Identification of HMGCR as the anticancer target of physapubenolide against melanoma cells by in silico target prediction.

Physapubenolide (PB), a withanolide-type compound extracted from the traditional herb Physalis minima L., has been demonstrated to exert remarkable cytotoxicity against cancer cells; however, its molecular mechanisms are still unclear. In this study, we demonstrated that PB inhibited cell proliferation and migration in melanoma cells by inducing cell apoptosis. The anticancer activity of PB was further verified in a melanoma xenograft model. To explore the mechanism underlying the anticancer effects of PB, we carried out an in silico target prediction study, which combined three approaches (chemical similarity searching, quantitative structure-activity relationship (QSAR), and molecular docking) to identify the targets of PB, and found that PB likely targets 3-hydroxy-methylglutaryl CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway, which promotes cancer cell proliferation, migration, and metastasis. We further demonstrated that PB interacted with HMGCR, decreased its protein expression and inhibited the HMGCR/YAP pathway in melanoma cells. In addition, we found that PB could restore vemurafenib sensitivity in vemurafenib-resistant A-375 cells, which was correlated with the downregulation of HMGCR. In conclusion, we demonstrate that PB elicits anticancer action and enhances sensitivity to vemurafenib by targeting HMGCR.

RSK2 protects human breast cancer cells under endoplasmic reticulum stress through activating AMPKα2-mediated autophagy.

Autophagy can protect stressed cancer cell by degradation of damaged proteins and organelles. However, the regulatory mechanisms behind this cellular process remain incompletely understood. Here, we demonstrate that RSK2 (p90 ribosomal S6 kinase 2) plays a critical role in ER stress-induced autophagy in breast cancer cells. We demonstrated that the promotive effect of RSK2 on autophagy resulted from directly binding of AMPKα2 in nucleus and phosphorylating it at Thr172 residue. IRE1α, an ER membrane-associated protein mediating unfolded protein response (UPR), is required for transducing the signal for activation of ERK1/2-RSK2 under ER stress. Suppression of autophagy by knockdown of RSK2 enhanced the sensitivity of breast cancer cells to ER stress both in vitro and in vivo. Furthermore, we demonstrated that inhibition of RSK2-mediated autophagy rendered breast cancer cells more sensitive to paclitaxel, a chemotherapeutic agent that induces ER stress-mediated cell death. This study identifies RSK2 as a novel controller of autophagy in tumor cells and suggests that targeting RSK2 can be exploited as an approach to reinforce the efficacy of ER stress-inducing agents against cancer.

UCH-L1-mediated Down-regulation of Estrogen Receptor α Contributes to Insensitivity to Endocrine Therapy for Breast Cancer.

Purpose: To determine the role of UCH-L1 in regulating ERα expression, and to evaluate whether therapeutic targeting of UCH-L1 can enhance the efficacy of anti-estrogen therapy against breast cancer with loss or reduction of ERα. Methods: Expressions of UCH-L1 and ERα were examined in breast cancer cells and patient specimens. The associations between UCH-L1 and ERα, therapeutic response and prognosis in breast cancer patients were analyzed using multiple databases. The molecular pathways by which UCH-L1 regulates ERα were analyzed using immunoblotting, qRT-PCR, immunoprecipitation, ubiquitination, luciferase and ChIP assays. The effects of UCH-L1 inhibition on the efficacy of tamoxifen in ERα (-) breast cancer cells were tested both in vivo and in vitro. Results: UCH-L1 expression was conversely correlated with ERα status in breast cancer, and the negative regulatory effect of UCH-L1 on ERα was mediated by the deubiquitinase-mediated stability of EGFR, which suppresses ERα transcription. High expression of UCH-L1 was associated with poor therapeutic response and prognosis in patients with breast cancer. Up-regulation of ERα caused by UCH-L1 inhibition could significantly enhance the efficacy of tamoxifen and fulvestrant in ERα (-) breast cancer both in vivo and in vitro. Conclusions: Our results reveal an important role of UCH-L1 in modulating ERα status and demonstrate the involvement of UCH-L1-EGFR signaling pathway, suggesting that UCH-L1 may serve as a novel adjuvant target for treatment of hormone therapy-insensitive breast cancers. Targeting UCH-L1 to sensitize ER negative breast cancer to anti-estrogen therapy might represent a new therapeutic strategy that warrants further exploration.

