LncGMDS-AS1 promotes the tumorigenesis of colorectal cancer through HuR-STAT3/Wnt axis.

Chronic inflammation promotes the tumorigenesis and cell stemness maintenance of colorectal cancer (CRC). However, the bridge role of long noncoding RNA (lncRNA) in linking chronic inflammation to CRC development and progression needs better understanding. Here, we elucidated a novel function of lncRNA GMDS-AS1 in persistently activated signal transducer and transcription activator 3 (STAT3) and Wnt signaling and CRC tumorigenesis. Interleukin-6 (IL-6) and Wnt3a induced lncRNA GMDS-AS1 expression, which was highly expressed in the CRC tissues and plasma of CRC patients. GMDS-AS1 knockdown impaired the survival, proliferation and stem cell-like phenotype acquisition of CRC cells in vitro and in vivo. We performed RNA sequencing (RNA-seq) and mass spectrometry (MS) to probe target proteins and identify their contributions to the downstream signaling pathways of GMDS-AS1. In CRC cells, GMDS-AS1 physically interacted with the RNA-stabilizing protein HuR, thereby protecting the HuR protein from polyubiquitination- and proteasome-dependent degradation. HuR stabilized STAT3 mRNA and upregulated the levels of basal and phosphorylated STAT3 protein, persistently activating STAT3 signaling. Our research revealed that the lncRNA GMDS-AS1 and its direct target HuR constitutively activate STAT3/Wnt signaling and promote CRC tumorigenesis, the GMDS-AS1-HuR-STAT3/Wnt axis is a therapeutic, diagnostic and prognostic target in CRC.

Critical role of histone H3 lysine 27 demethylase Kdm6b in the homeostasis and function of medullary thymic epithelial cells.

Medullary thymic epithelial cells (mTECs) play a central role in the establishment of T cell central immunological tolerance by promiscuously expressing tissue-restricted antigens (TRAs) and presenting them to developing T cells, leading to deletion of T cells responding to self-antigens. However, molecular mechanisms especially epigenetic regulation of mTEC homeostasis and TRA expression remain elusive. Here we show that the H3K27 demethylase Kdm6b is essential to maintain the postnatal thymic medulla by promoting mTEC survival and regulating the expression of TRA genes. Moreover, mice lacking Kdm6b developed pathological autoimmune disorders. Mechanically, Kdm6b exerted its function by reducing repressive H3K27 trimethylation (H3K27me3) at the promoters of anti-apoptotic gene Bcl2 and a set of Aire-dependent TRA genes. Thus, our findings reveal a dual role of Kdm6b in the regulation of mTEC-mediated T cell central tolerance.

Elevating H3K27me3 level sensitizes colorectal cancer to oxaliplatin.

Histone methylation is a context-dependent modification that regulates gene expression, and the trimethylation of histone H3 lysine 27 (H3K27me3) usually induces gene silencing. Overcoming colorectal cancer (CRC) chemoresistance is currently a huge challenge, but the relationship between H3K27me3 modification and chemoresistance remains largely unclear. Here, we found that H3K27me3 levels positively correlated with the metastasis-free survival of CRC patients and a low H3K27me3 level predicted a poor outcome upon chemotherapeutic drug treatment. Oxaliplatin stimulation significantly induced the expression of H3K27 lysine demethylase 6A/6B (KDM6A/6B), thus decreasing the level of H3K27me3 in CRC cells. Elevation of H3K27me3 level through KDM6A/6B depletion or GSK-J4 (a KDM6A/6B inhibitor) treatment significantly enhanced oxaliplatin-induced apoptosis. Conversely, when inhibiting the expression of H3K27me3 by EPZ-6438, an inhibitor of the histone methyltransferase EZH2, the proportion of apoptotic cells remarkably decreased. In addition, the combination of GSK-J4 and oxaliplatin significantly inhibited tumor growth in an oxaliplatin-resistant patient-derived xenograft model. Importantly, we revealed that oxaliplatin treatment dramatically induced NOTCH2 expression, which was caused by downregulation of H3K27me3 level on the NOTCH2 transcription initiation site. Thus, the activated NOTCH signaling promoted the expression of stemness-related genes, which resulted in oxaliplatin resistance. Furthermore, oxaliplatin-induced NOTCH signaling could be interrupted by GSK-J4 treatment. Collectively, our findings suggest that elevating H3K27me3 level can improve drug sensitivity in CRC patients.

Iron-dependent histone 3 lysine 9 demethylation controls B cell proliferation and humoral immune responses.

