Shunsuke Okumura

Prostaglandin I2 promotes recruitment of endothelial progenitor cells and limits vascular remodeling

Objective—Endothelial progenitor cells (EPCs) play an important role in the self-healing of a vascular injury by participating in the reendothelialization that limits vascular remodeling. We evaluated whether prostaglandin I2 plays a role in the regulation of the function of EPCs to limit vascular remodeling. Methods and Results—EPCs (Lin−cKit+Flk-1+ cells) were isolated from the bone marrow (BM) of wild-type (WT) mice or mice lacking the prostaglandin I2 receptor IP (IP−/− mice). Reverse transcription–polymerase chain reaction analysis showed that EPCs among BM cells specifically express IP. The cellular properties of EPCs, adhesion, migration, and proliferation on fibronectin were significantly attenuated in IP-deficient EPCs compared with WT EPCs. In contrast, IP agonists facilitated these functions in WT EPCs, but not in IP-deficient EPCs. The specific deletion of IP in BM cells, which was performed by transplanting BM cells of IP−/− mice to WT mice, accelerated wire injury–mediated neointimal hyperplasia in the femoral artery. Notably, transfused WT EPCs, but not IP-deficient EPCs, were recruited to the injured vessels, participated in reendothelialization, and efficiently rescued the accelerated vascular remodeling. Conclusion—These findings clearly indicate that the prostaglandin I2-IP system is essential for EPCs to accomplish their function and plays a critical role in the regulation of vascular remodeling.

Nicotinamide phosphoribosyltransferase: a potent therapeutic target in non-small cell lung cancer with epidermal growth factor receptor-gene mutation.

Background: Non-small cell lung cancer (NSCLC) often has an epidermal growth factor receptor (EGFR) gene mutation. Growth of EGFR-gene-mutated NSCLC depends predominantly on EGFR signaling and requires a large amount of intracellular ATP to activate EGFR signal transduction. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis, and it regulates intracellular ATP levels in mammalian cells. The effect of NAMPT inhibition on NSCLC has not been completely understood. Methods: We aimed to clarify the hypothesis that NAMPT inhibition suppresses growth of EGFR-gene-mutated NSCLC through reduction of intracellular ATP levels, using NAMPT-siRNA transfection and NAMPT inhibitor FK866. We used four lung adenocarcinoma cell lines, including H358 (Wild type EGFR), LC2 (EGFRL858R), PC9 (EGFRDel E746-A750), and H1975 (EGFRL858R+T790M), and evaluated the effect of FK866 on these cells and its mechanisms, using cell proliferation, Western blot, ATP, and apoptosis assay. Results: We found that (1) H358, LC2, and H1975 cell lines highly expressed NAMPT-mRNA; (2) NAMPT-specific siRNA and FK866 suppressed proliferation of these NSCLCs; (3) FK866 reduced intracellular ATP levels in H1975 cells; (4) FK866 dephosphorylated EGFR signal proteins, including EGFR, Akt, Map kinase kinase 1/2, and extracellular signal-regulated kinase 1/2 (ERK 1/2); (5) FK866 induced apoptosis of H1975 cells; and (6) FK866 suppressed growth of H1975 xenograft tumors and attenuated expression of phospho-ERK 1/2 in the tumors in a tumor-bearing mouse model. Conclusion: These findings indicate that NAMPT is a potent therapeutic target in the treatment of EGFR-gene-mutated NSCLC.

Prostaglandin I2 analog suppresses lung metastasis by recruiting pericytes in tumor angiogenesis.

Prostaglandin I2 (PGI2) agonist has been reported to reduce tumor metastasis by modifying tumor angiogenesis; however, the mechanisms of how PGI2 affects the endothelial cells or pericytes in tumor vessel maturation are still unclear. The purpose of this study was to clarify the effects of PGI2 on tumor metastasis in a mouse lung metastasis model using Lewis lung carcinoma (LLC) cells. The mice were treated continuously with beraprost sodium (BPS), a PGI2 analog, for 3 weeks and then examined for lung metastases. The number and size of lung metastases were decreased significantly by BPS treatment. In addition, scanning electron microscopy and immunohistochemistry revealed that BPS increased the number of tumor‑associated pericytes and improved intratumor hypoxia. Collectively, this study suggests that BPS attenuated vascular functional maturation in metastatic tumors.

