hF-LANa, a human homologue of Derlin family, regulating the expression of cancer-related genes promotes NIH3T3 cell transformation

Available online at www.sciencedirect.com

Cancer Letters 258 (2007) 171–180 www.elsevier.com/locate/canlet

hF-LANa, a human homologue of Derlin family, regulating the expression of cancer-related genes promotes NIH3T3 cell transformation Yihong Hu a, Hao Ying b, Yonghua Xu a

a,*

Laboratory of Molecular and Cellular Oncology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, 320 YueYang Road, Shanghai 200031, China b Laboratory of Molecular Biology, National Cancer Institute, 37 Convent Drive, Room 5128, Bethesda, MD 20892-4264, USA Received 21 February 2007; received in revised form 29 August 2007; accepted 31 August 2007

Abstract hF-LANa, a member of Derlin family, is a putative proto-oncogene and has a direct role in oncogenic transformation. hF-LANa over-expressed NIH3T3 cells grow 20% quicker than parent cells. Especially, hF-LANa over-expression promotes anchorage-independent growth of NIH3T3 cells in soft agar and tumorigenesis in nude mice. Analysis by cDNA microarray finds 252 up-regulated genes and 354 down-regulated genes in hF-LANa over-expressed NIH3T3 cells including 19 oncogenes/proto-oncogenes and 13 tumor suppressor genes/putative tumor suppressors. These experiments show the oncogenic transformation role of hF-LANa both in vitro and in vivo, and highlight the genes that might be related to hF-LANa’s oncogenic effects.  2007 Published by Elsevier Ireland Ltd. Keywords: hF-LANa; Transformation; Oncogene/proto-oncogene; Tumor suppressor gene; Mouse oligo cDNA expression assay

1. Introduction Hepatocellular carcinoma (HCC) accounts for more than 90% of all primary liver cancers. It ranks fifth in frequency among all malignancies worldwide and causes nearly 1 million deaths annually. At the same time, its incidence is increasing worldwide, especially in Asia and Africa [1,2]. HCC is a multistage disease whose occurrence is linked to environmental, dietary and life-style factors [3]. Studies of these factors provide evidence for prevention of *

Corresponding author. Tel./fax: +86 21 54921361. E-mail address: [email protected] (Y. Xu).

HCC. Aberrant gene expression is the most frequent molecular events in cancer. It has multiple forms and be due to genetic alterations – like point mutations, deletions in segments of genes or alternative splicing – or to epigenetic modifications that modulate the gene function, like gene methylation, without changing the DNA sequence but altering the function of the resulting protein. The activation of oncogenes and inactivation of tumor suppressor genes (TSG) play important roles in the mechanism of cancer development. These genes can induce malignant transformation when inappropriately expressed as a result of mutation, deletion, amplification or rearrangement [4]. The most common

0304-3835/$ - see front matter  2007 Published by Elsevier Ireland Ltd. doi:10.1016/j.canlet.2007.08.017

172

Y. Hu et al. / Cancer Letters 258 (2007) 171–180

genetic alterations in HCC have been revealed and grouped into 3 main routes, p53 pathway (p53 mutations, p14ARF promoter methylation), Wnt pathway (mutation of b-catenin) and Rb1 pathway (p16INK4a methylation, loss of Rb1 expression and cyclin D1 amplification) [5]. Many proteins of eukaryotic cells undergo folding and modification in the lumen of endoplasmic reticulum (ER). Properly folded polypeptides leave ER along the secretory pathway, whereas misfolded proteins or unaccessed protein complexes are retained. These proteins are eventually degraded by proteasome and must therefore be transported back into the cytosol by a multi-step process called ER-associated protein degradation (ERAD) [6,7]. Derlin protein family takes important roles in ERAD process. Three members of Derlin protein family have been found. They are Derlin-1, Derlin-2 and Derlin-3. All the members are evolutionarily conserved from yeast to human. They contain the Der1-like domain motif and localize to ER membrane. Previous study found that Der1p, a homologue of Derlin-1 in yeast (Saccharomyces cerevisiae), localized to ER and participated in ERAD [7]. Lilley’s work also proved that human Derlin-1 and Derlin-2 were ER-resident protein which participated in the degradation of proteins from ER [8]. However, Derlin family may have a role in HCC. hF-LANa (Derlin-2), a homologue of Derlin gene family, originally cloned by Hao Ying in our lab, was a cancer-related gene and upregulated in 71% (10 of 14 cases) of HCC [9]. Here, we report that the introduction of hFLANa into NIH3T3 cells through transfection with hF-LANa cDNA results in increased cell proliferation, anchorage-independent growth in soft agar, and formation of tumors after subcutaneous injection of nu/nu mice. Our results suggest that hFLANa is a putative human proto-oncogene. Analysis of mouse oligo cDNA expression assay shows the disordered expression of oncogenes or tumor suppressors including Wnt1 and Rb1, which are related to hF-LANa’s over-expression. 2. Materials and methods 2.1. Cell culture, expressing vectors and establishment of stable cell line NIH3T3 cells (Shanghai Cell Bank, Chinese Academy of Sciences) were grown at 37 C in 5% CO2 incubator in Dulbecco’s modified Eagle’s medium (DMEM, Gibco)

