Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease that shows minimal response to chemotherapy. Genetic changes involved in the progression of PDAC concern genes that encode proteins related to signal transduction networks. This fact reveals the importance in identifying the role and the relations between multiple signaling cascades in PDAC. One of the major factors that modulate signaling events is multidomain scaffold proteins that function by binding several proteins simultaneously, inducing their proximity and influencing the outcome of signaling. A particular group among them, containing multiple Src homology 3 (SH3) domains that can bind proteins containing proline-rich motifs, was associated to different aspects of cancer cell homeostasis. In this work, using a microarray-based analysis, we have shown that 13 multiple SH3 domain containing scaffold proteins are expressed in PDAC cells. Using a yeast two-hybrid approach, we have identified proteins that interact with these adaptor proteins. Among them we have found several molecules that modulate cell proliferation and survival (CIZ1, BIRC6, RBBP6), signaling (LTBP4, Notch2, TOM1L1, STK24) and membrane dynamics (PLSCR1, DDEF2, VCP). Our results indicate that interactions mediated by multi-SH3 domain-containing proteins could lead to the formation of dynamic protein complexes that function in pancreatic cancer cell signaling. The identification of such protein complexes is of paramount importance in deciphering pancreatic cancer biology and designing novel therapeutic approaches.
The INhibitor of Growth tumor suppressors (ING1-ING5) affect aging, apoptosis, DNA repair and tumorigenesis. Plant homeodomains (PHD) of ING proteins bind histones in a methylation-sensitive manner to regulate chromatin structure. ING1 and ING2 contain a polybasic region (PBR) adjacent to their PHDs that binds stress-inducible phosphatidylinositol monophosphate (PtIn-MP) signaling lipids to activate these INGs. ING1 induces apoptosis independently of p53 but other studies suggest proapoptotic interdependence of ING1 and p53 leaving their functional relationship unclear. Here we identify a novel ubiquitin-binding domain (UBD) that overlaps with the PBR of ING1 and shows similarity to previously described UBDs involved in DNA damage responses. The ING1 UBD binds ubiquitin with high affinity (Kd∼100 nM) and ubiquitin competes with PtIn-MPs for ING1 binding. ING1 expression stabilized wild-type, but not mutant p53 in an MDM2-independent manner and knockdown of endogenous ING1 depressed p53 levels in a transcription-independent manner. ING1 stabilized unmodified and six multimonoubiquitinated forms of wild-type p53 that were also seen upon DNA damage, but not p53 mutants lacking the six known sites of ubiquitination. We also find that ING1 physically interacts with herpesvirus-associated ubiquitin-specific protease (HAUSP), a p53 and MDM2 deubiquitinase (DUB), and knockdown of HAUSP blocks the ability of ING1 to stabilize p53. These data link lipid stress signaling to ubiquitin-mediated proteasomal degradation through the PBR/UBD of ING1 and further indicate that ING1 stabilizes p53 by inhibiting polyubiquitination of multimonoubiquitinated forms via interaction with and colocalization of the HAUSP-deubiquitinase with p53.
An age-associated isoform of ING1, ING1a, induces cell senescence by altering endocytosis, subsequently activating the retinoblastoma tumor suppressor.
BackgroundThe androgen receptor (AR) is a major driver of prostate cancer, and increased AR levels and co-activators of the receptor promote the development of prostate cancer. INhibitor of Growth (ING) proteins target lysine acetyltransferase or lysine deacetylase complexes to the histone H3K4Me3 mark of active transcription, to affect chromatin structure and gene expression. ING3 is a stoichiometric member of the TIP60 lysine acetyltransferase complex implicated in prostate cancer development.MethodsBiopsies of 265 patients with prostate cancer were stained for ING3, pan-cytokeratin, and DNA. LNCaP and C4-2 androgen-responsive cells were used for in vitro assays including immunoprecipitation, western blotting, Luciferase reporter assay and quantitative polymerase chain reaction. Cell viability and migration assays were performed in prostate cancer cell lines using scrambled siRNA or siRNA targeting ING3.ResultsWe find that ING3 levels and AR activity positively correlate in prostate cancer. ING3 potentiates androgen effects, increasing expression of androgen-regulated genes and androgen response element-driven reporters to promote growth and anchorage-independent growth. Conversely, ING3 knockdown inhibits prostate cancer cell growth and invasion. ING3 activates the AR by serving as a scaffold to increase interaction between TIP60 and the AR in the cytoplasm, enhancing receptor acetylation and translocation to the nucleus. Activation is independent of ING3's ability to target the TIP60 complex to H3K4Me3, identifying a previously unknown chromatin-independent cytoplasmic activity for ING3. In agreement with in vitro observations, analysis of The Cancer Genome Atlas (TCGA) data (n = 498) and a prostate cancer tissue microarray (n = 256) show that ING3 levels are higher in aggressive prostate cancers, with high levels of ING3 predicting shorter patient survival in a low AR subgroup. Including ING3 levels with currently used indicators such as the Gleason score provides more accurate prognosis in primary prostate cancer.ConclusionsIn contrast to the majority of previous reports suggesting tumor suppressive functions in other cancers, our observations identify a clear oncogenic role for ING3, which acts as a co-activator of AR in prostate cancer. Data from TCGA and our previous and current tissue microarrays suggest that ING3 levels correlate with AR levels and that in patients with low levels of the receptor, ING3 level could serve as a useful prognostic biomarker.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-017-0854-0) contains supplementary material, which is available to authorized users.
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