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Supplementary Figures 1-3 from An Antagonist of Dishevelled Protein-Protein Interaction Suppresses β-Catenin–Dependent Tumor Cell Growth

Background: Pleural mesothelioma, a devastating asbestos‐associated malignancy, urgently requires a novel effective therapy. Heat shock protein 70 (HSP70), which is synthesized in the cell response to protein damage, is expected to be a new target for antitumor treatment. In addition to its well‐known protein refolding function, HSP70 regulates cell proliferation through different pathways, including PI3K/AKT/mTOR, and autophagy in malignant cells. In this study, we attempted to clarify the effects of VER‐155008, an HSP70 inhibitor, on pleural mesothelioma. Methods: Human pleural mesothelioma cell lines 211H, H2452 and H28 were cultured with VER‐155008, and protein expression, cell proliferation, colony formation, cell cycle, synergistic effect with cisplatin, and autophagy induction were analyzed. Results: In mesothelioma cell lines, VER‐155008 (5.0 μM or more) inhibited cell growth and colony formation, accompanied by G1 cell cycle arrest. According to western blot analysis, VER‐155008 reduced p‐AKT expression. However, VER‐155008 failed to show a synergistic effect with cisplatin on cell growth. Mesothelioma cells transfected with the novel plasmid pMRX‐IP‐GFP‐LC3‐RFP‐LC3ΔG, which was developed for the quantitative and statistical estimation of macroautophagy, showed enhanced macroautophagy upon treatment with VER‐155008 and gefitinib which is an EGFR‐tyrosine kinase inhibitor. In addition, fetal bovine serum deprivation induced macroautophagy was further enhanced by VER‐155008. Conclusions: On the basis of these results, functional HSP70 inhibition by VER‐155008 suppressed cell growth in pleural mesothelioma cells, accompanied by enhanced macroautophagy. HSP70 inhibition is thus expected to become a new strategy for treating mesothelioma. Key points Significant findings of the study In pleural mesothelioma cells, inhibition of HSP70 function by VER‐155008 suppressed cell proliferation accompanied by induction of autophagy which was synergistically enhanced under the starvation condition, whereas gefitinib, an EGFR‐TKI, did not show the same synergistic effect in autophagy. What this study adds The inhibition of HSP70 induced autophagy and suppressed cell proliferation in mesothelioma cells., The effects of VER‐155008, gefitinib or FBS deprivation on macroautophagy in mesothelioma cells were assessed by a plasmid system (pMRX‐IP‐GFP‐LC3‐RFP‐ LC3ΔG). This plasmid produces light chain 3 (LC3) protein bound to green fluorescent protein (GFP), and LC3ΔG protein, an internal control, bound to red fluorescent protein (RFP).

Background Immune-checkpoint inhibitors (ICIs) have been increasingly used for non-small cell lung cancer (NSCLC) treatment in recent years. Although insufficient, the rate of programmed death-ligand 1 expression has been adopted as a predictor of ICI efficacy. We evaluated tumor growth rate as a clinically easy-to-use predictor of the therapeutic effect of ICIs. Methods This study is a single-institution retrospective study in Japan. NSCLC patients treated with nivolumab, pembrolizumab, or atezolizumab at Saitama Medical Center from January 1, 2016 to December 31, 2018 were enrolled, and followed until December 31, 2020. We defined and calculated the initial rapidity of tumor progression (IRP) as: the increase in the sum of the diameters of intrathoracic tumors and lymph nodes on two series of chest computed tomography (CT) scans (one obtained at an initial checkup and the other obtained immediately before the first treatment) divided by the number of days between these CT scans. Two coefficients were calculated: the maximal information coefficient (MIC) between IRP and time to treatment failure (TTF) using the Python package with minepy library, and the Spearman's rank correlation coefficient. Results A total of 55 patients (median age, 70 years; 47 men) were enrolled. The median TTF with ICIs was 126 days, and four patients continued to receive ICI treatment at the end of the follow-up. The MIC between IRP and TTF was 0.302 with weak correlation, and the Spearman's rank correlation coefficient was -0.347 (P=0.00938). Conclusions The initial tumor growth rate had a negative linear correlation with the therapeutic effect of ICIs.

Type III collagen is the major protein in the walls of blood vessels and hollow organs; it is decreased in patients with vascular Ehlers-Danlos syndrome (EDS). A 52-year-old man was admitted for severe back pain, and right hemothorax was suspected by chest computed tomography. Immediately after embolization for bleeding bronchial artery, aortic dissection occurred and was treated conservatively in the intensive-care unit. Vascular EDS with a mutation of COL3A1 cDNA (c.3175G>A) was diagnosed. When vascular EDS is suspected, the patient should be treated prophylactically, and a genetic examination should be performed to confirm the diagnosis.

Homeodomain-interacting protein kinase-2 (HIPK2) can either promote or inhibit transcription depending on cellular context. In this study, we show that a new HIPK2 isoform increases TEAD reporter activity in NSCLC cells. We detected HIPK2 copy number gain in 5/6 (83.3%) NSCLC cell lines. In NSCLC patients with high HIPK2 mRNA expression in the Human Protein Atlas, the five-year survival rate is significantly lower than in patients with low expression (38% vs 47%; p = 0.047). We also found that 70/78 (89.7%) of NSCLC tissues have moderate to strong expression of the N-terminal HIPK2 protein. We detected and cloned a novel HIPK2 isoform 3 and found that its forced overexpression promotes TEAD reporter activity in NSCLC cells. Expressing HIPK2 isoform 3_K228A kinase-dead plasmid failed to increase TEAD reporter activity in NSCLC cells. Next, we showed that two siRNAs targeting HIPK2 decreased HIPK2 isoform 3 and YAP protein levels in NSCLC cells. Degradation of the YAP protein was accelerated after HIPK2 knockdown in NSCLC cells. Inhibition of HIPK2 isoform 3 decreased the mRNA expression of YAP downstream gene CTGF. The specific HIPK2 kinase inhibitor TBID decreased TEAD reporter activity, reduced cancer side populations, and inhibited tumorsphere formation of NSCLC cells. In summary, this study indicates that HIPK2 isoform 3, the main HIPK2 isoform expressed in NSCLC, promotes YAP/TEAD transcriptional activity in NSCLC cells. Our results suggest that HIPK2 isoform 3 may be a potential therapeutic target for NSCLC.

