Involvement of Epithelial Cell Transforming Sequence-2 Oncoantigen in Lung and Esophageal Cancer Progression

Primary lung cancer is the leading cause of cancer deaths in most countries (1), and esophageal squamous cell carcinoma (ESCC) is one of the most common fatal malignancies of the digestive tract (2). In spite of improvements in surgical techniques and adjuvant chemoradiotherapy, patients with advanced lung or esophageal cancer often suffer fatal disease progression (1, 2). Therefore, it is extremely important to understand the biology of these two major thoracic cancers and to introduce more effective treatments to improve the survival of patients (3). The concept of specific molecular targeting has been applied to the development of innovative cancer-treatment strategies, and two main approaches are available at present in clinical practice: therapeutic monoclonal antibodies and small-molecule agents (4). To date, a number of targeted therapies such as bevacizumab, cetuximab, erlotinib, gefitinib, sorafenib, and sunitinib have been investigated in phase II and phase III trials for the treatment of advanced non–small-cell lung cancer (NSCLC; refs. 4–8). The addition of therapeutic antibodies against proangiogenic protein vascular endothelial growth factor (bevacizumab) or epidermal growth factor receptor (cetuximab) to conventional chemotherapy has a significant survival benefit in patients with NSCLC (4, 5). Two small-molecule epidermal growth factor receptor tyrosine kinase inhibitors, erlotinib and gefitinib, were shown to be effective for a subset of advanced NSCLC patients (6, 7). Phase II studies done with two oral multitargeted tyrosine kinase inhibitors, sorafenib (inhibitor for c-RAF, b-RAF, vascular endothelial growth factor receptors 2 and 3, platelet-derived growth factor receptor β, and KIT) and sunitinib (inhibitor for platelet-derived growth factor receptor, KIT, FLT3, and vascular endothelial growth factor receptor) suggested their efficacy in the treatment of advanced NSCLCs (8). However, issues of toxicity limit these treatment regimens to selected patients, and even if all kinds of available treatments are applied, the proportion of patients showing good response is still very limited (4–8).

To isolate potential molecular targets for diagnosis, treatment, and/or prevention of lung and esophageal carcinomas, we previously performed a genome-wide analysis of gene expression profiles of cancer cells from 101 lung-cancer and 19 ESCC patients by means of a cDNA microarray consisting of 27,648 genes or expressed sequence tags (3, 9–14). To verify the biological and clinicopathologic significance of the respective gene products, we have established a screening system by a combination of the tumor tissue microarray analysis of clinical lung and esophageal cancer materials with RNA interference technique (15–33). In this process, we identified epithelial cell transforming sequence-2 (ECT2) oncogene as a prognostic biomarker as well as a therapeutic target for lung and esophageal cancers.

ECT2 was isolated through an expression cloning strategy from a mouse epithelial cell line BALB/MK, which conferred in vitro transforming activity (34). ECT2 is a member of the Dbl family that possesses a Dbl homology/pleckstrin homology cassette in the COOH terminus and mediates the guanine nucleotide exchange of Rho GTPases (35). The NH₂ terminus of ECT2 contains tandem repeats of the BRCT domain, which is conserved in many proteins involved in cell cycle check point and DNA damage response (36). ECT2 is localized at the central spindle and equatorial cortex and triggers cytokinesis by activating RhoA (37–39). ECT2 was indicated to be overexpressed in glioma cells (40). In spite of the evidence of essential ECT2 function in cytokinesis, the significance of activation of ECT2 in human cancer progression and its clinical potential as a therapeutic target were not fully described.

We report here the identification of ECT2 as a predictive cancer biomarker in the clinic and as a potential therapeutic target for pulmonary and esophageal cancer. We also describe possible biological roles of ECT2 in cancer progression.

Cell lines and tissue samples. Fifteen human lung-cancer cell lines used in this study included five adenocarcinomas (NCI-H1781, NCI-H1373, LC319, A549, and PC-14), five squamous cell carcinomas (SK-MES-1, NCI-H2170, NCI-H520, NCI-H1703, and LU61), one large-cell carcinoma (LX1), and four small-cell lung cancers (SBC-3, SBC-5, DMS273, and DMS114). Ten human esophageal carcinoma cell lines were used in this study: nine squamous cell carcinoma cell lines (TE1, TE2, TE3, TE4, TE5, TE6, TE8, TE9, and TE10) and one adenocarcinoma cell line (TE7; ref. 41). All cells were grown in monolayer in appropriate media supplemented with 10% FCS and were maintained at 37°C in humidified air with 5% CO₂. Human small airway epithelial cells (SAEC) used as a normal control were grown in optimized medium from Cambrex Bioscience, Inc.

