SRC-3 coactivator regulates cell resistance to cytotoxic stress via TRAF4-mediated p53 destabilization.

Steroid receptor coactivator 3 (SRC-3) is an oncogenic nuclear receptor coactivator that plays a significant role in drug resistance. Using a lentiviral cDNA library rescue screening approach, we identified a SRC-3 downstream gene-TRAF4 (tumor necrosis factor [TNF] receptor associated-factor 4)-that functions in cell resistance to cytotoxic stress. TRAF4 expression is positively correlated with SRC-3 expression in human breast cancers. Similar to that observed for SRC-3 overexpression, breast cancer cells overexpressing TRAF4 are more resistant to stress-induced death. Here, we further dissected the underlying molecular mechanism for SRC-3 and TRAF4-mediated resistance to cytotoxic agents. We observed that SRC-3 expression is inversely correlated with the expression of p53-regulated proapoptotic genes in breast cancers and further found that SRC-3 and TRAF4 overexpression diminished cytotoxic stress-induced up-regulation of the tumor suppressor p53 protein. To determine the mechanism, we showed that the TRAF domain of TRAF4 bound to the N-terminal TRAF-like region of the deubiquitinase HAUSP (herpesvirus-associated ubiquitin-specific protease; also named USP7) and blocked the access of p53 to the same region of HAUSP. This TRAF4-mediated inhibition of HAUSP then led to the loss of p53 deubiquitination and its stabilization in response to cellular stress. Consistent with this cellular function, we also found that TRAF4 overexpression in breast cancer patients was associated significantly with poor prognosis. Because of SRC-3's ability to abrogate p53 function, our results suggest that SRC-3 overexpression may be especially important in tumors in which p53 is not mutated.

Analysis of Genomes and Transcriptomes of Hepatocellular Carcinomas Identifies Mutations and Gene Expression Changes in the Transforming Growth Factor-ß Pathway.

BACKGROUND & AIMS: Development of hepatocellular carcinoma (HCC) is associated with alterations in the transforming growth factor-beta (TGF-ß) signaling pathway, which regulates liver inflammation and can have tumor suppressor or promoter activities. Little is known about the roles of specific members of this pathway at specific of HCC development. We took an integrated approach to identify and validate the effects of changes in this pathway in HCC and identify therapeutic targets. METHODS: We performed transcriptome analyses for a total of 488 HCCs that include data from The Cancer Genome Atlas. We also screened 301 HCCs reported in the Catalogue of Somatic Mutations in Cancer and 202 from Cancer Genome Atlas for mutations in genome sequences. We expressed mutant forms of spectrin beta, non-erythrocytic 1 (SPTBN1) in HepG2, SNU398, and SNU475 cells and measured phosphorylation, nuclear translocation, and transcriptional activity of SMAD family member 3 (SMAD3). RESULTS: We found somatic mutations in at least 1 gene whose product is a member of TGF-ß signaling pathway in 38% of HCC samples. SPTBN1 was mutated in the largest proportion of samples (12 of 202, 6%). Unsupervised clustering of transcriptome data identified a group of HCCs with activation of the TGF-ß signaling pathway (increased transcription of genes in the pathway) and a group of HCCs with inactivation of TGF-ß signaling (reduced expression of genes in this pathway). Patients with tumors with inactivation of TGF-ß signaling had shorter survival times than patients with tumors with activation of TGF-ß signaling (P = .0129). Patterns of TGF-ß signaling correlated with activation of the DNA damage response and sirtuin signaling pathways. HepG2, SNU398, and SNU475 cells that expressed the D1089Y mutant or with knockdown of SPTBN1 had increased sensitivity to DNA crosslinking agents and reduced survival compared with cells that expressed normal SPTBN1 (controls). CONCLUSIONS: In genome and transcriptome analyses of HCC samples, we found mutations in genes in the TGF-ß signaling pathway in almost 40% of samples. These correlated with changes in expression of genes in the pathways; up-regulation of genes in this pathway would contribute to inflammation and fibrosis, whereas down-regulation would indicate loss of TGF-ß tumor suppressor activity. Our findings indicate that therapeutic agents for HCCs can be effective, based on genetic features of the TGF-ß pathway; agents that block TGF-ß should be used only in patients with specific types of HCCs.

