Collagen prolyl 4-hydroxylase subunit α member-induced head and neck squamous cell carcinoma aggressiveness is antagonized by LLGL2 via reduced expression of occludin

Abstract

There are three isoforms of human collagen prolyl 4-hydroxylases (C-P4Hs), each of which has been reported to play an important role in regulating the progression of a variety of human cancers. By analyzing TGCA datasets on human head and neck squamous cell carcinoma (HNSC), we find that a higher expression of all three C-P4HAs (the α subunit of C-P4Hs) is a superior prognostic indicator than a higher expression of two or a single C-P4HA. Unexpectedly, some patients with higher levels of three C-P4HAs survive longer than patients whose tumors have lower expression of C-P4HAs. Therefore, there may be molecule(s) that can negate the deleterious effects of overexpressing C-P4HAs during cancer progression. By constructing a functional protein interaction network of C-P4HAs and analyzing molecules whose expressions are correlated significantly with that of C-P4HAs, we identify scribble cell polarity complex component 2 (LLGL2) as a factor that antagonizes the effects of overexpressed C-P4HAs on HNSC. Silencing of LLGL2 in the human oral squamous cell line Cal-27 upregulates the expression of occludin and increases cancer cell invasion and migration. In contrast, knocking down C-P4HA alone inhibits cell migration and invasion. Furthermore, simultaneously downregulating three C-P4HAs has more pronounced effects on inhibiting cell migration and invasion. Accordingly, high LLGL2 expression is also a marker indicating improved prognosis in patients with HNSC. These results suggest that the interplay between LLGL2 and C-P4HAs may be targeted to mitigate HNSC tumorigenesis and progression.

1. Introduction

Head and neck squamous cell carcinoma (HNSC) accounted for approximately 870,000 new cases and 440,000 deaths worldwide in 2020 [1]. It includes cancers of the oral cavity (40%), pharynx (25%), and larynx (15%) [2]. The prognosis of patients with HNSC is generally poor, especially for those with recurrent or metastatic disease, and treatment options for these patients are limited. Therefore, a better understanding of the biology of HNSC may reveal novel targets for therapeutic interventions.

Histologically, HNSC is encased by a dense extracellular matrix, which is mainly composed of collagens in which the 4-hydroxyproline residues are essential for the stability of the collagen triple helix. In mammalian cells, three collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the hydroxylation of proline to 4-hydroxyproline at the Y position in the glycine-X-Y motif of the α chain. C-P4Hs are tetramers composed of two identical α chains and two identical β chains. The enzymatic activity resides in the α chain, and the chaperone activity is in the β chain. Although the catalytic properties of the isoenzymes are very similar, each isoform appears to have distinct peptide substrate binding properties and sensitivity to inhibitors. Additionally, the glycine-X-Y motif is also present in noncollagenous proteins; these proteins may be substrates of C-P4Hs [3,4].

Accumulating evidence shows that α subunits of C-P4Hs (C-P4HAs) are overexpressed in a variety of human cancers and contribute to cancer progression [5–7]. In addition to playing a role in collagen maturation and secretion [5], C-P4HAs have been reported to play other roles in cancer biology. For example, they have been reported to stimulate glycolysis in some tumor cells, which is important in tumor cell biology [8]. C-P4HA is also known to adjust the stability of hypoxia-inducible factor (HIF) and fine-tune the demethylase activity of Jumonji (JMJ), a histone demethylase, and ten-eleven translocation (TET), which modifies DNA by oxidizing 5-methylcytosine (5mC) in breast cancer. In addition, C-P4HA has been reported to alter the hydroxylation of proline on Carabin, an endogenous calcineurin inhibitor, in B-cell lymphoma [9–11]. Therefore, suppressing the activity of C-P4HAs has been considered a strategy to