C1 Expression Research Articles

KIR- Ligand Mismatching Is Associated with Inferior Survival in Patients with Acute Leukemia Following T-Cell Replete Peripheral-Blood Stem Cell Haplo-Identical Transplantation with Post-Transplant Cyclophosphamide. a Study from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation (EBMT)

Background: Haplo-identical stem cell transplantation is increasingly been used in recent years in the treatment of acute leukemia. In particular, the use of T-cell replete grafts with post-transplant cyclophosphamide (PTCy) for prevention of graft-vs-host disease (GVHD) is becoming a valid treatment option with encouraging outcome. The initial studies used bone-marrow as the stem-cell source, but more recently, peripheral blood stem cells (PBSC) are also used. There is more limited data on the prognostic factors following haplo-identical transplantation with PBSC. Natural killer (NK) allo-reactivity as predicted by missing killer-cell immunoglobulin-like receptor (KIR) ligands in the recipients that are present in the donors has been shown to be an important factor in the outcome of T-cell depleted haplo-identical transplants. NK alloreactivity is associated with reduced relapse risk and improved survival in patients with AML, but not in patients with adult ALL. In addition, patients with donor NK alloreactivity had better engraftment and lower risk of GVHD. The role of NK cell allo-reactivity in the setting of T-cell replete haplo-identical transplant with PBSC and PTCy is unknown.Patients and Methods: The study included 238 patients, 139 male, 99 female, median age 45 years (range, 18-78) with AML (n=176) or ALL (n=62) who had T-replete haplo-identical transplant with PTCy between the years 2010-2015. All patients were given PBSC as their stem cell source. Patients were in CR1 (38%), CR2 (24%) or active disease (38%). Conditioning was myeloablative (47%) or reduced-intensity (53%). GVHD prevention included cyclosporine or tacrolimus with mycophenolate, in addition to PTCy, in 82%. KIR-ligand mismatching was predicted according to patient and donor HLA typing and correlated with transplantation outcomes. In all, 89 patients (37%) had a KIR mismatch in the GVHD direction, based on missing expression of C1, C2 or Bw4 ligands in the recipients that were expressed in their donors. There was no significant difference in patient characteristics between the group with and without KIR mismatch.Results: The median follow-up was 14 months (range, 1-50). Ninety-four percent of patients engrafted. The 2-year relapse and non-relapse mortality (NRM) rates in the entire group were 36% (95%CI, 29-42) and 22% (17-27), respectively. The leukemia-free (LFS) and overall survival (OS) rates were 43% (36-50) and 49% (42-56), respectively. Acute GVHD grade II-IV and chronic GVHD occurred in 34% (29-40) and 32% (26-39), respectively. The 2-year OS was 35% (22-47) in patients with KIR mismatch and 51% (42-60) in patients with no KIR-ligand mismatching (P=0.02). 2-year LFS rates were 27% (16-38) and 47% (38-56), respectively (P=0.003). Multivariate analysis identified AML (compared to ALL) (HR 0.51, P=0.008), active disease at transplantation (HR 5.03, P<0.001) and KIR mismatching (HR 1.60, P=0.03) as factors associated with 2-year OS. A center effect was also identified (P=0.05). The same factors predicted for LFS. KIR mismatching was associated with a higher incidence of relapse, 47% (34-58) and 32% (24-41), respectively (P=0.05). Multivariate analysis identified active disease at transplantation (HR 6.38, P<0.001), KIR mismatching (HR 1.97, P=0.03), female donors (HR 0.58, P=0.03) and reduced-intensity conditioning (HR 1.71, P=0.05) as factors associated with 2-year relapse. KIR mismatching was not associated with NRM, acute or chronic GVHD.Conclusions: Haplo-identical transplant with PTCy and PBSC graft is associated with favorable outcome in patients with acute leukemia. KIR ligand mismatching may be associated with increased relapse rate and worse survival in these patients. Donors with KIR mismatching should be avoided when possible in the PBSC haplo-identical setting. [Display omitted] DisclosuresCiceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Mohty:Sanofi: Honoraria, Speakers Bureau.