Temporal-Spatial Establishment of Initial Niche for the Primary Spermatogonial Stem Cell Formation Is Determined by an ARID4B Regulatory Network.

During neonatal testis development, centrally located gonocytes migrate to basement membrane of the seminiferous cords, where physical contact with a niche established by Sertoli cells is essential for transition of gonocytes into spermatogonial stem cells (SSCs). To provide structural support and signaling stimuli for the gonocyte-to-SSC transition that occurs at a specific location during a finite phase, temporal-spatial establishment of the niche is critical. To date, the factors that guide Sertoli cells to establish the initial stem cell niche remain largely unknown. Using the Sertoli cell-specific Arid4b knockout (Arid4bSCKO) mice, we demonstrated that ablation of AT-rich interaction domain 4B (ARID4B) resulted in abnormal detachment of Sertoli cells from the basement membrane of seminiferous cords during the gonocyte-to-SSC transition phase, suggesting failure to establish a niche for the SSC formation. Without support by a niche environment, gonocytes showed disarranged cell distribution in the Arid4bSCKO testes and underwent apoptosis. The commitment of gonocytes to differentiate into the spermatogonial lineage was broken and the capability of SSCs to self-renew and differentiate was also impaired. Gene expression profiling revealed the molecular mechanisms responsible for the phenotypic changes in the Arid4bSCKO testes, by identifying genes important for stem cell niche function as downstream effectors of ARID4B, including genes that encode gap junction protein alpha-1, KIT ligand, anti-Müllerian hormone, Glial cell-line derived neurotrophic factor, inhibin alpha, inhibin beta, and cytochrome P450 family 26 subfamily b polypeptide 1. Our results identified ARID4B as a master regulator of a signaling network that governs the establishment of a niche during the critical gonocyte-to-SSC transition phase to control the fate of gonocytes and SSCs. Stem Cells 2017;35:1554-1565.

ARID4A and ARID4B regulate male fertility, a functional link to the AR and RB pathways.

ARID4A and ARID4B are homologous members of the ARID (AT-rich interaction domain) gene family. ARID4A and ARID4B physically interact with each other. ARID4A is a retinoblastoma (RB)-binding protein. Biological function of these interactions is still unknown. Here, we report that mice with complete deficiency of Arid4a, combined with haploinsufficiency of Arid4b (Arid4a(-/-)Arid4b(+/-)), showed progressive loss of male fertility, accompanied by hypogonadism and seminal vesicle agenesis/hypodysplasia. Arid4a and Arid4b are expressed mainly in Sertoli cells of testes, which implies that their roles in Sertoli cell function are to support spermatogenesis and create the impermeable blood-testis barrier. In fact, evaluation of germ cell development in the Arid4a(-/-)Arid4b(+/-) mice showed spermatogenic arrest at the stages of meiotic spermatocytes and postmeiotic haploid spermatids. Analysis of the integrity of the blood-testis barrier showed increased permeability of seminiferous tubules in the Arid4a(-/-)Arid4b(+/-) testes. Interestingly, phenotypic Sertoli cell dysfunction in the Arid4a(-/-)Arid4b(+/-) mice, including spermatogenic failures and the impaired blood-testis barrier, recapitulated the defects found in the Sertoli cell-specific androgen receptor (AR) knockout mice and the Sertoli cell-specific RB knockout mice. Investigation of the molecular mechanism identified several AR- and RB-responsive genes as downstream targets of ARID4A and ARID4B. Our results thus indicate that ARID4A and ARID4B function as transcriptional coactivators for AR and RB and play an integral part in the AR and RB regulatory pathways involved in the regulation of Sertoli cell function and male fertility.

ERK3 promotes endothelial cell functions by upregulating SRC-3/SP1-mediated VEGFR2 expression.

