Growth Arrest-specific 2 Research Articles

FOXA1, FOXA2, SOX10 and GAS2 Gene Expression in Oral Squamous Cell Carcinoma and Their Relationship with Clinicopathological Indices

Background: The use of molecular methods in cancer diagnosis has led to a better prognosis. One of the important gene families in the carcinogenic pathways of various cancers is the forkhead box (FOX) family genes. Moreover, developmental transcription factors and proapoptotic proteins play critical roles in cell function and carcinogenesis. Objectives: The current study aimed to evaluate the expression of A1 FOXA1, FOXA2, SOX10, and growth arrest specific 2 (GAS2) genes in oral squamous cell carcinoma (OSCC) tumors due to biomarker discovery and early diagnosis of cancer. Methods: To evaluate the expression of FOXA1, FOXA2, SOX10, and GAS2 genes, 30 OSCC samples and 30 normal specimens were obtained from Imam Khomeini Hospital Cancer Institute. RNA extraction and cDNA synthesis were done by relevant kits. After a specific primer design for FOXA1, FOXA2, SOX10, and GAS2 genes, real-time PCR was done to evaluate the genes’ expression for molecular biomarker discovery and validation. ANOVA and independent t-test were used to analyze the data. Results: Significant differences were observed in the expression of the studied genes in tumor and control tissues (P < 0.001). The results showed that FOXA1, GAS2, and SOX10 expressions in tumor and normal cells have significant differences (P < 0.001). Regardless of FOXA1, FOXA2 and SOX10, there was a significant difference in the expression of GAS2 genes in term patients’ age (P < 0.05) and overexpressed in patients over 55 years. SOX10 gene is upregulated in grade II OSCC tumors but there is no significant difference in expression of FOXA1, FOXA2, and GAS2 in different stages and grades. The ROC curve analysis, FOXA1, and FOXA2 showed AUC = 0.66 and AUC = 0.57 respectively. Meanwhile, SOX10 and GAS2 showed AUC = 0.9 and AUC = 1 respectively. Conclusions: In general, the expression of FOXA1, GAS2, and SOX10 genes in cancer and control tissues were different, and therefore the role of these genes in OSCC is confirmed. Also, in the present study, the biomarker potential of SOX10 and GAS2 genes for OSCC diagnosis was demonstrated. In the current study, the important role of the studied genes in OSCC diagnosis was shown. However, further studies are needed to confirm this.

MiR-520c-3p regulates IL-1\u03b2-stimulated human chondrocyte apoptosis and cartilage degradation by targeting GAS2

BackgroundMicroRNAs (miRNAs) have been shown to be associated with osteoarthritis (OA) progression. This study aimed to explore the role of miR-520c-3p in OA progression.MethodsExpression levels of miR-520c-3p and Growth arrest-specific 2 (GAS2) were detected using quantitative real-time PCR. The proliferation and apoptosis of cells were measured using cell counting kit 8 (CCK8) assay and flow cytometry. Furthermore, the protein levels of apoptosis-related markers, extracellular degradation markers, inflammatory response markers, and GAS2 were tested using quantitative real-time polymerase chain reaction (RT-PCR) and western blot (WB) analysis. In addition, the interaction between miR-520c-3p and GAS2 was examined using dual luciferase reporter assay.ResultsGAS2 was highly expressed, and miR-520c-3p was lowly expressed in OA cartilage tissues. miR-520c-3p could promote the proliferation and inhibit the apoptosis and inflammation of OA chondrocytes. miR-520c-3p could be sponged by GAS2, and its inhibitor could reverse the regulation of GAS2 on the biological functions of OA chondrocytes. GAS2 was a target of miR-520c-3p, which was identified by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of GAS2 could inhibit the proliferation and promoted the apoptosis and inflammation of OA chondrocytes.ConclusionOur data showed that miR-520c-3p might regulate the GAS2 to inhibit the progression of OA.

Growth arrest-specific 2 protein family: Structure and function.

