An analysis of semaphorin-mediated cellular interactions in the Caenorhabditis elegans epidermis using the IR-LEGO single-cell gene induction system.

One of the major functions of the semaphorin signaling system is the regulation of cell shape. In the nematode Caenorhabditis elegans, membrane-bound semaphorins SMP-1/2 (SMPs) regulate the morphology of epidermal cells via their receptor plexin, PLX-1. In the larval male tail of the SMP-PLX-1 signaling mutants, the border between two epidermal cells, R1.p and R2.p, is displaced anteriorly, resulting in the anterior displacement of the anterior-most ray, ray 1, in the adult male. To elucidate how the intercellular signaling mediated by SMPs regulates the position of the intercellular border, we performed mosaic gene expression analyses by using infrared laser-evoked gene operator (IR-LEGO). We show that PLX-1 expressed in R1.p and SMP-1 expressed in R2.p are required for the proper positioning of ray 1. The result suggests that SMP signaling promotes extension, rather than retraction, of R1.p. This is in contrast to a previous finding that SMPs mediate inhibition of cell extension of vulval precursor cells, another group of epidermal cells of C. elegans, indicating the context dependence of cell shape control via the semaphorin signaling system.

Comprehensive Proteomics Analysis of the Hemolymph Composition of Sugar-Fed Aedes aegypti Female and Male Mosquitoes.

In arthropods, hemolymph carries immune cells and solubilizes and transports nutrients, hormones, and other molecules that are involved in diverse physiological processes including immunity, metabolism, and reproduction. However, despite such physiological importance, little is known about its composition. We applied mass spectrometry-based label-free quantification approaches to study the proteome of hemolymph perfused from sugar-fed female and male Aedes aegypti mosquitoes. A total of 1403 proteins were identified, out of which 447 of them were predicted to be extracellular. In both sexes, almost half of these extracellular proteins were predicted to be involved in defense/immune response, and their relative abundances (based on their intensity-based absolute quantification, iBAQ) were 37.9 and 33.2%, respectively. Interestingly, among them, 102 serine proteases/serine protease-homologues were identified, with almost half of them containing CLIP regulatory domains. Moreover, proteins belonging to families classically described as chemoreceptors, such as odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), were also highly abundant in the hemolymph of both sexes. Our data provide a comprehensive catalogue of A. aegypti hemolymph basal protein content, revealing numerous unexplored targets for future research on mosquito physiology and disease transmission. It also provides a reference for future studies on the effect of blood meal and infection on hemolymph composition.

Hepatitis C virus E1 recruits high-density lipoprotein to support infectivity and evade antibody recognition.

Hepatitis C virus (HCV) is a member of the Flaviviridae family; however, unlike other family members, the HCV virion has an unusually high lipid content. HCV has two envelope glycoproteins, E1 and E2. E2 contributes to receptor binding, cell membrane attachment, and immune evasion. In contrast, the functions of E1 are poorly characterized due, in part, to challenges in producing the protein. This manuscript describes the expression and purification of a soluble E1 ectodomain (eE1) that is recognized by conformational, human monoclonal antibodies. eE1 forms a complex with apolipoproteins AI and AII, cholesterol, and phospholipids by recruiting high-density lipoprotein (HDL) from the extracellular media. We show that HDL binding is a function specific to eE1 and HDL hinders recognition of E1 by a neutralizing monoclonal antibody. Either low-density lipoprotein or HDL increases the production and infectivity of cell culture-produced HCV, but E1 preferentially selects HDL, influencing both viral life cycle and antibody evasion.IMPORTANCEHepatitis C virus (HCV) infection is a significant burden on human health, but vaccine candidates have yet to provide broad protection against this infection. We have developed a method to produce high quantities of soluble E1 or E2, the viral proteins located on the surface of HCV. HCV has an unusually high lipid content due to the recruitment of apolipoproteins. We found that E1 (and not E2) preferentially recruits host high-density lipoprotein (HDL) extracellularly. This recruitment of HDL by E1 prevents binding of E1 by a neutralizing antibody and furthermore prevents antibody-mediated neutralization of the virus. By comparison, low-density lipoprotein does not protect the virus from antibody-mediated neutralization. Our findings provide mechanistic insight into apolipoprotein recruitment, which may be critical for vaccine development.

SARS-CoV-2 infection of human lung epithelial cells induces TMPRSS-mediated acute fibrin deposition.

