Motoshi Suzuki

CERS6 is associated with metastasis and poor prognosis in non-small cell lung cancer (NSCLC) patients through d18:1/C16:0 ceramide (C16 ceramide)-mediated cell migration, though the detailed mechanism has not been elucidated. In the present study, examinations including co-immunoprecipitation, liquid chromatography, and tandem mass spectrometry analysis were performed to identify a novel binding partner of CERS6. Among the examined candidates, LASP1 was a top-ranked binding partner, with the LIM domain possibly required for direct interaction. In accord with those findings, CERS6 and LASP1 were found to co-localize on lamellipodia in several lung cancer cell lines. Furthermore, silencing of CERS6 and/or LASP1 significantly suppressed cell migration and lamellipodia formation, whereas ectopic addition of C16 ceramide partially rescued those phenotypes. Both LASP1 and CERS6 showed co-immunoprecipitation with actin, with those interactions markedly reduced when the LASP1–CERS6 complex was abolished. Based on these findings, it is proposed that LASP1–CERS6 interaction promotes cancer cell migration.

Non-coding RNAs have an integral regulatory role in numerous functions related to lung cancer development. Here, we report identification of a novel lncRNA, termed TP53-inhibiting lncRNA (TILR), which was found to function as a constitutive negative regulator of p53 expression, including activation of downstream genes such as p21 and MDM2, and induction of apoptosis. A proteomic search for TILR-associated proteins revealed an association with PCBP2, while the mid-portion of TILR was found to be required for both PCBP2 and p53 mRNA binding. In addition, depletion of PCBP2 resulted in phenocopied effects of TILR silencing. TILR was also shown to suppress p53 expression in a post-transcriptional manner, as well as via a positive feedback loop involving p53 and Fanconi anemia pathway genes. Taken together, the present findings clearly demonstrate that TILR constitutively inhibits p53 expression in cooperation with PCBP2, thus maintaining p53 transcriptional activity at a level sufficiently low for avoidance of spurious apoptosis induction.

The enzyme-labeled antigen method is an immunohistochemical technique detecting plasma cells producing specific antibodies in tissue sections. The probe is an antigen labeled with an enzyme or biotin. This immunohistochemical technique is appliable to frozen sections of paraformaldehyde (PFA)-fixed tissues, but it has been difficult to apply it to formalin-fixed, paraffin-embedded (FFPE) sections. In the current study, factors inactivating the antibody reactivity during the process of preparing FFPE sections were investigated. Lymph nodes of rats immunized with horseradish peroxidase (HRP) or a mixture of keyhole limpet hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) or biotinylated proteins in 4% PFA-fixed frozen sections, significantly decreased in unbuffered 10% formalin-fixed frozen sections. The positive cells were further decreased by paraffin embedding following formalin fixation. In paraffin-embedded sections fixed in precipitating fixatives such as ethanol and acetone and those prepared with the AMeX method, the antigen-binding reactivity of antibodies was preserved. Fixation in periodate-lysine-paraformaldehyde and Zamboni solution also kept the antigen-binding reactivity in paraffin to some extent. In conclusion, formalin fixation and paraffin embedding were major causes inactivating antibodies. Precipitating fixatives could retain the antigen-binding reactivity of antibodies in paraffin-embedded sections.

Alanine-serine-cysteine transporter 2 (ASCT2) has been associated with increased levels of metabolism in various malignant tumors. However, its biological significance in the proliferation of prostate cancer (PCa) cells remains under investigation. We used the cBioPortal database to assess the effect of ASCT2 expression on the oncological outcomes of 108 PCa patients. To evaluate the function of ASCT2 in castration-sensitive PCa (CSPC) and castration-resistant PCa (CRPC), LNCaP cells and the ARV7-positive PCa cell line, 22Rv1, were assessed using cell proliferation assays and Western blot analyses. The ASCT2 expression level was associated with biochemical recurrence-free survival after prostatectomy in patients with a Gleason score ≥ 7. In vitro experiments indicated that the growth of LNCaP cells after combination therapy of ASCT2 siRNA and enzalutamide treatment was significantly reduced, compared to that following treatment with enzalutamide alone or ASCT2 siRNA transfection alone (p < 0.01, 0.01, respectively). After ASCT2 inhibition by siRNA transfection, the growth of 22Rv1 cells was significantly suppressed as compared with negative control siRNA via downregulation of ARV7 both in fetal bovine serum and androgen-deprivation conditions (p < 0.01, 0.01, respectively). We demonstrated that ASCT2 inhibition significantly reduced the proliferation rates of both CSPC and CRPC cells in vitro.