Detection of immunoglobulin and T-cell receptor gene rearrangements in angioimmunoblastic T-cell lymphoma.

Objective: To assess the value of immunoglobulin and T-cell receptor gene rearrangements in the diagnosis and differential diagnosis of angioimmunoblastic T-cell lymphoma. Methods: We selected 55 cases of angioimmunoblastic T-cell lymphoma confirmed by histopathology and 15 cases of reactive lymph node hyperplasia. Using the IdentiClone gene rearrangement detection kit, BIOMED-2 primer system, and GeneScanning analysis, we tested for immunoglobulin and T-cell receptor gene rearrangements. Results: Among all 55 angioimmunoblastic T-cell lymphoma cases, 1 (2%) displayed the first type of angioimmunoblastic T-cell lymphoma, which has an intact lymphoid follicle structure. Five cases (9%) displayed the second type, which has an intact segmental lymphatic follicular structure. Forty-nine cases (89%) displayed the third type, which is characterized by a complete obliteration of the lymphatic follicular structure. Fifty-two cases (95%) had tumor cells that were positive for CD3, 50 cases (91%) were positive for CD4, 33 cases (60%) were positive for Bcl-6, 20 cases (36%) were positive for CD10, 44 cases (80%) were positive for CXCL13 to different degrees, and 53 cases (96%) showed a strong positive expression of CD21. Ki67 expression intensity was 30-80% in tumor T cells. Clonal gene rearrangements were identified in 48 of the 55 angioimmunoblastic T-cell lymphoma cases (87%), of which 30 (55%) displayed IG gene rearrangements, including IGHA (7 cases; 13%), IGHB (6 cases; 11%), IGHC (2 cases; 4%), IGKA (22 cases; 40%), IGKB (6 cases; 11%), and IGL (20 cases; 36%). TCR gene rearrangements were observed in 32 cases (58%), including TCRBA (6 cases; 11%), TCRBB (5 cases; 9%), TCRBC (10 cases; 18%), TCRD (7 cases; 13%), TCRGA (22 cases; 40%), and TCRGB (16 cases; 29%). IG and TCR gene rearrangements were concurrently observed in 14 cases (25%). Immunoglobulin or TCR clonal gene rearrangements were not detected in the 15 cases of reactive hyperplasia. Conclusions: Angioimmunoblastic T-cell lymphomas may be positive for immunoglobulin or T-cell receptor clone gene rearrangements or may express double rearrangements. The assessment of clonal gene rearrangements is valuable for the diagnosis and differential diagnosis of angioimmunoblastic T-cell lymphoma.

Chromatin remodeling factor LSH affects fumarate hydratase as a cancer driver.

Cancer metabolism and epigenetic alteration are two critical mechanisms for tumorigenesis and cancer progression; however, the dynamic interplay between them remains poorly understood. As reported in the article entitled "Chromatin remodeling factor LSH drives cancer progression by suppressing the activity of fumarate hydratase," which was recently published in Cancer Research, our group examined the physiological role of lymphocyte-specific helicase (LSH) in nasopharyngeal carcinoma (NPC) by focusing on cancer progression and the tricarboxylic acid cycle. We found that LSH was overexpressed in NPC, and its expression associated with Epstein-Barr virus infection. We also found that LSH directly suppressed fumarate hydratase (FH), a key component of the tricarboxylic acid cycle, in combination with euchromatic histone-lysine N-methyltransferase 2 (EHMT2), also known as G9a. Depletion of FH promoted epithelial-mesenchymal transition (EMT). Moreover, LSH controlled expression of tricarboxylic acid cycle intermediates that promote cancer progression, including EMT, through activation by inhibitor of nuclear factor kappa-B kinase alpha (IKKα), a chromatin modifier and transcriptional activator. Our study showed that LSH plays a critical role in cancer progression, which has important implications for the development of novel strategies to treat NPC.