Severe COVID-associated lung injury is a major confounding factor of hospitalizations and death with no effective treatments. Here, we describe a non-classical fibrin clotting mechanism mediated by SARS-CoV-2 infected primary lung but not other susceptible epithelial cells. This infection-induced fibrin formation is observed in all variants of SARS-CoV-2 infections, and requires thrombin but is independent of tissue factor and other classical plasma coagulation factors. While prothrombin and fibrinogen levels are elevated in acute COVID BALF samples, fibrin clotting occurs only with the presence of viral infected but not uninfected lung epithelial cells. We suggest a viral-induced coagulation mechanism, in which prothrombin is activated by infection-induced transmembrane serine proteases, such as ST14 and TMPRSS11D, on NHBE cells. Our finding reveals the inefficiency of current plasma targeted anticoagulation therapy and suggests the need to develop a viral-induced ARDS animal model for treating respiratory airways with thrombin inhibitors.

LASP1, CERS6, and Actin Form a Ternary Complex That Promotes Cancer Cell Migration.

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.

Long non-coding RNA TILR constitutively represses TP53 and apoptosis in lung cancer.

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.

Enzyme-labeled Antigen Method: Factors Influencing the Deterioration of Antigen-binding Activity of Specific Antibodies during Formalin Fixation and Paraffin Embedding.

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 Inhibition Suppresses Prostate Cancer Cell Growth In Vitro.

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.

Antitumor effect of polyphyllin D on liver metastases of neuroblastoma.

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.

Mosaic gene expression analysis of semaphorin-plexin interactions in Caenorhabditis elegans using the IR-LEGO single-cell gene induction system.

Genetic mosaic analysis is a powerful means of addressing the sites of gene action in multicellular organisms. In conventional genetic analysis, the generation of desired mosaic patterns is difficult to control due to the randomness of generating the genetic mosaic which often renders the analysis laborious and time consuming. The infrared laser-evoked gene operator (IR-LEGO) microscope system facilitates genetic mosaic analysis by enabling gene induction in targeted single cells in a living organism. However, the level of gene induction is not controllable due to the usage of a heat-shock promoter. Here, we applied IR-LEGO to examine the cell-cell interactions mediated by semaphoring-plexin signaling in Caenorhabditis elegans by inducing wild-type semaphorin/plexin in single cells within the population of mutant cells lacking the relevant proteins. We found that the cell contact-dependent termination of the extension of vulval precursor cells is elicited by the forward signaling mediated by the semaphorin receptor, PLX-1, but not by the reverse signaling via the transmembrane semaphorin, SMP-1. By utilizing Cre/loxP recombination coupled with the IR-LEGO system to induce SMP-1 at a physiological level, we found that SMP-1 interacts with PLX-1 only in trans upon contact between vulval precursor cells. In contrast, when overexpressed, SMP-1 exhibits the ability to cis-interact with PLX-1 on a single cell. These results indicate that mosaic analysis with IR-LEGO, especially when combined with an in vivo recombination system, efficiently complements conventional methods.

Cellular concentrations of plasmalogen species containing a polyunsaturated fatty acid significantly increase under hypoxia in human colorectal cancer, Caco2 cells.

Plasmalogen localized in the raft of mammalian cell membranes plays a role in the storage of polyunsaturated fatty acid (PUFA), and exists to a higher extent in malignant cells that survive, and even grow in hypoxic conditions. The biosynthesis of plasmalogen in mammalian cells has been reported to depend on aerobic conditions. Using liquid chromatography-tandem mass spectrometry, we found that the intracellular concentration of plasmalogen species containing a PUFA at the sn-2-position did not change for two days from the start of hypoxic culture in human colorectal cancer-derived Caco2 cells. At the third day of hypoxia, Caco2 cells showed the average increase rate of 2.6 times in ethanolamine plasmalogen and 2.9 times in choline plasmalogen depending on the molecular species compared with those in the second day of hypoxia. In normoxic culture, there was little quantitative change in any species of both ethanolamine and choline plasmalogens for three days. The up-regulations of mRNA of Ca2+-independent phospholipase A2ß and cytoplasmic phospholipase A2γ as well as the down-regulation of lysoplasmalogenase observed in hypoxia were suggested to be responsible for the increase of plasmalogen in Caco2 cells under hypoxia.

Adipsin-Dependent Secretion of Hepatocyte Growth Factor Regulates the Adipocyte-Cancer Stem Cell Interaction.

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.

Short stature and combined immunodeficiency associated with mutations in RGS10.

We report the clinical and molecular phenotype of three siblings from one family, who presented with short stature and immunodeficiency and carried uncharacterized variants in RGS10 (c.489_491del:p.E163del and c.G511T:p.A171S). This gene encodes regulator of G protein signaling 10 (RGS10), a member of a large family of GTPase-activating proteins (GAPs) that targets heterotrimeric G proteins to constrain the activity of G protein-coupled receptors, including receptors for chemoattractants. The affected individuals exhibited systemic abnormalities directly related to the RGS10 mutations, including recurrent infections, hypergammaglobulinemia, profoundly reduced lymphocyte chemotaxis, abnormal lymph node architecture, and short stature due to growth hormone deficiency. Although the GAP activity of each RGS10 variant was intact, each protein exhibited aberrant patterns of PKA-mediated phosphorylation and increased cytosolic and cell membrane localization and activity compared to the wild-type protein. We propose that the RGS10 p.E163del and p.A171S mutations lead to mislocalization of the RGS10 protein in the cytosol, thereby resulting in attenuated chemokine signaling. This study suggests that RGS10 is critical for both immune competence and normal hormonal metabolism in humans and that rare RGS10 variants may contribute to distinct systemic genetic disorders.

