Prognostic impact of desmoplastic reaction in esophageal squamous cell carcinoma patients with neoadjuvant therapy.

AIM: This study aimed to examine the prognostic value of desmoplastic reaction (DR) in esophageal squamous cell carcinoma (ESCC), particularly in patients who received neoadjuvant therapy, such as chemotherapy (NAC) or chemoradiotherapy (NACRT). METHOD: In total, 153 patients with pStage II/III ESCC were included in this study. Ninety-one patients received neoadjuvant therapy (NAC, 70; NACRT, 21). Patients were classified according to three DR categories based on the presence of keloid-like collagen and/or myxoid stroma. RESULTS: In total, 50, 50, and 53 patients were classified as having mature, intermediate, and immature DR, respectively. The weighted kappa coefficient was 0.623 in the patients with preoperative treatments and 0.782, in those without. The 5-year disease-specific survival (DSS) rates in patients with intermediate/immature DR was significantly worse than those with mature DR (40.7% vs. 73.3%, p < 0.001). Similarly, the 5-year DSS rate in patients with intermediate/immature DR was significantly worse than those with mature DR in a study of patients who received neoadjuvant therapy (46.7% vs. 71.2%, p = 0.009). Multivariate analysis revealed that DR (hazard ratio [HR]: 3.15, 95% confidence interval [CI] 1.58-6.27, p = 0.001), along with N factors, was an independent risk factor for DSS. Moreover, multivariate analysis of patients who received neoadjuvant therapy revealed only DR (HR: 2.47, 95% CI 1.02-5.96, p = 0.045) as independent risk factors for DSS. CONCLUSION: The DR classification was a valuable prognostic factor not only in the ESCC patients without neoadjuvant therapy but also in those with neoadjuvant therapy.

Synergistic upregulation of ADAMTS4 (aggrecanase-1) by cytokines and its suppression in knee osteoarthritic synovial fibroblasts.

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family includes nine members with aggrecan-degrading activity, i.e., ADAMTS1, 4, 5, 8, 9, 15, 16, 18, and 20. However, their systematic expression profile in knee osteoarthritis (OA) synovium and effects of cytokines and growth factors on the expression in OA synovial fibroblasts remain elusive. In this study, expression of all nine aggrecanolytic ADAMTS species was assessed by quantitative real-time PCR in OA and control normal synovial tissues. OA synovial fibroblasts were treated with interleukin-1α (IL-1α), IL-1ß, tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), vascular endothelial growth factor165, and heparin-binding epidermal growth factor, and analyzed for the expression of the ADAMTS species. The signaling pathways and inhibition of ADAMTS4 expression by high-molecular-weight hyaluronan, adalimumab, tocilizumab, and signaling molecule inhibitors were studied. ADAMTS1, 4, 5, 9, and 16 were expressed in OA synovium, but only ADAMTS4 expression was significantly higher in OA as compared to normal synovium. IL-1α, TNF-α, and TGF-ß markedly increased ADAMTS4 expression, while their effects were minimal for the other ADAMTS species. ADAMTS4 was synergistically upregulated by treatment with IL-1α and TNF-α, IL-1α and TGF-ß, or IL-1α, TNF-α and TGF-ß. The signaling molecules' inhibitors demonstrated that IL-1α-induced ADAMTS4 expression is predominantly through TGF-ß-associated kinase 1 (TAK1), and the TNF-α-stimulated expression is via TAK1 and nuclear factor-κB (NF-κB). The TGF-ß-promoted expression was through the activin receptor-like kinase 5 (ALK5)/Smad2/3, TAK1, and non-TAK1 pathways. Adalimumab blocked TNF-α-stimulated expression. ADAMTS4 expression co-stimulated with IL-1α, TNF-α and TGF-ß was abolished by treatment with adalimumab, TAK1 inhibitor, and ALK5/Smad2/3 inhibitor. These data demonstrate marked and synergistic upregulation of ADAMTS4 by IL-1α, TNF-α and TGF-ß in OA synovial fibroblasts, and suggest that concurrent therapy with an anti-TNF-α drug and inhibitor(s) may be useful for prevention against aggrecan degradation in OA.

Cancer-associated fibroblasts at the unfavorable desmoplastic stroma promote colorectal cancer aggressiveness: Potential role of ADAM9.