Histamine H1 antagonist levocetirizine as a potential cause of lung injury

Histamine H1 antagonists rarely cause drug‐induced lung injury (DLI). A woman in her 60s, who had been taking antihistaminic levocetirizine for 2 months, presented with progressive cough and shortness of breath. A chest radiograph showed patchy infiltrations on both lower lung fields. Chest computed tomography findings were consistent with non‐specific interstitial pneumonia. Serum markers associated with interstitial pneumonias were elevated. Room air arterial blood gas analysis revealed hypoxemia. Restrictive ventilatory impairment was noted with reduced diffusing capacity. Transbronchial lung biopsy specimens demonstrated unclassifiable alveolitis. Steroid pulse therapy was introduced for respiratory distress, but the initial response to treatment was poor. A drug lymphocyte stimulation test was positive for levocetirizine. The interstitial pneumonia improved following withdrawal of levocetirizine. Her illness has not recurred under steroid therapy and the discontinuation of levocetirizine. Antihistaminics may have a potential risk of DLI.

Rikkunshito for Preventing Chemotherapy-Induced Nausea and Vomiting in Lung Cancer Patients: Results from 2 Prospective, Randomized Phase 2 Trials

The herbal medicine rikkunshito has the potential to improve chemotherapy-induced nausea and vomiting (CINV) by stimulating ghrelin secretion. We aimed to evaluate the efficacy and safety of rikkunshito in preventing CINV for patients with lung cancer. Two separate prospective, randomized, phase II parallel design studies were conducted in patients with lung cancer. Fifty-eight and sixty-two patients scheduled to receive highly emetogenic chemotherapy (HEC) and moderately emetogenic chemotherapy (MEC), respectively, were randomized 1:1 to receive either standard antiemetic therapy in accordance with international guidelines (S group) or standard antiemetic therapy plus oral rikkunshito (R group). The primary endpoint was overall complete response (CR)—that is, no emesis and rescue medication in the first 120 h post-chemotherapy. Secondary endpoints included CR in the acute (0–24 h) and delayed (>24–120 h) phases and safety. Fifty-seven patients (S group, 28; R group, 29) receiving HEC and sixty-two patients (S group, 30; R group, 32) receiving MEC with comparable characteristics were evaluated. The CR rates were similar across the S and R groups for the HEC study in the overall (67.9% vs. 62.1%), acute (96.4% vs. 89.6%), and delayed (67.9% vs. 62.1%) phases, respectively, and for the MEC study in the overall (83.3% vs. 84.4%), acute (100% vs. 100%), and delayed (83.3% vs. 84.4%) phases, respectively. No severe adverse events were observed. Although rikkunshito was well tolerated, it did not demonstrate an additional preventative effect against CINV in lung cancer patients receiving HEC or MEC. Clinical Trial Registry Information: This study is registered with the University Hospital Medical Information Network (UMIN) Clinical Trial Registry1, identification numbers UMIN 000014239 and UMIN 000014240.

Abstract 3148: Activation of Src signaling mediates acquired resistance to ALK inhibition in lung cancer

Anaplastic lymphoma kinase (ALK) fusion oncogenes occur in around 3%-5% non-small cell lung cancer (NSCLC) cases. Various ALK inhibitors are in clinical use for the treatment of ALK-NSCLC, including the first generation ALK inhibitor, Crizotinib, and recently the more highly potent Alectinib and Ceritinib. However, most tumors eventually become resistant to ALK specific inhibitors. To address the mechanisms underlying the development of ALK inhibitor resistance, we used iTRAQ quantitative mass spectrometry and phosphor-receptor tyrosine kinase arrays to investigate intracellular signaling alterations in ALK inhibitor resistant NSCLC cell lines. Src signaling was identified as an Alectinib resistance mechanism, and combination treatment with ALK and Src inhibitors was highly effective for inhibiting the growth of ALK inhibitor resistant cells in vitro and in mouse xenograft models. Furthermore, phospho-receptor tyrosine kinase activation and downstream PI3K/AKT signaling was effectively blocked by inhibiting Src in Alectinib resistant cells. Finally, we showed that the combined use of ALK and Src inhibitors inhibited the growth of other ALK-NSCLC cell lines, including those that were Ceritinib or Lorlatinib resistant. Our data suggest that targeting Src signaling may be an effective approach to the treatment of ALK–NSCLC with acquired resistance to ALK inhibitors. Citation Format: Ryohei Yoshida, Takaaki Sasaki, Shunsuke Okumura, Yoshinobu Ohsaki. Activation of Src signaling mediates acquired resistance to ALK inhibition in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3148. doi:10.1158/1538-7445.AM2017-3148

Abstract 2267: Efficacy of pemetrexed-based chemotherapy in patients with NSCLC harboring KRAS mutations

