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Activation of the EGFR, KRAS, and ALK oncogenes defines 3 different pathways of molecular pathogenesis in lung adenocarcinoma. However, many tumors lack activation of any pathway (triple-negative lung adenocarcinomas) posing a challenge for prognosis and treatment. Here, we report an extensive genome-wide expression profiling of 226 primary human stage I–II lung adenocarcinomas that elucidates molecular characteristics of tumors that harbor ALK mutations or that lack EGFR, KRAS, and ALK mutations, that is, triple-negative adenocarcinomas. One hundred and seventy-four genes were selected as being upregulated specifically in 79 lung adenocarcinomas without EGFR and KRAS mutations. Unsupervised clustering using a 174-gene signature, including ALK itself, classified these 2 groups of tumors into ALK-positive cases and 2 distinct groups of triple-negative cases (groups A and B). Notably, group A triple-negative cases had a worse prognosis for relapse and death, compared with cases with EGFR, KRAS, or ALK mutations or group B triple-negative cases. In ALK-positive tumors, 30 genes, including ALK and GRIN2A, were commonly overexpressed, whereas in group A triple-negative cases, 9 genes were commonly overexpressed, including a candidate diagnostic/therapeutic target DEPDC1, that were determined to be critical for predicting a worse prognosis. Our findings are important because they provide a molecular basis of ALK-positive lung adenocarcinomas and triple-negative lung adenocarcinomas and further stratify more or less aggressive subgroups of triple-negative lung ADC, possibly helping identify patients who may gain the most benefit from adjuvant chemotherapy after surgical resection. Cancer Res; 72(1); 100–11. ©2011 AACR.

Nutlin-3, an MDM2 inhibitor, activates p53, resulting in several types of cancer cells undergoing apoptosis. Although p53 is mutated or deleted in ∼50% of all cancers, p53 is still functionally active in the other 50%. Consequently, nutlin-3 and similar drugs could be candidates for neoadjuvant therapy in cancers with a functional p53. Cellular senescence is also a phenotype induced by p53 activation and plays a critical role in protecting against tumor development. In this report, we found that nutlin-3a can induce senescence in normal human fibroblasts. Nutlin-3a activated and repressed a large number of p53-dependent genes, including those encoding microRNAs. mir-34a, mir-34b, and mir-34c, which have recently been shown to be downstream effectors of p53-mediated senescence, were up-regulated, and inhibitor of growth 2 (ING2) expression was suppressed by nutlin-3a treatment. Two candidates for a p53-DNA binding consensus sequence were found in the ING2 promoter regulatory region; thus, we performed chromatin immunoprecipitation and electrophoretic mobility shift assays and confirmed p53 binding directly to those sites. In addition, the luciferase activity of a construct containing the ING2 regulatory region was repressed after p53 activation. Antisense knockdown of ING2 induces p53-independent senescence, whereas overexpression of ING2 induces p53-dependent senescence. Taken together, we conclude that nutlin-3a induces senescence through p53 activation in normal human fibroblasts, and p53-mediated mir34a, mir34b, and mir34c up-regulation and ING2 down-regulation may be involved in the senescence pathway. [Cancer Res 2008;68(9):3193–203]

Vascular Endothelial Growth Factor (VEGF) is a key factor for tumor progression through induction of angiogenesis and other actions including immune alteration. Myeloid-derived suppressor cells (MDSC) are a major type of immune-suppressing cell that appear in cancer or inflammation and that have been reported to express the VEGF-receptor and to be activated by VEGF. To study the clinical importance of VEGF and MDSC for cancer, peripheral blood was collected from patients with gastrointestinal, ovarian, breast, thyroid and pulmonary cancers. Peripheral blood mononuclear cells (PBMC) were separated using a Ficoll-density gradient and were used for the detection of MDSC (CD11b+CD14-CD33+) using flow cytometry and for cytokine-production assays. For these assays, PBMC were stimulated with PHA and the production of cytokines including IL-12 (Th1 inducer), IL-10, (anti-inflammatory cytokine) and IL-17, (pro-inflammatory cytokine), were measured over 24 h using ELISAs. Serum concentrations of IL-10 and VEGF were also measured using ELISAs. The serum levels of both VEGF and MDSC were significantly increased in almost all types of cancer tested and significantly correlated with each other. Their levels also significantly correlated with neutrophil/lymphocyte ratios (NLR) (inflammation marker) and CRP levels, and were inversely correlated with the PHA-stimulation index (SI) (cell mediated immune responses marker) and serum concentrations of rapid-turnover protein (RTP) (nutrition marker). VEGF levels also correlated with serum concentrations of IL-10 and VEGF, and production of IL-17, and inversely correlated with production of IL-12. The prognosis of stage IV colorectal cancer with high VEGF was significantly worse than that with low VEGF. In thyroid cancer, the number of MDSC was significantly higher, NLR and CRP levels were higher, and RTP levels were lower in patients with undifferentiated carcinoma than in those with differentiated carcinoma including papillary and follicular carcinomas. Thus VEGF was increased in cancer and correlated with immune suppression driven by MDSC, inflammation and malnutrition. Although cancer immunotherapy is currently in use for a number of cancers, MDSC have been reported to be a major inhibitor of cancer immunotherapy even in cases in which an immune checkpoint inhibitor was used. An anti-VEGF treatment strategy has now been established in combination with chemotherapy for many types of cancer. Among various types of anti-MDSC trials, anti-VEGF treatment seems to be an effective adjuvant therapy of cancer immunotherapy. Citation Format: Masahiko Shibata, Kenji Gonda, Takahiro Nakajima, Koji Kono, Hiroyuki Suzuki, Seiichi Takenoshita. VEGF-A is increased and correlates with MDSC-driven immune suppression, systemic inflammation, nutritional impairment and poor prognosis in patients with 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 3679. doi:10.1158/1538-7445.AM2017-3679