The tumor microenvironment plays a key role in cancer aggressiveness. Desmoplastic reaction (DR), morphologically classified as Mature, Intermediate and Immature types, has previously been shown to be highly prognostic in colorectal cancer (CRC) and it consists to a large extent of cancer-associated fibroblasts (CAFs). The aim of our study was to characterize the molecular background of DR and understand the effects of CAFs in tumor aggressiveness. The prognostic significance of DR was initially examined in 1497 patients. Then CAFs originating from patient tissues with different DR types were isolated and their impact on tumor growth was examined both in vitro and in vivo. DR was shown to be highly prognostic, with patients within the Immature DR group conferring the worst relapse-free survival. The conditioned media of CAFs from tumor with Immature-type DR (CAFsImmature ) significantly increased proliferation and migration of CRC cell lines and growth of CRC-derived organoids compared to that of CAFs from Mature-type DR (CAFsMature ). Subcutaneous or orthotopic implantation of CRC cells together with CAFsImmature in mice significantly promoted tumor growth and dissemination compared to implantation with CAFsMature . Systematic examination of the expression of "a disintegrin and metalloproteinases" (ADAMs) in CAFs isolated from CRC tissues showed that the secreted isoform of ADAM9 (ADAM9s) was significantly higher in CAFsImmature than in CAFsMature . Knockdown of ADAM9s in CAFsImmature abrogated the promoting effects on CRC cell proliferation and migration. CAFs-derived ADAM9s is implicated in deteriorating survival in CRC patients with Immature-type DR by increasing tumor cell proliferation and dissemination.

Proposal for a tumor budding predictive score derived from endoscopic biopsy samples in colorectal cancer.

BACKGROUND: In colorectal cancer, tumor budding is highlighted as both a prognostic indicator and a predictor of chemosensitivity. However, tumor budding has a serious drawback because of unattainable preoperative assessment, thereby, making it not applicable to decision-making on treatment strategies. Recently, high expressions of seven genes (i.e., MSLN, SLC4A11, WNT11, SCEL, RUNX2, MGAT3, and FOXC1) were shown to be associated with high-budding colorectal cancers. This study aimed to propose a budding prediction system using selected RNAs extracted from biopsy specimens. METHODS: The RNA expression levels in 86 surgically resected samples and in 104 samples obtained by colonoscopy before surgery were investigated. RESULTS: The tumor surface expressions of four exclusive genes (i.e., MSLN, SLC4A11, SCEL, and MGAT3) were correlated with the tumor budding grade. Subsequently, the logit P value calculated by multiple logistic regression analysis using the four surface gene expressions was set as the following budding predictive score: Logit (P) = - 0.55 + 0.27*MSLN + 0.16*SLC4A11 + 0.06*MGAT3 + 0.21*SCEL. The effectiveness of the model using colorectal cancer biopsy samples was well corroborated prospectively. CONCLUSION: The budding predictive score that we developed using endoscopic biopsy specimens was clarified to have a high potential for preoperative use.

Mesothelin Expression is Correlated with Chemoresistance in Stage IV Colorectal Cancer.

BACKGROUND: Mesothelin (MSLN) is a cell-surface glycoprotein present on mesothelial cells; its expression in several epithelial cancers generally portends an unfavorable prognosis. We investigated MSLN as a surrogate chemopredictive biomarker and examined the impact of MSLN expression in stage IV colorectal cancer (CRC). METHODS: We recruited 254 patients with CRC who received systemic chemotherapy following primary tumor resection between 2000 and 2019. Resected specimens were immunostained for MSLN and stratified by MSLN expression. The associations of tumor MSLN expression with tumor response in metastatic lesions and survival were evaluated. RESULTS: Of the 247 patients with stage IV CRC, 41 (16.1%) and 213 (83.9%) had high and low MSLN expression, respectively. Based on the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria, the investigator-assessed objective response rate was 22.0% in the high MSLN expression group and 45.5% in the low MSLN expression group (p = 0.0050). The disease control rates in these groups were 65.9% and 85.9%, respectively (p = 0.00019). In the patients with high MSLN expression, the conversion rate among those with initially unresectable metastases was 0% versus 14% in the patients with low MSLN expression (p = 0.0053). The median overall survival (OS) was 1.5 years (95% confidence interval [CI] 1.1-2.8) in the high MSLN expression group versus 2.6 years (95% CI 2.2-3.0) in the low MSLN expression group. The 3-year OS rates in these groups were 23.5 and 41.5%, respectively (p = 0.0120). CONCLUSIONS: High MSLN expression is correlated with chemoresistance and poor prognoses in stage IV CRC.

Correlation Between Immunoinflammatory Measures and Periostin Expression in Esophageal Squamous Cell Carcinoma: A Single-Center, Retrospective Cohort Study.

