transient G(2) arrest and subsequently apoptosis. This p14(ARF)-induced G(2) arrest was correlated with inhibition of CDC2 activity, inactivation of CDC25C phosphatase and induction of the CDK inhibitor p21(WAFI). Apoptosis was demonstrated using Hoechst 33352 staining, proteolytic activation of caspase-3 and PARP cleavage. Similar results were obtained in experiments with cells synchronized by hydroxyurea block. Importantly, we were able to reproduce these effects 'in vivo' by showing that p14(ARF) inhibits the growth of p53 nullizygous human tumours in nude mice and induces the regression of p53 -/- established tumours. In these experiments, tumoral regression was associated with inhibition of cell proliferation as well as induction of apoptosis confirming the data obtained in cell lines.

The transcription factor E2F1 has a key function during S phase progression and apoptosis. It has been well-demonstrated that the apoptotic function of E2F1 involves its ability to transactivate pro-apoptotic target genes. Alternative splicing of pre-mRNAs also has an important function in the regulation of apoptosis. In this study, we identify the splicing factor SC35, a member of the SerRich Arg (SR) proteins family, as a new transcriptional target of E2F1. We demonstrate that E2F1 requires SC35 to switch the alternative splicing profile of various apoptotic genes such as c-flip, caspases-8 and -9 and Bcl-x, towards the expression of pro-apoptotic splice variants. Finally, we provide evidence that E2F1 upregulates SC35 in response to DNA-damaging agents and show that SC35 is required for apoptosis in response to these drugs. Taken together, these results demonstrate that E2F1 controls pre-mRNA processing events to induce apoptosis and identify the SC35 SR protein as a key direct E2F1-target in this setting. Cell Death and Differentiation (2008) 15, 1815-1823 doi:10.1038/cdd.2008 published online 19 September 2008 Pre-mRNA splicing is an essential step for the expression of most genes in higher eukaryotic cells. This process has emerged as an important mechanism of genetic diversity as about 74% of human genes undergo alternative splicing, leading to the production of various protein isoforms. 1 SC35 belongs to the serine/arginine-rich (SR) protein family, one of the most important class of splicing regulators. Members of the SR family have a modular structure consisting of one or two copies of an N-terminal RRM (RNA-recognition motif) followed by a C terminus rich in serine and arginine residues known as the RS domain. They act at multiple steps of spliceosome assembly and participate in both constitutive and alternative splicing. 2 Together with most of the other splicing factors, SR proteins localize to nuclear subregions termed nuclear speckles. 3 Extensive serine phosphorylation of the RS domain has an important function in the regulation of both the localization and the activities of SR proteins. 4 Although the splicing functions of SR proteins have been well documented in vitro, their roles and physiological targets in vivo are less well known. However, based on gene targeting experiments demonstrating that they are required for cell viability and/or animal development, SR proteins undoubtedly control essential biological functions.Apoptosis is one of the cellular processes in which alternative splicing has an important regulatory function. Indeed, a remarkable number of transcripts that encode proteins involved in the apoptotic pathway are subjected to alternative splicing. This usually drives the expression of proteins with opposite functions, either pro-or anti-apoptotic. 5 Interestingly, changes in SR protein phosphorylation have been observed upon apoptotic stimulation following activation of the Fas receptor. 6 In addition, in vitro overexpression experiments have suggested a potential role for ...

Splicing abnormalities frequently occur in cancer. A key role as splice site choice regulator is played by the members of the SR (Ser/Arg-rich) family of proteins. We recently demonstrated that SRSF2 is involved in cisplatin-mediated apoptosis of human lung carcinoma cell lines. In this study, by using immunohistochemistry, we demonstrate that the SR proteins SRSF1 and SRSF2 are overexpressed in 63% and 65% of lung adenocarcinoma (ADC) as well as in 68% and 91% of squamous cell lung carcinoma (SCC), respectively, compared to normal lung epithelial cells. In addition, we show that SRSF2 overexpression correlates with high level of phosphorylated SRSF2 in both ADC (p<0.0001) and SCC (p = 0.02), indicating that SRSF2 mostly accumulates under a phosphorylated form in lung tumors. Consistently, we further show that the SR-phosphorylating kinases SRPK1 and SRPK2 are upregulated in 92% and 94% of ADC as well as in 72% and 68% of SCC, respectively. P-SRSF2 and SRPK2 scores are correlated in ADC (p = 0.01). Using lung adenocarcinoma cell lines, we demonstrate that SRSF1 overexpression leads to a more invasive phenotype, evidenced by activation of PI3K/AKT and p42/44MAPK signaling pathways, increased growth capacity in soft agar, acquisition of mesenchymal markers such as E cadherin loss, vimentin and fibronectin gain, and increased resistance to chemotherapies. Finally, we provide evidence that high levels of SRSF1 and P-SRSF2 proteins are associated with extensive stage (III–IV) in ADC. Taken together, these results indicate that a global deregulation of pre-mRNA splicing regulators occurs during lung tumorigenesis and does not predict same outcome in both Non Small Cell Lung Carcinoma histological sub-types, likely contributing to a more aggressive phenotype in adenocarcinoma.

The transcription factor E2F1 is a key component of cell cycle that acts to transactivate genes required for S phase entry. Thus, it plays an important role in cellular proliferation, oncogenesis and di erentiation. In order to investigate its potential implication in human lung carcinogenesis, we studied E2F1 protein expression by Western blotting and immunohistochemistry in a series of 58 human lung tumours of all histological types. We showed that E2F1 product was overexpressed in 92% (24/26) of small cell lung carcinoma (SCLC) and in 50% (5/10) of large cell neuroendocrine carcinoma (LCNEC) whereas it was undetectable in 90% (10/11) of adenocarcinoma and 82% (9/11) of squamous carcinoma when compared to corresponding normal lung. No ampli®cation was found but an increase in E2F1 mRNA expression was detected in 75% (18/24) of SCLC overexpressing E2F1 product. In these tumours and in contrast with NSCLC, upregulation of E2F1 product was associated with its nuclear accumulation and with overexpression of several of its target-genes. Moreover, E2F1 overexpression in NE lung tumours was signi®-cantly associated with a high KI67 index (P50.0001) as well as a Bcl-2:Bax ratio 41 (P50.001). Overall, these results demonstrate a distinct pattern of E2F1 expression in human lung tumours and suggest that its deregulation could be involved in the carcinogenesis of SCLC.