It was previously revealed that Wnt signaling is activated in mesothelioma cells. Although epidermal growth factor receptor (EGFR) is expressed in mesothelioma cells, EGFR-tyrosine kinase inhibitors (TKIs) are not effective for mesothelioma treatment. However, in non-small cell lung cancer, the blocking of Wnt signaling has been identified to enhance the anticancer effect of EGFR-TKIs. To confirm the anticancer effect of blocking Wnt signaling in combination with EGFR-TKI treatment in mesothelioma, the present study evaluated the effect of simultaneous suppression of human dishevelled-3 (Dvl-3) expression with Dvl-3 small interfering RNA (siRNA) and of EGFR inhibition with gefitinib on mesothelioma cell viability. Mesothelioma cell lines with and without β-catenin gene expression were transfected with Dvl-3 siRNA and were cultured with gefitinib, and cell viability, colony formation and cell cycle analyses were performed. Dvl-3 siRNA downregulated the expression of Dvl-3 in mesothelioma cells. The combination of Dvl-3 siRNA with gefitinib acted synergistically to induce concomitant suppression of cell viability and colony formation, suggesting that inhibition of Wnt signaling by downregulating Dvl-3 with siRNA and inhibiting EGFR with gefitinib leads to significant antitumor effects.

Background Owing to the rarity of this tumor, there is limited information about second-line chemotherapy for patients with previously treated advanced thymic carcinoma. Material and Methods We performed a multi-institutional, retrospective study named NEJ023 for patients with advanced thymic carcinoma. Patients without indications for curative treatment were treated with chemotherapy from 1995 to 2014 at 40 institutions in the North East Japan Study Group. Demographic and clinicopathologic characteristics, data on treatment methods, and outcomes of second-line chemotherapy were obtained from medical records. Results In total, 191 patients were enrolled in this study. Second-line chemotherapy included platinum-based doublets in 57.6% of patients, other multidrug chemotherapy (e.g., cisplatin, doxorubicin, vincristine, and cyclophosphamide) in 13.6%, and monotherapy in 28.8%. The median follow-up time was 50.5 months, and the median overall survival (OS) from the start of second-line chemotherapy was 22.4 (95% confidence interval, 17.5-26.7) months. The average response rate (RR) was 20.0% overall; it was 21.6% for patients treated with platinum-based doublet chemotherapy, 13.6% for those treated with other multidrug chemotherapy, and 19.6% for those treated with single agent chemotherapy. There was no significant difference in OS between platinum-based doublet chemotherapy, other multidrug chemotherapy, and monotherapy (the median OS was 22.4, 25.7, and 21.4 months, respectively). Conclusion The median OS was 22.4 months in patients with advanced thymic carcinoma treated with second-line chemotherapy. There were no significant differences in RR and OS between monotherapy and multidrug chemotherapy in this study.

In the eighth edition of the TNM classification of lung cancer, the M1b and M1c descriptors are newly defined by the number of extrathoracic metastases. To verify the prognostic value of these descriptors in Japan, we reclassified our cases and re-evaluated prognosis in M1b and M1c patients. All non-small cell lung cancer (NSCLC) patients with extrathoracic metastases who visited Saitama Medical Center from 2010 to 2016 were evaluated, divided according to the eighth edition of the TNM classification criteria into two groups (M1b, patients with single extrathoracic metastasis, and M1c, patients with multiple extrathoracic metastases), and followed up until December 31, 2017. Survival time analysis was performed using the Kaplan–Meier method, and between-group differences in overall survival time (OS) were evaluated by the log-rank test. A total of 231 NSCLC patients were divided into 57 patients with M1b and 174 with M1c. Median OS was 15.2 months (95% confidence interval [CI]: 9.3–19.9) and 7.3 months (95% CI 5.7–10.7) for M1b and M1c, respectively, with no significant between-group difference (P = 0.239). However, after excluding patients with epidermal growth factor receptor (EGFR) mutation or echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4–ALK) fusion gene, median OS was 12.9 months (95% CI 7.2–19.9) for M1b and 5.4 months (95% CI 3.8–6.3) for M1c, respectively, showing a significant difference (P = 0.029). The effect of therapy directed toward EGFR mutation or EML4–ALK fusion gene might obscure the significant prognostic difference between M1b and M1c.

Pleural mesothelioma is a poor prognostic malignancy because the effect of therapies remains limited; and thus, it is urgent to elucidate the pathophysiology leading to newer treatment. Our previous investigation showed that growth of mesothelioma cells was inhibited by suppression of dishevelled-3 on Wnt signaling pathway, accompanied with reduction of heat shock protein 70 (HSP70), which works as a cell-protective molecular chaperon against heat stress, oxidative stress, heavy metals and others, by refolding proteins. Furthermore, HSP70 has been showed to affect cell signaling such as Akt/mTOR pathway, which negatively regulates macroautophagy. This study evaluated effects of VER-155008, an adenosine-derived functional inhibitor of HSP70, on cell growth and macroautophagy of mesothelioma cell lines. Mesothelioma cell lines including MSTO-211H (211H), NCI-H2452 (H2452) and NCI-H28 (H28) were cultured with 0.5-40 µM VER-155008 for cell viability analysis by water-soluble tetrazolium salt-8, colony formation assay, cell cycle analysis and the following analyses. Western blotting confirmed the expression of HSP70, Akt, phosphorylated (p-) Akt, JNK, p-JNK, light chain 3A (LC3A) and lysosome-associated membrane protein 2A (LAMP2A). For evaluation of macroautophagy, a plasmid, pMRX-IP-GFP-LC3-RFP-LC3ΔG, was transfected to mesothelioma cells. The transfected cells generate GFP-LC3-RFP-LC3ΔG, and it was divided into GFP-LC3 and RFP-LC3ΔG by inherent ATG4. GFP-LC3 decreases by macroautophagy activation, while RFP-LC3ΔG serves as an internal control. VER-155008 suppressed cell viability dose-dependently, and colony formation of 211H, H2452 and H28. In 211H and H28 cells, the proportions of cell count in G1 phase significantly increased, accompanied with inhibition of cell growth. In western blot analysis, VER-155008 reduced expression of p-Akt, while the expression of Akt, p-JNK, JNK, LC3A, LAMP2A and HSP70 were not altered. In macroautophagy analysis by the use of mesothelioma cells, which were transfected with the plasmid and stably expressed GFP-LC3-RFP-LC3ΔG, 20 µM VER-155008, 5.0 µM gefitinib and deprivation of fetal bovine serum (FBS) induced macroautophagy. In addition, VER-155008 enhanced FBS-deprivation-induced macroautophagy. From these results, functional inhibition of HSP70 by VER-155008 suppressed cell growth in pleural mesothelioma cells, accompanied with enhancement of macroautophagy. Citation Format: Kosuke Sakai, Maya Inoue, Hiroaki Nishimura, Shintaro Mikami, Yoshiki Kuwabara, Akitoshi Kojima, Maiko Toda, Yumiko Ogawa, Satoshi Kikuchi, Yusuke Hirata, Yuriko Mikami, Hiroyuki Kyoyama, Gaku Moriyama, Akihiko Gemma, Kazutsugu Uematsu. Evaluation of anti-tumor effects of VER-155008, a functional inhibitor of HSP70, through macroautophagy in mesothelioma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1830.