The Effect of Green Tea on the Expression of NPC1L1, ABCG5, and ABCG8 in the Intestine of High Fat Diets-induced Rats

BACKGROUND: Signaling pathways contributing to cholesterol efflux and inhibitory inflammation in atherosclerosis that has not been explored is the liver X receptor (LXR). Catechin as LXR agonist influences the expression of Niemann-pick C1 like 1 (NPC1L1) protein transporter that triggers the inhibition of cholesterol absorption. This study aimed to examine the effect of Catechins on the expression of intestinal transporters: NPC1L1, ATP-binding cassete-proteins G5 (ABCG5) and G8 (ABCG8).METHODS: Twenty-five experimental animals were divided into five treatment groups, with 5 rats in each group. The groups were normal diet rats (group 1), high fat diets-induced rats (group 2), high fat diets-induced rats treated with 30 mg/kg body weight (BW) of Catechins (group 3), high fat diets-induced rats treated with 60 mg/kg BW of Catechins (group 4), and high fat diets-induced rats treated with 120 mg/kg BW of Catechins (group 5). After one-month, all rats were sacrificed, blood and intestine were collected. Lipid profile were determined enzymatically, mRNA levels were determined by reversetranscriptase polymerase chain reaction (RT-PCR), while the expression of protein transporter were determined by immunohistochemistry.RESULTS: Catechins treatment decreased the expression of NPC1L1, but increased the expression of ABCG5 and ABG8.CONCLUSION: Catechins can be developed as a candidate for NPC1L1 inhibitor to mediate the inhibiting absorption of intestinal cholesterol, therefore increasing the inhibitory effect of atherogenesis.KEYWORDS: ABCG5, ABCG8, aterogenesis, Catechins, green tea, NPC1L1

USP28 promotes aerobic glycolysis of colorectal cancer by increasing stability of FOXC1.

Aerobic glycolysis is essential for cancer cell metabolism and growth. Deubiquitinase, USP28 (ubiquitin specific peptidase 28), could maintain stability of proteins involved in tumor progression. This study was performed to investigate the role of USP28 in aerobic glycolysis of colorectal cancer. Our data showed that USP28 mRNA and protein expressions were enhanced in colorectal cancer tissues and cells. Functional assays demonstrated that overexpression of USP28 promoted cell proliferation and aerobic glycolysis of colorectal cancer, while USP28 inhibition could reverse these effects. Protein expression of Forkhead Box C1 (FOXC1) was increased by USP28 over-expression, whereas knockdown of USP28 aggravated cycloheximide (CHX; protein synthesis inhibitor) stimulated decrease of FOXC1. Moreover, proteasome inhibitor, MG132, could rescue USP28 silence-induced degradation of FOXC1. Overexpression of FOXC1 counteracted the suppressive effects of USP28 interference on colorectal cancer cell viability and aerobic glycolysis. In conclusion, USP28 enhanced cell viability and aerobic glycolysis of colorectal cancer by stabilizing FOXC1, suggesting that USP28-FOXC1 might be a novel therapeutic avenue for colorectal cancer.

Knockdown of HCG18 Inhibits Cell Viability, Migration and Invasion in Pediatric Osteosarcoma by Targeting miR-188-5p/FOXC1 Axis.

Understanding the underlying mechanisms of pediatric osteosarcoma (OS) migration and invasion is important for prognosis and treatment. We tried to measure the expression of long non-coding RNA HLA complex group 18 (HCG18) in OS and reveal its function in the malignant behaviors of OS cells. This study detected the expression of HCG18, miR-188-5p and forkhead box C1 (FOXC1) in OS tissues and cell lines by quantitative real-time PCR (qRT-PCR). The relevance between miR-188-5p and HCG18 or FOXC1 was affirmed by dual-luciferase reporter (DLR) assay. Cell viability was analyzed by MTT assay. Transwell assay was utilized to test cell invasion and migration. FOXC1 protein expression was detected by western blot. HCG18 expression was elevated in OS tissues, and enhanced HCG18 expression was related to metastasis. HCG18 silencing repressed the viability, migration and invasion of OS cells. Moreover, HCG18 interacted with miR-188-5p. MiR-188-5p up-regulation repressed cell viability, invasion and migration in OS cells. FOXC1, a known target of miR-188-5p, was negatively modulated by miR-188-5p. Furthermore, miR-188-5p inhibition or FOXC1 over-expression partially abolished the reduced of cell viability, invasion and migration mediated by HCG18 silencing in OS cell lines. This study revealed that HCG18 knockdown repressed the viability, invasion and migration of OS cells by targeting miR-188-5p and regulating FOXC1 expression. Thus, HCG18/ miR-188-5p/FOX may be a hopeful target for OS therapy.

