019 | PDE5A promotes hypoxic hepatocellular carcinoma cell survival through ERK-dependent signalling

Lara Lizzi, Alberto Massimi, Mara Massimi | Dept. of Life, Health and Environmental Sciences, University of L’Aquila, Italy.

Hypoxia is a common feature of hepatocellular carcinoma (HCC) and shapes tumour behaviour by selecting for cells able to withstand low oxygen availability, redox imbalance, and nutrient limitation [1]. Although hypoxia-driven signalling is typically studied through HIF-dependent transcriptional programs, less is known about how cyclic nucleotide pathways contribute to these adaptations. Phosphodiesterase-5A (PDE5A), a key cGMP-hydrolyzing enzyme and the pharmacological target of sildenafil, is increasingly implicated in cancer biology, but its possible role in supporting HCC cell survival under hypoxic stress remains unclear. Under physiological conditions, PDE5A expression in the liver is restricted to the centrilobular zone, a naturally hypoxic region; conversely, HCC displays marked PDE5A upregulation with loss of zonal specificity, consistent with a functional role in tumour adaptation to low oxygen tension. Here, we investigated PDE5A regulation and function in HCC cell lines (HepaRG, HepG2, Huh7) exposed to hypoxia (1% O₂), hypoxia-mimicking conditions (CoCl₂, deferoxamine), and oxidative stress (H₂O₂). Across models, both real and simulated hypoxia significantly increased PDE5A protein expression and activity, while mitochondrial ROS-driven oxidative stress further enhanced PDE5A levels, indicating a dual regulatory mechanism involving both HIF activation and redox-dependent signals. Hypoxia was validated by HIF-1α stabilization and increased lactate dehydrogenase activity. More aggressive cell lines exhibited the highest PDE5A activity and expression, consistent with an association between PDE5A levels and malignant features. To strengthen the mechanistic link between hypoxia-responsive transcriptional programs and PDE5A, in silico analyses (TCGA-LIHC/GEPIA2) revealed positive correlations among HIF1A, SP1, and PDE5A expression, and motif scanning (FIMO) identified multiple high-confidence Sp1 binding sites within the PDE5A genomic region [2]. Consistent with this model, pharmacological inhibition of Sp1 with mithramycin reduced PDE5A expression under hypoxia, supporting a hypoxia-Sp1-PDE5A regulatory axis. To assess PDE5A’s role in hypoxia-mediated survival signalling, we inhibited PDE5A with sildenafil during hypoxia. PDE5A inhibition led to a clear reduction in MAPK/ERK pathway activation compared with hypoxia alone, accompanied by increased expression of pro-apoptotic markers, including BAX. Taken together, our data indicate that PDE5A supports hypoxic HCC cell survival by sustaining ERK-dependent pro-survival signaling, whereas PDE5A blockade shifts the balance toward apoptosis. These findings position PDE5A as a relevant mediator of hypoxia-driven adaptation in HCC and a potential therapeutic vulnerability to disrupt stress tolerance in the hypoxic tumour compartment. Ongoing work using CRISPR/Cas9-mediated PDE5A knockout will test causality and refine the PDE5A-ERK-apoptosis framework.