Members of the growth arrest–specific 2 (GAS2) protein family consist of a putative actin‐binding (CH) domain and a microtubule‐binding (GAR) domain and are considered miniversions of spectraplakins. There are four members in the GAS2 family, viz. GAS2, GAS2L1, GAS2L2 and GAS2L3. Although GAS2 is defined as a family of growth arrest–specific proteins, the significant differences in the expression patterns, interaction characteristics and biological issues or diseases among the different GAS2 family members have not been systemically reviewed to date. Therefore, we summarized the available evidence on the structures and functions of GAS2 family members. This review facilitates a comprehensive molecular understanding of the involvement of the GAS2 family members in an array of biological processes, including cytoskeleton reorganization, cell cycle, apoptosis and cancer development.

GAS2 Promotes Cell Proliferation and Invasion and Suppresses Apoptosis in Pediatric T-Cell Acute Lymphoblastic Leukemia and Activates Wnt/β-Catenin Pathway.

PurposeThis study aimed to investigate the effect of growth arrest specific 2 (GAS2) on T-cell acute lymphoblastic leukemia (T-ALL) and its potential molecular mechanism.MethodsThe GAS2 expression level was detected by qRT-RCR and Western blot in normal T lymphocytes and T-ALL cells Jurkat and CCRF-CEM. The proliferation and invasion of Jurkat and CCRF-CEM cells were detected by MTT and Transwell assay, respectively. Apoptosis and cell cycle were measured by flow cytometry. In addition, the chemotherapeutic sensitivity of Jurkat and CCRF-CEM cells was measured MTT assay and flow cytometry.ResultsGAS2 was highly expressed in Jurkat and CCRF-CEM cells. GAS2 could promote cell proliferation and invasion, and inhibit apoptosis of Jurkat and CCRF-CEM cells. GAS2 also promoted cell cycle changes from G0/G1 phase to S phase in Jurkat and CCRF-CEM cells. In addition, GAS2 could reduce the chemotherapeutic sensitivity of Jurkat and CCRF-CEM cells. GAS2 overexpression could promote the expression levels of ki67, proliferating cell nuclear antigen (PCNA), Bcl-2, c-myc, cyclin D1 and β-catenin, while GAS2 knockdown could inhibit their expression levels. Meanwhile, GAS2 overexpression could inhibit Bax expression. Moreover, Wnt/β-catenin pathway inhibitor XAV939 could inhibit the expressions of c-myc, cyclin D1 and β-catenin, but activator LiCl could promote their expression.ConclusionOur study demonstrated that GAS2 could promote cell proliferation and invasion, and induce cell cycle, as well as inhibit apoptosis and could activate the Wnt/β-catenin pathway in T-ALL cells.

Growth Arrest-Specific 2 Promotes the Growth of T-Cell Acute Lymphoblastic Leukemia Cells Via Its Regulation of CXCR4

Growth arrest-specific 2 (GAS2) has multiple functions including the regulation of cell morphology, cell cycle, apoptosis and calpain activity.GAS2 has a dual function in cancer cells, however its expression and underlying mechanism in human T-cell acute lymphoblastic leukemia (T-ALL) remain unclear.In the present study, qRT-PCR analysis showed that GAS2 has significantly higher expression (155.5-fold, P=0.0048) in CD3+ cells from T-ALL patients (n=25) than healthy donors (n=13). GAS2 was present in Jurkat cells, while absent in MOLT-4 or HPB-ALL cells. A tiny CpG island of GAS2 was almost fully methylated in both MOLT-4 (100%) and HPB-ALL cells (80%), while 40% methylation in Jurkat cells; suggesting that DNA methylation played a subtle role in regulating GAS2 expression.Two independent shRNA sequences were delivered into Jurkat cells with lentiviral vector. GAS2 silencing inhibited the growth and colony-forming cell (CFC) production significantly. Conversely, GAS2 overexpression enhanced the growth and CFC production of both MOLT-4 and HPB-ALL cells. In addition, GAS2 overexpression promoted HPB-ALL cell induced leukemia in a xenoengraftment model (5 mice in each control group). In GAS2 expressed group, the disease latency was shortened, the splenomegaly was more severe than control group (0.35±0.04g vs. 0.27±0.05g), and more leukemic cells were present in bone marrow (85±3% vs. 45±7%).To obtain the molecular insights of how GAS2 acts, RNA-seq data comparing GAS2 silenced Jurkat cells with control cells were generated. Several Notch signaling molecules were inhibited, including NOTCH1, HES1 and HES4. Despite the differential expression of these transcripts was validated in Jurkat cells, GAS2 overexpression did not elevated the expression of these transcripts in MOLT-4 or HPB-ALL cells, suggesting GAS2 did not have a consistent impact on Notch signaling. However, we found that GAS2 silencing reduced CXCR4 protein expression in Jurkat cells and GAS2 overexpression enhanced CXCR4 protein expression in MOLT-4 cells, while CXCR4 transcript was not altered upon GAS2 manipulation. Consequently, GAS2 silencing significantly reduced migration ability of Jurkat cells and GAS2 overexpression enhanced migration ability of MOLT-4 cells. Overexpression of CXCR4 “rescued” the inhibited CFC production and migration upon GAS2 silencing. A truncated GAS2 (Δ171-313) coined as GAS2DN (dominant negative form of GAS2) has been known to inhibit normal function of GAS2. Herein, we showed that GAS2DN inhibited the growth of Jurkat cells and the expression of CXCR4. To delineate the role of calpain1 and calpain2 in GAS2 function, shRNA sequences against calpain1 and calpain2 was delivered into GAS2DN expressed Jurkat cells respectively, the results showed that calpain2 but not calpain1 silencing was able to enhance the cell growth and CXCR4 expression.Taken together, the present study has demonstrated that GAS2 is aberrantly expressed in human T-ALL cells, which promotes the growth of T-ALL cells partially via its post-transcriptional regulation of CXCR4 depending on calpain2 activity. These data provide new insights of the pathogenesis of T-ALL and possibly new clues to improve the management of the disease. DisclosuresNo relevant conflicts of interest to declare.