Background: The serum level of carcinoembryonic antigen (CEA is a marker of malignant disease but can also be increased in benign diseases. Patients with allergic bronchopulmonary aspergillosis (ABPA have bronchial asthma showing a wide variety of radiological findings. We measured serum CEA levels in patients with ABPA and evaluated the relationships of serum CEA levels with peripheral blood eosinophil counts total blood levels of immunoglobin (Ig E and findings of computed tomography (CT in ABPA. Methods: The subjects were 13 patients (6 men and 7 women aged 34 to 76 years who had been treated for ABPA at our hospital. Serum levels of CEA peripheral blood eosinophil counts total blood IgE levels and CT findings were examined before and after treatment with prednisolone. Results: Before the start of the treatment 7 of 13 patients had serum CEA levels higher than the upper limit of normal. The serum CEA level was not correlated with eosinophil counts or total IgE values. Serum CEA levels were examined after treatment in 9 patients and were found to have significantly decreased as pulmonary consolidation improved. Furthermore serum CEA levels before treatment in patients with pulmonary consolidation were significantly higher than those in patients without consolidation. Conclusion: Serum CEA levels are elevated in some patients with ABPA; these elevations might be associated with consolidation in the lung. Elevated serum CEA levels decrease as the consolidation decreases after treatment. The elevation of serum CEA might be attributed to the presence of local inflammation in the lung. (J Nippon Med Sch 2013; 80: 404―409

A 60-year-old man was admitted to our hospital experiencing difficulty in opening his mouth, and diplopia. Chest computed tomography showed a mass on the right lung. Transbronchial biopsy and scrutiny of the whole body resulted in the diagnosis of small cell lung cancer, cT2N2M1a stage IV. With chemotherapy including carboplatin and etoposide, the primary mass of the right lung shrank, and the patient no longer had diplopia or trouble opening his mouth. After the first-line chemotherapy, itching, ptosis, and dysphagia appeared, accompanied by tumor relapse. These symptoms improved with chemotherapy for lung cancer. After four lines of chemotherapy, the lung cancer was not controlled. The patient died 19 months after the first chemotherapy had started. Difficulty in opening the mouth is a rare symptom for lung cancer patients, but chemotherapy for lung cancer clearly improved this symptom. Therefore, we diagnosed this rare symptom as paraneoplastic neurological syndrome.

Background:The use of a budesonide (BUD)/formoterol (FOR) combination (Symbicort®) for both maintenance and reliever therapy reduces the exacerbation of asthma better than the traditional fixed-dose regimens. In the early stage of exacerbation, the combination therapy could intervene with the development of subsequent airway inflammation. The objective of the study was to evaluate whether in the early stage of cell activation the use of BUD/FOR combination would modify the adhesion of eosinophils or production of cytokines from mononuclear cells. Methods:Human peripheral blood eosinophils, pretreated with BUD (0.1 µM), FOR (0.1 µM) or BUD/FOR combination for 30 min at 37°C, were stimulated with IL-5, leukotriene D4 or phorbol myristate acetate, and eosinophil adhesion was evaluated using an eosinophil peroxidase assay. BUD (0.1 µM), FOR (0.1 µM) or BUD/FOR combination was added to the peripheral blood mononuclear cells stimulated with ionomycin plus phorbol myristate acetate. Concentrations of IL-5 and RANTES in the cell supernatant were measured using ELISA. Results:BUD, FOR or BUD/FOR combination did not modify the eosinophil adhesion induced by any stimulators. In contrast, BUD or BUD/FOR combination significantly reduced the productions of IL-5 and RANTES in peripheral blood mononuclear cells (BUD: p < 0.0001, p = 0.027 vs. control; BUD/FOR: p < 0.0001, p = 0.031 vs. control) when the drugs were added 15 min, but not 4 h, following cell stimulation. Conclusion: In the early stage of exacerbation of asthma, inhaled BUD may suppress the progression of the allergic inflammatory cascade by inhibiting the mononuclear cells. These results also underline the importance of using BUD in rescue inhaler.

Summary Reports on the safety and efficacy of endobronchial argon plasma coagulation (APC) for the treatment of neoplastic airway obstruction in critically ill patients are limited. We describe a case of severe airway stenosis in a patient with esophageal cancer who required high-inspired oxygen concentrations, mechanical ventilatory and hemodynamic support. Relief of obstruction was achieved with APC in the absence of bedside complications. APC can be performed safely for palliative management of obstruction due to endobronchial tumor even in critically ill patients in whom the use of the Nd-YAG laser is precluded.