Despite a regain of interest recently in ERK3 kinase signaling, the molecular regulations of both ERK3 gene expression and protein kinase activity are still largely unknown. While it is shown that disruption of ERK3 gene causes neonatal lethality, cell type-specific functions of ERK3 signaling remain to be explored. In this study, we report that ERK3 gene expression is upregulated by cytokines through c-Jun in endothelial cells; c-Jun binds to the ERK3 gene and regulates its transcription. We further reveal a new role for ERK3 in regulating endothelial cell migration, proliferation and tube formation by upregulating SRC-3/SP-1-mediated VEGFR2 expression. The underlying molecular mechanism involves ERK3-stimulated formation of a transcriptional complex involving coactivator SRC-3, transcription factor SP-1 and the secondary coactivator CBP. Taken together, our study identified a molecular regulatory mechanism of ERK3 gene expression and revealed a previously unknown role of ERK3 in regulating endothelial cell functions.

Hepatitis B virus X protein stabilizes amplified in breast cancer 1 protein and cooperates with it to promote human hepatocellular carcinoma cell invasiveness.

UNLABELLED: Chronic infection of hepatitis B virus (HBV) is closely associated with the development of human hepatocellular carcinoma (HCC). HBV X protein (HBx) plays a key role in the progression of HCC. We recently found that amplified in breast cancer 1 (AIB1) protein is overexpressed in 68% of human HCC specimens and promotes HCC progression by enhancing cell proliferation and invasiveness. Given that both HBx and AIB1 play important oncogenic roles in HCC, we aimed to determine whether they could cooperatively promote human HCC development. Herein, we show that HBx-positive HCC tissues had a higher level of AIB1 protein, compared to HBx-negative HCC tissues. A positive correlation between HBx protein level and AIB1 protein level was established in HCC specimens. Without affecting its messenger RNA level, HBx induced a significant increase of the protein level of AIB1, which correlated with a significant extension of the half-life of AIB1 protein. Mechanistically, HBx could interact with AIB1 to prevent the interaction between envelope protein 3 ubiquitin ligase F-box and WD repeat domain containing 7 (Fbw7)α and AIB1, then inhibited the Fbw7α-mediated ubiquitination and degradation of AIB1. In addition, reporter assays and chromatin immunoprecipitation assays revealed that both HBx and AIB1 were recruited to matrix metalloproteinase-9 (MMP-9) promoter to enhance MMP-9 promoter activity cooperatively. Consistently, HBx and AIB1 cooperatively enhanced MMP-9 expression in HepG2 cells, which, in turn, increased cell-invasive ability. CONCLUSION: Our study demonstrates that HBx can stabilize AIB1 protein and cooperate with it to promote human HCC cell invasiveness, highlighting the essential role of the cross-talk between HBx and AIB1 in HBV-related HCC progression.

Reprogramming the posttranslational code of SRC-3 confers a switch in mammalian systems biology.

Here we demonstrate that reprogramming steroid receptor coactivator-3 (SRC-3) function by changing its posttranslational modification (PTM) code drastically influences systems biology. These findings support the physiological importance of PTMs in directing in vivo functions of a master coregulator. We previously reported that the transactivation potential of SRC-3 is controlled in part by PTMs, although this data emanated from in vitro studies. To test the physiological implications of PTMs on SRC-3, we developed a knock-in mouse model containing mutations at four conserved phosphorylation sites. These mice displayed a systems biology phenotype with increased body weight and adiposity, coupled with reduced peripheral insulin sensitivity. Collectively, these phenotypes result from increased IGF1 signaling, due to elevated IGFBP3 levels. We provide convincing evidence that these mutations in SRC-3 promoted enhanced transcription of the IGFBP3 gene and globally influenced growth and metabolism. Consequently, these mice displayed increased liver tumorigenesis, which likely results from elevated IGF1 signaling.

MicroRNA-34 Family in Cancers: Role, Mechanism, and Therapeutic Potential.