Co-infection of Epstein-Barr virus and human papillomavirus in human tumorigenesis.

Viral infections contribute to approximately 12% of cancers worldwide, with the vast majority occurring in developing countries and areas. Two DNA viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), are associated with 38% of all virus-associated cancers. The probability of one patient infected with these two distinct types of viruses is increasing. Here, we summarize the co-infection of EBV and HPV in human malignancies and address the possible mechanisms for the co-infection of EBV and HPV during tumorigenesis.

Aspirin induces lytic cytotoxicity in Epstein-Barr virus-positive cells.

Epstein-Barr virus (EBV) infection in tumor cells is generally restricted to the latent forms of viral infection. Switching the latent form of viral infection into the lytic form may induce tumor cell death. High levels of nuclear factor (NF)-kappaB can inhibit EBV lytic replication, and aspirin has the ability to inhibit NF-kappaB activity. The aims of the current study were to determine the effects of aspirin on inducing EBV lytic infection, and thus to reveal the possibility of targeting EBV-positive cancer cells by aspirin. Our results showed that aspirin depleted NF-kappaB (p65) in the nucleus and reactivated EBV into lytic replication. Cells exhibited decreased viability in a dose- and time-dependent manner when incubated with aspirin. When ganciclovir was used in combination with aspirin to treat EBV-positive B95.8 cells and Raji cells, the cytotoxic effect of aspirin was amplified. We demonstrated that aspirin reduced the viability of EBV-positive B lymphocytes due to its ability to induce EBV lytic replication.

Mutations of glucocorticoid receptor differentially affect AF2 domain activity in a steroid-selective manner to alter the potency and efficacy of gene induction and repression.

The transcriptional activity of steroid hormones is intimately associated with their structure. Deacylcortivazol (DAC) contains several features that were predicted to make it an inactive glucocorticoid. Nevertheless, gene induction and repression by complexes of glucocorticoid receptor (GR) with DAC occur with potency (lower EC 50) greater than and efficacy (maximal activity, or A max) equal to those of the very active and smaller synthetic glucocorticoid dexamethasone (Dex). Guided by a recent X-ray structure of DAC bound to the GR ligand binding domain (LBD), we now report that several point mutants in the LBD have little effect on the binding of either agonist steroid. However, these same mutations dramatically alter the A max and/or EC 50 of exogenous and endogenous genes in a manner that depends on steroid structure. In some cases, Dex is no longer a full agonist. These properties appear to result from a preferential inactivation of the AF2 activation domain in the GR LBD of Dex-bound, but not DAC-bound, receptors. The Dex-bound receptors display normal binding to, but a greatly reduced response to, the coactivator TIF2, thus indicating a defect in the transmission efficiency of GR-steroid complex information to the coactivator TIF2. In addition, all GR mutants that are active in gene induction with either Dex or DAC have greatly reduced activity in gene repression. This contrasts with the reports of GR mutations preferentially suppressing GR-mediated induction. The properties of these GR mutants in gene induction support the hypothesis that the A max and EC 50 of GR-controlled gene expression can be independently modified, indicate that the receptor can be modified to favor activity with a specific agonist steroid, and suggest that new ligands with suitable substituents may be able to affect the same LBD conformational changes and thereby broaden the therapeutic applications of glucocorticoid steroids.

Modulation of transcription parameters in glucocorticoid receptor-mediated repression.