BACKGROUND: Immunoinflammatory measures such as the neutrophil-lymphocyte ratio (NLR), the platelet-lymphocyte ratio (PLR), and the C-reactive protein (CRP)-albumin ratio (CAR) are useful prognostic measures in various malignancies. However, no study has investigated the correlation of these measures with microenvironmental inflammation. Periostin (POSTN), a small extracellular matrix protein, strongly associates with cancer microenvironmental inflammation. The current study investigated the correlation of NLR, PLR, and CAR with periostin expression in esophageal squamous cell carcinoma (ESCC). METHODS: The study retrospectively evaluated preoperative NLR, PLR, and CAR hematologically and POSTN immunohistochemically in 171 patients. The correlation of immunoinflammatory measures, POSTN expression, and survival outcomes was measured. RESULTS: The study showed a significant correlation of POSTN-positive expression with poor overall survival (OS) (P < 0.0001) and recurrence-free survival (RFS) (P = 0.03). The POSTN-positive group had higher PLR (189.6 ± 8 vs. 159.3 ± 12; P = 0.04) and CAR (0.36 ± 0.06 vs. 0.14 ± 0.09; P < 0.05) than the POSTN-negative group, whereas NLR did not differ between the two groups (3.27 ± 0.19 vs. 2.65 ± 0.28; P = 0.07). The uni- and multivariate analyses showed that POSTN-positive expression (hazard ratio [HR], 1.595; 95% confidence interval [CI], 0.770-3.031; P = 0.03), CAR (HR, 1.663; 95% CI, 1.016-2.764; P = 0.03), gender (HR, 2.303; 95% CI, 1.067-6.019; P = 0.03), and tumor depth (HR, 1.957; 95% CI, 1.122-3.526; P = 0.01) were independent prognostic factors. CONCLUSIONS: Because POSTN-positive expression strongly correlates with immunoinflammatory measures, especially PLR and CAR, it is an independent prognostic factor in ESCC.

Clinical Significance of a Gene Signature Generated from Tumor Budding Grade in Colon Cancer.

BACKGROUND: Tumor budding, a microscopic finding of dedifferentiation at the invasive margin, has been reported as a definite prognostic marker in colon cancer (CC). Herein, we aimed to generate a molecular budding signature (MBS) based on DNA microarray data and to examine its prognostic significance. METHODS: Frozen tissue samples from 85 patients with stage II/III CC were used for DNA microarray analyses. First, we selected candidate genes that were differentially expressed (twofold change) between the invasive frontal regions and corresponding tumor centers of three extremely high-grade budding tumors. Subsequently, using microarray data from whole-tissue sections of the 85 patients, we selected MBS-constituent genes from the candidates based on correlation to the pathological budding grade. The MBS score was calculated using the sum of the logarithm of the expression of each gene. RESULTS: We selected seven MBS-constituent genes: MSLN, SLC4A11, WNT11, SCEL, RUNX2, MGAT3, FOXC1. A comparison of relapse-free survival (RFS) rates revealed a significant impact of the MBS score [5-year RFS, 77.4% (score-high) vs. 95.1% (score-low); P = 0.044]. Analyses of public databases revealed that low MBS score patients exhibited better prognosis than those with high-score cancers (GSE14333: 5-year RFS, 83.1% vs. 66.6%, P = 0.028; GSE39582: 5-year disease-free survival, 72.2% vs. 58.1%, P = 0.0005). Multivariate analysis revealed that the MBS score is an independent prognostic indicator in GSE39582 (hazard ratio, 1.611; P = 0.013). CONCLUSIONS: We developed a new gene classification method, i.e., MBS, and demonstrated its clinical relevance as an indicator of high recurrence risk of CC.

Expression and Function of a Disintegrin and Metalloproteinases in Cancer-Associated Fibroblasts of Colorectal Cancer.