MicroRNA (miRNA) are small noncoding RNAs that play vital roles in post-transcriptional gene regulation by inhibiting mRNA translation or promoting mRNA degradation. The dysregulation of miRNA has been implicated in numerous human diseases, including cancers. miR-34 family members (miR-34s), including miR-34a, miR-34b, and miR-34c, have emerged as the most extensively studied tumor-suppressive miRNAs. In this comprehensive review, we aim to provide an overview of the major signaling pathways and gene networks regulated by miR-34s in various cancers and highlight the critical tumor suppressor role of miR-34s. Furthermore, we will discuss the potential of using miR-34 mimics as a novel therapeutic approach against cancer, while also addressing the challenges associated with their development and delivery. It is anticipated that gaining a deeper understanding of the functions and mechanisms of miR-34s in cancer will greatly contribute to the development of effective miR-34-based cancer therapeutics.

Differentiation of fetal hematopoietic stem cells requires ARID4B to restrict autocrine KITLG/KIT-Src signaling.

Balance between the hematopoietic stem cell (HSC) duality to either possess self-renewal capacity or differentiate into multipotency progenitors (MPPs) is crucial for maintaining homeostasis of the hematopoietic stem/progenitor cell (HSPC) compartment. To retain the HSC self-renewal activity, KIT, a receptor tyrosine kinase, in HSCs is activated by its cognate ligand KITLG originating from niche cells. Here, we show that AT-rich interaction domain 4B (ARID4B) interferes with KITLG/KIT signaling, consequently allowing HSC differentiation. Conditional Arid4b knockout in mouse hematopoietic cells blocks fetal HSC differentiation, preventing hematopoiesis. Mechanistically, ARID4B-deficient HSCs self-express KITLG and overexpress KIT. As to downstream pathways of KITLG/KIT signaling, inhibition of Src family kinases rescues the HSC differentiation defect elicited by ARID4B loss. In summary, the intrinsic ARID4B-KITLG/KIT-Src axis is an HSPC regulatory program that enables the differentiation state, while KIT stimulation by KITLG from niche cells preserves the HSPC undifferentiated pool.

Specific amino acid residues in the basic helix-loop-helix domain of SRC-3 are essential for its nuclear localization and proteasome-dependent turnover.

SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM-1 is a primary transcriptional coactivator for the estrogen receptor. Here we report that deletion of the SRC-3 basic helix-loop-helix (bHLH) domain blocks its proteasome-dependent turnover. We further identified two residues (K17 and R18) in the SRC-3 bHLH domain that are essential for its stability. Moreover, we found that the bHLH domain contains a bipartite nuclear localization signal (NLS). SRC-3 NLS mutants block its translocation into the nucleus, and this correlates with its insensitivity to proteasome-dependent turnover. SRC-3 shows a time-dependent decay in the presence of cycloheximide which is not apparent for the cytoplasmic mutant. Fusion of a simian virus 40 T antigen NLS to the cytoplasmic localized SRC-3 mutant drives it back into the nucleus and restores its proteasomal sensitivity. In addition, the cytoplasmic mutants are inactive for transcriptional coactivation and cancer cell growth. Taken together, our data indicate that proteasome-dependent turnover of SRC-3 occurs in the nucleus and that two amino acid residues in the bHLH domain provide a signal for its nuclear localization and proteasome-dependent degradation as well as for regulation of SRC-3 transcriptional coactivator capacity.

Regulation of SRC-3 intercompartmental dynamics by estrogen receptor and phosphorylation.