Primary NSCLC and ESCC tissue samples as well as their corresponding normal tissues adjacent to resection margins from patients having no anticancer treatment before tumor resection had been obtained earlier with informed consent (9, 13, 14, 17). All tumors were staged on the basis of the pathologic tumor-node-metastasis classification of the International Union Against Cancer (Tables 1 and 2; ref. 42). Formalin-fixed primary lung tumors and adjacent normal lung tissue samples used for immunostaining on tissue microarrays had been obtained from 242 patients (136 adenocarcinomas, 87 squamous cell carcinomas, 16 large-cell carcinomas, and 3 adenosquamous carcinomas; 76 female and 166 male patients; median age of 63.3 y with a range of 26-84 y) undergoing surgery at Hokkaido University and its affiliated hospitals (Sapporo, Japan). A total of 240 formalin-fixed primary ESCCs (21 female and 219 male patients; median age of 61.9 y with a range of 41-81 y) and adjacent normal esophageal tissue samples near to resection margins had been obtained from patients undergoing surgery at Keiyukai Sapporo Hospital (Sapporo, Japan). The patients undergoing surgery did not receive any preoperative treatment, and among them only patients with lymph node metastasis were treated with platinum-based adjuvant chemotherapies after their surgery. This study and the use of all clinical materials mentioned were approved by individual institutional ethics committees.

Semiquantitative reverse transcription-PCR. A total of 3 μg of mRNA aliquot from each sample were reverse transcribed to single-stranded cDNAs using random primer (Roche Diagnostics) and Superscript II (Invitrogen). Semiquantitative reverse transcription-PCR (RT-PCR) experiments were carried out with the following sets of synthesized primers specific for human ECT2 or with β-actin (ACTB)–specific primers as an internal control: ECT2, 5′-GCGTTTTCAAGATCTAGCATGTG-3′ and 5′-CAATTTTCCCATGGTCTTATCC-3′; ACTB, 5′-GAGGTGATAGCATTGCTTTCG-3′ and 5′-CAAGTCAGTGTACAGGTAAGC-3′. PCR reactions were optimized for the number of cycles to ensure product intensity to be within the linear phase of amplification.

Northern blot analysis. Human multiple tissue blots covering 23 tissues (BD Bioscience) were hybridized with an [α-³²P]dCTP–labeled, 719-bp PCR product of ECT2 that was prepared as a probe using primers 5′-TGGTGAAAGCTGGAAGGAAG-3′ and 5′-CAATTTTCCCATGGTCTTATCC-3′. Prehybridization, hybridization, and washing were done following manufacturer's recommendation. The blots were autoradiographed with intensifying screens at −80°C for 7 d.

Anti-ECT2 antibodies. Plasmids expressing COOH-terminal portion of ECT2 (codons 703-883) that contained His-tagged epitopes at their NH₂ termini were prepared using pET28 vector (Novagen). The recombinant proteins were expressed in Escherichia coli, BL21 codon-plus strain (Stratagene), and purified using Ni-NTA Superflow (Qiagen) according to the supplier's protocol. The protein was inoculated into rabbits; the immune sera were purified on affinity columns according to standard methods. The affinity-purified anti-ECT2 polyclonal antibodies were used for Western blotting and immunostaining. We confirmed that the antibody was specific to ECT2 on Western blots using lysates from cell lines that had been transfected with ECT2 expression vector and those from lung and esophageal cancer cell lines, either of which expressed ECT2 endogenously or not.

Western blotting. Tumor cells were lysed in lysis buffer; 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% sodium deoxycholate, and Protease Inhibitor Cocktail Set III (Calbiochem). The protein content of each lysate was determined by a Bio-Rad protein assay (Bio-Rad) with bovine serum albumin as a standard. Ten micrograms of each lysate were resolved on 10% to 12% denaturing polyacrylamide gels (with 3% polyacrylamide stacking gel) and transferred electrophoretically onto a nitrocellulose membrane (GE Healthcare Biosciences). After blocking with 5% nonfat dry milk in TBST, the membrane was incubated with a rabbit polyclonal anti-human ECT2 antibody (generated to recombinant ECT2; please see above) for 1 h at room temperature. Immunoreactive proteins were incubated with horseradish peroxidase–conjugated secondary antibodies (GE Healthcare Bio-sciences) for 1 h at room temperature. After washing with TBST, the reactants were developed using the enhanced chemiluminescence kit (GE Healthcare Bio-sciences).