BACKGROUND: Cancer tissues consist of cancer cells and stroma, the latter of which dictates cancer tissue microenvironment. We recently reported that the desmoplastic reaction (DR) pattern at the invasive front in colorectal cancer (CRC) is a promising prognostic indicator. However, the molecular mechanisms of DR formation and contribution to patients' prognosis remain unclear. SUMMARY: The tumor tissue microenvironment composed of extracellular matrix (ECM), soluble factors (growth factors/cytokine/cytokine), and stromal cells controls tumor growth and spread. Among stromal cells, cancer-associated fibroblasts (CAFs) play a key role in development of the cancer tissue microenvironment, and they are responsible for DR formation. CAFs express a disintegrin and metalloproteinases (ADAMs), which modulate cancer tissue microenvironmental factors. We isolated CAFs and normal fibroblasts from colon tissues of patients with CRC and characterized them. CAFs showed the increased expression of several ADAM species including ADAM9, ADAM10, ADAM12, and ADAM17, and the expression was further increased on the ECM-coated plates. Our in vitro and in vivo studies using CAFs and CRC cells suggest that ADAM expression is associated with the morphological DR category, and ADAMs may affect cancer malignancy through tumor proliferation in CRC. Key Message: This review summarizes the present knowledge on ADAMs in cancer and describes our recent findings regarding the molecular biological background of DR mainly by focusing on ADAMs.

Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization Is Up-Regulated and Involved in Hyaluronan Degradation in Human Osteoarthritic Cartilage.

Hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), also called cell migration-inducing protein (CEMIP; alias KIAA1199), plays a key role in the degradation of HA in skin and arthritic synovial fibroblasts, but its functions in osteoarthritic (OA) cartilage remain elusive. Here, we investigated the expression and roles of HYBID in human OA cartilage. HYBID was highly expressed by chondrocytes in the HA-depleted area of OA cartilage, and HYBID immunoreactivity was correlated with Mankin score, the histopathologic severity of OA lesions of cartilage. Real-time quantitative PCR indicated that HYBID expression was significantly higher in OA cartilage than in control cartilage. In addition, OA chondrocytes exhibited HA-degrading activity, which was abolished by knock-down of HYBID by siRNAs. Although OA chondrocytes also expressed certain levels of hyaluronidases 1 and 2 and CD44, knock-down of these molecules exhibited negligible effects on HA degradation. Double immunostaining of HYBID and clathrin heavy chain revealed that HYBID was localized in the clathrin-coated vesicles, and HA was endocytosed within the vesicles of OA chondrocytes. Among eight factors including cytokines and growth factors examined, only tumor necrosis factor α stimulated OA chondrocytes to overexpress HYBID. These data are the first to demonstrate that HYBID is up-regulated in OA cartilage, and suggest that tumor necrosis factor α-stimulated HYBID plays a role in HA degradation in OA cartilage.

Differential clinical impacts of tumour budding evaluated by the use of immunohistochemical and haematoxylin and eosin staining in stage II colorectal cancer.

AIMS: The aim of this study was to clarify the quantitative and qualitative differences in tumour budding identification between haematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining for cytokeratin, and to estimate the respective clinical impacts in stage II colorectal cancer. METHODS AND RESULTS: We retrospectively examined 314 surgically resected cases of stage II colorectal cancer, and assessed tumour budding on serial section slides with H&E staining and IHC staining for cytokeratin. Tumour budding counts based on cytokeratin-stained slides were strongly correlated with those based on H&E-stained slides, and had higher detection and reproducibility. On the basis of receiver operating characteristic analyses, the optimal cut-off values of budding counts for relapse-free survival (RFS) were 7 and 16 in a ×200 microscopic field with H&E and IHC staining, respectively. With these cut-off values, tumour budding based on H&E staining had a significant correlation with RFS (80.3% and 93.2% of 5-year RFS in the high-budding group and the low-budding group, respectively), and similar results were observed for IHC staining (79.9% and 91.7%, respectively). The Akaike Information Criterion value for RFS with H&E staining was favourable as compared with that with IHC staining. CONCLUSIONS: Tumour budding counts based on cytokeratin-stained slides showed higher detection and better reproducibility, but did not have as satisfactory clinical impacts as those based on H&E staining.

Differential Survival Benefits of 5-Fluorouracil-Based Adjuvant Chemotherapy for Patients With Microsatellite-Stable Stage III Colorectal Cancer According to the Tumor Budding Status: A Retrospective Analysis.