Trace elements play important roles in human health, but little is known about their functions in humoral immunity. Here, we show an important role for iron in inducing cyclin E and B cell proliferation. We find that iron-deficient individuals exhibit a significantly reduced antibody response to the measles vaccine when compared to iron-normal controls. Mice with iron deficiency also exhibit attenuated T-dependent or T-independent antigen-specific antibody responses. We show that iron is essential for B cell proliferation; both iron deficiency and α-ketoglutarate inhibition could suppress cyclin E1 induction and S phase entry of B cells upon activation. Finally, we demonstrate that three demethylases, KDM2B, KDM3B and KDM4C, are responsible for histone 3 lysine 9 (H3K9) demethylation at the cyclin E1 promoter, cyclin E1 induction and B cell proliferation. Thus, our data reveal a crucial role of H3K9 demethylation in B cell proliferation, and the importance of iron in humoral immunity.

A pilot study of new promising non-coding RNA diagnostic biomarkers for early-stage colorectal cancers.

Background Diagnostic biomarkers for the detection of colorectal cancers (CRCs) are lacking. Recent studies have demonstrated that circulating long non-coding RNAs have the potential to serve as biomarkers for the detection of cancers. We analyzed the significance of lncRNAs 91H, PVT-1 and MEG3 in the detection of CRC. Methods We examined the expression levels of 13 candidate lncRNAs in the plasma of 18 CRC patients and 20 non-cancerous controls. Then, we validated our findings by determining the expression levels of six promising lncRNAs in CRC tissues and normal colorectal tissues. Finally, we evaluated the clinical relevance of lncRNAs 91H, PVT-1 and MEG3 in the plasma of 58 CRC patients and 56 non-cancerous controls. Results Our data revealed that the expression levels of lncRNAs 91H, PVT-1 and MEG3 were significantly higher in plasma samples from CRC patients than in those from non-cancerous controls. The combination of 91H, PVT-1 and MEG3 could discriminate CRC patients from non-cancerous controls with an area under the receiver-operating curve (AUC) of 0.877 at a cut-off value of 0.3816, with a sensitivity of 82.76% and 78.57% specificity. More importantly, the combination of lncRNAs shows more sensitivity in the detection of early-stage CRC than the combination of CEA and CA19-9, biomarkers currently used for CRC detection (p < 0.0001). Conclusions lncRNAs 91H, PVT-1 and MEG3 are promising diagnostic biomarkers for early-stage CRC.

RelB/NF-κB links cell cycle transition and apoptosis to endometrioid adenocarcinoma tumorigenesis.

Dysfunction of nuclear factor-κB (NF-κB) signaling has been causally associated with numerous human malignancies. Although the NF-κB family of genes has been implicated in endometrial carcinogenesis, information regarding the involvement of central regulators of NF-κB signaling in human endometrial cancer (EC) is limited. Here, we investigated the specific roles of canonical and noncanonical NF-κB signaling in endometrial tumorigenesis. We found that NF-κB RelB protein, but not RelA, displayed high expression in EC samples and cell lines, with predominant elevation in endometrioid adenocarcinoma (EEC). Moreover, tumor cell-intrinsic RelB was responsible for the abundant levels of c-Myc, cyclin D1, Bcl-2 and Bcl-xL, which are key regulators of cell cycle transition, apoptosis and proliferation in EEC. In contrast, p27 expression was enhanced by RelB depletion. Thus, increased RelB in human EC is associated with enhanced EEC cell growth, leading to endometrial cell tumorigenicity. Our results reveal that regulatory RelB in noncanonical NF-κB signaling may serve as a therapeutic target to block EC initiation.

MicroRNAs as potential biomarkers for gastric cancer.

Gastric cancer is the fourth most common cancer in the world and the second leading cause of cancer-related death. More than 80% of diagnoses occur at the middle to late stage of the disease, highlighting an urgent need for novel biomarkers detectable at earlier stages. Recently, aberrantly expressed microRNAs (miRNAs) have received a great deal of attention as potential sensitive and accurate biomarkers for cancer diagnosis and prognosis. This review summarizes the current knowledge about potential miRNA biomarkers for gastric cancer that have been reported in the publicly available literature between 2008 and 2013. Available evidence indicates that aberrantly expressed miRNAs in gastric cancer correlate with tumorigenesis, tumor proliferation, distant metastasis and invasion. Furthermore, tissue and cancer types can be classified using miRNA expression profiles and next-generation sequencing. As miRNAs in plasma/serum are well protected from RNases, they remain stable under harsh conditions. Thus, potential functions of these circulating miRNAs can be deduced and may implicate their diagnostic value in cancer detection. Circulating miRNAs, as well as tissue miRNAs, may allow for the detection of gastric cancer at an early stage, prediction of prognosis, and monitoring of recurrence and/or lymph node metastasis. Taken together, the data suggest that the participation of miRNAs in biomarker development will enhance the sensitivity and specificity of diagnostic and prognostic tests for gastric cancer.