Overexpression of Apolipoprotein C1 (APOC1) in Clear Cell Renal Cell Carcinoma and Its Prognostic Significance

BackgroundThe aims of this study included 3 aspects: 1) assessing the expression of Apolipoprotein C1 (APOC1) in clear cell renal cell carcinoma (ccRCC) and normal groups; 2) evaluating the prognostic significance of APOC1 expression in the overall survival (OS) of ccRCC patients; and 3) exploring APOC1-related signaling pathways.Material/MethodsThe APOC1 expression value and clinical data of ccRCC patients were obtained from the cBioPortal database. We then evaluated the association of APOC1 expression with clinical characteristics of ccRCC patients. We also assessed the correlation between APOC1 expression and clinical outcome using Kaplan-Meier method. Our work then verified the independent prognostic factors of ccRCC by Cox regression analysis. Finally, the potential role of genes co-expressed with APOC1 was revealed via functional enrichment analysis.ResultsBioinformatic data revealed that APOC1 was expressed at higher levels in ccRCC tissue than in the normal group (all P<0.05). The high expression of APOC1 was associated with unfavorable prognosis of female patients (P<0.01), but not of male patients. APOC1 high expression also shortened the survival time of ccRCC patients age ≥60 years old (P<0.05). Cox regression analysis further indicated that APOC1 expression was an independent prognostic factor for OS of ccRCC patients. Additionally, we found that APOC1 expression was significantly associated with sex, grade, clinical stage, and T stage. Finally, enrichment analysis suggested that APOC1-associated pathways were involved in tumor growth and metastasis.ConclusionsThe current study indicated that APOC1 was highly expressed in ccRCC and was significantly associated with key clinical features. APOC1 appears to be an independent prognostic factor in patients with ccRCC. Importantly, APOC1 might be a potential therapeutic target for ccRCC via regulating pathways involved in cell growth and metastasis.

Dectin-1-mediated suppression of RANKL-induced osteoclastogenesis by glucan from baker's yeast.

Immunoreceptors expressed on osteoclast precursor cells modify osteoclast differentiation and bone resorption activity. Dectin-1 is a lectin receptor of β-glucan and is specifically expressed in osteoclast precursor cells. In this study, we evaluated the bioactivity of β-glucan on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and observed that glucan from baker's yeast inhibited this process in mouse bone marrow cells and dectin-1-overexpressing RAW264.7 (d-RAW) cells. In conjunction, RANKL-induced nuclear factor of activated T cell c1 expression was suppressed, subsequently downregulating TRAP and Oc-stamp. Additionally, nuclear factor-kappa B activation and the expression of c-fos and Blimp1 were reduced in d-RAW cells. Furthermore, glucan from baker's yeast induced the degradation of Syk protein, essential factor for osteoclastogenesis. These results suggest that glucan from baker's yeast suppresses RANKL-induced osteoclastogenesis and can be applied as a new treatment strategy for bone-related diseases.

MiR-138-5p knockdown promotes osteogenic differentiation through FOXC1 up-regulation in human bone mesenchymal stem cells.

This study examined the hypothesis that the microRNA miR-138-5p reduces the osteodifferentiation of human bone mesenchymal stem cells (hBMSCs) by downregulating the expression of forkhead box C1 (FOXC1). For this, hBMSCs were separated from bone marrow and osteogenic induction medium was added to stimulate osteogenic differentiation. Flow cytometric analysis was applied to evaluate the expression of cell-surface antigens associated with hBMSCs, including CD29, CD44, CD90, CD45, and CD34. qRT-PCR assays and Western blot assays were used to measure the mRNA and protein expression of miR-138-5p, osteocalcin, runt-related transcription factor 2, bone sialoprotein, alkaline phosphatase (ALP), and FOXC1. ALP staining assays and Alizarin Red staining (ARS) assays were used to confirm osteogenic differentiation. We used a luciferase assay to test the interaction between miR-138-5p and FOXC1. We demonstrated that miR-138-5p is downregulated in osteogenic differentiated hBMSCs. Further, overexpression of miR-138-5p reduced the expression of markers for osteodifferentiation, ALP activity, and ARS activity. Furthermore, we showed that FOXC1 is a downstream target gene of miR-138-5p, and that knockdown of miR-138-5p improves the osteogenesis differentiation of hBMSCs by upregulating FOXC1. The results from this study indicate miR-138-5p as a new target for osteogenic differentiation of hBMSCs and the treatment of bone defects.