BACKGROUND: Recent research has established tumor budding as a prognostic factor and a possible histomorphologic reflection of epithelial-mesenchymal transition in colorectal cancer, highlighting the ability of cancer cells exhibiting epithelial-mesenchymal transition to resist chemotherapy. OBJECTIVE: This study aimed to investigate the clinical benefits of adjuvant chemotherapy according to the tumor budding status in microsatellite-stable stage III colorectal cancer. DESIGN: This was a retrospective study of 2 cohorts. SETTINGS: The study was conducted at the National Defense Medical College in Japan. PATIENTS: We reviewed 2 data sets of patients with microsatellite-stable stage III colorectal cancer with curatively intended surgery (R0) from 1999 to 2005 (first cohort; n = 203) and 2006 to 2012 (second cohort; n = 346). In both cohorts, 128 and 203 patients received 5-fluorouracil-based adjuvant chemotherapy and 75 and 143 patients did not. MAIN OUTCOME MEASURES: We assessed the benefits of adjuvant chemotherapy according to the grades of tumor budding based on the cancer-specific survival. RESULTS: In low-budding tumors, the chemotherapy group exhibited better cancer-specific survival than the surgery-alone group (first cohort, 93.1% vs 65.5%, p = 0.001; second cohort, 94.0% vs 76.0%, p < 0.0001). Conversely, the prognostic difference between the chemotherapy and surgery-alone groups was statistically insignificant in high-budding tumors (first cohort, 59.7% vs 52.4%, p = 0.57; second cohort, 83.1% vs 75.6%, p = 0.19). The multivariate analysis corroborated the benefits of adjuvant chemotherapy in low-budding tumors (first cohort, p = 0.002, HR = 0.28; second cohort, p < 0.0001, HR = 0.23) but not in high-budding tumors. LIMITATIONS: Postoperative adjuvant chemotherapy and treatments for recurrence were not homogeneous, and the patient backgrounds differed between the chemotherapy and surgery alone groups. CONCLUSIONS: The high-budding group demonstrated resistance to 5-fluorouracil-based chemotherapy, whereas the low-budding group exhibited significant survival benefits from adjuvant chemotherapy in stage III colorectal cancer. See Video Abstract at http://links.lww.com/DCR/B14. BENEFICIOS DE SUPERVIVENCIA DIFERENCIAL DE LA QUIMIOTERAPIA ADYUVANTE BASADA EN 5-FLUOROURACILO PARA PACIENTES CON CÁNCER COLORRECTAL EN ESTADIO III ESTABLE CON MICROSATÉLITE SEGÚN EL ESTADO DE BROTACIÓN DEL TUMOR: UN ANÁLISIS RETROSPECTIVO:: Investigaciones recientes han establecido la aparición de tumores como un factor pronóstico y una posible reflexión histomorfológica de la transición epitelial-mesenquimatosa en el cáncer colorrectal, destacando la capacidad de las células cancerosas que presentan una transición epitelio-mesenquimática para resistir la quimioterapia.El objetivo de este estudio es investigar los beneficios clínicos de la quimioterapia adyuvante según el estado de brotación del tumor en el cáncer colorrectal en estadio III estable con microsatélite.Este fue un estudio retrospectivo de dos cohortes.El estudio se realizó en la Escuela de Medicina de la Defensa Nacional de Japón.Revisamos dos conjuntos de datos de pacientes con cáncer colorrectal en estadio III estable con microsatélite con cirugía de intención curativa (R0) de 1999 a 2005 (primera cohorte; n = 203) y 2006 a 2012 (segunda cohorte; n = 346). En ambas cohortes, 128 y 203 pacientes recibieron quimioterapia adyuvante basada en 5-fluorouracilo y 75 y 143 pacientes no, respectivamente.Evaluamos los beneficios de la quimioterapia adyuvante de acuerdo con los grados de brotación del tumor en función de la supervivencia específica del cáncer.n los tumores con brotes bajos, el grupo de quimioterapia mostró una mejor supervivencia específica al cáncer que el grupo con cirugía sola (primera cohorte, 93.1% vs. 65.5%, p = 0.001; segunda cohorte, 94.0% vs. 76.0%, p < 0.0001). A la inversa, la diferencia pronóstica entre los grupos de quimioterapia y cirugía sola fue estadísticamente insignificante en los tumores de brotes elevados (primera cohorte, 59.7% vs. 52.4%, p = 0.57; segunda cohorte, 83.1% vs. 75.6%, p = 0.19). El análisis multivariado corroboró los beneficios de la quimioterapia adyuvante en los tumores de brotes bajos (primera cohorte, p = 0,002, índice de riesgo: 0,28; segundo cohorte, p <0,0001, índice de riesgo: 0,23) pero no en los tumores de alto brote.a quimioterapia adyuvante postoperatoria y los tratamientos para la recurrencia no fueron homogéneos, y los antecedentes de los pacientes difirieron entre los grupos de quimioterapia y cirugía sola.El grupo de alto brote demostró resistencia a la quimioterapia basada en 5-fluorouracilo, mientras que el grupo de bajo brote mostró beneficios significativos de supervivencia de la quimioterapia adyuvante en el cáncer colorrectal en estadio III. Vea el Resumen del Video en http://links.lww.com/DCR/B14.

