Inactivation of tumor suppressor gene HIC1 in gastric cancer is reversed via small activating RNAs

HIC1 is a tumor suppressor gene that is down-expressed in different malignancies, in part, because of promoter hypermethylation. However, the biological function of HIC1 in gastric cancer remains unclear. It is known that small double-stranded RNAs can induce gene expression by targeting promoter sequences. In the present study, we examined the expression levels of HIC1 in gastric cancer tissue. Several pieces of small double-stranded RNAs were used for the activation of HIC1. Tissue microarray analysis of gastric cancer indicated that down-regulation of HIC1 in gastric cancer tissue was dramatic compared with the adjacent gastric mucosa. Gastric cancer cell lines also showed down-regulated HIC1 expression compared with a human immortalized gastric mucosa cell line. One out of four dsRNAs produced activation of HIC1 as assessed by real-time PCR and Western blotting. Use of a cell counting kit 8 and clonogenicity assays indicated that dsRNA-mediated re-expression of HIC1 inhibited cell proliferation and clonogenicity in gastric cancer. Reactivation of HIC1 suppressed cell migration and induced cell cycle arrest in the G0/G1 phase, as well as induced apoptosis. These results suggest that HIC1 is a potential target of gene therapy against gastric cancer, and that dsRNAs could function as a therapeutic option for up-regulating tumor suppressor genes in gastric cancer and other malignancies.     

Identification of novel subregions of LOH in gastric cancer and analysis of the HIC1 and TOB1 tumor suppressor genes in these subregions

Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R(1)-R(3) from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR(1)) and TOB1 (in SR(3)) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR(1)-SR(5) (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.     

Pro-apoptotic meiogynin A derivatives that target Bcl-xL and Mcl-1

The biological evaluation of a natural sesquiterpene dimer meiogynin A 1, is described as well as that of five non-natural analogues. Although active on a micromolar range on the inhibition of Bcl-xL/Bak and Mcl-1/Bid interaction, meiogynin A 1 is not cytotoxic on three cell lines that overexpress Bcl-xL and Mcl-1. Contrarily, one of its analogues 6 with an inverted configuration on the side chain and an aromatic moiety replacing the cyclohexane ring was active on both target proteins, cytotoxic on a micromolar range and was found to induce apoptosis through a classical pathway.     

Human VRK2 modulates apoptosis by interaction with Bcl-xL and regulation of BAX gene expression

VRK2 is a novel Ser-Thr kinase whose VRK2A isoform is located in the endoplasmic reticulum and mitochondrial membranes. We have studied the potential role that VRK2A has in the regulation of mitochondrial-mediated apoptosis. VRK2A can regulate the intrinsic apoptotic pathway in two different ways. The VRK2A protein directly interacts with Bcl-xL, but not with Bcl-2, Bax, Bad, PUMA or Binp-3L. VRK2A does not compete with Bax for interaction with Bcl-xL, and these proteins can form a complex that reduces apoptosis. Thus, high VRK2 levels confer protection against apoptosis. In addition, VRK2 knockdown results in an increased expression of BAX gene expression that is mediated by its proximal promoter, thus VRK2A behaves as a negative regulator of BAX. Low levels of VRK2A causes an increase in mitochondrial Bax protein level, leading to an increase in the release of cytochrome C and caspase activation, detected by PARP processing. VRK2A loss results in an increase in cell death that can be detected by an increase in annexinV+ cells. Low levels of VRK2A increase cell sensitivity to induction of apoptosis by chemotherapeutic drugs like camptothecin or doxorubicin. We conclude that VRK2A protein is a novel modulator of apoptosis.     

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.     

Design,synthesis and biological evaluation of new inhibitors of Bax/Bcl-xL interaction in cancer cells

We describe the synthesis of a series of new molecules containing phenol and triazoles moieties, compounds which have been evaluated for their ability to inhibit Bax/Bcl-xL interactions in cancer cells, by using BRET assays, and to induce cell death. Several derivatives exhibit a very promising activity, being more potent than the reference compounds acylpyrogallol A and ABT-737. These preliminary results demonstrate that derivatives of this family can be attractive to develop new molecules with potent anticancer activity.     

Physical and functional interaction of the p14ARF tumor suppressor with ribosomes

Alterations in the p14(ARF) tumor suppressor are frequent in many human cancers and are associated with susceptibility to melanoma, pancreatic cancer, and nervous system tumors. In addition to its p53-regulatory functions, p14(ARF) has been shown to influence ribosome biogenesis and to regulate the endoribonuclease B23, but there remains considerable controversy about its nucleolar role. We sought to clarify the activities of p14(ARF) by studying its interaction with ribosomes. We show that p14(ARF) and B23 interact within the nucleolar 60 S preribosomal particle and that this interaction does not require rRNA. In contrast to previous reports, we found that expression of p14(ARF) does not significantly alter ribosome biogenesis but inhibits polysome formation and protein translation in vivo. These results suggest a ribosome-dependent p14(ARF) pathway that regulates cell growth and thus complements p53-dependent p14(ARF) functions.     

