Dataset Information

Expression of the c-Met/HGF receptor in human melanocytic neoplasms: demonstration of the relationship to malignant melanoma tumour progression.

ABSTRACT: The c-MET proto-oncogene encodes the receptor for the Hepatocyte Growth Factor/Scatter Factor, which is known to mediate mitogenic, motogenic and invasive responses of several cell types. We have analysed by immunohistochemistry and biochemically the expression of c-MET in benign and malignant melanocytic lesions. The Met/HGF receptor which in the melanocytic lineage displays the structural features of the authentic receptor was undetectable in tissue melanocytes and in nevocytic nevi. Only four out of 23 primary melanomas scored positive. Expression was increased to a significant level in 17 out of the 44 metastatic lesions examined. The c-MET expression was homogeneous in multiple metastases from the same patients. Comparative analyses showed both lack of correlation with the expression of the tumour progression associated ICAM-1 adhesion molecule and, in 23% of cases, co-expression with the c-KIT encoded receptor. These findings show that the c-MET gene is expressed at late stages of melanoma progression and suggest that the presence of Met/HGF receptor may contribute to the acquisition of an invasive phenotype.

SUBMITTER: Natali PG

PROVIDER: S-EPMC1968608 | biostudies-other | 1993 Oct

REPOSITORIES: biostudies-other

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Expression of the c-Met/HGF receptor in human melanocytic neoplasms: demonstration of the relationship to malignant melanoma tumour progression.

Natali P G PG Nicotra M R MR Di Renzo M F MF Prat M M Bigotti A A Cavaliere R R Comoglio P M PM

British journal of cancer 19931001 4

The c-MET proto-oncogene encodes the receptor for the Hepatocyte Growth Factor/Scatter Factor, which is known to mediate mitogenic, motogenic and invasive responses of several cell types. We have analysed by immunohistochemistry and biochemically the expression of c-MET in benign and malignant melanocytic lesions. The Met/HGF receptor which in the melanocytic lineage displays the structural features of the authentic receptor was undetectable in tissue melanocytes and in nevocytic nevi. Only four ...[more]

PMID: 8104462

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Project description:BackgroundSustained activation of hepatocyte growth factor (HGF)/c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression, but underlying mechanism is unclear. ArfGAP With SH3 Domain, Ankyrin Repeat And PH Domain 2 (ASAP2) can reportedly activate GTPases and promote receptor tyrosine kinase signaling. However, the exact role of ASAP2 in HCC, especially for c-MET activation, also remains elusive.MethodsASAP2 expression levels in HCC tissues and cells were quantified using qRT-PCR, western blot (WB) analysis, and immunohistochemistry staining. Cell counting kit-8 (CCK-8) and colony formation assays were performed to evaluate cell proliferation rates. Flow cytometry assays were conducted to assess apoptosis rates. Wound healing and Transwell assays were performed to determine cell migration and invasion capacities. Epithelial-mesenchymal transition (EMT)-related marker expression levels were also examined. Subcutaneous implantation and tail vein injection models were applied for in vivo growth and metastasis evaluations, respectively. Bioinformatics analyses of The Cancer Genome Atlas and STRING datasets were performed to explore ASAP2 downstream signaling. Co-immunoprecipitation and Cycloheximide chasing experiments were performed to assess protein-protein interactions and protein half-life, respectively.ResultsASAP2 had higher expression levels in HCC tissues than in normal liver, and also predicted poor prognosis. Knocking down ASAP2 significantly impaired cell proliferation, migration, and invasion capacities, but promoted apoptosis in HCC cells in vitro. However, overexpression of ASAP2 achieved the opposite effects. In vivo experiments confirmed that ASAP2 could promote HCC cell growth and facilitate lung metastasis. Interestingly, ASAP2 was essential for triggering EMT. Gene Set Enrichment Analysis demonstrated that c-MET signaling was greatly enriched in ASAP2-high HCC cases. Additionally, c-MET signaling activity was significantly decreased following ASAP knockdown, evidenced by reduced c-MET, p-AKT, and p-ERK1/2 protein levels. Importantly, ASAP2 knockdown effectively attenuated HGF/c-MET signaling-induced malignant phenotypes. c-MET and ASAP2 expression levels were positively correlated in our cohort. Mechanistically, ASAP2 can directly bind to CIN85, thereby disrupting its interaction with c-MET, and can thus antagonize CIN85-induced c-MET internalization and lysosome-mediated degradation. Notably, knocking down CIN85 can rescue the observed inhibitory effects caused by ASAP2 knockdown.ConclusionsThis study highlights the importance of ASAP2 in sustaining c-MET signaling, which can facilitate HCC progression.

Project description:Perturbations in microRNA (miRNA) expression profiles has been reported for cutaneous malignant melanoma (CMM). With regards to a rapidly growing number of newly discovered miRNA sequences, the availability of up-to-date miRNA expression profiles for primary cutaneous malignant melanoma (PCMM), cutaneous malignant melanoma metastases (CMMM) and benign melanocytic naevi (BMN) is limited. Patients with PCMM (n=9), CMMM (n=4) and BMN (n=8) were included in the study. Specimens were obtained during surgery from the center of the tumors (lesional). An exploratory microarray analysis was performed by miRNA expression profiling based on miRBase V.16. Additionally, the expression levels of selected miRNA candidates were confirmed by TaqMan real-time quantitative polymerase chain reaction (RT-PCR). New miRNA candidates previously not described to be associated with CMM were found to be potentially dysregulated in CMM. Nine patients with PCMM (PCMM1-PCMM9), four patients with CMMM (CMMM1-CMMM4) and eight patients with BMN (BMN1-BMN8) were enrolled in the present study. All cutaneous specimens were harvested in the operating room, immediately stored in RNAlater (Quiagen, Hilden, Germany) and kept at - 80 °C until RNA isolation. Total RNA including miRNAs was isolated using the miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA concentration and purity was determined by NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany). RNA integrity control (RIN) was determined by means of capillary electrophoresis with the 2100 Bioanalyzer and the RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA). To be further considered for Microarray analysis a RIN ≥ 7.0 indicating medium to good RNA quality was defined as inclusion criteria. For assessment of labeling and hybridization efficiencies total RNA samples were spiked with in-vitro synthesized oligonucleotides by using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA). A total of 100 ng total RNA per sample were introduced into a labeling reaction. The spiked total RNA was treated with alkaline calf intestine phosphatase (CIP). The dephosphorylated RNA was labeled with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturer´s instructions using T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (pCp). The Cyanine-3-labeled miRNA samples were prepared for One-Color based hybridization with Complete miRNA Labeling and Hyb Kit (Agilent Technologies, Santa Clara, USA) according to the manufacturer´s instructions. Labeled miRNA samples were hybridized at 55°C for 20 hrs on separate Human miRNA Microarrays Release 16.0 (8x60K format, (Agilent Technologies, Santa Clara, USA). Afterwards, microarrays were washed with increasing stringency using Gene Expression Wash Buffers (Agilent Technologies, Santa Clara, USA) followed by drying with acetonitrile (Sigma-Aldich, St.Louis, USA). Fluorescent signal intensities were detected with Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) on the Agilent DNA Microarray Scanner and extracted from the images using Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA). All the steps described were carried out according to the manufacturer´s instructions.

Dataset Information

Expression of the c-Met/HGF receptor in human melanocytic neoplasms: demonstration of the relationship to malignant melanoma tumour progression.

Publications

Expression of the c-Met/HGF receptor in human melanocytic neoplasms: demonstration of the relationship to malignant melanoma tumour progression.

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