Cancer-induced spiculation on computed tomography: a significant preoperative prognostic factor for colorectal cancer.

PURPOSE: Cancer-induced spiculation (CIS) on computed tomography, which is reticular or linear opacification of the pericolorectal fat tissues around the cancer site, is generally regarded as cancer infiltration into T3 or T4, but its clinicopathological significance is unknown. This study examines the correlation between CIS and clinicopathological findings to establish its prognostic value. METHODS: The subjects of this retrospective study were 335 patients with colorectal cancer (CRC), who underwent curative surgery between January, 2010 and December, 2011, at the National Defense Medical College Hospital in Saitama Prefecture, Japan. RESULTS: The level of interobserver agreement in the evaluation of CIS was substantial (83%; kappa value, 0.65). The presence of CIS was specific for T3/T4 disease (positive predictive value, 88.3%), and was significantly associated with tumor size and venous invasion. The 5-year relapse-free survival rate was significantly lower in patients with CIS than in those without CIS (68.6% and 84.0%, respectively, p = 0.001). Subgroup analysis revealed remarkable prognostic differences in patients with stage III and T3 disease. Multivariate analysis revealed that CIS was a significant independent prognostic factor. CONCLUSIONS: CIS was a significant preoperative prognostic factor and could be useful in the selection of preoperative therapy for patients with CRC.

ADAM9 is over-expressed in human ovarian clear cell carcinomas and suppresses cisplatin-induced cell death.

ADAMs (a disintegrin and metalloproteinases) are involved in various biological events such as cell adhesion, migration and invasion, membrane protein shedding and proteolysis. However, there have been no systematic studies on the expression of ADAMs in human ovarian carcinomas. We therefore examined mRNA expression of all the proteolytic ADAM species including ADAM8, 9, 10, 12, 15, 17, 19, 20, 21, 28, 30, 33 and ADAMDEC1 in human ovarian carcinomas, and found that prototype membrane-anchored ADAM9m, but not secreted isoform ADAM9s, is significantly over-expressed in carcinomas than in control non-neoplastic ovarian tissue. Among the histological subtypes of serous, endometrioid, mucinous and clear cell carcinomas, ADAM9m expression was highest in clear cell carcinomas. Immunohistochemistry showed that all the clear cell carcinoma samples displayed ADAM9m primarily on the carcinoma cell membrane. By immunoblotting, ADAM9m was detected mainly in an active form in the clear cell carcinoma tissues. When two clear cell carcinoma cell lines (RMG-I and TOV21G cells) with ADAM9m expression were treated with cisplatin, viability was significantly reduced and apoptosis increased in ADAM9m knockdown cells compared with mock transfectants. In addition, treatment of the cells with neutralizing anti-ADAM9m antibody significantly decreased viability compared with non-immune IgG, whereas ADAM9m over-expression significantly increased viability compared with mock transfectants. Our data show, to the best of our knowledge, for the first time, that ADAM9m is over-expressed in an activated form in human ovarian clear cell carcinomas, and suggest that ADAM9m plays a key role in cisplatin resistance.

SOX4, an epithelial-mesenchymal transition inducer, transactivates ADAM28 gene expression and co-localizes with ADAM28 at the invasive front of human breast and lung carcinomas.

ADAM28 (a disintegrin and metalloproteinase 28) is abundantly expressed by carcinoma cells in the human breast and non-small cell lung carcinomas, and plays a role in carcinoma cell growth and metastasis. Although Src is an inducer of ADAM28 gene expression through the PI3K/AKT/mTOR and MEK/ERK pathways, direct transcriptional regulators for ADAM28 gene expression remain unknown. In this study, we performed the luciferase reporter assay and found that SOX4 (SRY-related HMG-box 4), an inducer of epithelial-mesenchymal transition (EMT), is a transcriptional activator for the ADAM28 gene. This activation required the SOX4-binding consensus sequence at the 5'-untranslated region of the mouse and human ADAM28 genes. Forced expression of SOX4 promoted the ADAM28 gene expression and migration in human breast and lung carcinoma cell lines. In the human breast and lung carcinoma tissues, ADAM28 and SOX4 were co-expressed at the invasive front of carcinoma cell nests. Our data demonstrate that SOX4 transactivates ADAM28 gene expression through direct binding to the ADAM28 promoter region and suggest the possibility that ADAM28 plays a role in invasion through SOX4-mediated EMT in the human breast and lung carcinomas.