Expression and network analysis of YBX1 interactors for identification of new drug targets in lung adenocarcinoma.

Y-Box Binding protein 1 (YBX-1) is known to be involved in various types of cancers. It's interactors also play major role in various cellular functions. Present work aimed to study the expression profile of the YBX-1 interactors during lung adenocarcinoma (LUAD). The differential expression analysis involved 57 genes from 95 lung adenocarcinoma samples, construction of gene network and topology analysis. A Total of 43 genes were found to be differentially expressed from which 17 genes were found to be down regulated and 26 genes were up-regulated. We observed that Polyadenylate-binding protein 1 (PABPC1), a protein involved in YBX1 translation, is highly correlated with YBX1. The interaction network analysis for a differentially expressed non-coding RNA Growth Arrest Specific 5 (GAS5) suggests that two proteins namely, Growth Arrest Specific 2 (GAS2) and Peripheral myelin protein 22 (PMP22) are potentially involved in LUAD progression. The network analysis and differential expression suggests that Collagen type 1 alpha 2 (COL1A2) can be potential biomarker and target for LUAD.

MiR‑342‑5p promotes Zmpste24‑deficient mouse embryonic fibroblasts proliferation by suppressing GAS2.

Cellular senescence is an irreversible growth arrest of cells that maintain their metabolic activities. Premature senescence can be induced by different stress factors and occurs in mouse embryonic fibroblasts (MEFs) derived from Zmpste24 metalloproteinase-deficient mice, a progeria mouse model of Hutchinson-Gilford Progeria Syndrome. Previous studies have shown that miR-342-5p, an intronic microRNA (miRNA/miR) reportedly involved in ageing associated diseases, is downregulated in Zmpste24−/− MEFs. However, whether miR-342-5p is associated with the premature senescence phenotype of Zmpste24−/− MEFs remains unclear. Thus, the present study investigated the effects of miR-342-5p on cellular senescence and cell proliferation in Zmpste24−/− MEFs. The results showed that miR-342-5p overexpression ameliorated the cellular senescence phenotype to a certain extent, promoted cell proliferation and increased the G2+M cell cycle phase in Zmpste24−/− MEFs. Nonetheless, it was difficult to observe the opposite cell phenotypes in wild-type (WT) MEFs transfected with the miR-342-5p inhibitor. Growth-arrest-specific 2 (GAS2) was identified as a target gene of miR-342-5p in Zmpste24−/− MEFs. In addition, miR-342-5p was identified to be downregulated in WT MEFs during replicative senescence, while Gas2 was upregulated. Taken together, these findings suggest that downregulated miR-342-5p is involved in regulating cell proliferation and cell cycles in Zmpste24−/− MEFs by suppressing GAS2 in vitro.