Oral cancer cell‑derived exosomes modulate natural killer cell activity by regulating the receptors on these cells.

Oral cancer (OC) is the most common type of head and neck malignant tumor. Tumor-derived exosomes induce a complex extracellular environment that affects tumor immunity. In the present study, exosomes were isolated from OC cell lines (WSU-HN4 and SCC-9) by ultrafiltration and the protein content of these oral cancer-derived exosomes (OCEXs) was analyzed by mass spectrometry, which revealed the enrichment of transforming growth factor (TGF)-β1. Natural killer (NK) cells were examined by flow cytometry following co-culture with OCEXs. The expression of killer cell lectin like receptor K1 (KLRK1; also known as NKG2D, as used herein) and natural cytotoxicity triggering receptor 3 (NCR3; also known as NKp30, as used herein) in NK cells was found to be significantly upregulated following co-culture with the OCEXs for 1 day, whereas this expression decreased at 7 days. Killer cell lectin like receptor C1 (KLRC1; also known as NKG2A; as used herein) expression exhibited an opposite trend at 1 day. In addition, NK cell cytotoxicity against the OC cells was enhanced at 1 day, but was attenuated at 7 days. TGF-β1 inhibited the function of NK cells at 7 days, whereas it had no obvious effects at 1 and 3 days. On the whole, the findings of the present study reveal changes in NK cell function and provide new insight into NK cell dysfunction.

Abstract 5306: Clinicopathological significance of KIFC1 in human colorectal cancer

Abstract Colorectal cancer (CRC) ranks third in terms of incidence and second in terms of mortality, more than 1.8 million new colorectal cancer cases and 881,000 deaths are reported in Global Cancer Statistics 2018. Therefore, there is an urgent need for a new molecular target in considering treatment strategy. In recent years, cancer has been recognized as a stem cell disease. Spheroid colony formation has been demonstrated as an effective method to characterize cancer stem cells (CSCs). Previously, we analyzed the gene expression profile of spheroid colonies from the gastric cancer cell lines by microarray analysis and found that expression of Kinesin Family Member C1 (KIFC1) was upregulated in gastric spheroid colonies. The kinesin superfamily is a group of proteins that share a motor domain and participate in several biological functions. KIFC1 (or HSET) has been reported to play a vital role in centrosome clustering in cancer cells and is related to pathogenesis in several kinds of cancers. However, the expression of KIFC1 in CRC has not been investigated. The aim of this study is to investigate the expression and the biological function of KIFC1 in CRC. We analyzed the expression and distribution of KIFC1 in 129 CRC cases by immunohistochemistry. In contrast to weak or no staining of KIFC1 in non-neoplastic mucosa, CRC tissue showed stronger, more extensive KIFC1 staining. In total, 67 (52%) of 129 CRC cases were positive for KIFC1. KIFC1-positive CRC cases were significantly associated with CD44-positive and aldehyde dehydrogenase 1 (ALDH1)-positive CRC cases. In the CRC cell proliferation analysis, KIFC1 siRNA-transfected cells showed significantly reduced cell growth ability relative to negative control siRNA-transfected cells. The expression level of phosphorylated Akt and Erk were lower in KIFC1 siRNA-transfected cells compared to negative control siRNA-transfected cells. In the sphere formation analysis, the number and the size of spheres from CRC cells were significantly reduced in KIFC1 siRNA transfected cells than in negative control siRNA-transfected cells. These results indicate that KIFC1 plays an important role in CRC pathogenesis and might be involved in cancer stemness. Although further in-depth studies to clarify the biological significance of KIFC1 in colorectal CSCs are needed, KIFC1 may be a promising therapeutic target for the treatment of CRC. Citation Format: Shintaro Akabane, Naohide Oue, Yuto Fujiki, Naohiro Uraoka, Naoya Sakamoto, Kazuhiro Sentani, Hiroyuki Egi, Hideki Ohdan, Wataru Yasui. Clinicopathological significance of KIFC1 in human colorectal cancer [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 5306.