The steroid receptor coactivator 3 gene (SRC-3) (AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family transcription coactivator and a known oncogene. Despite its importance, the functional regulation of SRC-3 remains poorly understood within a cellular context. Using a novel combination of live-cell, high-throughput, and fluorescent microscopy, we report SRC-3 to be a nucleocytoplasmic shuttling protein whose intracellular mobility, solubility, and cellular localization are regulated by phosphorylation and estrogen receptor alpha (ERalpha) interactions. We show that both chemical inhibition and small interfering RNA reduction of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by epidermal growth factor signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known participants in the phosphocode that regulates SRC-3 activity. Accordingly, the cytoplasmic localization of a nonphosphorylatable SRC-3 mutant further supported these results. In the presence of ERalpha, U0126 also dramatically reduces (i) ligand-dependent colocalization of SRC-3 and ERalpha, (ii) the formation of ER-SRC-3 complexes in cell lysates, and (iii) SRC-3 targeting to a visible, ERalpha-occupied and -regulated prolactin promoter array. Taken together, these results indicate that phosphorylation coordinates SRC-3 coactivator function by linking the probabilistic formation of transient nuclear receptor-coactivator complexes with its molecular dynamics and cellular compartmentalization. Technically and conceptually, these findings have a new and broad impact upon evaluating mechanisms of action of gene regulators at a cellular system level.

Targeting the E3 Ubiquitin Ligase PJA1 Enhances Tumor-Suppressing TGFß Signaling.

RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFß/SMAD signaling. SMAD3 and its adaptors, such as ß2SP, are important mediators of TGFß signaling and regulate gene expression to suppress stem cell-like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/ß2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3 +/-), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFß1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients.Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFß in liver carcinogenesis.

Transcriptional regulation by steroid receptor coactivator phosphorylation.

The basic mechanisms underlying ligand-dependent transcriptional activation by nuclear receptors (NRs) require the sequential recruitment of various coactivators. Increasing numbers of coactivators have been identified in recent years, and both biochemical and genetic studies demonstrate that these coactivators are differentially used by transcription factors, including NRs, in a cell/tissue type- and promoter-specific manner. However, the molecular basis underlying this specificity remains largely unknown. Recently, NRs and coregulators were shown to be targets of posttranslational modifications activated by diverse cellular signaling pathways. It is argued that posttranslational modifications of these proteins provide the basis for a combinatorial code required for specific gene activation by NRs and coactivators, and that this code also enables coactivators to efficiently stimulate the activity of other classes of transcription factors. In this review, we will focus on coactivators and discuss the recent progress in understanding the role of phosphorylation of the steroid receptor coactivator family and the potential ramifications of this posttranslational modification for regulation of gene expression.

Genetic screening reveals an essential role of p27kip1 in restriction of breast cancer progression.

The genetic changes and mechanisms underlying the progression of estrogen-dependent breast cancers to estrogen-independent, antiestrogen-resistant, and metastatic breast cancers are unclear despite being a major problem in endocrine therapy. To identify genes responsible for this progression, we carried out a genetic screening by an enhanced retroviral mutagen (ERM)-mediated random mutagenesis in the estrogen-dependent T47D breast cancer cells. We found that T47D cells contain only one p27kip1 (p27) allele coding for the p27 cyclin-dependent kinase (CDK) inhibitor. An ERM insertion into the p27 locus of T47D cells disrupted the p27 gene and created estrogen-independent and antiestrogen-resistant breast cancer cells that still maintained functional estrogen receptors. Disruption of p27 in T47D cells resulted in several changes, and most of these changes could be rescued by p27 restoration. First, CDK2 activity was increased in the absence of estrogen or in the presence of estrogen antagonists tamoxifen or ICI 182780; second, amplified in breast cancer 1 (AIB1), a cancer overexpressed transcriptional coactivator, was hyperphosphorylated, which made AIB1 a better coactivator for E2F1; and third, growth factor receptor binding protein 2-associated binder 2 (Gab2) and Akt activity were increased following E2F1 overactivation, leading to a significant enhancement of cell migration and invasion. Furthermore, the p27-deficient cells, but not T47D control cells, developed lung metastasis in an ovarian hormone-independent manner when they were i.v. injected into nude mice. In sum, loss of p27 activated AIB1, E2F1, Gab2, and Akt; increased cell migration and invasion; caused antiestrogen insensitivity; and promoted metastasis of breast cancer cells. These findings suggest that p27 plays an essential role in restriction of breast cancer progression.

Identification of the PTEN-ARID4B-PI3K pathway reveals the dependency on ARID4B by PTEN-deficient prostate cancer.