Growth Arrest Specific 2 (GAS2) is a Critical Mediator of Germ Cell Cyst Breakdown and Folliculogenesis in Mice.

In the mouse ovary, the primordial follicle pool is established through a diverse array of signaling pathways and tissue remodeling events. Growth arrest specific gene two (GAS2) is a highly conserved cytoskeleton-associated protein whose in vivo function remains unclear. In Drosophila, loss of the GAS2 homolog, Pigs, results in infertility. We demonstrate herein that, in the mouse ovary, GAS2 is expressed in the stromal cells surrounding the oocyte cysts on 16.5 dpc, and in stromal cells surrounding growing follicles during juvenile and adult life. We have generated genetically engineered mice with inactivated Gas2. Gas2 homozygous mutant mice are viable but have severely impaired fertility in females, in which oocyte cyst breakdown is disrupted and follicle growth is impaired, with significantly reduced numbers of large antral follicles and corpora lutea. In these mutant mice, the organization of the basal lamina surrounding developing follicles is severely defective at multiple stages of folliculogenesis. We also found that Notch signaling activity was altered in ovaries from Gas2 null mice around the time of birth and during follicular development later in life. These results indicate that GAS2 is a critical and novel regulator of tissue remodeling in the ovary during oocyte cyst breakdown and folliculogenesis.

Discovery of genes from feces correlated with colorectal cancer progression.

Colorectal cancer (CRC) is considered to develop slowly via a progressive accumulation of genetic mutations. Markers of CRC may serve to provide the basis for decision-making, and may assist in cancer prevention, detection and prognostic prediction. DNA and messenger (m)RNA molecules that are present in human feces faithfully represent CRC manifestations. In the present study, exogenous mouse cells verified the feasibility of total fecal RNA as a marker of CRC. Furthermore, five significant genes encoding solute carrier family 15, member 4 (SLC15A4), cluster of differentiation (CD)44, 3-oxoacid CoA-transferase 1 (OXCT1), placenta-specific 8 (PLAC8) and growth arrest-specific 2 (GAS2), which are differentially expressed in the feces of CRC patients, were verified in different CRC cell lines using quantitative polymerase chain reaction. The present study demonstrated that the mRNA level of SLC15A4 was increased in the majority of CRC cell lines evaluated (SW1116, LS123, Caco-2 and T84). An increased level of CD44 mRNA was only detected in an early-stage CRC cell line, SW1116, whereas OXCT1 was expressed at higher levels in the metastatic CRC cell line CC-M3. In addition, two genes, PLAC8 and GAS2, were highly expressed in the recurrent CRC cell line SW620. Genes identified in the feces of CRC patients differed according to their clinical characteristics, and this differential expression was also detected in the corresponding CRC cell lines. In conclusion, feces represent a good marker of CRC and can be interpreted through the appropriate CRC cell lines.

Upregulation of the growth arrest-specific-2 in recurrent colorectal cancers, and its susceptibility to chemotherapy in a model cell system

Colorectal cancer (CRC) is one of the most common life-threatening malignances worldwide. CRC relapse markedly decreases the 5-year survival of patients following surgery. Aberrant expression of genes involved in pathways regulating the cell cycle, cell proliferation, or cell death are frequently reported in CRC tumorigenesis. We hypothesized that genes involved in CRC relapse might serve as prognostic indicators. We first evaluated the significance of gene sequences in the feces of patients with CRC relapse by consulting a public database. Tumorigenesis of target tissues was tested through tumor cell growth, cell cycle regulation, and chemotherapeutic efficacy. We found a highly significant correlation between CRC relapse and growth arrest-specific 2 (GAS2) gene expression. Based on cell models, the overexpressed GAS2 was associated with cellular growth rate, cell cycle regulation, and with chemotherapeutic sensitivity. Cell division was impaired by treating cells with 2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]-propionic acid (XK469), even when the cells were overexpressing GAS2. Thus, downregulation of GAS2 expression might control CRC relapse after curative resection. GAS2 could serve as a noninvasive marker from the feces of patients with prediagnosed CRC. Our findings suggest that GAS2 could have potential clinical applications for predicting early CRC relapse after radical resection, and that XK469 might impair tumor cell division by reducing GAS2 expression or blocking its cellular translocation. This will help in selecting the best therapeutic option, 5-fluorouracil in combination with XK469, for patients overexpressing GAS2 in CRC cells. Thus, GAS2 might act as a prognostic biomolecule and potential therapeutic target in patients with CRC relapse.