supplemented with 10% new born calf serum (Evergreen, Hangzhou, P. R. China), 100 U/ml penicillin and 100 lg/ ml streptomycin. The open-reading frame of hF-LANa was inserted into eukaryotic expressing vector pcDNA3.1()/Myc-HisB and transfected into NIH3T3 cells by DOTAP Liposomal Transfection Reagent (Roche) in a ratio of 1:6. After selection for 3–4 weeks in G418 selection medium (G418, 400 lg/ml, Geneticin, Sigma), hF-LANa over-expressed stable cell lines were maintained in 100 lg/ml G418 containing medium. As a control, pcDNA3.1()/Myc-HisB was transfected into NIH3T3 cells. More than twenty clones were isolated and amplified. Pooled population cells and independent clone cells transfected with hF-LANa were used in subsequent experiments. The enforced expression of hF-LANa was detected by RT-PCR and Western blot analysis. 2.2. Semiquantitative RT-PCR, immunoprecipitation and Western blot analysis RNA was prepared with Trizol and RT-PCR was conducted according to the manufacturer’s instruction (Promega). One microliter reverse transcription product from 5 lg RNA was used for PCR. The primers for hFLANa sequence were: 5 0 -GCC TCG AGA TGG CGT ACC AGA GCT T-3 0 (sense) and 5 0 -GCC TCG AGA CCT CCA AGC CGC TGG CCC TCA C-3 0 (antisense), which amplified a 720 bp product. The amplification conditions were an initial denaturation at 94 C for 5 min, followed by 5 cycles at 94 C for 30 s, 58 C for 30 s, and 72 C for 45 s; then 25 cycles were run at 94 C for 30 s, 56 C for 30 s, and 72 C for 45 s. The primers for Rb1 sequence were: 5 0 -CAA CCC CCC CAA ACC ACT GA-3 0 (sense) and 5 0 -CCA GAT GTA GGG GGT CAG GA-3 0 (antisensse), which amplified a 499 bp product. The amplification conditions were an initial denaturation at 94 C for 2 min, followed by 30 cycles at 94 C for 20 s, 56 C for 30 s, and 72 C for 30 s. The primers for Wnt1 sequence were: 5 0 -CAT CGA GTC CTG CAC CTG3 0 (sense) and 5 0 -TGG GCG ATT TCT CGA AGT AG3 0 (antisense), which amplified a 449 bp product. The amplification conditions were an initial denaturation at 94 C for 2 min, followed by 30 cycles at 94 C for 20 s, 58 C for 30 s, and 72 C for 30 s. The PCR products were analyzed using 2% agarose and ethidium bromide. Cells were lysed in RIPA buffer (50 mM Tris–HCl pH7.5; 150 mM NaCl; 0.5% NaDOC; 1% NP40; 0.1% SDS) plus protease inhibitors. Cell extracts (2 mg/ml for each immunoprecipitation) were precleared 1 h at 4 C with 200 ll unspecific control hybridoma supernatant buffered with NET-gelatin buffer (50 mM Tris–HCl pH 7.5; 150 mM NaCl; 1% NP40; 1 mM EDTA; 0.25% gelatin and 0.02% NaN3 plus protease inhibitors) and protein G agrose. After centrifugation, the supernatants were removed and incubated overnight at 4 C with anti-c-myc mouse monoclonal antibody (sc-40, Santa Cruz), followed

Y. Hu et al. / Cancer Letters 258 (2007) 171–180

by incubation with beads for 1 h. Samples were centrifuged. And pellets were washed 4 times with RIPA buffer. The immunoprecipitated proteins (IP) were eluted from the beads by adding SDS sample buffer (50 mM Tris–HCl, pH 6.8, 1% 2-Mercaptoethanol, 3% SDS, 10% glycerol and 0.1% bromophenol blue) and heated in boiling water for 5 min. IPs were analyzed by Western blot using antihis mouse monoclonal antibody (sc-8036, Santa Cruz). For Western blot analysis, protein concentration was determined using Bradford Reagent. Equal amount of samples were separated on SDS–PAGE, transferred onto nitrocellulose membrane and probed with an appropriate dilution of primary and secondary antibodies, then detected using the enhanced chemiluminescence system. 2.3. Cell proliferation assay, soft agar assay and in vivo tumor growth assay Twenty-five microliters of the 5 mg/ml stock solution of MTT (Sigma) was added to each well and after 2 h of incubation at 37 C, 100 ll of extraction buffer was added. After an overnight incubation at 37 C, the optical densities at 570 nm were measured using a Titer-tech 96well microplate reader (Bio-Rad, Model 550), employing the extraction buffer as a blank [10]. Anchorage-independent cell growth was determined by analyzing the formation of colonies in soft agar. About 2 · 103 cells from parent cells, transfected cells including pooled population and independent clones were suspended in 0.3% agar in DMEM containing 10% new born calf bovine serum and plated on solidified agar (0.7%) in 35 mm dishes. After 28-day incubation, 1 ml of 0.5 mg/ ml p-iodonitrotetrazolium violet (Sigma) was added to each well and incubated overnight. Colonies with a diameter greater than 100 lm were counted. The experiments were performed in triplicate and repeated three times. We investigated the in vivo tumorigenicity potential of wild type NIH3T3 cells, vector-transfected cells (vector-4) and hF-LANa over-expressed clone cells (F-15) in three groups of nude mice. Cells growing at log phase were harvested by trypsinization and washed twice with PBS. Resuspended in PBS to a final concentration of 3 · 106/ml, cells were injected (6 · 105 cells in 200 ll PBS/site) subcutaneously (s.c.) into flanks of 5- to 6-week female BALB/c nu/ nu mice, 3 mice per group. Mice were sacrificed, and tumors were harvested after 4 weeks. Skin and connective tissues were dissected from tumors, and tumor volume was calculated from measurements of (length · width2) · 0.5. The experiments were repeated three times.