PURPOSE: We previously reported that polyphyllin D, a main component of the traditional Chinese medicinal herb Paris polyphylla, exhibited anticancer effects in vitro against human neuroblastoma cells. The aims of this investigation was to examine the presence or absence of in vivo anti-metastasis effects of polyphyllin D were to establish a liver metastasis model of neuroblastoma and to evaluate the anti-metastasis effects of polyphyllin D. METHODS: Subcutaneous and intraperitoneal tumors, and metastasis models were established in immune-deficient BALB/c nude and BALB/c Rag-2/Jak3 double-deficient (BRJ) mice using the human neuroblastoma cell lines IMR-32, LA-N-2, or NB-69. For evaluating polyphyllin D activity, we used a mouse model of liver metastasis with the IMR-32 cells line injected through the tail vein. We analyzed the livers number and area of liver tumors in of the phosphate buffer solution- and polyphyllin D-treated groups. RESULTS: Liver metastasis and intraperitoneal dissemination models were successfully established in immune-deficient BRJ mice using the three human neuroblastoma cell lines. In the liver metastasis, the model of IMR-32 cells, we found that polyphyllin D suppressed both the number and total area of metastatic foci the average number of metastatic foci, average focus areas, and number of cleaved caspase-3-positive cells were significantly lower in the polyphyllin D group (p = 0.016, 0.020, 0.043, respectively). CONCLUSIONS: We developed a mouse models of neuroblastoma metastasis and demonstrated for the first time that polyphyllin D has an antitumor effect on neuroblastoma liver metastases.

Adipose tissue is a component of the tumor microenvironment and is involved in tumor progression. We have previously shown that adipokine adipsin (CFD) functions as an enhancer of tumor proliferation and cancer stem cell (CSC) properties in breast cancers. We established the Cfd-knockout (KO) mice and the mammary adipose tissue-derived stem cells (mADSCs) from them. Cfd-KO in mADSCs significantly reduced their ability to enhance tumorsphere formation of breast cancer patient-derived xenograft (PDX) cells, which was restored by the addition of Cfd in the culture medium. Hepatocyte growth factor (HGF) was expressed and secreted from mADSCs in a Cfd-dependent manner. HGF rescued the reduced ability of Cfd-KO mADSCs to promote tumorsphere formation in vitro and tumor formation in vivo by breast cancer PDX cells. These results suggest that HGF is a downstream effector of Cfd in mADSCs that enhances the CSC properties in breast cancers.

Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations.

Ceramide synthase 6 (CERS6) promotes lung cancer metastasis by stimulating cancer cell migration. To examine the underlying mechanisms, we performed luciferase analysis of the CERS6 promoter region and identified the Y-box as a cis-acting element. As a parallel analysis of database records for 149 non-small-cell lung cancer (NSCLC) cancer patients, we screened for trans-acting factors with an expression level showing a correlation with CERS6 expression. Among the candidates noted, silencing of either CCAAT enhancer-binding protein γ (CEBPγ) or Y-box binding protein 1 (YBX1) reduced the CERS6 expression level. Following knockdown, CEBPγ and YBX1 were found to be independently associated with reductions in ceramide-dependent lamellipodia formation as well as migration activity, while only CEBPγ may have induced CERS6 expression through specific binding to the Y-box. The mRNA expression levels of CERS6, CEBPγ, and YBX1 were positively correlated with adenocarcinoma invasiveness. YBX1 expression was observed in all 20 examined clinical lung cancer specimens, while 6 of those showed a staining pattern similar to that of CERS6. The present findings suggest promotion of lung cancer migration by possible involvement of the transcription factors CEBPγ and YBX1.

We have previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a direct transcriptional target of TTF-1/NKX2-1, a lineage-survival oncogene in lung adenocarcinoma. ROR1 sustains prosurvival signaling from multiple receptor tyrosine kinases including epidermal growth factor receptor, MET, and insulin-like growth factor 1 receptor in part by maintaining the caveolae structure as a scaffold protein of cavin-1 and caveolin-1. In this study, a high throughput screening of the natural product library containing 2560 compounds was undertaken using a cell-based FluoPPI assay detecting ROR1-cavin-1 interaction. As a result, geldanamycin (GA), a known inhibitor of heat shock protein 90 (HSP90), was identified as a potential inhibitor of ROR1. Geldanamycin, as well as two GA derivatives tested in the clinic, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreased ROR1 protein expression. We found that ROR1 physically interacted with HSP90α, but not with other HSP90 paralogs, HSP90β or GRP94. Geldanamycin in turn destabilized and degraded ROR1 protein in a dose- and time-dependent manner through the ubiquitin/proteasome pathway, resulting in a significant suppression of cell proliferation in lung adenocarcinoma cell lines, for which the kinase domain of ROR1, but not its kinase activity or N-glycosylation, was required. Our findings indicate that HSP90 is required to sustain expression of ROR1 crucial for lung adenosarcoma survival, suggesting that inhibition of HSP90 could be a promising therapeutic strategy in ROR1-positive lung adenocarcinoma.

Sphingolipids constitute a class of bio-reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non-small-cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR-101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1-positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.