GAS2-Calpain2 axis contributes to the growth of leukemic cells.

Growth arrest specific 2 (GAS2) modulates cell cycle, apoptosis, and Calpain activity. GAS2-Calpain2 axis is required for the growth of BCR-ABL(+) hematopoietic cells and chronic myeloid leukemia cells. However, the expression of GAS2 in acute leukemia patients remains unclear and what role GAS2-Calpain2 axis plays in these leukemic cells is not known yet. In this study, GAS2 was found to have significantly higher expression in 16 various leukemic cell lines than in control cells. Using THP-1 cells (from acute myeloid leukemia patient, AML) and Jurkat cells (from acute lymphoid leukemia patient, ALL) as models, we found that GAS2 silence led to elevated Calpain activity, decreased cellular growth, and inhibition of colony-forming cell (CFC) production; and these effects could be rescued by GAS2 re-expression. Moreover, GAS2 silence prevented tumor formation of THP-1 cells in nude mice. In both THP-1 and Jurkat cells, GAS2 interacted with Calpain2 rather than Calpain1. The dominant negative form of GAS2 (GAS2DN, GAS2Δ171-313) had similar effects on leukemic cells through the activation of Calpain. Importantly, Calpain2 silence abolished the proliferation inhibition induced by GAS2 targeting. We also found that GAS2 was aberrantly expressed and Calpain activity was decreased in clinical isolates from acute leukemia patients. Taken together, our results demonstrated the deregulation of GAS2 in both AML and ALL and the requirement of GAS2-Calpain2 axis for the growth of leukemic cells, which will help to understand the molecular pathogenesis of hematological malignancies and possibly to develop novel approaches to treat these deadly diseases.

Abstract 814: Truncated HBx-dependent silencing of growth arrest-specific 2 promotes hepatocarcinogenesis through inhibition of p53-mediated apoptosis

Abstract Hepatocellular carcinoma (HCC) is a worldwide threat to public health, especially in China where chronic hepatitis B virus (HBV) infection is found in 80-90% of all HCCs. The HBV encoded X antigen (HBx) is a trans-regulatory protein involved in the virus-induced hepatocarcinogenesis. Although the carboxyl-terminus truncated HBx, rather than the full-length counterpart, is frequently overexpressed in human HCCs, its functional mechanisms have yet been fully defined. In this study, we investigated the molecular pathogenesis of a naturally-occurring HBx variant which has 35 amino acids deleted at C-terminus (HBxΔ35). By performing genome-wide scanning analysis and PCR validation, we identified growth arrest-specific 2 (GAS2) as a direct target of HBxΔ35 at transcriptional level in human immortalized liver cells. HBxΔ35 was found to bind the promoter region of GAS2 and suppress its expression to promote hepatocellular proliferation and tumorigenicity. Further functional assays showed that overexpression of GAS2 inhibited tumor growth both in vitro and in vivo by inducing p53-dependent apoptosis. Conversely, siRNA-mediated knockdown of GAS2 in hepatocytes attenuated apoptotic response and reduced cell susceptibility to etoposide-induced apoptosis. Notably, GAS2 expression was significantly down-regulated in truncated HBx-expressing HCCs compared with the corresponding normal tissues. In conclusion, our integrated study uncovered a novel viral mechanism in hepatocarcinogenesis, wherein HBxΔ35 ablates p53-driven apoptosis via direct silencing of GAS2, and thereby provides survival advantage for pre-neoplastic hepatocytes to facilitate cancer development. Citation Format: Alfred S. L. Cheng, Ranxu Zhu, Myth T.S. Mok, Wai Kang, Ka-Fai To, Joseph J.Y. Sung, Henry L.Y. Chan. Truncated HBx-dependent silencing of growth arrest-specific 2 promotes hepatocarcinogenesis through inhibition of p53-mediated apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 814. doi:10.1158/1538-7445.AM2015-814

Truncated HBx-dependent silencing of GAS2 promotes hepatocarcinogenesis through deregulation of cell cycle, senescence and p53-mediated apoptosis.