Immunohistochemistry and tissue microarray. To investigate clinicopathologic significance of the ECT2 protein in clinical samples that had been formalin fixed and embedded in paraffin blocks, we stained the sections using Envision+ Kit/horseradish peroxidase (DakoCytomation) in the following manner. For antigen retrieval, slides were immersed in Target Retrieval Solution High pH (DakoCytomation) and boiled at 108°C for 15 min in an autoclave. Rabbit polyclonal anti-human ECT2 antibody (3.3 μg/mL; generated to recombinant ECT2; please see above) was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with horseradish peroxidase–labeled antirabbit IgG [Histofine Simple Stain MAX PO (G), Nichirei] as the secondary antibody. Substrate-chromogen was added, and the specimens were counterstained with hematoxylin.

Tumor tissue microarrays were constructed with 242 formalin-fixed primary NSCLCs and 240 primary ESCCs, each of which had been obtained by a single institutional group (please see above) with an identical protocol to collect, fix, and preserve the tissues after resection (43–45). Considering the histologic heterogeneity of individual tumors, tissue area for sampling was selected based on visual alignment with the corresponding H&E-stained section on a slide. Three, four, or five tissue cores (diameter, 0.6 mm; depth, 3-4 mm) taken from a donor tumor block were placed into a recipient paraffin block with a tissue microarrayer (Beecher Instruments). A core of normal tissue was punched from each case, and 5-μm sections of the resulting microarray block were used for immunohistochemical analysis. Three independent investigators semiquantitatively assessed ECT2 positivity without prior knowledge of clinicopathologic data. Because the intensity of staining within each tumor tissue core was mostly homogeneous, the intensity of ECT2 staining was semiquantitatively evaluated using the following criteria: strong positive (scored as 2+), dark brown staining in >50% of tumor cells completely obscuring nucleus and cytoplasm; weak positive (1+), any lesser degree of brown staining appreciable in tumor cell nucleus and cytoplasm; absent (scored as 0), no appreciable staining in tumor cells. Cases were accepted as strongly positive only if reviewers independently defined them as such.

Statistical analysis. Statistical analyses were done using the StatView statistical program (SaS). Strong ECT2 immunoreactivity was assessed for association with clinicopathologic variables such as age, gender, pathologic tumor-node-metastasis stage, and histologic type using the Fisher exact test. Tumor-specific survival curves were calculated from the date of surgery to the time of death related to NSCLC or ESCC, or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for ECT2 expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were done with the Cox proportional hazard regression model to determine associations between clinicopathologic variables and cancer-related mortality. First, we analyzed associations between death and possible prognostic factors including age, gender, histology, pT classification, and pN classification, taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced strong ECT2 expression into the model, along with any and all variables that satisfied an entry level of P < 0.05. As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05.

RNA interference assay. To evaluate the biological functions of ECT2 in lung and esophageal cancer cells, we used small interfering RNA (siRNA) duplexes against the target genes (Dharmacon). The target sequences of the synthetic oligonucleotides for RNA interference were as follows: control 1 (luciferase), Photinus pyralis (luciferase gene), 5′-CGUACGCGGAAUACUUCGA-3′; control 2 (scramble), chloroplast Euglena gracilis gene coding for 5S and 16S (rRNAs), 5′-GCGCGCUUUGUAGGAUUCG-3′; si-ECT2-#1, 5′-GAUAAAGGAUGAUCUUGAA-3′; si-ECT2-#2, 5′-CAGAGGAGAUUAAGACUAU-3′. A lung cancer cell line, A549, and an esophageal cancer cell line, TE9, were plated onto 10-cm dishes (1.5 × 10⁶ per dish), and transfected with either of the siRNA oligonucleotides (100 nmol/L) using 24 μL of Lipofectamine 2000 (Invitrogen) according to the manufacturers' instructions. After 7 d of incubation, these cells were stained by Giemsa solution to assess colony formation, and cell numbers were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Flow cytometry. Cells transfected with siRNA oligonucleotides were plated at densities of 5 × 10⁵ per 100-mm dish. Cells were trypsinized 2 or 3 d after transfection, collected in PBS, and fixed in 70% cold ethanol for 30 min. After treatment with 100 μg/mL RNase (Sigma-Aldrich), the cells were stained with 50 μg/mL propidium iodide (Sigma-Aldrich) in PBS. Flow cytometry was done on a Becton Dickinson FACScan and analyzed by ModFit software (Verity Software House, Inc.). The cells selected from at least 20,000 ungated cells were analyzed for DNA content.