Glucocorticoid receptors (GRs) affect both gene induction and gene repression. The disparities of receptor binding to DNA and increased vs. decreased gene expression have suggested significant mechanistic differences between GR-mediated induction and repression. Numerous transcription factors are known to modulate three parameters of gene induction: the total activity (Vmax) and position of the dose-response curve with glucocorticoids (EC50) and the percent partial agonist activity with antiglucocorticoids. We have examined the effects on GR-mediated repression of five modulators (coactivators TIF2 [GRIP1, SRC-2] and SRC-1, corepressor SMRT, and comodulators STAMP and Ubc9), a glucocorticoid steroid (deacylcortivazol [DAC]) of very different structure, and an inhibitor of histone deacetylation (trichostatin A [TSA]). These factors interact with different domains of GR and thus are sensitive topological probes of GR action. These agents altered the Vmax, EC50, and percent partial agonist activity of endogenous and exogenous repressed genes similarly to that previously observed for GR-regulated gene induction. Collectively, these results suggest that GR-mediated induction and repression share many of the same molecular interactions and that the causes for different levels of gene transcription arise from more distal downstream steps.

Doubling the size of the glucocorticoid receptor ligand binding pocket by deacylcortivazol.

A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 A(3), effectively doubling the size of the GR dexamethasone-binding pocket of 540 A(3) and yet leaving the structure of the coactivator binding site intact. DAC occupies only approximately 50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

[Cross-talk between c-Jun/Ets1 involved in EB virus-encoded latent membrane protein 1 regulates expression of matrix metalloproteinase-9 in nasopharyngeal carcinoma].

OBJECTIVE: To investigate effect of AP-1 and Ets binding site adjacent to matrix metalloproteinase-9 (MMP-9) promoter on activation of MMP-9 transcription of nasopharyngeal carcinoma cells transfected with EBV-encoded latent membrane protein 1 (LMP1), and to ascertain if cross-talk between c-Jun and Ets1 is involved in LMP1-regulating expression of MMP-9. METHODS: Site-directed mutagenesis technique was used to establish a series of mutants, including MMP-9-CAT-Ets(-540)mt, MMP-9-CAT-AP-1(-533)mt and MMP-9-CAT-AP-1(-533)/Ets(-540)mt. After the mutants were transfected into LMP1-expressing NPC HNE2 cells regulated by Tet-on system (pTet-on-LMP1 HNE2), CAT activity of these mutants were assayed with induction of LMP1. With blockade of c-Jun or Ets1 antisense oligonucleotides, the activity of MMP-9 induced by LMP1 was assayed with gelatin zymography. RESULTS: The CAT activity of MMP-9-Ets(-540)mt-CAT, MMP-9-AP-1(-533)mt-CAT, MMP-9-AP-1(-533)/Ets(-540) mt-CAT decreased significantly compared to MMP-9-CAT wt. After blockade with c-Jun or Ets1 antisense oligonucleotides, activity of MMP-9 induced by LMP1 decreased significantly, especially with combined blockade of c-Jun and Ets1. CONCLUSION: The results suggest that transcription factor AP-1 and Ets play an crucial role in activation of MMP-9 transcription induced by LMP1, and cross-talk between c-Jun/Ets1 is involved in expression of MMP-9 mediated by LMP1.

Epstein-Barr virus encoded latent membrane protein 1 modulates nuclear translocation of telomerase reverse transcriptase protein by activating nuclear factor-kappaB p65 in human nasopharyngeal carcinoma cells.

Sustained proliferation of cancer cells requires that telomerase maintain chromosomal stability and prolong telomere length-regulated cell replication. Human telomerase reverse transcriptase (hTERT), the human telomerase catalytic subunit, and also the key determinant of the enzymatic activity of human telomerase, is regulated both at the transcriptional level and via phosphorylation and translocation. In this study, we show that latent membrane protein 1 (LMP1), the principal oncoprotein of Epstein-Barr virus (EBV), modulates telomerase activity by inducing the direct binding of hTERT to nuclear factor kappaB (NF-kappaB) p65 and translocation of both proteins from the cytoplasm to the nucleus in nasopharyngeal carcinoma cells (NPC). Conversely, a NF-kappaB nuclear translocation inhibitor, (benzylcarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO), and a dominant negative mutant of inhibitor of NFkappaB (IkappaBalpha), can block LMP1-induced hTERT nuclear translocation. These studies suggest a novel function of LMP1 and confirm that NF-kappaB plays an important role in regulating the activation and nuclear translocation of telomerase in NPC cells.