PTEN is frequently mutated in prostate cancer. The tumor suppressor function of PTEN is attributed to its lipid phosphatase activity that counters PI3K action. Here, we report a PTEN-ARID4B-PI3K axis in which PTEN inhibits expression of ARID4B, while ARID4B is a transcriptional activator of the PI3K subunit genes PIK3CA and PIK3R2 that are crucial for activation of the PI3K/AKT pathway. Reciprocal binding of ARID4B and histone H1 to the PIK3CA and PIK3R2 promoters modulates chromatin condensation, suggesting a mechanism by which ARID4B activates these promoters. Functional analyses reveals that ARID4B is required for prostate tumorigenesis when PTEN is deficient. The biological significance is further substantiated by the existence of a PTEN/ARID4B/PIK3CA three-gene signature that improves the predictive power for prostate cancer recurrence in patients. In summary, we identify ARID4B as a master regulator in the PTEN-PI3K pathway, thus providing a potential therapeutic target for prostate cancer carrying PTEN mutations.

Rapid estrogen-induced phosphorylation of the SRC-3 coactivator occurs in an extranuclear complex containing estrogen receptor.

SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 is a primary transcriptional coregulator for estrogen receptor (ER). Six SRC-3 phosphorylation sites have been identified, and these can be induced by steroids, cytokines, and growth factors, involving multiple kinase signaling pathways. Using phosphospecific antibodies for six phosphorylation sites, we investigated the mechanisms involved in estradiol (E2)-induced SRC-3 phosphorylation and found that this occurs only when either activated estrogen receptor alpha (ERalpha) or activated ERbeta is present. Both the activation function 1 and the ligand binding domains of ERalpha are required for maximal induction. Mutations in the coactivator binding groove of the ERalpha ligand binding domain inhibit E2-stimulated SRC-3 phosphorylation, as do mutations in the nuclear receptor-interacting domain of SRC-3, suggesting that ERalpha must directly contact SRC-3 for this posttranslational modification to take place. A transcriptionally inactive ERalpha mutant which localizes to the cytoplasm supports E2-induced SRC-3 phosphorylation. Mutations of the ERalpha DNA binding domain did not block this rapid E2-dependent SRC-3 phosphorylation. Together these data demonstrate that E2-induced SRC-3 phosphorylation is dependent on a direct interaction between SRC-3 and ERalpha and can occur outside of the nucleus. Our results provide evidence for an early nongenomic action of ER on SRC-3 that supports the well-established downstream genomic roles of estrogen and coactivators.

Peptidyl-prolyl isomerase 1 (Pin1) serves as a coactivator of steroid receptor by regulating the activity of phosphorylated steroid receptor coactivator 3 (SRC-3/AIB1).

Steroid receptor coactivator 3 (SRC-3/AIB1) interacts with steroid receptors in a ligand-dependent manner to activate receptor-mediated transcription. A number of intracellular signaling pathways initiated by growth factors and hormones induce phosphorylation of SRC-3, regulating its function and contributing to its oncogenic potential. However, the range of mechanisms by which phosphorylation affects coactivator function remains largely undefined. We demonstrate here that peptidyl-prolyl isomerase 1 (Pin1), which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, interacts selectively with phosphorylated SRC-3. In addition, Pin1 and SRC-3 activate nuclear-receptor-regulated transcription synergistically. Depletion of Pin1 by small interfering RNA (siRNA) reduces hormone-dependent transcription from both transfected reporters and an endogenous steroid receptor target gene. We present evidence that Pin1 modulates interactions between SRC-3 and CBP/p300. The interaction is enhanced in vitro and in vivo by Pin1 and diminished when cellular Pin1 is reduced by siRNA or in stable Pin1-depleted cell lines. Depletion of Pin1 in MCF-7 human breast cancer cells reduces the endogenous estrogen-dependent recruitment of p300 to the promoters of estrogen receptor-dependent genes. Pin1 overexpression enhanced SRC-3 cellular turnover, and depletion of Pin1 stabilized SRC-3. Our results suggest that Pin1 functions as a transcriptional coactivator of nuclear receptors by modulating SRC-3 coactivator protein-protein complex formation and ultimately by also promoting the turnover of the activated SRC-3 oncoprotein.