Matrigel invasion assay. NIH3T3 and COS-7 cells transfected either with p3XFLAG-tagged (COOH-terminal) plasmid designed to express ECT2 or with mock plasmid were grown to near confluence in DMEM containing 10% FCS. The cells were harvested by trypsinization, washed in DMEM without addition of serum or proteinase inhibitor, and suspended in DMEM at a concentration of 1 × 10⁵/mL. Before preparing the cell suspension, the dried layer of Matrigel matrix (Becton Dickinson Labware) was rehydrated with DMEM for 2 h at room temperature. DMEM (0.75 mL) containing 10% FCS was added to each lower chamber in 24-well Matrigel invasion chambers, and 0.5 mL (5 × 10⁴ cells) of cell suspension was added to each insert of the upper chamber. The plates of inserts were incubated for 24 h at 37°C. Then the chambers were processed, and cells invading through the Matrigel were fixed and stained by Giemsa as directed by the supplier (Becton Dickinson Labware).

ECT2 expression in lung and esophageal cancers and normal tissues. We previously performed the genome-wide expression profile analysis of 101 lung carcinomas (86 NSCLCs or 15 small-cell lung cancers) and 19 ESCCs, as well as 30 normal organs, using cDNA microarray consisting of 27,648 genes or expressed sequence tags (9–14). We identified elevated expression (≥3-fold) of ECT2 transcript in cancer cells in the great majority of the lung and esophageal cancer samples examined. Moreover, we observed no ECT2 expression in any of 29 normal tissues except testis (data not shown). Therefore, we considered ECT2 to be a good molecular candidate for further analyses. We confirmed its overexpression by means of semiquantitative RT-PCR experiments in 12 of 15 lung cancer tissues, in 10 of 15 lung-cancer cell lines, in 8 of 10 ESCC tissues, and in 4 of 10 ESCC cell lines examined (Fig. 1A and B). We subsequently generated rabbit polyclonal antibodies specific for human ECT2 and confirmed by Western blot analysis the overexpression of ECT2 protein in five of six lung cancer cell lines and two of four ESCC cell lines (Fig. 1C).

Northern blot analysis with an ECT2 cDNA as a probe identified a 4.3-kb transcript only in testis among 23 normal human tissues examined (Fig. 1D, top). We subsequently examined expression of ECT2 protein in five normal tissues (liver, heart, kidney, lung, and testis) as well as lung cancers using anti-ECT2 antibody. We found that ECT2 expression was hardly detectable in the former four tissues whereas positive ECT2 staining appeared in the nucleus and cytoplasm of testis and lung cancer cells (Fig. 1D, bottom).

Association of ECT2 overexpression with poor prognosis for NSCLC and ESCC patients. To investigate the biological and clinicopathologic significance of ECT2 in pulmonary and esophageal carcinogenesis, we carried out immunohistochemical staining on tissue microarray containing tissue sections from 242 NSCLC and 240 ESCC cases who had undergone surgical resection. ECT2 staining with the anti-ECT2 polyclonal antibody was mainly observed at the nucleus and cytoplasm of lung tumor cells, but not detected in their surrounding normal lung cells (Fig. 2A, top). We classified ECT2 expression levels on the tissue array ranging from absent (scored as 0) to weak/strong positive (scored as 1+ ∼ 2+). Of the 242 NSCLCs, ECT2 was strongly stained in 112 cases (46%; score 2+), weakly stained in 91 cases (38%; score 1+), and not stained in 39 cases (16%: score 0; details are shown in Table 1). We next examined a correlation of ECT2 expression levels (strong positive versus weak positive/absent) with various clinicopathologic variables and found that strong expression of ECT2 in NSCLCs was significantly associated with non-adenocarcinoma histology (P = 0.0389, Fisher's exact test; Table 1) and with tumor-specific 5-year survival after the resection of primary tumors (P = 0.0004, log-rank test; Fig. 2A, bottom). We also applied univariate analysis to evaluate associations between patient prognosis and several factors including age (≥65 versus <65 years), gender (male versus female), histology (non-adenocarcinoma versus adenocarcinoma), pT stage (tumor size; T₂-T₃ versus T₁), pN stage (lymph node metastasis; N₁-N₂ versus N₀), and ECT2 expression (score 2+ versus 0, 1+). All those parameters were significantly associated with poor prognosis (Table 3). Multivariate analysis using the Cox proportional hazard model indicated that pT stage, pN stage, age, and strong ECT2 staining were independent prognostic factors for NSCLC (Table 3).