Purpose This multicenter, phase II study was conducted to evaluate the activity of amrubicin, a topoisomerase II inhibitor, against refractory or relapsed small-cell lung cancer (SCLC). Patients and Methods SCLC patients with measurable disease who had been treated previously with at least one platinum-based chemotherapy regimen and had an Eastern Cooperative Oncology Group performance status of 0 to 2 were eligible. Two groups of patients were selected: patients who experienced first-line treatment failure less than 60 days from treatment discontinuation (refractory group), and patients who responded to first-line treatment and experienced disease progression ≥ 60 days after treatment discontinuation (sensitive group). Amrubicin was administered as a 5-minute daily intravenous injection at a dose of 40 mg/m2 for 3 consecutive days, every 3 weeks. Results Between June 2003 and December 2004, 60 patients (16 refractory and 44 sensitive) were enrolled. The median number of treatment cycles was four (range, one to eight). Grade 3 or 4 hematologic toxicities comprised neutropenia (83%), thrombocytopenia (20%), and anemia (33%). Febrile neutropenia was observed in three patients (5%). Nonhematologic toxicities were mild. No treatment-related death was observed. The overall response rates were 50% (95% CI, 25% to 75%) in the refractory group, and 52% (95% CI, 37% to 68%) in the sensitive group. The progression-free survival, overall survival, and 1-year survival in the refractory group and the sensitive group were 2.6 and 4.2 months, 10.3 and 11.6 months, and 40% and 46%, respectively. Conclusion Amrubicin exhibits significant activity against SCLC, with predictable and manageable toxicities; this agent deserves to be studied more extensively in additional trials.

Activation of the Wnt/β-catenin signaling pathway has been associated with human cancers. To test whether Wnt-2 signal is a survival factor in human melanoma cells and thus represents a potential therapeutic target, we investigated the effects of inhibition of Wnt-2 signaling in human melanoma cell lines. We have developed a novel monoclonal antibody against the NH2 terminus of the human Wnt-2 ligand that induces apoptosis in human melanoma cells overexpressing Wnt-2. Whereas incubation of this antibody with normal cells lacking Wnt-2 expression does not induce apoptosis, Wnt-2 signaling blockade by the ligand-binding antibody is confirmed by down-regulation of Dishevelled and β-catenin. Wnt-2 small interfering RNA treatment in these cells yielded similar apoptotic effects and downstream changes. Down-regulation of an inhibitor of apoptosis family protein, survivin, was observed in both the Wnt-2 antibody-treated and small interfering RNA-treated melanoma cell lines, suggesting that the antibody induces apoptosis by inactivating survivin. In an in vivo study, this monoclonal anti-Wnt-2 antibody suppresses tumor growth in a xenograft model. These findings suggest that the anti-Wnt-2 monoclonal antibody may be useful for the treatment of patients with malignant melanoma.

In this report, we have demonstrated that Wnt-2 protein is overexpressed in freshly resected human non-small-cell lung cancer (NSCLC) tissues. We have also developed a monoclonal antibody against the N-terminus of human Wnt-2 protein. This monoclonal antibody induces apoptosis in human NSCLC cell lines that overexpress Wnt-2 protein. Incubation of this antibody with normal human airway cells lacking Wnt-2 expression does not induce apoptosis. Wnt-2 signaling blockade by the anti-Wnt-2 antibody is confirmed by downregulation of cytosolic beta-catenin and reduction in TCF-dependent transcriptional activity (TOPFLASH assay). In addition, Wnt-2-specific small interfering RNA (siRNA) treatment in the NSCLC cell line A549 also downregulated cytosolic beta-catenin and induced apoptosis. Moreover, downregulation of an inhibitor of apoptosis family protein, Survivin, was noticed both in the Wnt-2 antibody- and siRNA-treated NSCLC cells, suggesting that inhibition of Wnt-2-mediated signaling induces apoptosis through inactivating Survinin.

It is known that Wnt-1 signaling inhibits apoptosis by activating β-catenin/tcf-mediated transcription. Here, we show that blocking Wnt-1 signaling in β-catenin-deficient mesothelioma cell lines H28 and MS-1 induces apoptotic cell death. Both Wnt-1 small interfering RNA (siRNA) and Dishevelled siRNA induced significant apoptosis in these cell lines. A small molecule inhibitor of c-Jun NH2-terminal kinase inhibited the apoptotic cell killing induced by either Wnt-1 siRNA or Dishevelled siRNA in these cells. Our data suggest that β-catenin-independent noncanonical pathway(s), i.e., Wnt/JNK pathway, may play a role in the apoptotic inhibition caused by Wnt-1 signaling.

Aberrant activation of the Wingless-type (Wnt)/β-catenin signaling pathway is associated with a variety of human cancers. Little is known regarding the role that Wnt ligands play in human carcinogenesis. To test whether a Wnt-1 signal is a survival factor in human cancer cells and thus may serve as a potential cancer therapeutic target, we investigated the effect of inhibition of Wnt-1 signaling in a variety of human cancer cell lines, including non small cell lung cancer, breast cancer, mesothelioma, and sarcoma. Both monoclonal antibody and RNA interference (RNAi) were used to inhibit Wnt-1 signaling. We found that incubation of a monoclonal anti-Wnt-1 antibody induced apoptosis and caused downstream protein changes in cancer cells overexpressing Wnt-1. In contrast, apoptosis was not detected in cells lacking or having minimal Wnt-1 expression after the antibody incubation. RNAi targeting of Wnt-1 in cancer cells overexpressing Wnt-1 demonstrated similar downstream protein changes and induction of apoptosis. The antibody also suppressed tumor growth in vivo. Our results indicate that both monoclonal anti-Wnt-1 antibody and Wnt-1 siRNA inhibit Wnt-1 signaling and can induce apoptosis in human cancer cells. These findings hold promise as a novel therapeutic strategy for cancer.

Lung cancer is the leading cause of cancer death in the world, but the molecular mechanisms for its development have not been well characterized. The suppressors of cytokine signaling (SOCS) are inhibitors of cytokine signaling that function via the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Eight SOCS proteins with similar structures have been identified so far. SOCS family members, however, have distinct mechanisms of inhibition of JAK/STAT signaling. Abnormalities of the JAK/STAT pathway are associated with cancer. Inhibition of signaling results in growth suppression in various cell types. Recently, the involvement of SOCS-1 in carcinogenesis has been reported. Here, we report identification of frequent hypermethylation in CpG islands of the functional SOCS-3 promoter that correlates with its transcription silencing in cell lines (lung cancer, breast cancer, and mesothelioma) and primary lung cancer tissue samples. Restoration of SOCS-3 in lung cancer cells where SOCS-3 was methylation-silenced resulted in the down-regulation of active STAT3, induction of apoptosis, and growth suppression. Our results suggest that methylation silencing of SOCS-3 is one of the important mechanisms of constitutive activation of the JAK/STAT pathway in cancer pathogenesis. The data also suggest that SOCS-3 therapy may be useful in the treatment of cancer.