Abstract 5425: Novel molecular subtypes identified in prostate cancer: Results from the SPARTAN study

Abstract Background: Results from SPARTAN, a phase III placebo (PBO)-controlled study in patients (pts) with nonmetastatic castration-resistant prostate cancer (nmCRPC), show that apalutamide (APA) plus ongoing androgen deprivation therapy (ADT) significantly improves metastasis-free survival (MFS) compared with PBO + ADT. This analysis investigated the effects of APA in biologically distinct molecular subclasses of prostate cancer defined by gene expression profiles. Methods: Gene expression profiles (DECIPHER® prostate test, San Diego, CA) were generated from 233 archival primary prostate tumors; data were summarized based on 160 predefined gene signatures indicative of clinical prognosis and prostate cancer-related biology. Unsupervised consensus clustering identified sets of co-regulated expression signatures, and associations between signature expression, treatment, and signature-treatment interaction with MFS were evaluated using Kaplan-Meier analysis and Cox proportional hazards models. Results: Four co-regulated expression signature classes, each with distinct biological pathway functions, were identified. Class C1 included prognosis-related (risk) signatures; C2 included steroid homeostasis-related signatures; C3 included hormonal therapy nonresponsive basal and neuroendocrine-like signatures; C4 included immune and stromal signatures. Increased C1 expression was associated with shorter MFS in the PBO group (HR [95% CI], 2.18 [1.11-4.28], p = 0.02), while it was associated with longer MFS in the APA group (interaction HR [95% CI], APA vs PBO, 0.36 [0.14-0.95], p = 0.04). Similarly, increased C2 expression was associated with shorter MFS in the PBO group (HR [95% CI], 1.42 [1.02-1.98], p = 0.04), while it was associated with longer MFS in the APA group (interaction HR [95% CI], APA vs PBO, 0.57 [0.35-0.93], p = 0.02). Although there is no significant interaction effect between signature and treatment in C3, pts with low expression of C3 (adeno-like) showed longer MFS on APA vs PBO (HR [95% CI], 0.23 [0.13-0.40], p &amp;lt; 0.0001) compared with high-C3-expressing neuroendocrine-like tumors. Increased C4 expression was associated with decreased risk of metastasis in the APA group (HR [95% CI], 0.55 [0.35-0.86], p = 0.008) compared with PBO (interaction HR [95% CI], APA vs PBO, 0.53 [0.28-0.98], p = 0.04). Conclusion: Expression signatures were clustered into 4 correlated unique biological subclasses. Clinical benefit of APA + ADT was observed in pts with expression profiles with high risk (C1), C2, C3, or C4 compared with PBO + ADT. Citation Format: Clemente Aguilar, Michael Gormley, Shibu Thomas, Paul N. Mainwaring, David Olmos, Fred Saad, Simon Chowdhury, Elai Davicioni, Yang Liu, Deborah S. Ricci, Angela Lopez-Gitlitz, Margaret K. Yu, Matthew R. Smith, Eric J. Small, Felix Feng. Novel molecular subtypes identified in prostate cancer: Results from the SPARTAN study [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 5425.

TNNC1 Reduced Gemcitabine Sensitivity of Nonsmall-Cell Lung Cancer by Increasing Autophagy.

BackgroundAs we know, chemotherapy resistance is a critical factor leading to recurrence and metastasis of nonsmall-cell lung cancer (NSCLC). To clarify the key target and potential mechanism of resistance to gemcitabine (GEM) in NSCLC, we selected Gene Expression Omnibus Data Set and statistically analyzed a parent cell group and a GEM-resistant cell group. Results showed that the expression of troponin C1, slow skeletal and cardiac type (TNNC1) in GEM-resistant cells was higher than in parent cells, which implies that TNNC1 was associated with GEM resistance in lung cancer cells.Material/MethodsTNNC1 expression level was detected by reverse transcription-quantitative polymerase chain reaction or western blot in GEM-resistant patient serum and cell lines. It could reduce or increase autophagy response and GEM resistance accordingly by inhibition of the short interfering ribonucleic acid or by forced overexpression of TNNC1 viruses in A549 cell line and GEM-resistant cell line (A549/GemR) respectively. Blocking autophagy with 3-methyladenine increased the sensitivity of chemotherapy confirmed by flow cytometry and microtubule-associated protein 1A/1B – light chain 3 punctate assay. What’s more, in a loss-of-function model, silencing of forkhead box 03 (FOXO3) in A549/GemR cells could rescue the autophagy weakened by TNNC1.ResultsTNNC1 promoted GEM chemoresistance of NSCLC by activating cytoprotective autophagy, regulated negatively by FOXO3. This research may provide a completely new strategy for NSCLC treatment.ConclusionsTargeting the TNNC1/FOXO3 signaling pathway in NSCLC may be a novel strategy to combat GEM resistance.

