Protumorigenic Role of HAPLN1 and Its IgV Domain in Malignant Pleural Mesothelioma

Abstract

Purpose Tumor extracellular matrix (ECM) plays a crucial role in cancer progression mediating and transforming host-tumor interactions. Targeting the ECM is becoming an increasingly promising therapeutic approach in cancer treatment. We find that one of the ECM proteins, HAPLN1, is overexpressed in the majority of mesotheliomas. This study was designed to characterize the protumorigenic role of HAPLN1 in mesothelioma. Experimental Design Overexpression of HAPLN1was assessed and validated on a large set of normal/mesothelioma specimens on the RNA and protein levels. We also analyzed DNA copy number alterations in the HAPLN1 genomic locus using the array-based comparative genomic hybridization representational oligonucleotide microarray analysis tool. Tumorigenic activities of the HAPLN1 domains were evaluated in vitro on mesothelioma cells transfected with HAPLN1-expressing constructs. Results We found that HAPLN1 is 23-fold overexpressed in stage Imesothelioma and confirmed it for 76% samples (n = 53) on RNA and 97% (n = 40) on protein levels. The majority of lung cancers showed no differential expression of HAPLN1. Analysis of DNA copy number alterations identified recurrent gain in the 5q14.3 HAPLN1 locus in ~27% of tumors. Noteworthy, high expression of HAPLN1negatively correlated with time to progression (P = 0.05, log-rank test) and overall survival (P = 0.006). Proliferation, motility, invasion, and soft-agar colony formation assays on mesothelioma cells overexpressing full-length HAPLN1 or its functional domains strongly supported the protumorigenic role of HAPLN1 and its SP-IgV domain. Conclusion Overexpression of HAPLN1 and its SP-IgV domain increases tumorigenic properties of mesothelioma. Thus, targeting the SP-IgV domain may be one of the therapeutic approaches in cancer treatment.

Pro-tumorigenic Effects of miR-31 Loss in Mesothelioma

“…Transfection media were removed after 4 h of incubation and replaced with fresh Dulbecco's modified Eagle's medium, 10% fetal bovine serum medium. 48 h post-transfection, miR-31-transfected cells were trypsinized, counted, and assayed for proliferation, colony formation in soft agar, wound closure, and Matrigel invasion in triplicate experiments as described previously (16,17).…”

“…Validation of Putative miR-31 Targets-For clinical validation of putative miR-31 targets, we used our U133 Plus 2.0 Affymetrix expression array data from 32 MM and 7 normal peritoneal specimens as described previously (10,16). Western analysis was performed with antibodies to PPP6C (NB100-2883) and MCM2 H00004171-M01) from Novus Biologicals (Littleton, CO).…”

“…Regulation-To evaluate the clinical significance of KEGG Cell Cycle genes suppressed by miR-31 introduction, we assessed their expression in our collection of 32 specimens of MM and 7 healthy peritoneum controls, which were used in our previous studies (10,16). This comparison showed that half of the proposed miR-31 targets are, indeed, up-regulated in MM (see column 5 in Table 1) as compared with normal mesothelium and may therefore be important for tumor progression.…”

TrkC signaling is activated in adenoid cystic carcinoma and requires NT-3 to stimulate invasive behavior

“…Cell motility, migration, invasion and colony formation in soft agar Scratch (wound healing) assay was performed as described, 69 following the inhibition of proliferation using mitomycin C at 5 mg/ml. Migration and invasion assays used 8.0 mm polycarbonate membrane inserts for 24-well plates (BD Biosciences, Franklin Lakes, NJ, USA).…”

Acquisition of useful sero-diagnostic autoantibodies using the same patients’sera and tumor tissues

“…Takashima et al reported proteomic analysis of AAbs in patients with hepatocellular carcinoma using autologous tissues and sera, but did not focus on AAbs derived from a tumor-associated stroma (28). Solid tumors are composed of neoplastic cells and surrounding tumor-associated stroma, and the latter is essential for the survival and proliferation of tumor cells (10). The invasion of cancer cells into surrounding tissues is the hallmark of malignancy, and the invasion process is associated with the marked destruction and regeneration of stromal elements, composed of inflammatory cells, including lymphocytes, granulocytes, and macrophages, vascular endothelial cells and pericytes, and fibroblasts (9).…”