SOCS-3 is a member of a newly discovered protein family that inhibits LIF-activated Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling in a negative auto-regulatory manner. In this study, we have cloned and characterized the promoter region of the human SOCS-3 gene. This region is approximately 1.1 kbp in length and consists of two putative STAT-binding elements, a G-rich element, and a putative TATA box. These elements are highly conserved in both murine and rat SOCS-3 promoters. Functional analysis of this region shows that the whole fragment (approximately 1.1 kbp) has high basal promoter activity and is responsive to growth factors. We also found that the wild type SOCS-3 promoter construct has significantly greater activity in non-small-cell lung cancer cell lines than in normal cells in accordance with STAT3 disregulation in these cells. Cloning of the human SOCS-3 promoter should help uncover mechanisms of regulation of the JAK-STAT pathway in human cancer.

Mitotic checkpoint impairment is present in human lung cancers with chromosomal instability (CIN). Spindle-checkpoint genes have been reported to be mutated in several human cancers, but these mutations are infrequent. Recent reports suggest that the hBUBR1 gene may play an important role in mitotic checkpoint control and in mitotic checkpoint impairment in human cancers. We analyzed the expression of hBUBR1 in lung cancer cell lines using real time quantitative RT-PCR. The expression of BUBR1 was found to be up-regulated in all of these cell lines. In addition, we cloned and characterized the promotor region of hBUBR1 and determined its genomic structure, which includes 23 exons. The open reading frame (ORF) of the hBUBR1 gene comprises exons 1 through 23. There are GC-rich regions located at the flanking region and about 150 bp upstream from exon 1. The promoter region (424 bp upstream from exon 1) showed promoter activity and includes multiple transcription factor consensus binding motifs, including those for Sp1, Nkx-2, CdxA, SRY, MyoD, Ik-2, HNF-3b, Staf, Oct-1, Nkx-2, v-Myb, and AML 1a. Multiple pathways leading to activation of those binding factors may contribute to hBUBR1 gene transcription. Knowledge of the genomic structure and the promoter region of the hBUBR1 gene will facilitate investigation of its role in mitotic checkpoint control and tumor progression in human cancers.

Xenon-enhanced dual-energy computed tomography (xenon-enhanced CT) can provide lung ventilation maps that may be useful for assessing structural and functional abnormalities of the lung. Xenon-enhanced CT has been performed using a multiple-breath-hold technique during xenon washout. We recently developed xenon-enhanced CT using a single-breath-hold technique to assess ventilation. We sought to evaluate whether xenon-enhanced CT using a single-breath-hold technique correlates with pulmonary function testing (PFT) results. Twenty-six patients, including 11 chronic obstructive pulmonary disease (COPD) patients, underwent xenon-enhanced CT and PFT. Three of the COPD patients underwent xenon-enhanced CT before and after bronchodilator treatment. Images from xenon-CT were obtained by dual-source CT during a breath-hold after a single vital-capacity inspiration of a xenon–oxygen gas mixture. Image postprocessing by 3-material decomposition generated conventional CT and xenon-enhanced images. Low-attenuation areas on xenon images matched low-attenuation areas on conventional CT in 21 cases but matched normal-attenuation areas in 5 cases. Volumes of Hounsfield unit (HU) histograms of xenon images correlated moderately and highly with vital capacity (VC) and total lung capacity (TLC), respectively (r = 0.68 and 0.85). Means and modes of histograms weakly correlated with VC (r = 0.39 and 0.38), moderately with forced expiratory volume in 1 second (FEV1) (r = 0.59 and 0.56), weakly with the ratio of FEV1 to FVC (r = 0.46 and 0.42), and moderately with the ratio of FEV1 to its predicted value (r = 0.64 and 0.60). Mode and volume of histograms increased in 2 COPD patients after the improvement of FEV1 with bronchodilators. Inhalation of xenon gas caused no adverse effects. Xenon-enhanced CT using a single-breath-hold technique depicted functional abnormalities not detectable on thin-slice CT. Mode, mean, and volume of HU histograms of xenon images reflected pulmonary function. Xenon images obtained with xenon-enhanced CT using a single-breath-hold technique can qualitatively depict pulmonary ventilation. A larger study comprising only COPD patients should be conducted, as xenon-enhanced CT is expected to be a promising technique for the management of COPD.

Non-small cell lung cancer is associated with approximately 85% mortality due to its high metastatic potential. Therapeutic efforts have failed to produce a significant improvement in prognosis. In this situation, a better understanding of the key factors of metastasis may be useful for designing new molecular targets of therapy. In order to identify these factors, we compared the expression profiles of two subpopulations of an adenocarcinoma cell line with a high metastatic potential, PC9/f9 and PC9/f14, with the parent cell line, PC9, using a cDNA array. The expression of 15 genes was found to be significantly enhanced or reduced in the highly metastatic subpopulations. The expression of matrix metalloproteinase-2 (MMP-2), plasminogen activator inhibitor-1 (PAI-1) and interleukin-1 (IL-1 alpha) were upregulated in the highly metastatic subpopulations, while the expression of carcinoembryonic antigen (CEA), caspase-5, Fas ligand, Prk/FNK, cyclin E, cyclin B1, Ki-67, proliferating cell nuclear antigen (PCNA), Smad4, macrophage proinflammatory human chemokine-3 alpha (MIP-3 alpha)/LARC, Met and CD44 were downregulated. Data from the literature suggest that the altered expression of MMP-2, PAI-1, IL-1 alpha, CEA, caspase-5, Fas ligand, Prk/FNK and Smad4 promotes the highly metastatic phenotype. The differential expression of these genes was confirmed by Northern blot analysis, standard reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR. This analysis in subpopulations of a lung cancer cell line indicated that the highly metastatic potential of lung cancer may be induced not by an alteration in the expression of a single gene, but by the accumulation of alterations in the expression of several genes involved in extracellular matrix (ECM) adhesion disruption, ECM degradation, escape from apoptosis, and resistance to transforming growth factor-beta(1) (TGF-beta(1)). Strategies for inhibiting metastasis of pulmonary adenocarcinoma should be designed accordingly.

