Suping Ren

Identification of novel epigenetically modified genes in human melanoma via promoter methylation gene profiling

The inactivation of tumor‐related genes through the aberrant methylation of promoter CpG islands is thought to contribute to tumor initiation and progression. We therefore investigated promoter methylation events involved in cutaneous melanoma by screening 30 genes of interest for evidence of promoter hypermethylation, examining 20 melanoma cell lines and 40 freshly procured melanoma samples. Utilizing quantitative methylation‐specific PCR, we identified five genes (SOCS1, SOCS2, RAR‐beta 2, TNFSF10C, and TNFSF10D) with hypermethylation frequencies ranging from 50% to 80% in melanoma cell lines as well as freshly procured tissue samples. Eighteen genes (LOX, RASSF1A, WFDC1, TM, APC, TFPI2, TNFSF10A, CDKN2A, MGMT, TIMP3, ASC, TPM1, IRF8, CIITA‐PIV, CDH1, SYK, HOXB13, and DAPK1) were methylated at lower frequencies (2–30%). Two genes (CDKN1B and PTEN), previously reported as methylated in melanoma, and five other genes (RECK, IRF7, PAWR, TNFSF10B, and Rb) were not methylated in the samples screened here. Daughter melanoma cell lines showed identical methylation patterns when compared with original samples from which they were derived, as did synchronous metastatic lesions from the same patient. We identified four genes (TNFSF10C, TNFSF10D, LOX, and TPM1) that have never before been identified as hypermethylated in melanoma, with an overall methylation frequency of 60, 80, 50, and 10%, respectively, hypothesizing that these genes may play an important role in melanoma progression.

Epigenetics in human melanoma.

BACKGROUND Recent technological advances have allowed us to examine the human genome in greater detail than ever before. This has opened the door to an improved understanding of the gene expression patterns involved with cancer. METHODS A review of the literature was performed to determine the role of epigenetic modifications in human melanoma. We focused the search on histone deacetylation, methylation of gene promoter regions, demethylation of CpG islands, and the role of microRNA. We examined the relationship between human melanoma epigenetics and their importance in tumorigenesis, tumor progression, and inhibition of metastasis. The development and clinical application of select pharmacologic agents are also discussed. RESULTS We identified several articles that have extensively studied the role of epigenetics in melanoma, further elucidating the complex processes involved in gene regulation and expression. Several new agents directly affect epigenetic mechanisms in melanoma, with divergent affects on the metastatic potential of melanoma. CONCLUSIONS Epigenetic mechanisms have emerged as having a central role in gene regulation of human melanoma, including the identification of several putative tumor suppressor genes and oncogenes. Further research will focus on the development of novel therapeutics that will likely target and alter such epigenetic changes.

Chidamide, a novel histone deacetylase inhibitor, synergistically enhances gemcitabine cytotoxicity in pancreatic cancer cells.

Pancreatic cancer is a lethal human malignancy with an extremely poor prognosis and urgently requires new therapies. Histone deacetylase inhibitors (HDACIs) represent a new class of anticancer agents and have shown promising antitumor activities in preclinical models of pancreatic cancer. In this study, we sought to determine the antitumor effects of a novel HDACI, chidamide (CS055), in pancreatic cancer cells alone or in combination with gemcitabine. Treatments of BxPC-3 or PANC-1 pancreatic cancer cell lines with chidamide resulted in dose- and time-dependent growth arrest, accompanied by induction of p21 expression. When combined in a sequential schedule, chidamide synergistically enhanced gemcitabine-induced cell growth arrest and apoptosis, accompanied by cooperative downregulation of Mcl-1 and loss of mitochondrial membrane potential (ΔΨm). Chidamide enhanced gemcitabine-induced DNA double-strand breaks and S phase arrest, and abrogated the G2/M cell cycle checkpoint, potentially through suppression of CHK1 expression. Our results suggest that chidamide has a therapeutic potential for treating pancreatic cancer, especially in combination with gemcitabine.