Forkhead Box C1 (FOXC1) Expression in Stromal Cells within the Microenvironment of T and NK Cell Lymphomas: Association with Tumor Dormancy and Activation.

PurposeForkhead box C1 (FOXC1) is critical for maintaining bone marrow microenvironments during hematopoiesis, but its role in hematological malignancies remains obscure. Here, we investigated whether FOXC1 regulates tumor dormancy and activation in the microenvironments of T and natural killer (NK) cell lymphomas.Materials and MethodsOne hundred and twenty cases of T and NK cell lymphomas were included; the immunohistochemical expression of FOXC1 was investigated in stromal cells, and numbers of FOXC1+ stromal cells were counted. Furthermore, the expression of phosphorylated p38 (p-p38) and phosphorylated ERK1/2 (p-ERK1/2) in tumor cells was investigated using immunohistochemistry.ResultsFOXC1 was variably expressed in C-X-C motif chemokine 12–associated reticular stromal cells, histiocytes, (myo)fibroblasts, and endothelial cells. The phenotypes of cases were categorized as dormant (high p-p38/low p-ERK1/2; n=30, 25.0%), active (high p-ERK1/2/low p-p38; n=25, 20.8%), or intermediate (others; n=65, 54.2%). Lower FOXC1+ stromal cell infiltration was associated with the dormant phenotype, the precursor T lymphoblastic leukemia/lymphoma subtype, and inferior overall survival rates, whereas higher FOXC1+ stromal cell infiltration was associated with the active phenotype and favorable patient prognosis (p < 0.05 for all).ConclusionThese results suggested that FOXC1+ stromal cells within the microenvironments of T and NK cell lymphomas might be related to tumor phenotypes.

SOX2 is a positive regulator of osteoclast differentiation

Elucidating the mechanism underlying osteoclast differentiation is important to improve our understanding of the pathophysiologies related to skeletal diseases and osteolytic metastasis in cancer. Sex-determining region Y-box containing gene 2 (SOX2), a stemness marker, is known to affect osteoblast differentiation and cancer metastasis. However, its role in osteoclastogenesis has not been investigated to date. Here, we report that SOX2 protein and mRNA expression was upregulated during osteoclast differentiation. The overexpression or knockdown of SOX2 in osteoclast precursor cells enhanced or suppressed, respectively, receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation and migration, and nuclear factor of activated T-cell c1 (NFATc1) and factor-associated suicide ligand (FASL) expression. In addition, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) activation were regulated by SOX2 expression; both EGFR and ERK inhibitors abrogated the SOX2 overexpression-induced increase in osteoclast differentiation and NFATc1 expression under RANKL stimulation. Overall, these results suggest SOX2 as a positive regulatory factor during osteoclast differentiation partly through the EGFR and ERK signaling pathways, highlighting a new potential target for restoring abnormal osteoclast activation.

Multi-cancer V-ATPase molecular signatures: A distinctive balance of subunit C isoforms in esophageal carcinoma.

BackgroundV-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expression of these proton pumps has been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the expression of V-ATPase genes in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers.MethodsExpression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples.FindingsA differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence.InterpretationAltogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the H+ pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in specific subunit isoforms.FundingThis work was supported by grants from CNPq and FAPERJ-Brazil.

Forkhead box C1 promotes the pathology of osteoarthritis by upregulating β-catenin in synovial fibroblasts.

Osteoarthritis (OA) is a joint disease characterized by articular cartilage degeneration, and no effective treatment is available. The OA classification has shifted from a cartilage-only disease to a whole-joint disease, and the synovial membrane plays an important role. Therefore, studies are needed to identify additional genes that regulate the pathologicalchanges in the synovial membrane to develop a promising therapeutic strategy for OA. Here, we validated that the expression of forkhead box protein C1 (FoxC1) and β-catenin was upregulated in OA synovial membranes and synovial fibroblasts (SFs). Gain- and loss-of-function studies revealed that FoxC1 overexpression promoted, whilst silencing inhibited OA synovial fibroblast (OASF) proliferation and pro-inflammatory cytokine [interleukin 6 (IL-6), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α)] production. FoxC1 overexpression increased β-catenin mRNA, total and nuclear protein expression in OASFs and upregulated a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS-5), fibronectin, matrix metalloproteinase 3 (MMP3) and matrix metalloproteinase 13 (MMP13) mRNA and total protein expression in OASFs. Conversely, FoxC1 knockdown reduced β-catenin mRNA, total and nuclear protein expression in OASFs and reduced ADAMTS-5, fibronectin, MMP3 and MMP13 mRNA and total protein expression in OASFs. β-catenin mediates FoxC1-induced pathologicalchanges (proliferation, catabolic regulation and inflammation) in OASFs. MicroRNA-200a-3p (miR-200a-3p) binds to the 3'-UTR of FoxC1 and mediates FoxC1 expression. Intra-articular FoxC1-specific siRNA transfection hindered OA development in mice. Therefore, our results demonstrate the key role FoxC1 plays invivo and invitro in OA synovial pathology, possibly identifying a potential novel therapeutic target for OA.