Some of the many human cancers that exhibit chromosomal instability also carry mutations in mitotic checkpoint genes and/or reveal reduced expression of some of those genes, such as hMAD2. To facilitate investigation of alterations of hMAD2, we determined its genomic structure and intronic primers designed to amplify the entire coding region. Since general impairment of the mitotic checkpoint is frequently reported in lung cancers, and reduced expression of hMAD2 has been reported in breast cancers as well, we searched for mutations throughout the coding sequence of this gene in the genomic DNA of 30 primary lung tumors, 30 lung-cancer cell lines and 48 primary breast cancers. Our approach, which involved polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing, revealed nucleotide variants in only two of the 108 specimens. One was a cytosine-to-adenine substitution 3 bp upstream of exon 4 that occurred in one lung cancer cell line and one primary breast tumor, a change that did not alter transcriptional sequence. The other was an adenine-to-guanine substitution within exon 4, of the same lung cell line; this change already had been reported as a polymorphism. The results suggested that the hMAD2 gene is not commonly mutated in either lung nor breast cancers. Further studies should focus on other mechanisms that might account for reduced expression of the hMAD2 gene, and/or pursue analyses of other mitotic checkpoint genes for mutations in human cancer. Nevertheless, the genomic structure, the intronic primer sequences, and polymorphisms of the hMAD2 gene presented here will facilitate future studies to determine the full spectrum and frequency of the genetic events that can affect expression of the hMAD2 gene in human tumors.

Mutations in mitotic checkpoint genes have been detected in several human cancers, and these cancers exhibit chromosomal instability. Aneuploid stem cells seem to result from chromosomal instability and have been reported in many lung cancers. To determine whether alteration of mitotic checkpoint regulators is involved in carcinogenesis and tumor progression in primary lung cancer, we screened the genomic DNA sequence of 30 human lung cancer cell lines and 30 primary lung cancer tumors for a mutation in the hBUB1 mitotic checkpoint gene. First, we designed 26 sets of intron-based primers to amplify each of the 25 exons of the hBUB1 gene to examine the entire coding region of the hBUB1 gene. Using these primers, we performed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis as well as direct sequencing in the mutation analysis of the hBUB1 gene. Three different nucleotide substitutions were detected in the coding region of the hBUB1 gene in some of the cancer cell lines and primary tumors as follows. The hBUB1 gene of one adenocarcinoma tumor contained a somatic missense mutation, a cytosine-to-guanine substitution in codon 51 of exon 5 that resulted in a histidine-to-aspartic acid amino acid substitution. The hBUB1 gene of three lung cancer cell lines contained a thymine-to-cytosine substitution in codon 430 of exon 12, which did not result in an amino-acid substitution. We were unable to determine whether the nucleotide substitution in exon 12 was a polymorphism or a silent mutation because matched normal tissue was not available. A polymorphism in codon 93 of exon 4, a guanine-to-thymine substitution, in hBUB1 was found in one lung cancer cell line and one primary lung tumor. This is the first report of a somatic missense mutation of a gene involved in a mitotic checkpoint in primary lung cancer. The presence of a point mutation in the hBUB1 gene is consistent with the hypothesis that alteration of mitotic checkpoint genes is involved in the development of primary lung cancers. Because the frequency of hBUB1 gene mutations was low, future studies should focus on other mechanisms of inactivation of the hBUB1 gene as well as mutation analysis of other mitotic checkpoint genes in lung cancers. © 2000 Wiley-Liss, Inc.

Summary We report a case of spontaneous regression of a pulmonary sarcoma. A 76-year-old-woman was found to have a mass arising from the left pulmonary artery and extending into the left lung. Seven months later, the mass of the left lung lesion had shrunk without treatment, but metastases appeared in the left lung. Sarcoma was diagnosed with histological examination of tissue from the pulmonary metastases. The patient died after metastases appeared in the brain 22 months after presentation.

8569Background: Thymic carcinoma is a rare neoplasm. Minimal information exists regarding second-line chemotherapy efficacy in patients with previously treated advanced thymic carcinoma. Methods: W...

Malignant pleural mesothelioma (MPM) has a poor prognosis, and conventional imaging modalities do not reflect the prognosis of MPM. In this study, the clinical significance of 18F-fluorodeoxyglucose positron emission tomography/computed tomography fusion imaging (18F-FDG PET/CT) was evaluated for the differential diagnosis, staging and prognosis in MPM patients. Ninety patients who underwent 18F-FDG PET/CT scanning due to a clinical diagnosis or suspicion of MPM prior to therapy were reviewed. Of 90 patients, 31 were pathologically diagnosed as MPM. Maximum standardized uptake values (SUVmax) were semi-quantitatively obtained from PET/CT 60 min (early phase) and 120 min (delayed phase) after injection of 18F-FDG, and the clinicopathological correlations with the level of SUVmax obtained from PET/CT were examined. The survival curves of MPM patients were plotted according to the methods of Kaplan-Meier. The prognostic implications of the level of SUVmax were estimated by t-test. PET/CT scan showed intense abnormal FDG uptake (SUVmax>2.0) in the pleural lesions of all 31 MPM patients at delayed phase, while it showed abnormal FDG uptake in 30 (97%) patients at early phase. In all 31 MPM patients, the values of SUVmax at delayed phase were higher than those at the early phase. PET/CT also indicated metastasis in the lymph node in 7 patients (23%) and in the systemic lesions in 8 patients (26%) with MPM. Twenty-three MPM patients with high SUVmax, whose prognosis was apparent, showed significantly poorer prognosis in both early and delayed phase (respectively, p=0.03 and p=0.01, t-test). The results showed that 18F-FDG PET/CT at delayed phase is very useful for the diagnosis of pleural diseases, and SUVmax on PET/CT in the delayed phase is a more reliable prognostic factor than that in the early phase. High uptake of 18F-FDG PET/CT may be a predictive factor of prognosis in MPM patients.