Prognostic tests for patients with early-stage lung cancer may provide needed guidance on postoperative surveillance and therapeutic decisions. We used a novel strategy to develop and validate a prognostic classifier for early-stage lung cancer. Specifically, we focused on 42 genes with roles in lung cancer or cancer prognosis. Expression of these biologically relevant genes and their association with relapse-free survival (RFS) were evaluated using microarray data from 148 patients with stage I lung adenocarcinoma. Seven genes associated with RFS were further examined by quantitative reverse transcription PCR in 291 lung adenocarcinoma tissues from Japan, the United States, and Norway. Only BRCA1, HIF1A, DLC1, and XPO1 were each significantly associated with prognosis in the Japan and US/Norway cohorts. A Cox regression-based classifier was developed using these four genes on the Japan cohort and validated in stage I lung adenocarcinoma from the US/Norway cohort and three publicly available lung adenocarcinoma expression profiling datasets. The results suggest that the classifier is robust across ethnically and geographically diverse populations regardless of the technology used to measure gene expression. We evaluated the combination of the four-gene classifier with miRNA miR-21 (MIR21) expression and found that the combination improved associations with prognosis, which were significant in stratified analyses on stage IA and stage IB patients. Thus, the four coding gene classifier, alone or with miR-21 expression, may provide a clinically useful tool to identify high-risk patients and guide recommendations regarding adjuvant therapy and postoperative surveillance of patients with stage I lung adenocarcinoma. Cancer Res; 73(13); 3821–32. ©2013 AACR.

PAICS (Phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase) is an important bifunctional enzyme in de novo purine biosynthesis. It could be an attractive target for anticancer drug design, since rapidly dividing cancer cells rely on the purine through de novo pathway for synthesis of adenine and guanine, whereas normal cells favor the salvage pathway. Previous reports demonstrated that PAICS expression was up-regulated in ALL, lung cancer, and glioblastoma. In the present study, we first performed the comparison of PAICS mRNA expression between cancer tissue and normal colonic mucosa in 83 colorectal cancer (CRC) patients by realtime RT-PCR. We further investigated PAICS expression by immunohistochemical staining in 258 CRC patients and analyzed the association between PAICS expression and clinicopathological factors and prognosis. To understand the biological significance of PAICS expresssion, knockdown experiments of PAICS expression were performed by a siRNA method using the colon cancer cell line, HCT116. We analyzed the relationship between PAICS mRNA expression and genetic status and prognosis using several databases. PAICS mRNA expression in CRC tissue was significantly enhanced when compared to that in normal tissue. In immunohistochemical examination, PAICS expression was observed in cytoplasm of cancer cells. PAICS expression was detected in 55% cases of colorectal cancer patients. There were significant association between PAICS expression and histology (p = 0.049). There was not significant correlation between PAICS expression and overall survival in CRC patients. Regarding CRC patients with stage III, patients with positive expression of PAICS had significantly good prognosis as compared to those with negative expression (p = 0.015). Knockdown of PAICS expression acquired the ability of cellular invasion using colon cancer cell lines, HCT116 and SW480. In all CRC databases we analyzed, stage III patients with high expression of PAICS had good prognosis as compared to those with low expression of PAICS. We concluded that PAICS expression was up-regulated in approximately 55% CRC, and higher level of PAICS expression might serve as a good prognostic marker in advanced CRC patients. Citation Format: Yusuke Kobayashi, Kensuke Kumamoto, Yoshiko Matsumoto, Motonobu Saito, Tatsuo Shimura, Seiichi Takenoshita. PAICS is the prognostic marker in colorectal cancer patients with stage III. [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 3415. doi:10.1158/1538-7445.AM2015-3415

