Study of the role of neuropilin-1 in the development, prognosis and cellular response to lenvatinib in human hepatocellular carcinoma

Resumen

Hepatocellular carcinoma (HCC), the main type of primary liver cancer, constitutes the sixth most common and third most deadly cancer worldwide, respectively. Due to the late diagnosis and the molecular heterogeneity of this tumor, the clinical onset of HCC patients remains complex, with a high rate of therapeutic failure and recurrence. Although novel findings have been accomplished in recent years, the precise mechanisms underlying the processes of tumor development, progression and drug responsiveness remain unclear. In this line, autophagy, responsible for the maintenance of cell homeostasis, acts as a double-edged process in cancer, displaying a key function in the progression and cellular response to targeted drugs. Furthermore, increasing evidence highlights the crucial role of the hypoxic microenvironment in the chemoresistance development in solid tumors, and mainly in HCC, where the hypoxia-inducible factor 1α (HIF-1α) acts as the main mediator of the cellular response to hypoxia. The interplay between these mechanisms is closely associated to the therapeutic use of tyrosine kinase inhibitors (TKIs), such as lenvatinib, that primarily act by disrupting tumor angiogenesis through inhibition of tyrosine kinase receptors (RTKs) activity. The transmembrane glycoprotein neuropilin-1 (NRP1) has exhibited an interesting function in the modulation of tumor-associated signaling pathways and processes by interacting with key RTKs, such as vascular endothelial growth factor receptor (VEGFR) or platelet-derived growth factor receptors (PDGFR) among others, and their corresponding ligands. Despite the interesting role that NRP1 seems to have in cancer cell survival and drug efficacy, fewer studies have evaluated the potential value of NRP1 as a tumor biomarker or therapeutic target in HCC. For this reason, we aimed at assessing the clinical significance of the receptor NRP1 in the prognosis, diagnosis and other tumor-associated features in HCC patients, as well as to determine the role of NRP1 in the underlying mechanisms of lenvatinib efficacy with potential implication in the loss of cellular sensitivity to lenvatinib. To accomplish these purposes, we conducted a systematic review with meta-analysis including all the articles that evaluate the clinical correlation of NRP1 overexpression with tumor prognosis, development and/or other clinicopathological features in patients diagnosed with HCC. On the other hand, we confirmed these results using datasets of human HCC samples from publicly available databases, and we also assessed the potential role of NRP1 in the lenvatinib efficacy employing three HCC cell lines, HepG2, Hep3B and Huh-7 as the in vitro model for the experimental study. Initially, a total of 1305 patients from seven articles were included in the quantitative analysis, where approximately 53.81% of patients exhibited NRP1 overexpression. After meta-analysis, increased levels of NRP1 showed to be significantly correlated with poor prognosis, represented by shorter overall survival (OS), as well as with tumor pathogenesis by observing higher NRP1 expression in tumor tissue samples from 692 HCC patients. In addition, a significant association was found between NRP1 overexpression and patient’s age, younger than 50 years old, and increased risk of venous invasion, highlighting the potential of NRP1 as a tumor biomarker in HCC. Meanwhile, results from the experimental study showed higher NRP1 levels in HCC samples from public databases compared to normal liver tissue, and a strong correlation with advanced tumor stages and nodal metastasis status. When analyzed in an in vitro model of HCC, NRP1 was overexpressed in the Hep3B and Huh-7 cell lines in comparison to the HepG2 HCC line, also showing a higher susceptibility to lenvatinib the Hep3B and Huh-7 cells. Analysis of cell proliferation and migration revealed that NRP1 downregulation derived from lenvatinib treatment was involved in the antitumor effects of this drug. To deeply evaluate the potential role of NRP1 in the underlying mechanisms of the lenvatinib effectiveness, we further analyzed the modulation exerted by lenvatinib in NRP1 expression. The double-edged process of autophagy was revealed as the main mechanism responsible for the lenvatinib-derived downregulation of NRP1 in the Hep3B and Huh-7 cell lines, where autophagy blockade restrained the lenvatinib efficacy, being prevented by specific NRP1 targeting. Furthermore, after hypoxia induction, protein levels of NRP1 were significantly diminished through a hypoxia-related autophagy increase. Interestingly, NRP1 expression was also modulated by HIF-1α, showing lower levels of NRP1 after HIF-1α silencing. Even though autophagy blockade increased cell survival even after hypoxia induction and lenvatinib treatment, gene silencing of HIF-1α achieved to prevent this loss of lenvatinib effectiveness and the recovery of protein NRP1 expression in Hep3B and Huh-7 cell lines. Altogether, results from this study suggest that NRP1 might constitute a valuable biomarker for prognosis, diagnosis and risk of invasion in patients with HCC, together with the potential role that NRP1 seems to play in the underlying mechanisms of the lenvatinib efficacy in HCC cells, where both the double-edged autophagy and the HIF-1α-related hypoxia response are involved. Therefore, NRP1 could be a novel tumor biomarker and therapeutic target for improving the clinical landscape of advanced HCC.