S6 kinase 2 is bound to chromatin-nuclear matrix cellular fractions and is able to phosphorylate histone H3 at threonine 45 in vitro and in vivo.

The activity of S6 kinases (S6K) is highly induced in cancer cells highlighting an essential role in carcinogenesis. The S6K family has two members: S6K1 and S6K2 which bear common as well as distinct features. In an attempt to identify S6K2 unique sequence features compared to S6K1, we applied extensive bioinformatic analysis and motif search approaches. Interestingly, we identified 14 unique protein signatures which are present in proteins directly connected to chromatin and/or involved in transcription regulation. Using chromatin binding assay, we biochemically showed that S6K2 is bound to chromatin as well as nuclear matrix cellular fractions in HEK293 cells. The presence of S6K2 in chromatin fractions raised the possibility that it may be in close proximity to a number of chromatin substrates. For that, we then searched for S6K phosphorylation consensus sites RXRXXT/S in mammalian proteins using the SWISS-PROT database. Interestingly, we identified some potential phosphorylation sites in histone H3 (Thr45). Using in vitro kinase assays and siRNA-based knockdown strategy; we confirmed that S6K2 but not S6K1 or AKT is essential for histone H3-Thr45 phosphorylation in HEK293 cells. Furthermore, we show that the nuclear localisation sequence in the S6K2 C-terminus is essential for this modification. We have found that, H3-Thr45 phosphorylation correlates to S6K activation in response to mitogens and TPA-induced cell differentiation of leukaemic cell lines U937, HL60 and THP1. Overall, we demonstrate that S6K2 is a novel kinase that can phosphorylate histone H3 at position Thr45, which may play a role during cell proliferation and/or differentiation.

Identification of the general transcription factor Yin Yang 1 as a novel and specific binding partner for S6 kinase 2.

S6 kinase is a member of the AGC family of serine/threonine kinases and plays a key role in diverse cellular processes including cell growth and metabolism. Although, the high degree of homology between S6K family members (S6K1 and S6K2) in kinase and kinase-extension domains, the two proteins are highly divergent in the N- and C-terminal regulatory regions, hinting at differential regulation, downstream signalling and cellular function. Deregulated signalling via S6Ks has been linked to various human pathologies, such as diabetes and cancer. Therefore, S6K has emerged as a promising target for drug development. Much of what we know about S6K signalling in health and disease comes from studies of S6K1, as molecular cloning of this isoform was reported a decade earlier than S6K2. In this study, we report for the first time, the identification of the general transcription factor Yin Yang 1 (YY1) as a novel and specific binding partner of S6K2, but not S6K1. The interaction between YY1 and S6K2 was demonstrated by co-immunoprecipitation of transiently overexpressed and endogenous proteins in a number of cell lines, including HEK293, MCF7 and U937. Furthermore, direct association between S6K2 and YY1 was demonstrated by GST pull-down assay using recombinant proteins. A panel of deletion mutants was used to show that the C-terminal regulatory region of S6K2 mediates the interaction with YY1. Interestingly, the complex formation between S6K2 and YY1 can be detected in serum-starved cells, but the interaction is strongly induced in response to mitogenic stimulation. The induction of S6K2/YY1 complex formation in response to serum stimulation is abolished by pre-treatment of cells with the mTOR inhibitor, rapamycin. Furthermore, mTOR is also detected in complex with YY1 and S6K2 in serum-stimulated cells. We utilized size exclusion chromatography along with co-immunoprecipitation analysis to demonstrate the existence of the mTOR/S6K2/YY1 complex in high molecular weight fractions, which might also involve other cellular proteins. The physiological significance of the mTOR/S6K2/YY1 complex, which is induced in response to mitogenic stimulation, remains to be further investigated.

Overexpression of s6 kinase 1 in brain tumours is associated with induction of hypoxia-responsive genes and predicts patients' survival.

mTOR/S6K pathway is a crucial regulator of cell growth and metabolism. Deregulated signalling via S6K has been linked to various human pathologies, including metabolic disorders and cancer. Many of the molecules signalling upstream of S6K have been shown to be either mutated or overexpressed in tumours, leading to S6K activation. The role of S6K1 in brain tumours is not fully investigated. In this study, we investigated the gene expression profile of S6 kinases in brain and CNS tumours using the publically available Cancer Microarray Database. We found that S6K1 but not S6K2 gene is overexpressed in brain tumours and this upregulation is associated with patients' poor survival. Furthermore, we interrogated Oncomine database for the expression profile of hypoxia-induced genes using a literature-defined concept. This gene list included HIF1A, VEGFA, SOX4, SOX9, MMP2, and NEDD9. We show that those genes are upregulated in all brain tumour studies investigated. Additionally, we analysed the coexpression profile of S6K1 and hypoxia responsive genes. The analysis was done across 4 different brain studies and showed that S6K1 is co-overexpressed with several hypoxia responsive genes. This study highlights the possible role of S6K1 in brain tumour progression and prediction of patients' survival. However, new epidemiological studies should be conducted in order to confirm these associations and to refine the role of S6K1 in brain tumours as a useful marker for patients' survival.

FHIT gene and flanking region on chromosome 3p are subjected to extensive allelic loss in Egyptian breast cancer patients.

The fragile histidine triad gene (FHIT) is a candidate tumor suppressor gene at chromosome 3p14.2. Deletions in FHIT gene were reported in different types of cancer including breast cancer. In this study, we investigated the loss of heterozygosity (LOH) incidence that target FHIT genomic structure and chromosome 3p in cancerous and pre-neoplastic lesions of Egyptian breast patients. Genomic DNA was isolated from tumor tissues and their normal counterparts of 55 Egyptian patients diagnosed with breast cancer and 11 patients diagnosed with preneoplastic breast lesions. LOH was detected in 51% of breast cancer cases in at least one microsatellite marker of the four investigated markers. While, none of the markers showed LOH among the pre-neoplastic breast lesions. We also observed a significant association between LOH and invasive ductal carcinoma (IDC) histopathological type while no association observed between LOH and patients' age, tumor grade, or lymph node involvement. We also investigated FHIT gene expression profiles in breast cancer using Oncomine database. We found that FHIT is significantly reduced in all investigated studies. We conclude that, FHIT is underexpressed in breast cancer tissues compared to their normal counterparts due to the extensive allelic loss that is observed in its gene structure.

Multiple Patterns of FHIT Gene Homozygous Deletion in Egyptian Breast Cancer Patients.

Fragile histidine triad (FHIT) gene encodes a putative tumour suppressor protein. Loss of Fhit protein in cancer is attributed to different genetic alterations that affect the FHIT gene structure. In this study, we investigated the pattern of homozygous deletion that target the FHIT gene exons 3 to 9 genomic structure in Egyptian breast cancer patients. We have found that 65% (40 out of 62) of the cases exhibited homozygous deletion in at least one FHIT exon. The incidence of homozygous deletion was not associated with patients' clinicopathological parameters including patients' age, tumour grade, tumour type, and lymph node involvement. Using correlation analysis, we have observed a strong correlation between homozygous deletions of exon 3 and exon 4 (P < 0.0001). Deletions in exon 5 were positively correlated with deletions in exon 7 (P < 0.0001), Exon 8 (P < 0.027), and exon 9 (P = 0.04). Additionally, a strong correlation was observed between exons 8 and exon 9 (P < 0.0001).We conclude that FHIT gene exons are homozygously deleted at high frequency in Egyptian women population diagnosed with breast cancer. Three different patterns of homozygous deletion were observed in this population indicating different mechanisms of targeting FHIT gene genomic structure.