Although a causal relationship between inflammation and innate immunity of cancer is more widely accepted today, many of the precise cellular mechanisms mediating this relationship remain unclear. Th17 cells, which produce the proinflammatory cytokine IL-17, have been recognized as one of the key factors in regulation of inflammatory bowel disease and rheumatoid arthritis. IL-17 also has recently been reported to play an important role in induction of MDSC (myeloid-derived suppressor cells). This study demonstrated that, in patients with gastric and colorectal cancers, IL-17 production was correlated with MDSC level and with markers for nutritional impairment, immune suppression and chronic inflammation. IL-17 was significantly higher in patients with gastrointestinal cancer than in normal volunteers. In addition, IL-17 levels were significantly correlated with neutrophil counts and the neutrophil/lymphocyte ratio, and significantly inversely correlated with cell mediated immune response indicators (lymphocyte PHA-blastogenesis and IL-12 induction) and patients’ nutritional status (prealbumin levels). These patients were separated into two groups with the production of IL-17 and the overall survival of the patients with higher production of IL-17 was significantly worse than those with lower production of it. These results suggest that, in human gastrointestinal cancers, chronic inflammation involving IL-17 may be an important mechanism for disease progression through enhancement of induction of MDSC resulting immune suppression, or cachexia. The production of IL-17 was also demonstrated as one of the effective prognostic indicators in these patients. Controlling activation of Th17 cells may prove to be a valuable strategy for treatment of patients with gastrointestinal cancer. Further studies are warranted to explore this possibility. Citation Format: Masahiko Shibata, Kenji Gonda, Takahiro Nakajima, Shinji Ohki, Izumi Nakamura, Tatsuo Shimura, Koji Kono, Seiichi Takenoshita. Production of interleukin-17 is increased in patients with gastrointestinal cancer and correlates with immune suppression involving MDSC, nutritional impairment, and poor prognosis. [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 1285. doi:10.1158/1538-7445.AM2015-1285

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasm of the gastrointestinal tract and highly express c-kit. Although surgical removal and molecularly targeted therapy are known to be effective, specific markers other than c-kit would be beneficial for histopathological classification, risk assessment, and treatment decision. To this end, we conducted a DNA-microarry analysis using approximately 900 tumor tissues obtained in Fukushima Medical University Hospital from 2007 to present. The database contained more than 30 kinds of tumors including 10 cases of GIST. We searched ∼32,000 genes on the array that showed expression ratio of >2.0 in GIST samples compared to others, and finally extracted 54 GIST-specific genes. In this study, we identified that cathepsin L mRNA is highly expressed in GISTs. Moreover, immunohistochemical analysis of 43 GIST specimens revealed that 86.0% (n = 37) were cathepsin-L positive, and this positivity was significantly correlated with c-kit positivity. From these results, we conclude that cathepsin L is highly expressed in GISTs. Citation Format: Manabu Iwadate, Kohtaro Miyamoto, Emi Ito, Jun-ichi Imai, Naoki Sawada, Izumi Nakamura, Shinji Ohki, Shinya Watanabe, Satoshi Waguri, Seiichi Takenoshita. GIST-specific markers identified by comprehensive gene expression profiles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 20. doi:10.1158/1538-7445.AM2013-20

ING2 (inhibitor of growth family, member 2) plays pivotal roles in the regulation of cellular senescence, apoptosis, DNA damage repair, gene transcription and chromatin modification. Our previous in vitro studies on cellular senescence suggested that ING2 functionally interplays with the p53 tumor suppressor protein in two different manners: endogenous ING2 inhibits senescence and the transcriptional repression of ING2 by p53 abrogates this inhibition; and overexpressed ING2 enhances p53 acetylation and stability to induce senescence. ING2, as a subunit of the mSin3A-HDAC1 (histone deacetylase 1) complex, specifically binds to tri-methylated lysine 4 of histone H3 (H3K4me3) via its plant homeodomain (PHD) finger and regulates gene expression through chromatin modifications in response to DNA damage. This study investigates the in vivo developmental and physiological functions of ING2. Abundant expression of ING2 in mouse and human testes and its decreased expression associated with male infertility and defective spermatogenesis in humans suggested an essential role of ING2 in spermatogenesis, which is a process tightly regulated by chromatin modifications. Consistently, male mice deficient for Ing2 were infertile. Their testes had degeneration of seminiferous tubules, which became more severe with age, and showed enhanced p53-dependent and -independent apoptosis. Spermatogenesis arrest at meiotic phase in Ing2−/− testes was due to impaired HDAC1 accumulation and deregulated chromatin acetylation. This study establishes ING2 as a novel mammalian regulator of spermatogenesis, suggests that an HDAC1/ING2/H3K4me3-regulated, stage-specific coordination of chromatin modifications is essential to normal spermatogenesis, and provides a model system to study idiopathic and iatrogenic infertility in men, including those who had cancer chemotherapy and radiotherapy. Spontaneous tumor incidence and spectrum were similar in wild-type and Ing2-deficient mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4890.