Genome-wide microRNA profiles identify miR-378 as a serum biomarker for early detection of gastric cancer.

Recent studies demonstrated that in several human malignancies aberrant expression profiles of circulating microRNAs (miRNAs) anticipate great cancer diagnostic potential. Here we showed that serum miR-378 could serve as a novel noninvasive biomarker in gastric cancer (GC) detection. Genome-wide miRNA expression profiles followed with Real-Time quantitative RT-PCR (qRT-PCR) assays revealed that miR-187(*), miR-371-5p and miR-378 were significantly elevated in GC patients. Further validation indicated that miR-378 alone could yields a ROC curve area of 0.861 with 87.5% sensitivity and 70.73% specificity in discriminating GC patients from healthy controls. Collectively, these data support our contention that serum miR-378 has strong potential as a novel noninvasive biomarker in gastric cancer detection.

PKCδ regulates Mdm2 independently of p53 in the apoptotic response to DNA damage.

Apoptosis is the key process in which cells with defective genome can be eliminated. Dys-regulation of apoptosis causes accumulation of irreparable mutation arisen from DNA damage and is the underlying cause of carcinogenesis. PKCδ is a multifunctional kinase involved in signal transduction of genotoxic-induced apoptosis. Previous studies have demonstrated that PKCδ transactivates p53 in response to DNA damage. These findings led us to determine if Mdm2, a nuclear phospho-protein and negative regulator of p53, could also be a PKCδ-modulated substrate. We discovered that inhibition of PKCδ down-regulates Mdm2 protein expression regardless of p53 status. Given that Mdm2 mRNA change was detected in p53-proficient, but not deficient cells, PKCδ affected Mdm2 on the post-translational level. Interestingly, treatment of MG132 restored Mdm2 expression to the steady-state level. Further investigation showed that PKCδ inhibited Mdm2 ubiquitination in p53-deficient cells and loss of PKCδ resulted in an increase in Mdm2 proteosomal degradation. Moreover, P300/CBP-associated factor (PCAF), an ubiquitin ligase 3 for Mdm2, was observed to participate in Mdm2 ubiquitination by PKCδ inhibition and knock-down of PCAF rescued Mdm2 diminution. Finally, as shown for PKCδ, Mdm2 was also required to exert pro-apoptotic response caused by genotoxic agents in p53-null cells. In addition, overexpression of Mdm2 restored inhibitory effect of apoptosis in cells silenced for PKCδ. Taken together, we conclude that PKCδ regulates Mdm2 expression distinctively of p53 pathway by affecting Mdm2 ubiquitination and maintenance of Mdm2 expression by PKCδ is important to ensure normal genotoxic cell death response in human cancer cells.

Identification of Evi-1 as a novel effector of PKCδ in the apoptotic response to DNA damage.

Protein kinase C delta (PKCδ), a PKC family isoform, regulates diverse signal transduction pathways during DNA damage to induce apoptosis. To explore the apoptosis mechanism that PKCδ modulates, we sought to uncover transcription factor targets of PKCδ by devising a screening strategy that utilizes ChIP-cloning and microarray analysis. Transcription factor candidates were generated with the application of public access data-mining tools and this resulted in the identification of Evi-1 as a novel PKCδ-mediated DNA damage responsive molecule. The results demonstrated that PKCδ is constitutively associated with Evi-1. PKCδ regulated Evi-1 to activate PLZF transcription upon genotoxic stress. Furthermore, both Evi-1 and PLZF were associated with DNA damage-stimulated apoptosis. Taken together, we have discovered a novel regulation of Evi-1, which transactivates PLZF, by PKCδ to induce cell death in response to genotoxic stress.

DNA damage signalling recruits RREB-1 to the p53 tumour suppressor promoter.

Transcriptional regulation of the p53 tumour suppressor gene plays an important role in the control of the expression of various target genes involved in the DNA damage response. However, the molecular basis of this regulation remains obscure. In the present study we demonstrate that RREB-1 (Ras-responsive-element-binding protein-1) efficiently binds to the p53 promoter via the p53 core promoter element and transactivates p53 expression. Silencing of RREB-1 significantly reduces p53 expression at both the mRNA and the protein levels. Notably, disruption of RREB-1-mediated p53 transcription suppresses the expression of the p53 target genes. We also show that, upon exposure to genotoxic stress, RREB-1 controls apoptosis in a p53-dependent manner. These findings provide evidence that RREB-1 participates in modulating p53 transcription in response to DNA damage.

Protein kinase Cdelta activates RelA/p65 and nuclear factor-kappaB signaling in response to tumor necrosis factor-alpha.