MicroRNA-34 family in breast cancer: from research to therapeutic potential.

MicroRNA (miRNA)-34 family (miR-34s), including miR-34a/b/c, is the most well studied non-coding RNAs that regulate gene expression post-transcriptionally. The miR-34s mediates the tumor suppressor function of p53 in the pathogenesis of breast cancer by targeting different oncogenes. This review focuses on the anti-oncogenic regulation of the miR-34s, emphasizing the major signaling pathways that are involved in the modulation of miR-34s in breast cancer. Moreover, it highlights how epigenetic modification by the p53/miR-34s axis regulates the proliferation, invasiveness, chemoresistance, and sternness of breast cancer. A better understanding of the molecular mechanisms of miR-34s will open new opportunities for the development of novel therapeutic strategies and define a new approach in identifying potential biomarkers for early diagnosis of breast cancer.

Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) Coactivator activity by I kappa B kinase.

In the past few years, many nuclear receptor coactivators have been identified and shown to be an integral part of receptor action. The most frequently studied of these coactivators are members of the steroid receptor coactivator (SRC) family, SRC-1, TIF2/GRIP1/SRC-2, and pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3. In this report, we describe the biochemical purification of SRC-1 and SRC-3 protein complexes and the subsequent identification of their associated proteins by mass spectrometry. Surprisingly, we found association of SRC-3, but not SRC-1, with the I kappa B kinase (IKK). IKK is known to be responsible for the degradation of I kappa B and the subsequent activation of NF-kappa B. Since NF-kappa B plays a key role in host immunity and inflammatory responses, we therefore investigated the significance of the SRC-3-IKK complex. We demonstrated that SRC-3 was able to enhance NF-kappa B-mediated gene expression in concert with IKK. In addition, we showed that SRC-3 was phosphorylated by the IKK complex in vitro. Furthermore, elevated SRC-3 phosphorylation in vivo and translocation of SRC-3 from cytoplasm to nucleus in response to tumor necrosis factor alpha occurred in cells, suggesting control of subcellular localization of SRC-3 by phosphorylation. Finally, the hypothesis that SRC-3 is involved in NF-kappa B-mediated gene expression is further supported by the reduced expression of interferon regulatory factor 1, a well-known NF-kappa B target gene, in the spleens of SRC-3 null mutant mice. Taken together, our results not only reveal the IKK-mediated phosphorylation of SRC-3 to be a regulated event that plays an important role but also substantiate the role of SRC-3 in multiple signaling pathways.

Transforming growth factor-ß in liver cancer stem cells and regeneration.

Cancer stem cells have established mechanisms that contribute to tumor heterogeneity as well as resistance to therapy. Over 40% of hepatocellular carcinomas (HCCs) are considered to be clonal and arise from a stem-like/cancer stem cell. Moreover, HCC is the second leading cause of cancer death worldwide, and an improved understanding of cancer stem cells and targeting these in this cancer are urgently needed. Multiple studies have revealed etiological patterns and multiple genes/pathways signifying initiation and progression of HCC; however, unlike the transforming growth factor ß (TGF-ß) pathway, loss of p53 and/or activation of ß-catenin do not spontaneously drive HCC in animal models. Despite many advances in cancer genetics that include identifying the dominant role of TGF-ß signaling in gastrointestinal cancers, we have not reached an integrated view of genetic mutations, copy number changes, driver pathways, and animal models that support effective targeted therapies for these common and lethal cancers. Moreover, pathways involved in stem cell transformation into gastrointestinal cancers remain largely undefined. Identifying the key mechanisms and developing models that reflect the human disease can lead to effective new treatment strategies. In this review, we dissect the evidence obtained from mouse and human liver regeneration, and mouse genetics, to provide insight into the role of TGF-ß in regulating the cancer stem cell niche. (Hepatology Communications 2017;1:477-493).