173

pared by reverse transcription. Vector-transfected control sample was labeled with Cy5, and hF-LANa-transfected sample (F-15) was labeled with Cy3. Briefly, total RNA was heat denatured in the presence of 0.1 g/L oligo(dT) (Bioasia) for 10 min at 70 C. Reverse transcription was performed in 1 · first-strand buffer (Invitrogen Corp.) in the presence of 1.0 ll reverse transcriptase (200 U/ll SuperScriptTM II reverse transcriptase; Invitrogen Corp.), 2.0 ll DTT (0.1 M), 1.5 ll dNTP mixture (dATP, dGTP, dTTP at 20 mM), 1.0 ll Cy3/Cy5-dCTP (1 mM), and 1.0 ll RNasin (Takara) for 120 min at 42 C. Probes were purified by QIAquick Nucleotide Removal Kit, quantified and denatured at 99 C for 2 min before use. Mouse 16K gene expression library oligonucleotide arrays (V1.0, Shanghai Biochip Co., LTD.) containing 16463 genes and 24 comparisons were used. The probes in 40ll hybridization buffer including 2 ll mouse Cot-I and 20 ll formamide were denatured at 95 C for 2 min and prehybridized at 70 C for 10– 20 min, added on the chips and covered with glass. The chips were hybridized in a sealed chamber at 40 C for 12–16 h. After removing the cover-glass, the chips were washed at 55 C in 1 · SSC + 0.2%SDS, 0.1 SSC + 0.2%SDS and 0.1 · SSC, 5–10 min for two times each, respectively. After a quick rinse in ddH2O, slides were air-dried for scanning. We used two batches of RNA to conduct the experiments and repeated two times with each batch of RNA. Arrays were scanned using Agilent scanner (G2655AA, Agilent) at two wavelengths to detect emission from both Cy3 and Cy5. Acquired images were analyzed using Imagene 4.0 software (BioDiscovery, USA). The resulting data were then normalized, filtered, and analyzed using GeneSpring 6.1, Silicon Genetics (Redwood City, CA). The fluorescent intensity of each spot at the two wavelengths that represent the quantity of Cy3-dCTP and Cy5-dCTP was normalized and calculated. Experiments were normalized to a fold change ratio, and two-color normalizations automatically displayed after background subtraction based on negative controls and per spot/per chip intensity dependent normalization (LOWESS). The criteria for screening out each differentially expressed gene were defined as the ratio of Cy3/Cy5 > 2.0 or <0.5. To classify the functional roles of the identified differentially expressed genes, we used the free online tools, FatiGO (www.fatigo.org), which utilize the Gene Ontology (GO) database provided by the GO Consortium (www.geneontology.org). Fisher’s exact test was used to compare the percentage distribution of genes with GO annotation in each category. Genes involved in biological pathway were analyzed according to GenMAPP (www.genmapp.org).

2.4. cDNA array analysis 3. Results Prepared RNA was purified with QIAGEN RNeasy Kit. RNA concentration was determined spectroscopically, and its quality was checked by agarose gel and lab-on-chip electrophoresis. The fluorescent cDNA probes were pre-

We have found that hF-LANa antisense oligonucleotide suppressed hF-LANa expression in human HCC BEL-7404 cells and significantly inhibited cell growth