Nuclear factor-kappaB (NF-kappaB) is tightly modulated by IkappaB kinases and IkappaBalpha in the cytoplasm. On stimulation, NF-kappaB translocates into the nucleus to initiate transcription; however, regulation of its transcriptional activity remains obscure. Here, we show that protein kinase C (PKC) delta controls the main subunit of NF-kappaB, RelA/p65. On exposure to tumor necrosis factor-alpha (TNF-alpha), the expression of RelA/p65 target genes such as IkappaBalpha, RelB, and p100/p52 is up-regulated in a PKCdelta-dependent manner. The results also show that PKCdelta is targeted to the nucleus and forms a complex with RelA/p65 following TNF-alpha exposure. Importantly, kinase activity of PKCdelta is required for RelA/p65 transactivation. In concert with these results, PKCdelta activates RelA/p65 for its occupancy to target-gene promoters, including IkappaBalpha and p100/p52. Moreover, functional analyses show that inhibition of PKCdelta is associated with substantial attenuation of NF-kappaB activity in response to TNF-alpha. These findings provide evidence that PKCdelta orchestrates RelA/p65 transactivation, a requisite for NF-kappaB signaling pathway in the nucleus.

A functional genome-wide RNAi screen identifies TAF1 as a regulator for apoptosis in response to genotoxic stress.

Evasion from apoptotic cell death is a characteristic of cancer; genes that modulate this process may be optimal for therapeutic attack. Identifying key regulators of apoptosis is thus a central goal in cancer therapy. Here, we describe a loss-of-function screen that uses RNA interference libraries to identify genes required for induction of apoptosis. We used a short-hairpin RNA expressing vector with high gene-expression silencing activity that contained fetal brain cDNAs. Survived cells from genotoxic stress were isolated to determine knock-down of molecules that are crucial for induction of apoptosis. We identified TBP-associated factor 1 (TAF1), a gene previously implicated as an essential component of transcription machinery. Depletion of TAF1 was associated with substantial attenuation of apoptosis induced by oxidative as well as genotoxic stress. Microarray analysis further demonstrated that a number of genes were transcriptionally declined in cells silenced for TAF1. Surprisingly, knocking down TAF1 exhibited a marked decrease in p27(Kip1) expression, allowing cells resistant from oxidative stress-induced apoptosis. These results suggest that TAF1 regulates apoptosis by controlling p27(Kip1) expression. Our system provides a novel approach to identifying candidate genes that modulate apoptosis.

Protein kinase C delta induces transcription of the TP53 tumor suppressor gene by controlling death-promoting factor Btf in the apoptotic response to DNA damage.

Expression of the TP53 tumor suppressor is tightly controlled for its ability to function as a critical regulator of cell growth, proliferation, and death in response to DNA damage. However, little is known about the mechanisms and contributions of the transcriptional regulation of TP53. Here we report that protein kinase C delta (PKCdelta), a ubiquitously expressed member of the novel subfamily of PKC isoforms, transactivates TP53 expression at the transcriptional level. Reporter assays demonstrated that PKCdelta induces the promoter activity of TP53 through the TP53 core promoter element (CPE-TP53) and that such induction is enhanced in response to DNA damage. The results also demonstrate that, upon exposure to genotoxic stress, PKCdelta activates and interacts with the death-promoting transcription factor Btf to co-occupy CPE-TP53. Inhibition of PKCdelta activity decreases the affinity of Btf for CPE-TP53, thereby reducing TP53 expression at both the mRNA and the protein levels. In concert with these results, we show that disruption of Btf-mediated TP53 gene transcription by RNA interference leads to suppression of TP53-mediated apoptosis following genotoxic stress. These findings provide evidence that activation of TP53 gene transcription by PKCdelta triggers TP53-dependent apoptosis in response to DNA damage.

Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor suppressor in the apoptotic response to DNA damage.

The p53 tumor suppressor is activated in the cellular response to genotoxic stress. Transactivation of p53 target genes dictates cell cycle arrest and DNA repair or induction of apoptosis; however, a molecular mechanism responsible for these distinct functions remains unclear. Recent studies revealed that phosphorylation of p53 on Ser(46) was associated with induction of p53AIP1 expression, resulting in the commitment of the cell fate into apoptotic cell death. Moreover, upon exposure to genotoxic stress, p53DINP1 was expressed and recruited a kinase(s) to p53 that specifically phosphorylated Ser(46). Here, we show that the pro-apoptotic kinase, protein kinase C delta (PKCdelta), is involved in phosphorylation of p53 on Ser(46). PKCdelta-mediated phosphorylation is required for the interaction of PKCdelta with p53. The results also demonstrate that p53DINP1 associates with PKCdelta upon exposure to genotoxic agents. Consistent with these results, PKCdelta potentiates p53-dependent apoptosis by Ser(46) phosphorylation in response to genotoxic stress. These findings indicate that PKCdelta regulates p53 to induce apoptotic cell death in the cellular response to DNA damage.