The Metalloproteinase ADAM28 Promotes Metabolic Dysfunction in Mice.

Obesity and diabetes are major causes of morbidity and mortality globally. The current study builds upon our previous association studies highlighting that A Disintegrin And Metalloproteinase 28 (ADAM28) appears to be implicated in the pathogenesis of obesity and type 2 diabetes in humans. Our novel study characterised the expression of ADAM28 in mice with the metabolic syndrome and used molecular inhibition approaches to investigate the functional role of ADAM28 in the pathogenesis of high fat diet-induced obesity. We identified that ADAM28 mRNA and protein expression was markedly increased in the livers of mice with the metabolic syndrome. In addition, noradrenaline, the major neurotransmitter of the sympathetic nervous system, results in elevated Adam28 mRNA expression in human monocytes. Downregulation of ADAM28 with siRNA technology resulted in a lack of weight gain, promotion of insulin sensitivity/glucose tolerance and decreased liver tumour necrosis factor-α (TNF-α) levels in our diet-induced obesity mouse model as well as reduced blood urea nitrogen, alkaline phosphatase and aspartate aminotransferase. In addition, we show that ADAM28 knock-out mice also displayed reduced body weight, elevated high density lipoprotein cholesterol levels, and reductions in blood urea nitrogen, alkaline phosphatase, and aspartate aminotransferase. The results of this study provide important insights into the pathogenic role of the metalloproteinase ADAM28 in the metabolic syndrome and suggests that downregulation of ADAM28 may be a potential therapeutic strategy in the metabolic syndrome.

ADAM23 is downregulated in side population and suppresses lung metastasis of lung carcinoma cells.

Cancer cells contain a small population of cancer stem cells or cancer initiating cells, which can be enriched in the side population (SP) after fluorescence activated cell sorting. To examine the members of the ADAM, ADAMTS and MMP gene families related to phenotypes of the SP and the main population (MP), we screened the expression of all the members in the propagated SP and MP of A549 lung adenocarcinoma cells, and found that the relative expression ratio of ADAM23 in the MP to the SP is most highly increased, but none of them are increased in the SP. A similar result on the ADAM23 expression was obtained with another cell line, Calu-3 cells. Overexpression of ADAM23 inhibited colony formation, cell adhesion and migration, and knockdown of ADAM23 by shRNA showed the reverse effects. ADAM23-mediated suppression of colony formation, cell adhesion and migration was greatly reduced by treatment with neutralizing anti-ADAM23 antibody, anti-αvß3 integrin antibody and/or ADAM23 disintegrin peptide. Expression of cancer stem cell-related genes, including AKRC1/2, TM4SF1 and NR0B1, was increased by knockdown of ADAM23. In addition, lung metastasis of A549 transfectants with different levels of ADAM23 expression was negatively regulated by the ADAM23 expression levels. Our data provide evidence that ADAM23 plays a role in suppression of cancer cell progression through interaction with αvß3 integrin, and suggest that downregulation of ADAM23 in SP cells may contribute toward providing a cancer stem cell phenotype by facilitating the activity of integrin αvß3.

Development of human neutralizing antibody to ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2).

ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2), members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, are considered to play a key role in aggrecan degradation of articular cartilage in human osteoarthritis. Here, we developed a neutralizing antibody to these aggrecanases by screening human combinatorial antibody library. Among the five candidate antibodies, one antibody was immunoreactive with both ADAMTS4 and ADAMTS5, showing no or negligible cross-reactivity with 10 different related metalloproteinases of the ADAMTS, ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) gene families. This antibody almost completely and partially inhibited aggrecanase activity of ADAMTS4 and ADAMTS5, respectively. It also suppressed the aggrecanase activity derived from interleukin-1-stimulated osteoarthritic chondrocytes. These data demonstrate that the antibody is specific to ADAMTS4 and ADAMTS5 and inhibits their aggrecanase activity at molecular and cellular levels, and suggest that this antibody may be useful for treatment of pathological conditions such as osteoarthritis.

Src plays a key role in ADAM28 expression in v-src-transformed epithelial cells and human carcinoma cells.