174

Y. Hu et al. / Cancer Letters 258 (2007) 171–180

[9]. Here, we transfected hF-LANa full-length open-reading frame expressing vector into NIH3T3 cells in which F-LANa mRNA expression was not detectable. The expression of hF-LANa in stable cell lines was examined by RT-PCR and Western blot analysis. As shown in Fig. 1A, the 720 bp products were detected in pcDNA3.1()-hF-LANa-Myc-HisB transfected NIH3T3 pooled population cells, F-LANa and representative independent clone cells, F-12 and F-15, while no signal could be found in NIH3T3 vector cells, vector-4 cells and parent cells. Immunoprecipitation assay showed that the 28 kDa his-tagged protein were obviously over-expressed in pcDNA3.1()-hF-LANa-Myc-His B transfectants (Fig. 1A, lane F-LANa, F-12 and F-15). Growth status of NIH3T3 cells with enforced expression of hF-LANa was measured by MTT assay. The growth rate of hF-LANa over-expressed pooled population cells and independent clone cells increased 20% (p < 0.05, Fig. 1B) in comparison with NIH3T3 parent cells, but no change was found in vector-transfected cells. This result implied the growth regulation activities of hFLANa gene in NIH3T3 cells was consistent with the inhibition role of hF-LANa antisense oligonucleotide in human HCC cells [9]. Anchorage-independent growth potentials correlate strongly with tumorigenicity and invasiveness of cancer cells. The ability to grow in soft agar is an indication of anchorage independent growth of cells. To investigate the transformation ability of hF-LANa, we examined the anchorage-independent growth potential in soft agar of parent NIH3T3 cells, vector-transfected NIH3T3 cells and hF-LANa over-expressed NIH3T3 pooled population cells and independent clone cells. As shown in Fig. 2, there were 486.33 ± 46.61, 462.00 ± 32.05 and 604.00 ± 48.22 clones in F-LANa cells, F-12 and F-15 cells, respectively; almost no clones were formed in soft agar by parent NIH3T3 cells, NIH3T3 vector cells and vector-4 cells. The results indicated over-expression of hF-LANa resulted in high-colony formation in soft agar. By subcutaneously injecting nude mice with parent NIH3T3 cells, vector-4 and F-15 cells, we examined the effect of hF-LANa on tumor formation in vivo. We found that nude mice injected with hF-LANa over-expressed NIH3T3 cells developed bulky tumors in four weeks after injection (Fig. 3); mice injected with parent cells or vectortransfected cells only developed baby-size nodes. The tumor weight and volume measured at the end of experiments were shown in Table 1. The experiments suggest that hF-LANa might induce tumor formation of NIH3T3 cells in nude mice. To explore the molecular events in cell transformation activated by hF-LANa, mouse 16K gene expression library oligonucleotide arrays were used to investigate gene expression in transformed NIH3T3 cells. All the differentially expressed genes between vector-transfected NIH3T3 cells and hF-LANa over-expressed NIH3T3

Fig. 1. Identification of transfectants and effects of hF-LANa on NIH3T3 cell growth. (a) RT-PCR and Western blot analysis of hF-LANa over-expressed NIH3T3 stable cell lines. 3T3, NIH3T3 parent cells; vector, pcDNA3.1()/Myc-HisB empty vectortransfected cells; F-LANa and F, pcDNA3.1()-hF-LANaMyc-HisB transfected pooled population cells and independent clone cells; Arabic numerals are the serial numbers of different clones; M, 100-bp DNA ladder. The 720 bp hF-LANa products have been aligned with G3DPH as a control. The 28 kDa overexpressed hF-LANa proteins were detected by using anti-his antibody. (b) The growth curve of different cell lines. hF-LANa accelerated cell growth of NIH3T3 cells, p < 0.05. Cell growth was measured by MTT method. Three wells were taken from each sample for MTT assay at 1, 2, 3, 4 and 5 days. The value shown is the mean of three wells. Data are expressed as the means ± SE. The experiments were repeated three times independently.

cells were listed in Supplementary Table S1 (up-regulated genes) and S2 (down-regulated genes). There were 252 genes up-regulated and 354 genes down-regulated in hF-LANa over-expressed NIH3T3 cells. According to biological process, molecular function and cellular component, we classified the altered genes by FatiGO with results shown in Fig. 4. There are sets of genes influenced by hF-LANa over-expression in NIH3T3 cells. In 252 up-regulated genes, there are 9, 4, 4, 7, 5, 18 and 28 genes involved in cell proliferation, glycoprotein

Y. Hu et al. / Cancer Letters 258 (2007) 171–180

175

Fig. 2. Transformation ability of hF-LANa in NIH3T3 cells detected by soft agar assay. Colonies were counted and calculated. The data are expressed as the means ± SE. It is the representative results from three independent experiments are shown. *High colony-formation ability.

Table 1 Weight and volume of the tumors in nude mice Transfectants

NIH3T3

Vector-4

F-15

Weight (g) Volume (cm3)

0.03 ± 0.02 0.08 ± 0.01

0.03 ± 0.01 0.08 ± 0.01

3.47 ± 0.88* 4.61 ± 0.56*

*

Fig. 3. Tumor formation of NIH3T3 cells induced by hF-LANa in vivo. hF-LANa over-expressed NIH3T3 cells produced bulky tumors in nude mice. Results are representative of three independent experiments. *Bulky tumors.

metabolic process, biopolymer glycosylation, neurogenesis, carrier activity, hydrolase activity and cytoplasm proteins, respectively. As for the 354 down-regulated genes, there are 3, 8, 2, 8 and 13 genes severally related

Significant difference.