[Background] Biomarkers like epidermal growth factor receptor (EGFR) gene mutations or anaplastic lymphoma kinase (ALK) rearrangement have been discovered in lung cancer, leading to targeted therapies developed in non-small cell lung cancer (NSCLC). Several oncogenes have been reported to be negative biomarkers for response to tyrosine kinase inhibitors (TKIs), for example, NSCLC with KRAS mutations are resistant to EGFR-TKIs. A preclinical study showed that KRAS mutant lung cancer cells were sensitive to pemetrexed, while a retrospective study reported that pemetrexed-based chemotherapy didn9t show a favorable outcomes in patients with KRAS mutant NSCLC. We, therefore, studied efficacy of pemetrexed-based chemotherapy in patients with NSCLC, and compared the efficacy between KRAS and wild-type NSCLC. [Methods] We retrospectively studied the medical records of patients diagnosed with NSCLC between 2013 to 2015. Genomic DNA (gDNA) was extracted from FFPE tissue samples (QIAamp DNA FFPE Tissue Kit, QIAGEN). KRAS mutations were evaluated using Ion Torrent NGS systems (Ion PGM, Thermo Fischer Scientific) or PCR-rSSO. AmpliSeq Colon and Lung Cancer Research Panel v2 was used in the present study. We used Ion Reporter software for next-generation sequencing data analysis. [Results] We analyzed the data of 226 patients with NSCLC and successfully extracted gDNA from 94 tissue samples. Among them, mutation analysis by Ion PGM system was performed in 48 samples and PCR-rSSO in 46 samples. We will report response rate and progression free survival in the patients treated with pemetrexed-based chemotherapy. Citation Format: Shunsuke Okumura, Takaaki Sasaki, Shin-ichi Chiba, Masatoshi Sado, Naoyuki Miyokawa, Hiroshi Funakoshi, Yoshinobu Ohsaki. Efficacy of pemetrexed-based chemotherapy in patients with NSCLC harboring KRAS mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2267.

Abstract 2121: Src mediates acquired resistance to ALK inhibitor in ALK-rearranged non-small cell lung cancers

ALK tyrosine kinase inhibitors (TKIs) play a significant role in the treatment of ALK-rearranged non-small cell lung cancer patients.After the significant initial response to ALK-TKIs, the emergence of acquired resistance ultimately occur for all patients. Understanding the origin of resistance mechanisms within a patient is crucial to guide treatment and developing drugs/combinations to circumvent resistance. There are reported that some of the tyrosine kinases are involving by bypassing ALK signaling, but comprehensive identification of proteins that are significant for acquired resistance in ALK-TKIs is still required. Quantitative proteomic analysis provides a powerful approach in screening for alterations in protein levels. An analysis of the differential proteins expression between original cancer cell and acquired resistance cell would be useful for identification of proteins drug resistance. To investigate the changes of chaperon holding proteins, ALK rearranged-cell line H3122 were used. ALK-TKI Alectinib resistant cell lines were established by exposing increased dose of alectinib to ALK rearranged H3122 cell line (AFR). Luminespib (AUY922, Novartis Pharm.) was used as HSP90 inhibitor and Saracatinib (Selleck Chemicals.) was used as Src inhibitor in this study. We used iTRAQ (isobaric tags for relative and absolute quantitation) technology using nano-LC-MS/MS analysis, to identify alterations in H3122 protein levels of pre/post HSP90 inhibitor treatment. For further analysis, protein expression was measured by western blot. To assess the cancer cell growth inhibition for ALK and/or Src inhibitors, we used colony formation assay. Our results revealed that Crk-Src related proteins were overexpressed in ALK TKI resistance cells and had a significant protein expression changes after HSP90 inhibitor treatment. To address the impact of overcoming resistance by blocking Crk-Src pathway, Drugs/combinations with ALK inhibitor and/or Src inhibitors showed that significant treatment effect in vitro and in vivo study using tumor-bearing mouse model. Conclusion Src is one of the significant signaling pathways in the ALK-rearranged NSCLCs resistant to ALK-TKIs including alectinib, and inhibiting Src signaling could be a potential target for ALK-rearranged NSCLC after ALK-TKI resistance. Citation Format: Ryohei Yoshida, Shunsuke Okumura, Takaaki Sasaki, Yoshinobu Osaki. Src mediates acquired resistance to ALK inhibitor in ALK-rearranged non-small cell lung cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2121.