ADAM28, a disintegrin and metalloproteinase 28, is overexpressed by carcinoma cells with direct correlations with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, the molecular mechanisms of ADAM28 gene expression in carcinoma cells remain elusive. Herein, we investigated the expression of ADAM28 in Madin-Darby canine kidney epithelial cells transformed by oncogenes, including v-src, LMP1, ErbB2, Ha-Ras, and c-Fos, and found that v-src transformants selectively induce ADAM28. Implantation of the v-src transformants showed a progressively growing tumor, which was significantly suppressed by local injections of anti-ADAM28 antibody. ADAM28 expression in v-src transformants was partially inhibited by treatment with inhibitors to Src kinase, mitogen-activated protein kinase kinase (MEK), phosphatidylinositol 3-kinase (PI3K), or mammalian target of rapamycin, and abrogated by v-Src kinase inhibitor, radicicol, or a mixture of MEK and PI3K inhibitors. Human carcinoma cell lines of the lung, breast, ovary, kidney, and colon showed ADAM28 expression, which was correlated with phosphorylation of c-Src and suppressed by the inhibitors in a similar way to v-src transformants. IHC of the human tumor tissues demonstrated co-expression of ADAM28 and phosphorylated Src in neoplastic cells of the breast, lung, and colon carcinomas and some adenomas of the colon, but not in nonneoplastic colon mucosa. Our data provide, to the best of our knowledge, the first evidence that Src is an inducer of ADAM28 gene expression through the MEK/extracellular signal-regulated kinase and PI3K/mammalian target of rapamycin pathways.

Regulatory role of dendritic cells in postinfarction healing and left ventricular remodeling.

BACKGROUND: Inflammation and immune responses are integral components in the healing process after myocardial infarction. We previously reported dendritic cell (DC) infiltration in the infarcted heart; however, the precise contribution of DC in postinfarction healing is unclear. METHODS AND RESULTS: Bone marrow cells from CD11c-diphtheria toxin receptor/green fluorescent protein transgenic mice were transplanted into lethally irradiated wild-type recipient mice. After reconstitution of bone marrow-derived cells, the recipient mice were treated with either diphtheria toxin (DC ablation) or vehicle (control), and myocardial infarction was created by left coronary ligation. CD11c(+) green fluorescent protein-positive DCs expressing CD11b and major histocompatibility complex class II were recruited into the heart, peaking on day 7 after myocardial infarction in the control group. Mice with DC ablation for 7 days showed deteriorated left ventricular function and remodeling. The DC-ablated group demonstrated enhanced and sustained expression of inflammatory cytokines such as interleukin-1ß, interleukin-18, and tumor necrosis factor-α, prolonged extracellular matrix degradation associated with a high level of matrix metalloproteinase-9 activity, and diminished expression level of interleukin-10 and endothelial cell proliferation after myocardial infarction compared with the control group. In vivo analyses revealed that DC-ablated infarcts had enhanced monocyte/macrophage recruitment. Among these cells, marked infiltration of proinflammatory Ly6C(high) monocytes and F4/80(+) CD206(-) M1 macrophages and, conversely, impaired recruitment of anti-inflammatory Ly6C(low) monocytes and F4/80(+) CD206(+) M2 macrophages in the infarcted myocardium were identified in the DC-ablated group compared with the control group. CONCLUSIONS: These results suggest that the DC is a potent immunoprotective regulator during the postinfarction healing process via its control of monocyte/macrophage homeostasis.

Periostin derived from cancer-associated fibroblasts promotes esophageal squamous cell carcinoma progression via ADAM17 activation.

Periostin (POSTN) is a matricellular protein that was originally identified in osteoblasts. Past studies have shown that POSTN is also preferentially expressed in cancer-associated fibroblasts (CAFs) in various types of cancer. We previously demonstrated that the increased expression of POSTN in stromal tissues is associated with an unfavorable clinical outcome in esophageal squamous cell carcinoma (ESCC) patients. In this study, we aimed to elucidate the role of POSNT in ESCC progression and its underlying molecular mechanism. We found that POSTN is predominantly produced by CAFs in ESCC tissues, and that CAFs-cultured media significantly promoted the migration, invasion, proliferation, and colony formation of ESCC cell lines in a POSTN-dependent manner. In ESCC cells, POSTN increased the phosphorylation of ERK1/2 and stimulated the expression and activity of a disintegrin and metalloproteinase 17 (ADAM17), which is critically involved in tumorigenesis and tumor progression. The effects of POSTN on ESCC cells were suppressed by interfering with the binding of POSTN to integrin αvß3 or αvß5 using neutralizing antibody against POSTN. Taken together, our data show that CAFs-derived POSTN stimulates ADAM17 activity through activation of the integrin αvß3 or αvß5-ERK1/2 pathway and thereby contributes to the progression of ESCC.