The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior

Abstract

Overactivation of the c-MET/HGF system is a feature of many cancers. We previously reported that type II testicular germ cell tumor (TGCT) cells express the c-MET receptor, forming non-seminomatous lesions that are more positive compared with seminomatous ones. Notably, we also demonstrated that NT2D1 non-seminomatous cells (derived from an embryonal carcinoma lesion) increase their proliferation, migration, and invasion in response to HGF. Herein, we report that HGF immunoreactivity is more evident in the microenvironment of embryonal carcinoma biopsies with respect to seminomatous ones, indicating a tumor-dependent modulation of the testicular niche. PI3K/AKT is one of the signaling pathways triggered by HGF through the c-MET activation cascade. Herein, we demonstrated that phospho-AKT increases in NT2D1 cells after HGF stimulation. Moreover, we found that this pathway is involved in HGF-dependent NT2D1 cell proliferation, migration, and invasion, since the co-administration of the PI3K inhibitor LY294002 together with HGF abrogates these responses. Notably, the inhibition of endogenous PI3K affects collective cell migration but does not influence proliferation or chemotactic activity. Surprisingly, LY294002 administered without the co-administration of HGF increases cell invasion at levels comparable to the HGF-administered samples. This paradoxical result highlights the role of the testicular microenvironment in the modulation of cellular responses and stimulates the study of the testicular secretome in cancer lesions.

ERK Signaling Pathway Is Constitutively Active in NT2D1 Non-Seminoma Cells and Its Inhibition Impairs Basal and HGF-Activated Cell Proliferation

“…In this cell line, we already demonstrated that c-Src and phosphatidylinositol 3-kinase (PI3K) pharmacological inhibition abrogates the HGF-dependent increase of cell proliferation, polarized and collective migration, as well as cell invasion. We also found that, surprisingly, c-Src or PI3K pharmacological inhibition, when administered in basal culture conditions, increases NT2D1 invasiveness via an HGF-independent way, highlighting the importance of the microenvironmental cues in modulating cellular responses to pharmacological stimuli [5,8].…”

“…NT2D1 embryonal carcinoma cells (American Type Culture Collection (ATCC) were used in this study and cultured as reported in [4,5,8]. Cells, cultured in DMEM (Sigma Aldrich, cat.…”

“…An amount of 9 × 10 4 NT2D1 were cultured and treated with HGF and U0126 for 24 h. After that, samples were fixed in glutaraldehyde 2.5% overnight (O.N.). Sample preparation was described in detail in [5]. Supra 40 FESEM (Zeiss, Jena, Germany) (Department.…”

“…In previous papers we analyzed c-MET and HGF expression in biopsies derived from patients affected by testicular germ cell tumors (TGCTs), concluding that non-seminomas (embryonal carcinoma, yolk sac tumor, and teratoma) present the highest expression level of this receptor on the plasma membrane [4] and, besides this, embryonal carcinoma has the highest expression of HGF in the tumor microenvironment with respect to seminoma lesions [5]. We studied c-MET and HGF expression even in seminoma and non-seminoma derived cell lines (TCam-2, NCCIT, NT2D1), demonstrating that HGF is not expressed by all these cell lines [4].…”

Oncofetal reprogramming in tumor development and progression: novel insights into cancer therapy

“…The modulation of cell cannibalism is determined by Jun N‐terminal kinase, EMT, and stem cell‐like markers, which lead to the activation of deceleration programs and drug resistance in vitro 178–180 . In addition, CAF‐derived secretory proteins, such as hepatocyte growth factor (HGF), the ligand of MET, trigger the activation of the PI3K–AKT signaling pathway 181–183 . This pathway endows resistance to BRAF inhibition in melanoma, colorectal, and glial tumor cells 184–187 .…”