The Impact of Genomics in Understanding Human Melanoma Progression and Metastasis

BACKGROUND Recent technological advances in the analysis of the human genome have opened the door to improving our primitive understanding of the gene expression patterns in cancer. For the first time, we have an overview of the complexities of tumorigenesis and metastatic progression of cancer. The examination of the phenotypic and (epi)genetic changes in cutaneous melanoma has identified several genes deemed central to the development and progression of melanoma. METHODS A review of the recent literature was performed to determine the role of array-based high-throughput gene expression analysis in understanding the specific genes involved as well as the pathways and the comparative gene expression patterns of primary and metastatic melanoma. RESULTS Most studies utilizing gene microarray analysis and other whole genome approaches reveal a wide array of genes and expression patterns in human melanoma. Furthermore, several of the same genes have been found in comparative studies, with some studies attempting correlation with clinical outcome. Several genes have been identified as potential prognostic markers of tumor progression and overall clinical outcome. CONCLUSIONS High-throughput gene expression analysis has had a major impact in melanoma research. Several gene expression platforms have provided insight into the gene expression patterns in melanoma. Such data will provide the foundations for the future development of prognostic markers and improved targeted therapies for patients with melanoma.

Cytoplasmic translocation of p21 mediates NUPR1-induced chemoresistance: NUPR1 and p21 in chemoresistance

The expression of Nuclear Protein 1 (NUPR1) is associated with chemoresistance in multiple malignancies. We previously reported that NUPR1 functions as a transcriptional cofactor for the p300–p53 complex and transcriptionally regulates p21 expression. In the present study we investigated the activity of NUPR1 in p53‐deficient, triple‐negative, inflammatory SUM159 breast cancer cells. Our studies reveal that NUPR1 confers growth benefit and chemoresistance by causing Akt‐mediated phosphorylation and subsequent cytoplasmic re‐localization of p21 and activation of the anti‐apoptotic Bcl‐xL protein. Our findings elucidate a NUPR1‐PI‐3‐K/Akt‐phospho‐p21 axis that functions in p53‐negative, inflammatory breast cancer cells to enhance chemoresistance in breast cancer.

Expression and functional analysis of the WAP four disulfide core domain 1 gene in human melanoma

The exact cellular and molecular mechanisms involved in melanoma tumorigenesis remain obscure. Previous gene expression profiling analyses performed upon NHEM and human melanoma samples identified WFDC1 as one of the most frequently down-regulated genes. Here we further showed that NHEM readily express WFDC1 but expression is reduced or completely lost in 80% of the patients-derived melanoma cell lines and tissue samples examined. Furthermore, we show that promoter hypermethylation accounts for the silencing of the WFDC1 gene in 20% of the melanoma cell lines examined. The over-expression of WFDC1 in two metastatic melanoma cell lines, A375 and LOX, resulted in a significant delay of tumor growth in a murine xenograft model, despite a non-significant difference in tumor cell growth in vitro. Gene expression microarray analysis and further expression validation suggests that the Dickkopf-1 (Dkk1) gene is up-regulated in WFDC1 over-expressing cell lines, suggesting that the tumor suppressive function of WFDC1 may be partially a result of up-regulated Dkk1 gene expression, which is known to be a potent inhibitor of the Wnt signaling pathway.

Functional characterization of the progestagen-associated endometrial protein gene in human melanoma

Utilizing gene microarray profiling of melanoma samples, we have recently identified a novel gene overexpressed in both thick primary and metastatic melanomas. This gene, progestagen‐associated endometrial protein (PAEP), has never before been implicated in the oncogenic processes of melanoma, with its true function in oncogenesis and tumour progression relatively unknown. Overexpression of the PAEP gene in freshly procured thick primary and metastatic melanoma samples (58%) and daughter cell lines (77%) is confirmed by quantitative RT‐PCR, immunohistochemistry, Western blotting and mass spectrometric analysis. We suggest that PAEP gene overexpression is involved with melanoma tumour progression as well as an aggressive phenotype. Transfection of melanoma cells with PAEP small interfering RNA (siRNA) reveals a significant decrease in soft agar colony formation and a marked inhibition of both cell migration and cell invasion. Furthermore, we establish stable melanoma transfectants via PAEP lentiviral small hairpin RNA (shRNA), examine their growth characteristics in a murine xenograft model and reveal that tumour growth is significantly inhibited in two separate melanoma cell lines. Our data strongly implicate the PAEP gene as a tumour growth promoter with oncogenic properties and a potential therapeutic target for patients with advanced melanoma.