Abstract 1770: A role of nicotinamide phosphoribosyltransferase in growth of KRAS mutant non-small cell lung cancer

Background: KRAS mutations are frequently found in non-small cell lung cancer (NSCLC). The KRAS mutations could be predictive of resistance to targeted therapy like epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) or a prognostic factor in NSCLC. No successful targeted therapy currently exists to treat patients with KRAS mutant NSCLC, and novel therapeutic strategies are needed to prolong survival in patients with KRAS mutant NSCLC. Nicotinamide phosphoribosyltransferase (nampt) is a rate-limiting enzyme in NAD+ salvage, and we previously reported that nampt is a potent therapeutic target in NSCLC. Based on the analysis result of GEO database, we found that expression of nampt is associated with RAS-MAPK signaling pathway in NSCLC, and we therefore, conducted an in vitro study to evaluate a role of nampt in growth of KRAS mutant NSCLC. Methods: We used a panel of KRAS mutant NSCLC cell lines (A427, A549, H157, H23, H1792, H2009, H358, H441, Calu6, Calu1). The cells were treated with a nampt inhibitor, FK866, and acid phosphatase (APH) assay (Thermo SCIENTIFIC) was used to evaluate cell viability of the KRAS mutant cells. Changes in expression of MAPK signaling pathway and BCL2 family proteins (antibodies from Cell Signaling and Santa Cruz) were evaluated by western blot. Apoptosis of the cells treated with FK866 was detected using an Annexin V FITC apoptosis detection kit (BD Biosciences). FACS analysis was performed using a BD FACSAria II cytometer (BD Bioscience). Autophagic proteolysis was studied by Cyto-ID autophagy detection kit (Cosmo Bio), and the expression of the fluorescent signal was visualized by a FV1000 confocal microscope (OLYMPUS JAPAN). Results: We found that the sensitivity to FK866 was different among the KRAS mutant cell lines, and for instance, A427 cells were sensitive while H157 cells were resistant. FK866 suppressed growth of KRAS mutant NSCLC and decreased expression of p-ERK expression in the cell line sensitive to FK866. We will report alteration of apoptosis and autophagy in the KRAS mutant NSCLC treated with FK866. Conclusion: Nampt could be a potent therapeutic target in KRAS mutant NSCLC. Citation Format: Shunsuke Okumura, Takaaki Sasaki, Yoshinobu Ohsaki. A role of nicotinamide phosphoribosyltransferase in growth of KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1770. doi:10.1158/1538-7445.AM2015-1770

Abstract 4379: Activating the prostaglandin I2-IP signaling suppresses metastasis in lung cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The mechanism by which bone marrow (BM)-derived cells might contribute to tumor metastasis and angiogenesis is controversial. We have reported that bone marrow derived angiogenic progenitor cells (Lin- cKit+ Flk-1+ cells) express prostaglandin I2 (PGI2) specific receptor; IP, and the PGI2-IP system is necessary for vascular remodeling and angiogenesis. Additionally, we have reported that the specific knockdown of IP in bone marrow derived cells (BMDCs) increases tumor metastasis in mouse models. Objectives: The purpose of this study was to test the hypothesis that activating the PGI2-IP signaling suppresses tumor metastasis. Materials & Methods: We employed both a mouse lung metastatic model and a mouse bone marrow transplanted model. Mouse-derived Lewis lung carcinoma (LLC) cells labeled with DsRed were used for a mouse lung metastatic model to distinguish cancer cells from BMDCs. The LLCs were injected into the tail vein of mice (c57BL/6J), which bone marrow cells were transplanted from GFP-labeled mice. Beraprost sodium (BPS; IP receptor agonist) or PBS control was continuously administered for 3 weeks by subcutaneous osmotic pumps. Tumor metastasis to lung was assessed by using hematoxylin-eosin staining. The a-SMA as a pericyte marker and the CD31 as an endothelial cell marker were studied by immunofluorescence to evaluate angiogenesis in metastatic lung tumors. Results: The size and number of tumor metastasis were significantly decreased in BPS group compared with in PBS control group assessed by the mice lung metastasis model. Immunofluorescence analysis revealed that the pericytes derived from bone marrow were scattered within tumors in PBS control group, while those were observed along with tumor micro vessels by supporting the endothelial cells within tumors in BPS group. Conclusions: The present study demonstrated that activating the PGI2-IP signaling suppresses metastasis in lung cancer. These results also suggested that the maturation of tumor angiogenesis by pericytes may decrease tumor metastasis. Finally, we propose that PGI2-IP system would be a novel therapeutic target in lung metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4379. doi:1538-7445.AM2012-4379