Of the 240 ESCC cases examined, ECT2 was strongly stained in 81 cases (34%; score 2+), weakly stained in 135 cases (56%; score 1+), and not stained in 24 cases (10%; score 0; Fig. 2B, top; details are shown in Table 2). We found a significant correlation of strong ECT2 positivity (score 2+) with pT stage (higher in deeper tumor invasion cases; P = 0.0124) and pN stage (higher in lymph node metastasis positive cases; P = 0.0442, Fisher's exact test; Table 2). ESCC patients whose tumors showed strong ECT2 expression revealed shorter tumor-specific survival periods compared with those with absent/weak ECT2 expression (P = 0.0088, log-rank test; Fig. 2B, bottom). Univariate analysis evaluating associations between ESCC prognosis and several factors including age (≥65 versus <65), gender (male versus female), pT stage (tumor depth; T₂, T₃ versus T₁), pN stage (N₁ versus N₀), and ECT2 expression (score 2+ versus 0, 1+) revealed that all of those parameters except age were significantly associated with poor prognosis (Table 4). In multivariate analysis, strong ECT2 expression did not reach the statistically significant level as an independent prognostic factor for surgically treated ESCC patients enrolled in this study (P = 0.1872), whereas pT and pN stages as well as gender did so, suggesting the relevance of ECT2 expression to these clinicopathologic factors in esophageal cancer (Table 4).

Inhibition of growth of cancer cells by siRNA for ECT2. To assess whether ECT2 is essential for growth or survival of lung and esophageal cancer cells, we transfected synthetic oligonucleotide siRNAs against ECT2 into A549 and TE9 cells in which ECT2 was endogenously overexpressed. The levels of ECT2 in the cells transfected with si-ECT2-#1 or si-ECT2-#2 were significantly decreased in comparison with those transfected with either control siRNAs (Fig. 3A, top). MTT and colony formation assays revealed a drastic reduction in the number of cells transfected with si-ECT2-#1 or si-ECT2-#2 (Fig. 3A, middle and bottom). To clarify the mechanism of tumor suppression by siRNAs against ECT2, we performed flow cytometric analysis of the tumor cells transfected with these siRNAs. We found a significant increase of the cells at the G₂-M phase at 48 h and a subsequent increase of the cells of sub-G₁ fraction at 72 h after the treatment (Fig. 3B).

Activation of mammalian cellular invasion by ECT2. Because ECT2 is a guanine nucleotide exchange factor for Rho GTPases, which may be associated with cell motility, and the immunohistochemical analysis on tissue microarray had indicated that lung and esophageal cancer patients with strong ECT2-positive tumors showed shorter cancer-specific survival period than those with ECT2-weak positive/negative tumors, we examined a possible role of ECT2 in cellular invasion by Matrigel assays using two mammalian cells (NIH3T3 and COS-7). Transfection of ECT2 cDNA into either of the cells significantly enhanced their invasive activity through Matrigel (Fig. 4). This result also suggested that ECT2 could contribute to the highly malignant phenotype of cells.

Aerodigestive tract cancer, including carcinomas of the lung, esophagus, oral cavity, pharynx, and larynx, accounts for one third of all cancer deaths in the United States and is the most common cancer in some areas of the world (3). Despite the use of modern surgical techniques combined with various adjuvant treatment modalities such as radiotherapy and chemotherapy, the overall 5-year survival rate of ESCC patients remains at ∼40%, and that of lung cancer patients is only 15% (1, 2). Therefore, further development of new cancer diagnostics and therapeutics by targeting specific oncogenic pathways is urgently awaited. We performed a genome-wide expression profile analysis of 101 lung cancers and 19 ESCCs after enrichment of cancer cells by laser microdissection using a cDNA microarray containing 27,648 genes. We systematically analyzed the protein expression of candidate targets among hundreds of clinical samples on tissue microarrays, investigated loss-of-function phenotypes using RNA interference systems, and further defined biological functions of the proteins. Through these analyses, we have identified and validated a total of 24 oncoproteins that were up-regulated in cancer cells but not expressed in normal organs, except testis, placenta, and/or fetus, and considered them to be good candidates for the development of novel diagnostic biomarkers, therapeutic drugs, and/or immunotherapy (3, 9–33, 46). In this study, we report that ECT2, encoding a guanine nucleotide exchange factor for Rho GTPases, is frequently transactivated in the majority of lung and esophageal cancer samples, and that its gene products play indispensable roles in the growth/invasion of the cancer cells.