to cell proliferation, vesicle-mediated transport, neurogenesis, lipid binding and hydrolase activity. The results indicated that the GO distribution, especially in biological process including cell proliferation and neurogenesis, was different in hF-LANa over-expressed NIH3T3 cells and vector-transfected NIH3T3 cells. GenMAPP analysis outlined in Supplementary Table S3 (up-regulated genes) and Table S4 (down-regulated genes) showed that the major pathways related to NIH3T3 cell transformation were Wnt signaling pathway, cell cycle (Rb pathway) and G protein coupled receptors activated pathway. In addition, a number of the differentially expressed genes were divided into two classes: tumor suppressor and oncogenes/proto-oncogenes. As shown in Table 2,

176

Y. Hu et al. / Cancer Letters 258 (2007) 171–180

4. Discussion

Fig. 4. Gene ontology analysis of the altered genes induced by hF-LANa over-expression in mouse NIH3T3 cells. The percentage distribution of up- and down-regulated genes in each category was shown. *Significant GO annotation difference (p < 0.05, Fisher’s exact test).

in the 252 up-regulated genes, Wnt1, Th, Siat1, Rab4b, Pea3, Gfi1 and Dri1 are oncogenes/proto-oncogenes, while Rb1, Rbl1, Zac1, Ube4b, Slc22a1l, Ptch, Hic2 and Brca1 are tumor suppressor or putative suppressor. In the 354 down-regulated genes, Ebag9, Eef1a2, Frat1, Gli, Lmyc1, Mas1, Mell1, Ptn, Rrad, Spam, Tert and Vav3 are oncogenes/proto-oncogenes, while Tcfl1, Serpinb5, Pla2g2a, Lox and Copeb are tumor suppressors or putative suppressors (refer to www.atlasgeneticsoncology.org). Taken together, 19 oncogenes/proto-oncogenes and 13 tumor suppressor genes/putative tumor suppressors were involved in transformed NIH3T3 cells with over-expressed hF-LANa. In correspondence with the above-mentioned results in GO and GenMAPP analysis, we propose that Rb1 and Wnt1 might be important genes related to hFLANa’s transformation role. Alteration of Rb1 and Wnt1 in hF-LANa over-expressed NIH3T3 cells was confirmed in hF-LANa over-expressed pooled population cells and independent clone cells by RT-PCR. As shown in Fig. 5, Rb1 and Wnt1 were up-regulated in hF-LANa over-expressed NIH3T3 cells. The expression levels of Rb1 in F-LANa, F-12 and F-15 cells were 2.83, 1.80 and 2.06 times as much as in parent cells, vector and vector-4 cells. At the same time, Wnt1 was up-regulated 3.44, 2.49 and 2.79 times, respectively, in F-LANa, F-12 and F-15 compared with 3T3, vector and vector-4. These results matched with the chip analysis. So we considered that hF-LANa might regulate the expression level of those cancer-related genes in Rb1 and Wnt1 pathways, and transformed NIH3T3 cells.

From 2003 to 2005, cancer has surpassed heart disease as number one death cause in America, Canada, England and China, although death from cancer is decreasing. Human HCC is one of the most common causes of cancer death, and its survival rate is poor [11,12]. Hope has been focused on new approaches including targeted therapies, immune stimulants, and the emerging area of gene therapy except surgical section, orthotopic liver transplantation, chemotherapy, hormonal therapy, and regional intra-arterial treatments [13,14]. Scientists try to find some evolutionarily conserved genes or differentially expressed genes with basilic function. Genome-wide gene analysis has been performed in human genome project (HGP) using biological technology. Usually by whole-genome shotgun assembly (called WGSA), comparative proteomics or DNA array, largescale genes have been found as a basis for functional genomics. Cancer-related genes, whether known or unknown, are one of the major targets in functional genome research. In the past decades, at least 200 genes that may promote or prevent cancer have been identified in human genome, which become the groundwork of clinical treatment [15]. hF-LANa, a new cancer-related gene, belongs to Derlin gene family. The structure and functions of Derlin proteins are conserved among species. Previous study showed that Derlin-1 which localized to ER, participated in ERAD in various species from yeast (singlecellular organism) to human being (multicellular organism) [6,7,16], even in maize (Zea mays) [17]. Derlin proteins possess multiple functions. Derlin-1 has been proved to be involved in ERAD catabolic process, intracellular transport of viral protein in host cell, retrograde protein transport and unfolded protein response [6,18,19]. Zhihua Yang’s lab connected Derlin-1 with ER expansion [20] and proved that Derlin-1 was a novel growth factor-responsive endothelial antigen that promoted endothelial cell survival and growth [21]. F-LANa (Derlin-2) is also an ER-resident protein that, similar to Derlin-1, participates in the degradation of proteins from ER [8]. While Lilley’s recent work proved that F-LANa was required by murine polyomavirus to initiate infection [22]. We are interested in hF-LANa, because its stimulation to cell growth and transformation suggests that it might be a proto-oncogene. If possible, it could be used as a tumor marker in early HCC

Table 2 Differentially expressed genes with GenBank identity number in hF-LANa over-expressed NIH3T3 cells screened by cDNA array Gene_Symbol