CEBPγ facilitates lamellipodia formation and cancer cell migration through CERS6 upregulation.

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.

Increased chemosensitivity via BRCA2-independent DNA damage in DSS1- and PCID2-depleted breast carcinomas.

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.

Combined α-methylacyl-CoA racemase inhibition and docetaxel treatment reduce cell proliferation and decrease expression of heat shock protein 27 in androgen receptor-variant-7-positive prostate cancer cells.

BACKGROUND: Disease progression in castrate-resistant prostate cancer (PCa) is most commonly driven by the reactivation of androgen receptor (AR) signaling and involves AR splice variants including ARV7. MATERIALS AND METHODS: We used the ARV7-positive PCa cell line, 22Rv1, to study the relationship of the PCa marker α-methylacyl-CoA racemase (AMACR), AR, and ARV7 in PCa. RESULTS: Docetaxel addition but not AMACR inhibition decreased the proliferation of 22Rv1 cells. The combination of AMACR inhibition and docetaxel treatment resulted in a maximum reduction of cell proliferation. The Western blotting analysis revealed that both AR and ARV7 expression were significantly decreased with the use of charcoal-stripped serum following AMACR inhibition and docetaxel treatment. AMACR inhibition and docetaxel treatment in the charcoal-stripped serum condition reduced the proliferation of 22Rv1, possibly via the downregulation of the heat shock protein 27. CONCLUSION: Using cell proliferation and Western blot analysis, we demonstrated that AMACR inhibition and docetaxel treatment, under androgen deprivation conditions, significantly reduced the proliferation of ARV7 positive cancer cells and decreased the levels of AR and ARV7 expression, possibly via downregulation of heat shock protein 27.

Conditional Ror1 knockout reveals crucial involvement in lung adenocarcinoma development and identifies novel HIF-1α regulator.

We previously reported that ROR1 is a crucial downstream gene for the TTF-1/NKX2-1 lineage-survival oncogene in lung adenocarcinoma, while others have found altered expression of ROR1 in multiple cancer types. Accumulated evidence therefore indicates ROR1 as an attractive molecular target, though it has yet to be determined whether targeting Ror1 can inhibit tumor development and growth in vivo. To this end, genetically engineered mice carrying homozygously floxed Ror1 alleles and an SP-C promoter-driven human mutant EGFR transgene were generated. Ror1 ablation resulted in marked retardation of tumor development and progression in association with reduced malignant characteristics and significantly better survival. Interestingly, gene set enrichment analysis identified a hypoxia-induced gene set (HALLMARK_HYPOXIA) as most significantly downregulated by Ror1 ablation in vivo, which led to findings showing that ROR1 knockdown diminished HIF-1α expression under normoxia and clearly hampered HIF-1α induction in response to hypoxia in human lung adenocarcinoma cell lines. The present results directly demonstrate the importance of Ror1 for in vivo development and progression of lung adenocarcinoma, and also identify Ror1 as a novel regulator of Hif-1α. Thus, a future study aimed at the development of a novel therapeutic targeting ROR1 for treatment of solid tumors such as seen in lung cancer, which are frequently accompanied with a hypoxic tumor microenvironment, is warranted.

Inhibition of heat shock protein 90 destabilizes receptor tyrosine kinase ROR1 in lung adenocarcinoma.

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, HSP90ß 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.

CERS6 required for cell migration and metastasis in lung cancer.

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.

Upregulation of S100A10 in metastasized breast cancer stem cells.

Metastatic progression remains the major cause of death in human breast cancer. Cancer cells with cancer stem cell (CSC) properties drive initiation and growth of metastases at distant sites. We have previously established the breast cancer patient-derived tumor xenograft (PDX) mouse model in which CSC marker CD44+ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, the expression levels of S100A10 and its family proteins were much higher in the CD44+ cancer cells metastasized to the liver than those at the primary site. Knockdown of S100A10 in breast cancer cells suppressed and overexpression of S100A10 in breast cancer PDX cells enhanced their invasion abilities and 3D organoid formation capacities in vitro. Mechanistically, S100A10 regulated the matrix metalloproteinase activity and the expression levels of stem cell-related genes. Finally, constitutive knockdown of S100A10 significantly reduced their metastatic ability to the liver in vivo. These findings suggest that S100A10 functions as a metastasis promoter of breast CSCs by conferring both invasion ability and CSC properties in breast cancers.