The small Rho GTPase is known to play important roles in essential cellular processes such as the regulation of actin cytoskeleton, gene transcription, cell motility, cell adhesion, and cytokinesis (47). ECT2 contains a Dbl homology domain in tandem with a pleckstrin homology domain and catalyzes guanine nucleotide exchange on the small GTP-binding protein, such as RhoA and Cdc42 (47). ECT2 expression was directly regulated by E2Fs (38), and ECT2 protein was phosphorylated at Thr341 by cyclin-dependent kinase 1 during G₂-M phase, resulting in an increase of guanine nucleotide exchange factor activity and regulation of cytokinesis (39). It was proposed that late mitotic Plk1 activity promotes recruitment of ECT2 to the central spindle, triggering the initiation of cytokinesis and contributing to cleavage plane specification in human cells (37).

In this study, we confirmed that the treatment of cancer cells with specific siRNA for ECT2 resulted in inhibition of cancer cell growth through G₂-M arrest at 48 h after siRNA transfection and subsequent apoptosis at 72 hours. We also obtained additional evidence supporting the significance of ECT2 in human carcinogenesis. The expression of ECT2 resulted in the significant promotion of the cellular invasion. Moreover, clinicopathologic evidence obtained through our tissue microarray experiments indicated that NSCLC or ESCC patients with ECT2-positive tumors had shorter cancer-specific survival periods than those with ECT2-negative tumors. The expression of ECT2 might enhance the guanine nucleotide exchange factor and Rho GTPase activity and cytokinesis, resulting in an increase in the invasive ability of cancer cells. Although the molecular mechanisms underlying increased ECT2 expression levels in lung and esophageal cancer cells have not been elucidated, the results obtained by in vitro and in vivo assays strongly suggested that ECT2 is likely to be an important growth factor and might be associated with a highly malignant phenotype of cancer cells. Because ECT2 should be classified as a typical cancer-testis antigen that has been recognized as a group of highly attractive targets for cancer therapy (32, 46), selective inhibition of ECT2 enzymatic activity by small-molecule compounds or its sequence-specific gene silencing by a new type of drugs could be a promising therapeutic strategy that is expected to have a powerful biological activity against cancer with a minimal risk of adverse events. In fact, the development of nucleic acid drugs for the treatment of various diseases has shown great promise, although systemic delivery of them has been more problematic due to degradation in serum and poor cellular uptake (48–50). Randomized phase III trials to evaluate the combined use of oblimersen sodium (Genasense), an 18-mer phosphorothioate antisense oligonucleotide designed to bind to the first six codons of the human BCL2 mRNA with standard treatment, improved multiple clinical outcomes relative to standard treatment alone in chronic lymphocytic leukemia as well as malignant melanoma (48, 49). On the other hand, systemic delivery of apolipoprotein B (APOB)–specific siRNAs that were encapsulated in stable nucleic acid lipid particles could silence the APOB expression in the liver of nonhuman primates and result in significant and lasting reductions in APOB protein, serum cholesterol, and low-density lipoprotein levels (50). The antitumor activity of siRNAs targeting ECT2 coupled with a variety of drug delivery systems and chemical modification techniques may hold great promise for the development of a new class of anticancer drugs. Moreover, ECT2 oncoantigen may be useful for screening of HLA-restricted epitope peptides for cancer vaccine that can induce specific immune responses by CTLs against cancer cells with ECT2 expression. Because these ECT2 could have fundamental functions that may be responsible for cancer cell survival, vaccination with the peptides from this protein should reduce the risk of the emergence of immune escape variant of tumors that have lost their antigen expression.

In conclusion, our data strongly raise the possibility of designing new anticancer drugs to specifically target the oncogenic activity of ECT2 for the treatment of cancer patients. ECT2 overexpression in resected specimens may be a useful index for application of adjuvant therapy to lung and esophageal cancer patients who are likely to have poor clinical outcome.

No potential conflicts of interest were disclosed.