Description

Classification

Fold changes

NM_007880 NM_010278 NM_008815 BC007147 NM_009175 NM_009377 BC005449 U36475 AF117382 NM_008957 NM_009029 U27177 NM_008767 NM_022022 AF147785 NM_019480 NM_007906 NM_008043 NM_010296 NM_008506 AK015036 NM_013783 NM_008973 NM_019662 NM_009241 NM_009354 NM_020505 BC020042 NM_010728 NM_011108 BC005434 NM_009336

Dri1 Gfi1 Pea3 Rab4b Siat1 Th Wnt1 Brca1 Hic2 Ptch Rb1 Rbl1 Slc22a1l Ube4b Zac1 Ebag9 Eef1a2 Frat1 Gli Lmyc1 Mas1 Mell1 Ptn Rrad Spam Tert Vav3 Copeb Lox Pla2g2a Serpinb5 Tcfl1

Dead ringer homolog 1 (Drosophila) Growth factor independent 1 Polyomavirus enhancer activator 3 RAB4B, member RAS oncogene family Sialyltransferase 1 (beta-galactoside alpha-2,6-sialyltransferase) Tyrosine hydroxylase Wingless-related MMTV integration site 1 Breast cancer 1 Hypermethylated in cancer 2 Patched homolog Retinoblastoma 1 Retinoblastoma-like 1 (p107) Solute carrier family 22 (organic cation transporter), member 1-like Ubiquitination factor E4B, UFD2 homolog (S. cerevisiae) Zinc finger protein regulator of apoptosis and cell cycle arrest Estrogen receptor-binding fragment-associated gene 9 Eukaryotic translation elongation factor 1 alpha 2 Frequently rearranged in advanced T-cell lymphomas GLI-Kruppel family member GLI Lung carcinoma myc related oncogene 1 MAS1 oncogene Mel transforming oncogene-like 1 Pleiotrophin Ras-related associated with diabetes Sperm adhesion molecule Telomerase reverse transcriptase Vav 3 oncogene Core promoter element binding protein Lysyl oxidase Phospholipase A2, group IIA (platelets, synovial fluid) Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 5 Transcription factor-like 1

Oncogene Oncogene Protooncogene Protooncogene Tumor marker Tumor marker Oncogene Tumor suppressor Putative tumor suppressor Tumor suppressor Tumor suppressor Putative tumor suppressor Putative tumor suppressor Putative tumor suppressor Tumor suppressor Protooncogene Putative oncogene Protooncogene Oncogene Protooncogene Oncogene Protooncogene Oncogene Protooncogene Protooncogene Protooncogene Oncogene Putative tumor suppressor Putative tumor suppressor Tumor suppressor Candidate tumor suppressor Putative tumor suppressor

"2.30 "3.07 "2.92 "2.42 "3.54 "2.78 "2.34 "3.39 "3.42 "2.64 "3.67 "2.80 "2.51 "2.74 "3.18 #3.08 #2.12 #2.85 #3.62 #3.60 #2.62 #2.63 #3.05 #2.19 #2.42 #2.42 #2.47 #5.49 #2.62 #2.27 #2.48 #2.33

Y. Hu et al. / Cancer Letters 258 (2007) 171–180

GenBank ID

", up-regulated; #, down-regulated.

177

178

Y. Hu et al. / Cancer Letters 258 (2007) 171–180

Fig. 5. Up-regulation of Rb1 and Wnt1 in hF-LANa transfectants. The 499 bp products of Rb1 and 449 bp products of Wnt1 were lined out with G3DPH as a control. M, 100-bp DNA ladder. Results were quantified by densitometry and the value from parent cells was taken as unity.

and as a target in gene therapy using antisense oligonucleotide treatment and RNA interference to improve the level of HCC surveillance. In addition to the ERAD function described by Lilley and Ye [6,8,16], we revealed an important role of hF-LANa in cancer and examined its transformation activity as a proto-oncogene. Our results are coincident with Yuliang’s BBRC work [21] which found that Derlin-1 promoted cell survival and growth. Former wok in our lab, up-regulation of hF-LANa (Derlin-2) in HCC, suggest that it might play a role in multistage carcinogenesis [9]. The inhibition of BEL-7404 cell growth by hF-LANa antisense oligonucleotide and the stimulation effect on NIH3T3 cell growth induced by hF-LANa overexpression proved that hF-LANa did participate in regulating cell growth and accelerating cell proliferation as Derlin-1 did in human vascular endothelial cells [21]. The forced hF-LANa over-expression significantly transformed NIH3T3 cells in vitro, and push NIH3T3 cells into malignancy growth in anchorage-independent manner, as the capability of soft agar colony formation has been considered as evidence for fibroblast cell transformation [23]. Further accessed experiments in nude mice proved that NIH3T3 cells expressing hF-LANa could