Manganese Superoxide Dismutase Gene Therapy Protects Against Irradiation- Induced Intestinal Injury

Radiation-induced intestinal injury is a common complication in radiotherapy for solid organ malignancies in abdomen or pelvis. However, currently there are no approved medical countermeasures for radiation-induced intestinal injury. Therefore, it is urgent to develop new treatments for radiation-induced intestinal injury. In the present study, we demonstrated that bone marrow derived mesenchymal stem cells (MSCs) and overexpression of human manganese superoxide dismutase (MnSOD) could ameliorate radiation-induced intestinal syndrome. NOD/SCID mice received abdominal irradiation at a selected dose of 5 Gy, and then infused intravenously with MnSOD-MSCs. Mice body weight, survival and diarrhea were monitored for 30-days. Colonization and differentiation of MnSOD-MSCs in the irradiated intestine were analyzed by histological and immunohistochemical methods. Consequently, our data demonstrated that intravenous administration of MnSOD-MSCs improved survival, decreased diarrhea occurrence and protected the small intestinal structural integrity of irradiated mice. Moreover, intravenously transplanted MnSOD-MSCs could colonize the irradiated intestine and repair injured sites. These findings suggested that MnSOD-MSCs may be an attractive and potential option for radiation-induced intestinal injury.

Chidamide Inhibits Aerobic Metabolism to Induce Pancreatic Cancer Cell Growth Arrest by Promoting Mcl-1 Degradation

Pancreatic cancer is a fatal malignancy worldwide and urgently requires valid therapies. Previous research showed that the HDAC inhibitor chidamide is a promising anti-cancer agent in pancreatic cancer cell lines. In this study, we elucidate a probable underlying anti-cancer mechanism of chidamide involving the degradation of Mcl-1. Mcl-1 is frequently upregulated in human cancers, which has been demonstrated to participate in oxidative phosphorylation, in addition to its anti-apoptotic actions as a Bcl-2 family member. The pancreatic cancer cell lines BxPC-3 and PANC-1 were treated with chidamide, resulting in Mcl-1 degradation accompanied by induction of Mcl-1 ubiquitination. Treatment with MG132, a proteasome inhibitor reduced Mcl-1 degradation stimulated by chidamide. Chidamide decreased O2 consumption and ATP production to inhibit aerobic metabolism in both pancreatic cancer cell lines and primary cells, similar to knockdown of Mcl-1, while overexpression of Mcl-1 in pancreatic cancer cells could restore the aerobic metabolism inhibited by chidamide. Furthermore, chidamide treatment or Mcl-1 knockdown significantly induced cell growth arrest in pancreatic cancer cell lines and primary cells, and Mcl-1 overexpression could reduce this cell growth inhibition. In conclusion, our results suggest that chidamide promotes Mcl-1 degradation through the ubiquitin-proteasome pathway, suppressing the maintenance of mitochondrial aerobic respiration by Mcl-1, and resulting in inhibition of pancreatic cancer cell proliferation. Our work supports the claim that chidamide has therapeutic potential for pancreatic cancer treatment.

Valproic acid-mediated myocardial protection of acute hemorrhagic rat via the BCL-2 pathway.

BACKGROUND Hemorrhage is a major cause of morbidity and mortality among trauma patients. The pathophysiologic changes following acute severe hemorrhage and tissue hypoxia lead to an imbalance of protein acetylation. Histone deacetylase inhibitors (HDACIs) were reported to restore the acetylation imbalance and serve as potential drugs for treating severe hemorrhage. However, the molecular mechanism of HDACI-mediated cytoprotection remains unclear. In this study, we examined the myocardial protective effects and respective mechanism of the HDACI valproic acid (VPA) administered during hemorrhagic and hypoxic stress in vivo and in vitro. METHODS In vivo, the therapeutic effect of VPA was evaluated in acute severe hemorrhagic rats, and the expressions of BCL-2 signal pathway molecules were observed in rat heart tissues. To explore the molecular mechanism of VPA-mediated myocardial protection, a cobalt chloride (CoCl2)–induced hypoxia model of rat H9c2 cardiomyoblasts was applied to mimic hypoxic injury raised by acute hemorrhage. RESULTS VPA administration significantly improved the 4-hour survival rate of hemorrhagic animals from 55% to 100% and protected H9c2 cells against CoCl2-induced hypoxic injury at a dose of between 12.5 &mgr;M and 100 &mgr;M. Increased expression of BCL-2 messenger RNA was observed following VPA treatment in the heart tissues of hemorrhagic rats (approximately 4.9-fold) and in H9c2 cells that survived CoCl2-induced hypoxia (approximately 4.9-fold). Western blot analysis showed a concomitant increase in BCL-2 protein expression and Akt phosphorylation following VPA treatment. The cytoprotective activity of VPA was diminished by triciribine-mediated inhibition of Akt activation and by silencing of BCL-2 gene expression. CONCLUSION These findings suggest that VPA protects myocardial cells from hemorrhagic and hypoxic stress through the Akt/BCL-2 survival pathway, indicating a potential use of HDACIs for acute severe hemorrhage treatment.