[EB virus encoded latent membrane protein 1 mediated expression of transcription factor Ets-1 in nasopharyngeal carcinoma cells].

OBJECTIVE: To elucidate the expression of tanscription factor Ets-1 mediated by EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma (NPC) cells. METHODS: LMP1-expressing NPC HNE2 cells regulated by Tet-on system (pTet-on-LMP1 HNE2) were used. Expression of LMP1 and Ets-1 was observed after induction with Doxycycline (Dox). Expression of Ets-1 mRNA and protein was detected by RT-PCR and Western blot, respectively. The phosphorylation level of Ets-1 protein was examined by co-immunoprecipitation. The DNA binding activity of Ets-1 was detected by electrophoretic-mobility shift assay (EMSA). RESULTS: After induction with Dox in pTet-on-LMP1 HNE2 cells, to some extent, the expression of Ets-1 mRNA and protein, its phosphorylation level and DNA binding activity were increased with enhancement of LMP1 expression. CONCLUSION: LMP1 induces transcriptional activation and expression of Ets-1 which may contribute to the development of NPC.

Heterodimer formation between c-Jun and Jun B proteins mediated by Epstein-Barr virus encoded latent membrane protein 1.

Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is essential for the immortalization of human B cells and is linked etiologically to several human tumors. LMP1 is an integral membrane protein which acts like a constitutively active receptor. It binds tumor necrosis factor (TNF)-receptor-associated factors (TRAFs), activates NFkappaB and triggers the transcription factor activating protein-1 (AP-1) via the c-Jun N-terminal kinase (JNK) cascade, but its specific contribution to AP-1 has not been elucidated fully. Members of AP-1 family, the Jun and fos related protein, have been shown to directly interact and form heterodimeric complexes. In this report, using a Tet-on LMP1 HNE2 cell line which is a dual-stable LMP1 integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 in which could be regulated by Tet-on system, we show that Jun B can efficiently form a new heterodimeric complex with the c-Jun protein under the regulation of LMP1, phosphorylation of c-Jun (ser63, ser73) and Jun B involved in the process of the new heterodimeric form. We also find that this heterodimeric form can bind to the AP-1 consensus sequence. Transfection studies suggest that JNK interaction protein (JIP) could inhibit the heterodimer form of c-Jun and Jun B through blocking the AP-1 signaling pathway triggered by LMP1. The interaction and function between c-Jun protein and Jun B protein increase the repertoire of possible regulatory complexes by LMP1 that could play an important role in the regulation of transcription of specific cellular genes in the process of genesis of nasopharyngeal carcinoma.

Nuclear factor kappa B (NFkappaB) dependent modulation of Epstein-Barr virus latent membrane protein 1 (LMP1) in epidermal growth factor receptor (EGFR) promoter activity.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncoprotein may cause multiple cellular changes, including the induction of epidermal growth factor receptor (EGFR) expression and the activation of the nuclear factor kappa B (NFkappaB) transcription factor. LMP1 increases the levels of both EGFR protein and mRNA but does not stabilize EGFR mRNA. Thus the effects of LMP1 are likely to be mediated by direct activation of the EGFR promoter. In this study, induction of LMP1 increased the EGFR in both protein and promoter levels in a dose dependent manner using tetracycline-regulated LMP1 expression in nasopharyngeal carcinoma (NPC) cell line. Mutational analysis of the LMP1 protein indicated that the C-terminal activation region-1 (CTAR1) domain was mainly involved in the EGFR promoter induction, while CTAR2 was necessary but not sufficient to induce EGFR promoter. Inhibition of LMP1 mediated NFkappaB activation by constitutive repressive inhibitory kappa B alpha (IkappaBalpha) marginally decreased EGFR promoter activity using transiently transfected IkappaBalpha dominant negative mutant. Promoter mutagenesis analysis demonstrated that two putative NFkappaB binding sites of EGFR promoter were very necessary for the transcriptional activity of EGFR induced by LMP1, the proximal NFkappaB binding site was more important than the distal NFkappaB binding site. Taken together, Epstein-Barr virus latent membrane protein 1 modulated the EGFR promoter activity in a NFkappaB dependent manner.