induce tumor formation in vivo. The in vitro and in vivo experiments mutually proved the oncogenic transformation role of hF-LANa in NIH3T3 cells. It is doubtless that hF-LANa has the transformation ability besides its stimulation to cell growth. Those evidences convinced us that hF-LANa might contribute to human cell malignancies and tumorigenesis. However, over-expression of hF-LANa also altered the glycoprotein metabolic process, biopolymer glycosylation, vehicle-mediated transport, carrier activity and hydrolase activity. These biological process or molecular function may joint hF-LANa’s oncogenic transformation role with its ERAD role. It is reasonable that hF-LANa might be a multi-function protein with transformation ability in fibroblast cells. Among the 19 oncogenes and 13 tumor suppressor genes, there were more oncogenes down-regulated (12 vs. 7) and more tumor suppressor genes up-regulated (8 vs. 5). It is not contradictory to the oncogenic properties of hF-LANa transfected cells. On one hand, many genes possess various functions in different cells and systems. On the other hand, 252 up-regulated genes and 354 down-regulated genes are involved in different biological process and molecular functions in cell proliferation and neurogenesis. Here, we focus on Wnt signaling and Rb1 pathways, two main routes in HCC. Wnt signaling pathway has been identified as one of the key signaling pathways in cancer, regulating cell growth, motility and differentiation. Wnt ligand over-expression has been found in a range of human tumors [24]. It works through the molecules in the pathway, target genes and crosstalk with other pathways and plays an important role in tumor formation [25,26]. Our research found the altered expression of Wnt1, Mmp11 and Lmyc1 and the invariable change of Apc, Axin, b-catenin and CyclinD1 in Wnt pathway in hF-LANa over-expressed NIH3T3 cells (Supplementary Tables S1 and S2). It suggests that hF-LANa might function in cell transformation by the connection with Wnt pathway. The synergistic action of Myc, Wnt1 pathway and Rb/E2F pathway is crucial in human cancer [27]. Although Rb1 is a well-known tumor suppressor gene and inhibits cell proliferation through the E2F family of transcription factors, new roles for Rb were found as a key regulator of cellular senescence, a homeostasis balance maintainer in tissues and an apoptosis suppressor [27–30]. Recent studies have found the novel role of Rb1 in attenuating cell apoptosis [28,31]. The ability of Rb to suppress

Y. Hu et al. / Cancer Letters 258 (2007) 171–180

apoptosis has been contributed to its repression of a distinct proapoptotic set of E2F target genes [28]. On the contrary to being a tumor suppressor, Rb was also found to be required for Ras-induced oncogenic transformation [32]. With the opposite functions in cell proliferation and apoptosis, the effects of up-regulated Rb1 in hF-LANa-transformed NIH3T3 cells might contribute to the malignant growth and tumorigenesity of NIH3T3 cells, especially when Rb works as an apoptosis suppressor or plays a necessary role in oncogenic transformation. So the cellular and regulation mechanisms of hF-LANa need to be lucubrated, and the role of Rb1 and Wnt pathway in the oncogenic transformation role of hF-LANa are still unclear as well. In summary, our present study show that overexpression of hF-LANa could accelerate proliferation of NIH3T3 cells. Importantly, hF-LANa could transform NIH3T3 cells in vitro and in vivo with altered expression of many cancer-related genes. Our findings suggest hF-LANa might be a putative proto-oncogene/oncogene. hF-LANa could be an initiator that regulates other oncogenes and tumor suppressor genes including Wnt1 and Rb1 during cell transformation and hepatocarcinogenesis. All those genes may function in different signaling pathways, and work on NIH3T3 cell transformation. Rb1 and Wnt signaling pathways could be two of the next targets for further study which help us to understand the mechanisms of NIH3T3 cell transformation induced by hF-LANa. It is hopeful that future study of ERAD process would contribute to our understanding in cell proliferation and tumor formation mechanisms in hF-LANa expressed cells. Acknowledgements We thank Dr Caroline Kim for critical reading and helpful suggestions of the manuscript. This work was supported by research grants from the Special Grant for Human Genomics Program of the Chinese Academy of Sciences and the Special Funds for Major State Basic Research of China (Grant G1999053905) and National Natural Science Foundation of China (30170207). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2007.08.017.