胸水型肺腺癌に対する治療法として胸腔ドレナージ後に各種薬剤の胸腔内投与を行う事の有用性については多く報告されているが, 全身化学療法の有用性はいまだ不明である.そこで今回, 我々は胸水型肺腺癌に対する全身化学療法の有用性の有無を検討した.チューブドレナージとOK-432胸腔内投与のみで, 全身化学療法は行わない群 (26例) と, CDDP+VDSあるいはCDDP+VDS+MMC併用全身化学療法を加えた群 (24例) の50%生存期間は, ともに9ヵ月で, 2年生存率は, ともに4%であった.また, 胸水制御に対する効果は, 両群とも同程度であった.胸水型肺腺癌に対する全身化学療法の生存への寄与は認められないと推測された.

Mutation in the epidermal growth factor receptor (EGFR) is frequently seen in non-small cell lung cancers (NSCLCs), especially in Asian females with adenocarcinoma. The frequency of mutation and the factors associated requires to be elucidated by analyzing a large number of consecutive clinical samples. We summarized the result of the EGFR mutation analysis for 1,176 patients performed at the time of diagnosis or relapse. The PNA-LNA PCR clamp, a highly sensitive detection method for the EGFR mutation, was employed. For fresh cases a portion of samples isolated to establish the diagnosis of lung cancer was used. For cases with a relapsed disease archival tissue were tested. The variables associated with the EGFR mutation after removing the confound factors were investigated by the logistic analysis using the samples collected in our university (n = 308) where detailed information on patients were available. The frequency of the EGFR mutation and its subtypes were investigated using all samples (n = 1,176). The EGFR mutation was significantly associated with adenocarcinoma (p = 0.006) and light-smoking (p < 0.0001), but not gender. The deletions in exon 19 were more frequently associated with male gender while exon 21 deletions were with female gender (p = 0.0011). The overall frequency of the EGFR mutation was 31%. Our result suggests that the female predominance in the EGFR mutation rate is a reflection of a higher frequency of adenocarcinoma in females. The gender difference in the mutation subtypes may provide a clue for the mechanism of the occurrence of the EGFR mutation.

We have previously found that Wnt signaling is activated in mesothelioma cells. To clarify the effect of blocking Wnt signaling in mesothelioma, the expression of dishevelled (Dvl), an intermediator of Wnt signaling, was down-regulated by a reformed type of small interfering RNA (siRNA), stealth RNAi, which can reduce the cytotoxic interferon response unlike conventional siRNA.Mesothelioma cell lines were transfected with stealth RNAi of Dvl, and cell growth and colony formation were examined. The synergistic effect on cell growth of Dvl stealth RNAi and cisplatin in combination was evaluated.Dvl stealth RNAi down-regulated the expression of Dvl-3 in mesothelioma cells and induced cell cycle aberration which caused suppression of cell growth. Colony formation was also suppressed. Dvl stealth RNAi and cisplatin in combination suppressed cell growth synergistically.Our data suggest that inhibition of Wnt signaling leads to significant antitumor effects.

Patients with idiopathic pulmonary fibrosis (IPF) have an increased risk of developing lung cancer. To identify key molecules involved in malignant transformation in IPF, we analyzed the expression profiles of lung and lung tumor tissue from patients with lung cancer and IPF (lung cancer/IPF) using cDNA arrays and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Reduced expression of the Smad4 gene was identified in all eight tumor samples from the lung cancer/IPF patients using real-time RT-PCR. Expression levels of Smad4 were significantly lower in tumors from lung cancer/IPF patients than in those from lung cancer patients without IPF or in lung cancer cell lines (p

Recent progress in the development of inhibitors of protein-protein interactions has opened the door for developing drugs that act by novel and selective mechanisms. Building on that work, we designed a small-molecule inhibitor of the Wnt signaling pathway, which is aberrantly activated across a wide range of human tumors. The compound, named FJ9, disrupts the interaction between the Frizzed-7 Wnt receptor and the PDZ domain of Dishevelled, down-regulating canonical Wnt signaling and suppressing tumor cell growth. The antitumorigenic effects of FJ9 were pronounced, including induction of apoptosis in human cancer cell lines and tumor growth inhibition in a mouse xenograft model. FJ9 is thus among the first non-peptide inhibitors to show therapeutic efficacy through disruption of PDZ protein-protein interactions. [Cancer Res 2007;67(2):573–9]

The patient was a 57-year-old man with a chief complaint of anterior chest pain who was diagnosed with clinical stage IV (c-T2N2M1) non-small-cell lung cancer (adenocarcinoma). Tenderness in the sternoclavicular joint, acne, periodontitis, and palmoplantar pustulosis were evident, and SAPHO syndrome was diagnosed. SAPHO syndrome is a rare disorder that results in synovitis, acne, pustulosis, hyperostosis, and osteomyelitis. Bone scintigraphy showed tracer accumulation in the costal cartilage, sternoclavicular joint, and cervical vertebrae 6-7. Although the bone lesions of SAPHO syndrome were difficult to differentiate from bone metastasis of pulmonary adenocarcinoma, metastatic bone tumors were ruled out by magnetic resonance imaging, computed tomography, and fluorodeoxyglucose positron emission tomography. There have been no previously reported cases of lung cancer with comorbid SAPHO syndrome. We report such a case and discuss the relevant literature, particularly that concerned with the evaluation of bone lesions.

A 55-year-old man was treated with gefitinib for disseminated pleural lesions, 1 year after resection of the left lower lobe for non-small cell lung cancer. After 6 weeks of continuous daily treatment with oral gefitinib, he developed dyspnoea on exertion and a non-productive cough. CXR and CT revealed focal areas of ground-glass opacity (GGO) in the right upper lobe. Despite gefitinib being discontinued, high-resolution CT revealed extension of GGO and restructuring of lung parenchyma, suggesting acute interstitial pneumonia. Transbronchial biopsy revealed acute-phase diffuse alveolar damage. After administration of methylprednisolone pulse therapy (1 g/day intravenously) for three consecutive days, the areas of GGO shrank on high-resolution CT and symptoms resolved. Diffuse alveolar damage caused by gefitinib can be successfully treated in the early phase with high-dose corticosteroids. Patients receiving gefitinib should be carefully examined for symptoms and undergo CT if their condition deteriorates.