Physical and functional interaction between the HCMV IE2 protein and the Wilms' tumor suppressor WT1

Human cytomegalovirus (HCMV) is a major renal pathogen in congenitally infected infants and renal allograft recipients. It has been shown that human kidney cells of glomerular, tubular, and vascular origin were all infected by HCMV in vitro. It has previously been demonstrated that the IE2 protein of HCMV directly associates with the zinc finger domain of Egr-1. The zinc finger region of WT1 is a sequence-specific DNA-binding domain which also recognizes the consensus DNA binding site (5'-CGCCCCCGC-3') of Egr-1, thus suggesting a possible interaction between WT1 and IE2. Here we demonstrate that HCMV IE2 binds to the C-terminal region of WT1 containing zinc finger domain in vivo as well as in vitro and that WT1 can inhibit IE2-driven transactivation of the responsive promoter. Our results suggest that WT1 may be able to regulate the functional activity of HCMV IE2. Furthermore, these data may provide new insights into the possible involvement of HCMV in WT1-related pathogeneses.     

Inhibitor of Nrf2 (INrf2 or Keap1) protein degrades Bcl-xL via phosphoglycerate mutase 5 and controls cellular apoptosis

INrf2 (Keap1) is an adaptor protein that facilitates INrf2-Cul3-Rbx1-mediated ubiquitination/degradation of Nrf2, a master regulator of cytoprotective gene expression. Here, we present evidence that members of the phosphoglycerate mutase family 5 (PGAM5) proteins are involved in the INrf2-mediated ubiquitination/degradation of anti-apoptotic factor Bcl-xL. Mass spectrometry and co-immunoprecipitation assays revealed that INrf2, through its DGR domain, interacts with PGAM5, which in turn interacts with anti-apoptotic Bcl-xL protein. INrf2-Cul3-Rbx1 complex facilitates ubiquitination and degradation of both PGAM5 and Bcl-xL. Overexpression of PGAM5 protein increased INrf2-mediated degradation of Bcl-xL, whereas knocking down PGAM5 by siRNA decreased INrf2 degradation of Bcl-xL, resulting in increased stability of Bcl-xL. Mutation of PGMA5-E79A/S80A abolished INrf2/PGAM5/Bcl-xL interaction. Therefore, PGAM5 protein acts as a bridge between INrf2 and Bcl-xL interaction. Further studies showed that overexpression of INrf2 enhanced degradation of PGAM5-Bcl-xL complex, led to etoposide-mediated accumulation of Bax, increased release of cytochrome c from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. In addition, antioxidant (tert-butylhydroquinone) treatment destabilized the Nrf2-INrf2-PGAM5-Bcl-xL complex, which resulted in release of Nrf2 in cytosol and mitochondria, release of Bcl-xL in mitochondria, increase in Bcl-xL heterodimerization with Bax in mitochondria, and reduced cellular apoptosis. These data provide the first evidence that INrf2 controls Bcl-xL via PGAM5 and controls cellular apoptosis.     

Expression of Parkin,APC, APE1, and Bcl-xL in Colorectal Polyps

Colorectal cancer can develop through molecular, chromosomal, and epigenetic cumulative changes that transform the normal intestinal epithelium into the colorectal polyps, called conventional adenomas (CAs) or serrated polyps (SPs), recognized as precursors of invasive colorectal neoplasia. These benign lesions need to explore the morphology, histological diagnosis, and biomarkers profile to accurately characterize lesions with potential for evolution to cancer. This study aimed to correlate the immunohistochemical expression of Parkin and Adenomatous Polyposis Coli (APC; tumor suppressors), Human Apurinic/Apyrimidinic endonuclease 1 (APE1), and B-cell lymphoma-extra-large (Bcl-xL; oncogenic proteins) in sporadic colorectal polyps with clinical, endoscopic, and diagnostic data. Immunohistochemical analysis was performed on tissue microarray samples of 306 polyps. Based on the Allred score, the expressions were graduated in the cytoplasm and nucleus of superficial and cryptic cells. There was higher Parkin nuclear expression (p=0.006 and 0.010) and APC cytoplasmic expression in cryptic cells (p<0.001) in SPs. CAs, APE1 (p<0.001) and Bcl-xL (p<0.001) were more expressed in the nuclei and cytoplasms, respectively. These results are related to the biological role proposed for these proteins in cellular functions. They can contribute to the diagnosis criteria for polyps and improve the knowledge of biomarkers that could predict cancer development:     

Morphological changes and nuclear translocation of DLC1 tumor suppressor protein precede apoptosis in human non-small cell lung carcinoma cells

We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer.     

Molecular interaction between HAX-1 and XIAP inhibits apoptosis

Caspase-3 is an important executor caspase that plays an essential role in apoptosis. Recently, HS1-associated protein X1 (HAX-1) was found to be a substrate of caspase-3. Although HAX-1 has serve multifunctional roles in cellular functions such as cell survival and calcium homeostasis, the detailed functional mechanism of HAX-1 remains still unclear. In this study, we performed proteomic experiments to identify the HAX-1 interactome. Through immunoprecipitation and 2D gel electrophoresis, we identified X-linked inhibitor of apoptosis protein (XIAP) as a novel HAX-1-interacting protein. By performing the GST pull-down assay, we defined the interaction domains in HAX-1 and XIAP, showing that HAX-1 binds to the BIR2 and BIR3 domains of XIAP whereas XIAP binds to the C-terminal domain of HAX-1. In addition, surface plasma resonance experiments showed that both BIR2 and BIR3 domains of XIAP bind to HAX-1 with affinity similar to that of full-length XIAP, indicating that either domain is necessary and sufficient for tight binding to HAX-1. Taken together with the observation that HAX-1 suppresses the polyubiquitination of XIAP, the cell viability assay results suggest that the formation of the HAX-1-XIAP complex inhibits apoptosis by enhancing the stability of XIAP against proteosomal degradation.