Hepatocellular carcinoma (HCC) is a worldwide threat to public health, especially in China, where chronic hepatitis B virus (HBV) infection is found in 80-90% of all HCCs. The HBV-encoded X antigen (HBx) is a trans-regulatory protein involved in virus-induced hepatocarcinogenesis. Although the carboxyl-terminus-truncated HBx, rather than the full-length counterpart, is frequently overexpressed in human HCCs, its functional mechanisms are not fully defined. We investigated the molecular function of a naturally occurring HBx variant which has 35 amino acids deleted at the C-terminus (HBxΔ35). Genome-wide scanning analysis and PCR validation identified growth arrest-specific 2 (GAS2) as a direct target of HBxΔ35 at transcriptional level in human immortalized liver cells. HBxΔ35 was found to bind the promoter region of GAS2 and attenuate its expression to promote hepatocellular proliferation and tumourigenicity. Further functional assays demonstrated that GAS2 induces p53-dependent apoptosis and senescence to counteract HBxΔ35-mediated tumourigenesis. Notably, GAS2 expression was significantly down-regulated in HCCs compared with the corresponding normal tissues. In conclusion, our integrated study uncovered a novel viral mechanism in hepatocarcinogenesis, wherein HBxΔ35 deregulates cell growth via direct silencing of GAS2 and thereby provides a survival advantage for pre-neoplastic hepatocytes to facilitate cancer development.

Growth Arrest Specific 2 Is Up-Regulated in Chronic Myeloid Leukemia Cells and Required for Their Growth

Although the generation of BCR-ABL is the molecular hallmark of chronic myeloid leukemia (CML), the comprehensive molecular mechanisms of the disease remain unclear yet. Growth arrest specific 2 (GAS2) regulates multiple cellular functions including cell cycle, apoptosis and calpain activities. In the present study, we found GAS2 was up-regulated in CML cells including CD34+ progenitor cells compared to their normal counterparts. We utilized RNAi and the expression of dominant negative form of GAS2 (GAS2DN) to target GAS2, which resulted in calpain activity enhancement and growth inhibition of both K562 and MEG-01 cells. Targeting GAS2 also sensitized K562 cells to Imatinib mesylate (IM). GAS2DN suppressed the tumorigenic ability of MEG-01 cells and impaired the tumour growth as well. Moreover, the CD34+ cells from CML patients and healthy donors were transduced with control and GAS2DN lentiviral vectors, and the CD34+ transduced (YFP+) progeny cells (CD34+YFP+) were plated for colony-forming cell (CFC) assay. The results showed that GAS2DN inhibited the CFC production of CML cells by 57±3% (n = 3), while affected those of normal hematopoietic cells by 31±1% (n = 2). Next, we found the inhibition of CML cells by GAS2DN was dependent on calpain activity but not the degradation of beta-catenin. Lastly, we generated microarray data to identify the differentially expressed genes upon GAS2DN and validated that the expression of HNRPDL, PTK7 and UCHL5 was suppressed by GAS2DN. These 3 genes were up-regulated in CML cells compared to normal control cells and the growth of K562 cells was inhibited upon HNRPDL silence. Taken together, we have demonstrated that GAS2 is up-regulated in CML cells and the inhibition of GAS2 impairs the growth of CML cells, which indicates GAS2 is a novel regulator of CML cells and a potential therapeutic target of this disease.

GAS2-like proteins mediate communication between microtubules and actin through interaction with end-binding proteins

ABSTRACTCrosstalk between the microtubule (MT) and actin cytoskeletons is fundamental to many cellular processes including cell polarisation and cell motility. Previous work has shown that members of the growth-arrest-specific 2 (GAS2) family mediate the crosstalk between filamentous actin (F-actin) and MTs, but the molecular basis of this process remained unclear. By using fluorescence microscopy, we demonstrate that three members of this family, GAS2-like 1, GAS2-like 2 and GAS2-like 3 (G2L1, G2L2 and G2L3, also known as GAS2L1, GAS2L2 and GAS2L3, respectively) are differentially involved in mediating the crosstalk between F-actin and MTs. Although all localise to actin and MTs, only the exogenous expression of G2L1 and G2L2 influenced MT stability, dynamics and guidance along actin stress fibres. Biochemical analysis and live-cell imaging revealed that their functions are largely due to the association of these proteins with MT plus-end-binding proteins that bind to SxIP or SxLP motifs located at G2L C-termini. Our findings lead to a model in which end-binding (EB) proteins play a key role in mediating actin–MT crosstalk.