Leukemia cells remodel marrow adipocytes via TRPV4-dependent lipolysis.

Remodeling of adipocyte morphology and function plays a critical role in prostate cancer development. We previously reported that leukemia cells secrete growth differentiation factor 15 (GDF15), which remodels the residual bone marrow (BM) adipocytes into small adipocytes and is associated with a poor prognosis in patients with acute myeloid leukemia. However, little is known about how GDF15 drives BM adipocyte remodeling. In this study, we examined the role of the transient receptor potential vanilloid (TRPV) channels in the remodeling of BM adipocytes exposed to GDF15. We found that TRPV4 negatively regulated GDF15-induced remodeling of BM adipocytes. Furthermore, transforming growth factor-β type II receptor was identified as the main receptor for GDF15 on BM adipocytes. PI3K inhibitor treatment reduced GDF15- induced pAKT, identifying PI3K/AKT as the downstream stress response pathway. Subsequently, GDF15 reduced the expression of the transcription factor Forkhead box C1 (FOXC1) in BM adipocytes subjected to RNAsequencing screening and western blot analysis. Moreover, it was also confirmed that FOXC1 combined with the TRPV4 promoter by chromatin immunoprecipitation with quantitative polymerase chain reaction experiments, which suggests that FOXC1 mediates GDF15 regulation of TRPV4. In addition, an acute myeloid leukemia mouse model exhibited smaller BM adipocytes, whereas the TRPV4 activator 4a-phorbol 12,13-didecanoate partly rescued this process and increased survival. In conclusion, TRPV4 plays a critical role in BM adipocyte remodeling induced by leukemia cells, suggesting that targeting TRPV4 may constitute a novel strategy for acute myeloid leukemia therapy.

MiR-338 regulates NFATc1 expression and inhibits the proliferation and epithelial-mesenchymal transition of human non-small-cell lung cancer cells.

BackgroundIt is well known that nuclear factor of activated T cells c1 (NFATc1) expression is closely associated with progression of many cancers. And we found that miR‐338 could directly target the NFATc1. However, the precise mechanisms of miR‐338 in non‐small‐cell lung cancer (NSCLC) have not been well clarified. Our study aimed to explore the interaction between NFATc1 and miR‐338 in NSCLC.MethodsQuantitative RT‐PCR was utilized to determine the expressions of NFATc1 and miR‐338 in NSCLC tissues and cell lines. And the cell proliferation and epithelial‐mesenchymal transition (EMT) were assessed to determine the functional roles of miR‐338 and NFATc1 in NSCLC cells. NFATc1 expression was detected using quantitative RT‐PCR and western blotting, respectively. Luciferase reporter assays were performed to validate NFATc1 as a target of miR‐338 in NSCLC cells.ResultsIn this study, our results showed that NFATc1 expression was significantly up‐regulated in NSCLC tissues and cell lines, and the miR‐338 level was dramatically down‐regulated. Moreover high NFATc1 expression was closely associated with low miR‐338 level in NSCLC tissues. Moreover introduction of miR‐338 significantly inhibited proliferation and EMT of NSCLC cells. Bioinformatics analysis predicted that the NFATc1 was a potential target gene of miR‐338. We demonstrated that miR‐338 could directly target NFATc1 by using luciferase reporter assay. Besides, knockdown of NFATc1 had the similar effects with miR‐338 overexpression on NSCLC cells. Up‐regulation of NFATc1 in NSCLC cells partially abolished the inhibitory effects of miR‐338 mimic.ConclusionsOverexpression of miR‐338 inhibited cell proliferation and EMT of NSCLC cells by directly down‐regulating NFATc1 expression.

P38-regulated FOXC1 stability is required for colorectal cancer metastasis.