179

References [1] A.S. Yu, E.B. Keeffe, Management of hepatocellular carcinoma, Rev. Gastroenterol. Disord. 3 (2003) 8–24. [2] J.M. Llovet, A. Burroughs, J. Bruix, Hepatocellular carcinoma, Lancet 362 (2003) 1907–1917. [3] R. Romeo, M. Colombo, The natural history of hepatocellular carcinoma, Toxicology 181–182 (2002) 39–42. [4] M.E. Nita, V.A. Alves, F.J. Carrilho, S.K. Ono-Nita, E.S. Mello, J.J. Gama-Rodrigues, Molecular aspects of hepatic carcinogenesis, Rev. Inst. Med. Trop. Sao Paulo 44 (2002) 39–48. [5] E. Szabo, C. Paska, P. Kaposi Novak, Z. Schaff, A. Kiss, Similarities and differences in hepatitis B and C virus induced hepatocarcinogenesis, Pathol. Oncol. Res. 10 (2004) 5–11. [6] Y. Ye, Y. Shibata, C. Yun, D. Ron, T.A. Rapoport, A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol, Nature 429 (2004) 841–847. [7] R. Hitt, D.H. Wolf, Der1p, a protein required for degradation of malfolded soluble proteins of the endoplasmic reticulum: topology and Der1-like proteins, FEMS Yeast Res. 4 (2004) 721–729. [8] B.N. Lilley, H.L. Ploegh, Multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane, Proc. Natl. Acad. Sci. USA 102 (2005) 14296–14301. [9] H. Ying, Y. Yu, Y. Xu, Cloning and characterization of FLANa, upregulated in human liver cancer, Biochem. Biophys. Res. Commun. 286 (2001) 394–400. [10] M.B. Hansen, S.E. Nielsen, K. Berg, Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill, J. Immunol. Methods 119 (1989) 203–210. [11] M.H. Nguyen, E.B. Keeffe, Screening for hepatocellular carcinoma, J. Clin. Gastroenterol. 35 (2002) S86–S91. [12] T. Parvez, B. Parvez, K. Pervaiz, A.A. Gumgumji, S. Al Ahmadi, A.A. Sabir, et al., Screening for hepatocellular carcinoma, J. Coll. Phys. Surg. Pak. 14 (2004) 570–575. [13] A.P. Venook, Hepatocellular carcinoma, Curr. Treat Options Oncol. 1 (2000) 407–415. [14] B. Sangro, G. Mazzollini, J. Prieto, Future therapies for hepatocellular carcinoma, Eur. J. Gastroenterol. Hepatol. 17 (2005) 515–521. [15] R.A. Gatenby, P.K. Maini, Mathematical oncology: cancer summed up, Nature 421 (2003) 321. [16] B.N. Lilley, H.L. Ploegh, A membrane protein required for dislocation of misfolded proteins from the ER, Nature 429 (2004) 834–840. [17] M.E. Kirst, D.J. Meyer, B.C. Gibbon, R. Jung, R.S. Boston, Identification and characterization of endoplasmic reticulum-associated degradation proteins differentially affected by endoplasmic reticulum stress, Plant Physiol. 138 (2005) 218– 231. [18] Y. Ye, Y. Shibata, M. Kikkert, S. van Voorden, E. Wiertz, T.A. Rapoport, Inaugural article: recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane [see comment], Proc. Natl. Acad. Sci. USA 102 (2005) 14132–14138. [19] F. Sun, R. Zhang, X. Gong, X. Geng, P.F. Drain, R.A. Frizzell, Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator

180

[20]

[21]

[22]

[23]

[24]

Y. Hu et al. / Cancer Letters 258 (2007) 171–180 (CFTR) and CFTR folding mutants, J. Biol. Chem. 281 (2006) 36856–36863. D. Hu, Y.L. Ran, X. Zhong, H. Hu, L. Yu, J.N. Lou, et al., Overexpressed Derlin-1 inhibits ER expansion in the endothelial cells derived from human hepatic cavernous hemangioma, J. Biochem. Mol. Biol. 39 (2006) 677–685. Y. Ran, Y. Jiang, X. Zhong, Z. Zhou, H. Liu, H. Hu, et al., Identification of Derlin-1 as a novel growth factor-responsive endothelial antigen by suppression subtractive hybridization, Biochem. Biophys. Res. Commun. 348 (2006) 1272–1278. B.N. Lilley, J.M. Gilbert, H.L. Ploegh, T.L. Benjamin, Murine polyomavirus requires the endoplasmic reticulum protein Derlin-2 to initiate infection, J. Virol. 80 (2006) 8739–8744. M.A. Cifone, I.J. Fidler, Correlation of patterns of anchorage-independent growth with in vivo behavior of cells from a murine fibrosarcoma, Proc. Natl. Acad. Sci. USA 77 (1980) 1039–1043. M.J. Smalley, T.C. Dale, Wnt signaling and mammary tumorigenesis, J. Mammary Gland Biol. Neoplasia 6 (2001) 37–52.

[25] A.M. Brown, Wnt signaling in breast cancer: have we come full circle?, Breast Cancer Res 3 (2001) 351–355. [26] J. Behrens, B. Lustig, The Wnt connection to tumorigenesis, Int. J. Dev. Biol. 48 (2004) 477–487. [27] J.R. Nevins, The Rb/E2F pathway and cancer, Hum. Mol. Genet. 10 (2001) 699–703. [28] H. Liu, B. Dibling, B. Spike, A. Dirlam, K. Macleod, New roles for the RB tumor suppressor protein, Curr. Opin. Genet. Dev. 14 (2004) 55–64. [29] M. Classon, E. Harlow, The retinoblastoma tumour suppressor in development and cancer, Nature Rev. Cancer 2 (2002) 910–917. [30] O. Stevaux, N.J. Dyson, A revised picture of the E2F transcriptional network and RB function, Curr. Opin. Cell Biol. 14 (2002) 684–691. [31] B.N. Chau, J.Y. Wang, Coordinated regulation of life and death by RB, Nat. Rev. Cancer 3 (2003) 130–138. [32] J.P. Williams, T. Stewart, B. Li, R. Mulloy, D. Dimova, M. Classon, The retinoblastoma protein is required for Rasinduced oncogenic transformation, Mol. Cell. Biol. 26 (2006) 1170–1182.