Summary Background Angiogenesis plays an important role in tumorigenesis and has attracted interest as a potential target in cancer treatment. Patients and methods We examined the prognostic value of the expression of vascular endothelial growth factor (VEGF) and of the VEGF receptors (VEGFRs) fms-like tyrosine kinase receptor-1 (flt-1) and kinase insert domain-containing receptor (KDR) in non-small-cell lung cancer (NSCLC). Sixty patients with surgical stage I NSCLC who had not undergone induction therapy or adjuvant therapy were selected from among 170 patients with NSCLC who had undergone surgery from January to December 1995. Specimens obtained at surgical resection were subjected to immunohistological staining, and the relationship between postoperative outcome and the expression of VEGF and its receptors was investigated. All patients included in the analysis had been followed up for 5 years or longer or until death. Results Patients with tumors expressing VEGF or KDR tended to have poorer outcomes, and VEGF expression and KDR expression were positively correlated. In contrast, flt-1 expression was not correlated with VEGF expression or outcome. Outcomes were poor in patients with tumors positive for both VEGF and VEGFRs. Multivariate analysis identified expression of both flt-1 and KDR and VEGF and KDR as possible independent prognostic factors. Conclusions Our results suggest that expression of VEGF and VEGFR are associated with a poor prognosis via autocrine and paracrine growth stimulation of cancer cells. Moreover, tumors expressing both flt-1 and KDR may have greater malignant potential and are associated with a poor prognosis.

Background Sarcomas are one of the most refractory diseases among malignant tumors. More effective therapies based on an increased understanding of the molecular biology of sarcomas are needed as current forms of therapy remain inadequate. Recently, it has been reported that Wnt-1/β-catenin signaling inhibits apoptosis in several cancers. In this study, we investigated the efficacy of a monoclonal anti-Wnt-1 antibody in sarcoma cells. Methods We treated cell lines A-204, SJSA-1, and fresh primary cultures of lung metastasis of sarcoma with a monoclonal anti-Wnt-1 antibody. Wnt-1 siRNA treatment was carried out in A-204. We assessed cell death using Crystal Violet staining. Apoptosis induction was estimated by flow cytometry analysis (Annexin V and PI staining). Cell signaling changes were determined by western blotting analysis. Results We detected Wnt-1 expression in all tissue samples and cell lines. Significant apoptosis induction was found in monoclonal anti-Wnt-1 antibody treated cells compared to control monoclonal antibody treated cells (p < 0.02). Similarly, we observed increased apoptosis in Wnt-1 siRNA treated cells. Blockade of Wnt-1 signaling in both experiments was confirmed by analyzing intracellular levels of Dishevelled-3 and of cytosolic β-catenin. Furthermore, the monoclonal anti-Wnt-1 antibody also induced cell death in fresh primary cultures of metastatic sarcoma in which Wnt-1 signaling was active. Conclusion Our results indicate that Wnt-1 blockade by either monoclonal antibody or siRNA induces cell death in sarcoma cells. These data suggest that Wnt-1 may be a novel therapeutic target for the treatment of a subset of sarcoma cells in which Wnt-1/β-catenin signaling is active.

Non small cell lung cancer (NSCLC) is the leading cause of cancer deaths in the United States and worldwide. Unfortunately, standard therapies remain inadequate. An increased understanding of the molecular biology of lung cancer biology is required to develop more effective new therapies. In this report, we show that the Wnt pathway is activated through Dishevelled (Dvl) overexpression in NSCLC. Analysis of freshly resected tumors and lung cancer cell lines demonstrate that Dvl-3, a critical mediator of Wnt signaling, is overexpressed. Specifically, Dvl-3 was overexpressed significantly in 75% of fresh NSCLC microdissected samples compared to control paired matched normal lung samples. To evaluate the biological significance of Wnt signaling and, in particular, Dvl function in lung cancer, we transfected siRNA (designed to inhibit selectively human Dvl-1, -2, and -3), to the NSCLC cell line H1703, which is known to have beta-catenin-mediated Tcf-dependent transcriptional activity. Here, we demonstrate that Dvl-specific siRNA treatment in H1703 decreases significantly Dvl and beta-catenin expression, resulting in reduction of Tcf-dependent transcriptional activity, and, importantly, growth inhibition. Taken together, these data support the novel hypothesis that Dvl overexpression is critical to Wnt signaling activation and cell growth in NSCLC.

Malignant pleural mesothelioma is a relatively uncommon and yet incurable tumor. The pathogenesis of mesothelioma remains poorly understood. This study evaluated the role of Wnt signaling in mesothelioma. Western blot analysis was conducted to confirm the expression of Dishevelled (Dvl) and cytosolic beta-catenin in matched autologous tissue samples (tumor and normal pleura), malignant pleural effusions, and in established mesothelioma cell lines LRK1A, REN and H513. Thirteen of 15 mesotheliomas examined showed consistent overexpression of Dvl and increased cytosolic beta-catenin levels as compared with controls. To evaluate T-cell factor (Tcf)-dependent transcriptional activity of beta-catenin, luciferase assays were conducted. Fresh mesothelioma cells (effusion derived), as well as LRK1A, REN, and H513 cell lines showed a significant fold increase (1.5-2.4-fold, P0.01) in Tcf-dependent transcriptional activity of beta-catenin. To evaluate the biological significance of Dvl function in mesothelioma, a PDZ domain deletion mutant (DeltaPDZ-Dvl) was created and stably transfected into LRK1A, REN, and H513. The effect of DeltaPDZ-Dvl on mesothelioma growth was assayed in vitro (colony formation assay in soft agar) and in vivo (s.c. implantation in athymic mice NCRNU-M). In mesothelioma cells tested, DeltaPDZ-Dvl-mediated inhibition of Dvl decreased cytosolic beta-catenin levels, diminished Tcf-mediated transcription, and suppressed tumorigenesis of LRK1A and REN in vitro and in vivo. DeltaPDZ-Dvl also down-regulated expression of c-myc in REN and COX-2 in H513. Our data suggest that in malignant pleural mesothelioma, Wnt signaling is activated through Dvl overexpression and downstream signaling through beta-catenin. Inhibition of this signaling leads to significant antitumor effects. These results demonstrate Dvl overexpression in human cancer and, specifically, that Wnt signaling plays a role in mesothelioma pathogenesis. These data offer possible new avenues for therapeutic intervention.