Epstein-Barr virus latent membrane protein 1 modulates epidermal growth factor receptor promoter activity in a nuclear factor kappa B-dependent manner.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncoprotein may cause multiple cellular changes including the induction of epidermal growth factor receptor (EGFR) expression and activation of the NFkappaB transcription factor. LMP1 increases the levels of both EGFR protein and mRNA, but does not stabilize EGFR mRNA. Thus, the effects of LMP1 are likely to be mediated by the direct activation of the EGFR promoter. In this study, induction of LMP1 increased the EGFR in both protein and promoter levels in a dose-dependent manner using tetracycline-regulated LMP1 expression in nasopharyngeal carcinoma (NPC) cell line. Mutational analysis of the LMP1 protein indicated that the C-terminal activation region-1 (CTAR1) domain was mainly involved in the EGFR promoter induction, while CTAR2 was necessary but not sufficient to induce EGFR promoter. Inhibition of LMP1-mediated NFkappaB activation by constitutive repressive IkappaBalpha marginally decreased EGFR promoter activity using transiently transfected IkappaBalpha dominant negative mutant. Promoter mutagenesis analysis demonstrated that two putative NFkappaB binding sites of EGFR promoter were very necessary for the transcriptional activity of EGFR induced by LMP1, the proximal NFkappaB binding site was more important than the distal NFkappaB binding site, and both NFkappaB binding sites played a cooperative role. Taken together, Epstein-Barr virus latent membrane protein 1 modulated the EGFR promoter activity in a NFkappaB-dependent manner.

[Effect of JIP on the proliferation and apoptosis of nasopharyngeal carcinoma cells].

BACKGROUND & OBJECTIVE: Previous studies showed that JNK signaling pathway activated by LMP1 plays an important role in carcinogenesis of nasopharyngeal carcinoma (NPC). JNK interacting protein (JIP) can inhibit JNK signaling pathway in NPC cell. This study was designed to elucidate the effect of JIP on the proliferation and apoptosis of NPC cells. METHODS: After treatment with JIP at different concentrations and time points, the number of proliferating cells were determined by MTT assay; the ability of proliferation of NPC cells was measured by the rate of cloning formation; cell cycle and the apoptotic rate of NPC cells was assayed by flow cytometry. RESULTS: 1. MTT assay showed that cell proliferation was significantly inhibited by JIP in a dose- and time-dependent manner. After treatment with JIP for 24, 48, and 72 hours, the rate of survival cells were 77.8%, 59.2%, and 61.8%, respectively. 2. The number and volume of colonies were decreased after transfection with JIP. 3. The number of cells in S phase was decreased from 25.87% to 19.96%, and the number of cells in G0/G1 phase was elevated from 66.24% to 71.89% after treatment with JIP. 4. In contrast to the control group, the 24 hours apoptotic rate was elevated from 1.25% to 8.25% (about 6.6 times); the 48 hours apoptotic rate was elevated from 1.04% to 31.45% (about 30 times). CONCLUSIONS: The results demonstrated that JIP inhibit the growth of nasopharyngeal carcinoma through arresting the cell cycle at G1/S checkpoint and triggering the apoptosis of cells. Data suggest that JIP is a potent molecular drug for the treatment of the patients with nasopharyngeal carcinoma.