Aberrant expression of forkhead box C1 (FOXC1) promotes tumor metastasis in multiple human malignant tumors. However, the upstream modulating mode and downstream molecular mechanism of FOXC1 in metastasis of colorectal cancer (CRC) remain unclear. Herein we describe a systematic analysis of FOXC1 expression and prognosis in CRC performed on our clinical data and public databases, which indicated that FOXC1 upregulation in CRC samples was significantly associated with poor prognosis. FOXC1 knockdown inhibited migration and invasion, whereas FOXC1 overexpression caused the opposite phenotype in vitro and in vivo. Furthermore, MMP10, SOX4 and SOX13 were verified as the target genes of FOXC1 for promoting CRC metastasis. MMP10 was demonstrated as the direct target and mediator of FOXC1. Interestingly, Ser241 and Ser272 of FOXC1 were identified as the key sites to interact with p38 and phosphorylation, which were critically required for maintaining the stability of FOXC1 protein. Moreover, FOXC1 was dephosphorylated by protein phosphatase 2A and phosphorylated by p38, which maintained FOXC1 protein stability through inhibiting ubiquitination. Expression of p38 was correlated with FOXC1 and MMP10 expression, indirectly indicating that FOXC1 was regulated by p38 MAPK. Therefore, FOXC1 is strongly suggested as a pro-metastatic gene in CRC by transcriptionally activating MMP10, SOX4 and SOX13; p38 interacts with and phosphorylates the Ser241 and ser272 sites of FOXC1 to maintain its stability by inhibiting ubiquitination and degradation. In conclusion, the protein stability of FOXC1 mediated by p38 contributes to the metastatic effect in CRC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Multi-Cancer V-ATPase Molecular Signatures: A Distinctive Balance of Subunit C Isoforms in Esophageal Carcinoma

Background: V-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expressions of these proton pumps have been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the V-ATPase genes expression in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers. Methods: Expression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples. Findings: A differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. Interpretation: Altogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in single subunit/isoforms moved by standard cutoffs. Funding Statement: This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil). This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - Brasil (CAPES) - Finance Code 001 (PhD fellowship to JCVCS and Post-doctoral fellowship to EPC). JCVCS received Postdoctoral fellowship from Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF) and FFF received a PhD fellowship from FAPERJ. Declaration of Interests: The authors state that they have no conflicts of interest. Ethics Approval Statement: Human subjects: The use of the human samples was approved by the Ethic Committee of the institution (INCA - 116/11). All patients, who kindly agreed to participate in the study, signed an informed consent form prior to study enrollment.

Cysteine and methionine contribute differentially to regulate alternative oxidase in leaves of poplar (Populus deltoides x Populus euramericana ‘Nanlin 895’) seedlings exposed to different salinity

The effects of different doses of NaCl on the expression profiles of genes involved in the mitochondrial electron transport chain (miETC), H2O2 and O2− levels, and antioxidant enzymes and amino acid metabolism were investigated in the leaves of poplar (Populus deltoides x Populus euramericana 'Nanlin 895'). In the miETC, complexes II and III and bypasses of the cytochrome c pathway including AOX and UCP displayed higher transcript abundance, whereas COX6b encoding cytochrome c oxidase were suppressed at 200 and 400 mM. H2O2 accumulated at 200 mM NaCl but O2− was generated at 400 mM. Accordingly, CAT was enhanced at 200 and 400 mM, while G-POD strengthened only at 400 mM. In addition, cysteine was reduced at 400 mM but did not change at 200 mM, although methionine was accumulated at 200 mM but not altered at 400 mM. Exogenous cysteine accumulated H2S and methionine increased ACC at 200 mM NaCl. At 400 mM NaCl, cysteine elevated the expression of CGS encoding cystathionine gamma-synthase and MS2 encoding methionine synthase as well as ACC and H2S levels, and methionine increased ACC content with repressed CGS and MS2. Moreover, exogenous KCN decreased cysteine levels, with an augment in H2S and up-regulation of CYS C1 encoding β-cyanoalanine synthase at all salinity conditions, whereas antimycin A (AA) and salicylhydroxamic acid (SHAM) affected neither the levels of cysteine or H2S, nor the CYS C1 expression. However, neither KCN, AA nor SHAM affected ACC content. AOX1b was induced both by exogenous cysteine and methionine as well as KCN and AA but suppressed by SHAM at 200 and 400 mM NaCl, in negative correlation with MDA content. These results suggest that poplar leaf evolved diverse strategies in amino acid metabolism of manipulating the AOX pathway to defend against different levels of salt stress.