T315I mutation found in chronic myelogenous leukemia (CML) and Ph + ALL patients is the most serious one among resistance against BCR/ABL kinase inhibitors including imatinib and is only responsive to ponatinib (PNT). However, the novel strategy is required to reduce life-threatening adverse effects of PNT including ischemic cardiovascular disease. We examined the mechanism of PNT-induced cytotoxicity against a T315I(+) Ph + ALL cell line, TccY/Sr. PNT induced apoptosis (increased sub G1 cells, and cleaved caspase3 and PARP), and suppressed protein expression of MCL1, cyclin D2 and c-myc, which were reversed by a proteasome inhibitor, MG132, suggesting enhanced proteasomal degradation by PNT. Among BCL2 family inhibitors, MCL1 inhibitors (maritoclax and AZD5991) robustly induced cell death, showing the MCL1-dependent survival of TccY/Sr cells. Decreased MCL1 and c-myc expression by PNT was also observed in T315I(+) MEGA2/STIR cells. PNT suppressed PI3K activation followed by AKT inhibition and GSK3 dephosphorylation. PI3K/AKT inhibitors mimicked PNT, suggesting that PI3K/AKT signaling is important for survival of TccY/Sr cells. Moreover, GSK3 inhibitor (SB216763) reduced PNT-induced cytotoxicity and degradation of c-myc and MCL1. AZD5991 exhibited the synergistic action with PNT, anti-cancer drugs and venetoclax (BCL2 inhibitor), suggesting the utility of MCL1 inhibitor alone or in combination as a future clinical option for Ph + leukemia patients.

Resveratrol (RSV) has recently attracted keen interest because of its pleiotropic effects. It exerts a wide range of health-promoting effects. In addition to health-promoting effects, RSV possesses anti-carcinogenic activity. However, a non-physiological concentration is needed to achieve an anti-cancer effect, and its in vivo bioavailability is low. Therefore, the clinical application of phytochemicals requires alternative candidates that induce the desired effects at a lower concentration and with increased bioavailability. We previously reported a low IC50 of vaticanol C (VTC), an RSV tetramer, among 12 RSV derivatives (Ito T. et al, 2003). However, the precise mechanism involved remains to be determined. Here, we screened an in-house chemical library bearing RSV building blocks ranging from dimers to octamers for cytotoxic effects in several leukemia and cancer cell lines and their anti-cancer drug-resistant sublines. Among the compounds, VTC exhibited the highest cytotoxicity, which was partially inhibited by a caspase 3 inhibitor, Z-VAD-FMK. VTC decreased the expression of sphingosine kinase 1, sphingosine kinase 2 and glucosylceramide synthase by transcriptional or post-transcriptional mechanisms, and increased cellular ceramides/dihydroceramides and decreased sphingosine 1-phosphate (S1P). VTC-induced sphingolipid rheostat modulation (the ratio of ceramide/S1P) is thought to be involved in cellular apoptosis. Indeed, exogenous S1P addition modulated VTC cytotoxicity significantly. A combination of SPHK1, SPHK2, and GCS chemical inhibitors induced sphingolipid rheostat modulation, cell growth suppression, and cytotoxicity similar to that of VTC. These results suggest the involvement of sphingolipid metabolism in VTC-induced cytotoxicity, and indicate VTC is a promising prototype for translational research.

Hodgkin lymphoma (HL) is one of the most difficult neoplasms in terms of cytopathological research owing to the lack of established cytological murine models. Although HL is believed to be of lymphoid germinal center B-cell origin, HL cells exhibit unique biphenotypic characteristics of B cells and macrophages. B-cell/macrophage biphenotypic cells have also been identified in the spleen of Lyn-deficient mice. Moreover, Lyn-targeting germinal center-associated nuclear protein (GANP)-transgenic mice (Ig-ganpTg mice) spontaneously develop a lymphoid tumor. We aimed to investigate whether the lymphoid tumor developed in Ig-ganpTg mice exhibit biphenotypic characteristics of B cells/macrophages that correspond to human HL. Here, we demonstrated GANP overexpression in human HL cells and found that it may regulate transdifferentiation between B cells and macrophages. We also demonstrated that tumors were comparable with B-cell/macrophage biphenotypic Hodgkinoid lymphomas. The tumor cells expressed macrophage-related F4/80, CD68, and CD204 as well as cytoplasmic B220 and µ-/κ-chains; in addition, these cells exhibited phagocytic activity. These cells also expressed transcripts of CD30; c-fms; and the cytokines monocyte chemoattractant protein (MCP)-1, MCP-5, RANTES, tumor necrosis factor-α and thrombopoietin associated with macrophages as well as granulocyte/macrophage colony-stimulating factor, interleukin (IL)-4, IL-10, IL-12, and IL-13. Ig-ganpTg mice represent a novel cytological model for the study of cytopathological etiology and oncogenesis of HL.