Abstract 1341: Identification of significant linkage evidence for lethal prostate cancer on chromosome arm 11p15.

Abstract We performed genome wide linkage analysis in a set of high-risk prostate cancer pedigrees, each with 3 or more sampled cases whose death certificate indicated a contribution of prostate cancer to death (lethal prostate cancer). All prostate cancer cases were present in the Utah SEER Cancer Registry (pathology confirmed); all “lethal” prostate cancer cases were also required to have a Utah death certificate indicating prostate cancer as a contributing cause of death. A set of markers with no linkage disequilibrium was selected for linkage analysis. We took the intersection of SNP markers from five Illumina genotyping platforms used (550K, 610K, 1M,Omni_express (720k), and Omni_1M), resulting in selection of 301,646 markers. We began with the first marker on each chromosome and then skipped markers until one satisfied all criteria (min 0.1 cM distance, heterozygosity 0.35, R-squared < 0.16 -using HapMap's LD files based on CEPH). This resulted in selection of a set of 25,436 SNP LD-free genome wide markers for linkage. We have performed genome-wide linkage analysis for 21 of the genotyped high-risk lethal prostate cancer pedigrees with the 25,436 selected SNP markers. Genome wide hetLODs showed one significant linkage peak for the recessive model on chromosome 14 (LOD = 4.20). The pedigree with the highest LOD in this region has a +2.69; we are attempting to expand sampling for other pedigrees with linkage evidence in this region. A single pedigree with 16 prostate cancer cases, at least 4 of whom have already died from prostate cancer and were sampled, provided a LOD score = +3.56 on chromosome arm 11p15. An 11p15 haplotype inherited in common from the founder to 6 generations of descendants is shared in at least 9 of the prostate cancer cases, 3 of whom died from prostate cancer. The 1-LOD drop defined support interval for the pedigree linked to chromosome 11 occurs at approximately 34-46 cM and contains 13 currently documented genomic features, several of which may be reasonable candidates for lethal prostate cancer including the gene PRMT3 (arginine methyltransferase 3), DAL-1, NELL1, FANCF (Fanconi anemia, complementation group F), and GAS2 (growth arrest-specific 2). Sequencing of multiple haplotype sharing cases in the pedigree is underway. Citation Format: Lisa A. Cannon-Albright, Craig C. Teerlink, Neeraj Agarwal. Identification of significant linkage evidence for lethal prostate cancer on chromosome arm 11p15. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1341. doi:10.1158/1538-7445.AM2013-1341

Interferon Consensus Sequence Binding Protein (ICSBP) Decreases β-Catenin Activity in Myeloid Cells by Repressing GAS2 Transcription

The interferon consensus sequence binding protein (ICSBP) is an interferon regulatory transcription factor, also referred to as IRF8. ICSBP acts as a suppressor of myeloid leukemia, although few target genes explaining this effect have been identified. In the current studies, we identified the gene encoding growth arrest specific 2 (GAS2) as an ICSBP target gene relevant to leukemia suppression. We find that ICSBP, Tel, and histone deacetylase 3 (HDAC3) bind to a cis element in the GAS2 promoter and repress transcription in myeloid progenitor cells. Gas2 inhibits calpain protease activity, and beta-catenin is a calpain substrate in these cells. Consistent with this, ICSBP decreases beta-catenin protein and activity in a Gas2- and calpain-dependent manner. Conversely, decreased ICSBP expression increases beta-catenin protein and activity by the same mechanism. This is of interest, because decreased ICSBP expression and increased beta-catenin activity are associated with poor prognosis and blast crisis in chronic myeloid leukemia (CML). We find that the expression of Bcr/abl (the CML oncoprotein) increases Gas2 expression in an ICSBP-dependent manner. This results in decreased calpain activity and a consequent increase in beta-catenin activity in Bcr/abl-positive (Bcr/abl(+)) cells. Therefore, these studies have identified a Gas2/calpain-dependent mechanism by which ICSBP influences beta-catenin activity in myeloid leukemia.