Aspartate-β-hydroxylase and hypoxia marker expression in head and neck carcinomas: implications for HPV-associated tumors

Abstract

Background: A proportion of head and neck carcinomas (HNSCCs) are induced by high-risk human papillomaviruses (HPVs) and are associated with better patient outcomes compared to patients with HNSCCs related to tobacco and alcohol abuse. In the microenvironment of solid tumors, including HNSCCs, oxygen levels are often reduced, and a hypoxic state is induced. This can lead to a poor treatment response and a worse patient prognosis. One of the hypoxia-responsive genes is aspartate-β-hydroxylase (ASPH), whose activity promotes the growth, invasiveness, and metastasis of many types of solid tumors.

Methods: In our study, HNSCC samples were analyzed for the expression of ASPH and selected endogenous hypoxia markers by real-time PCR and/or multiplex fluorescence immunohistochemistry.

Results: Except for the EPAS1 gene, which had higher mRNA expression in the HPV-negative group of HNSCC (p < 0.05), we found no other differences in the expression of the tested genes that were related to HPV status. On the contrary, a statistically significantly higher number of cells producing ASPH (p < 0.0001), HIF1A (p < 0.0001), GLUT1 (p < 0.0001), and MMP13 (p < 0.05) proteins were detected in the HPV-positive tumor group than in the HPV-negative sample group. All the evaluated markers, except for MMP9/13, were more abundant in the tumor parenchyma than in the tumor stroma. The Cox proportional hazard models showed that increased numbers of cells with GLUT1 and HIF1A protein expression were positive prognostic markers for overall and disease-specific survival in patients independent of HPV tumor status.

Conclusion: The study examined HNSCC samples and found that elevated ASPH and hypoxia marker proteins, typically associated with poor prognosis, may actually indicate active HPV infection, the strongest prognostic factor in HNSCC patients. In cases where HPV status is uncertain, increased expression of HIF1A and GLUT1 can serve as positive prognostic factors.

Keywords: Aspartate-β-hydroxylase; Head and neck cancer; Human papillomavirus; Hypoxia; Prognosis.

Fig. 1

mRNA expression in groups of HPV-positive (HPV+) and HPV-negative (HPV−) HNSCCs. The median values are indicated, the box borders show the upper and lower quartiles, the whiskers show the variability, and outliers are indicated. *p < 0.05

Fig. 2

Protein markers detected by mIHC in the parenchyma and stroma of HPV-positive (HPV+) and HPV-negative (HPV−) HNSCCs. Graphs show the number of cells producing (A) ASPH, HIF1A, GLUT1, and MMP13, and (B) VEGFA and MMP9. The median values are indicated; the box borders show the upper and lower quartiles; the whiskers show the variability, and outliers are indicated. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Correlation of the number of cells producing ASPH and hypoxia markers in the parenchyma (A) and the stroma (B) of HNSCCs. The viral etiology of the tumors is indicated by the color. Spearman correlation coefficient is determined for the whole cohort of HNSCCs (Corr), and separately according to viral etiology of tumors (HPV+, HPV−). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Association of ASPH and hypoxia markers in the parenchyma (A) and the stroma (B) of HNSCCs according to HPV etiology of tumors and patient outcomes