DNp73-induced degradation of tyrosinase links depigmentation with EMT-driven melanoma progression

Highlights

• In invasive melanoma tumors with DNp73 overexpression, depigmentation occurs.

• Hypopigmentation is due to tyrosinase ER-arrest and proteosomal degradation.

• Tyrosinase degradation is a consequence of an active DNp73/IGF1R/PI3K/AKT pathway.

• Tyrosinase loss triggers EMT marker expression and invasiveness.

• A DNp73/IGF1R/Slug signature is a potential marker of progression-related MAH.

Abstract

Melanoma is an aggressive cancer with poor prognosis, requiring personalized management of advanced stages and establishment of molecular markers. Melanomas derive from melanocytes, which specifically express tyrosinase, the rate-limiting enzyme of melanin-synthesis. We demonstrate that melanomas with high levels of DNp73, a cancer-specific variant of the p53 family member p73 and driver of melanoma progression show, in contrast to their less-aggressive low-DNp73 counterparts, hypopigmentation in vivo. Mechanistically, reduced melanin-synthesis is mediated by a DNp73-activated IGF1R/PI3K/AKT axis leading to tyrosinase ER-arrest and proteasomal degradation. Tyrosinase loss triggers reactivation of the EMT signaling cascade, a mesenchymal-like cell phenotype and increased invasiveness. DNp73-induced depigmentation, Slug increase and changes in cell motility are recapitulated in neural crest-derived melanophores of Xenopus embryos, underscoring a previously unnoticed physiological role of tyrosinase as EMT inhibitor. This data provides a mechanism of hypopigmentation accompanying cancer progression, which can be exploited in precision diagnosis of patients with melanoma-associated hypopigmentation (MAH), currently seen as a favorable prognostic factor. The DNp73/IGF1R/Slug signature in colorless lesions might aid to clinically discriminate between patients with MAH-associated metastatic disease and those, where MAH is indeed a sign of regression.

Introduction

Melanoma is a skin cancer type originating from melanocytes, that produce melanin-synthetic enzymes, mainly tyrosinase (TYR), as well as its downstream-acting enzymes tyrosinase-related protein 1 (TRP-1) and dopachrome tautomerase (DCT) [1], which lead to step-wise conversion of tyrosine to melanin. Tyrosinase, a type I membrane-bound melanosomal glycoprotein enzyme, catalyzes hydroxylation of l-tyrosine and oxidation of l-DOPA, the rate limiting step of the melanin synthesis cascade. Thus, it prevents long-term accumulation of the melanin precursors that act as deleterious reactive oxygen species (ROS) and uses them as substrates to produce melanin, protecting cells from UVR-induced damage [2].

Correct protein folding and post-translational processing of tyrosinase ensures enzyme stability and activity. To this end, the 60-kDa core polypeptide is transported cotranslationally to the endoplasmic reticulum (ER). There, it undergoes addition of N-linked glycans, producing a 70-kDa immature form. The N-linked glycans are trimmed by ER glucosidases I and II, and the resulting monoglucosylated oligomannosidic glycans interact with ER-resident chaperone molecules for correct folding. At this stage, the immature forms are sensitive to endoglycosidase H (Endo-H), which cleaves asparagine-linked mannose-rich oligosaccharides added in the ER, but not the complex oligosaccharides from glycoproteins that are added later in the Golgi apparatus. In Golgi, the oligomannosidic N-linked glycans are processed to complex glycans, which finally produce an 80-kDa protein, resistant to Endo-H digestion. ER-retention followed by accelerated degradation of the immature, misfolded forms has been described for albinos who bear loss-of-function TYR mutant variants [3,4]. However, ER-arrest and proteasome degradation has been also reported for wild-type tyrosinase in some melanoma cell lines [5].

Tyrosinase is specifically expressed, together with other melanocytic-differentiation antigens (MDAs), in normal and malignant melanocytes [6], is highly immunogenic and provokes immune responses against melanocytes, which sometimes lead to spontaneous disease regression. Immunological destruction of malignant melanocytes may cause hypopigmentation in a percentage of melanoma patients and is interpreted as favorable clinical sign of regression [7]. Nevertheless, there are hints that depigmentation is not in all cases a sign of melanocyte destruction by tyrosinase-boosted anti-melanoma cascades. Instead, depigmentation may indicate acquisition of invasive features [[8], [9], [10]]. An inverse correlation between the expression of tyrosinase and other MDAs and epithelial-mesenchymal transition (EMT) markers has been observed, where cells with low MDA levels demonstrate a mesenchymal-like phenotype and epithelial-like cells have high MDAs [11]. This leads to the hypothesis that EMT activation during melanoma progression might be facilitated by loss of expression and/or activity of MDA(s), however, comprehensive evidence is missing.

Melanoma progression is causatively associated with alterations in the TP73 gene, a functional and structural homologue of the p53 tumor suppressor [12,13]. TP73 synthesizes the transactivation-competent, anti-oncogenic TAp73 isoforms which derive from an external (P1) promoter; and the oncogenic DNp73 isoforms, which lack an intact transactivation domain. DNp73 further encompass the ΔNp73 isoforms arising from an internal (P2) promoter, and the ΔTAp73 isoforms derived from P1 after aberrant splicing in the N terminus [14,15]. ΔN's are also detected in normal tissues, while the ΔΤΑ’s are expressed exclusively in tumors and consistently predict poor patient outcomes [16]. We and others have demonstrated that the expression pattern of p73 isoforms is altered in melanoma, favoring upregulation of a specific splice-variant of the ΔTA class, namely p73ΔEx2/3 (hereafter also called DNp73) [17,18].

We have previously documented that DNp73 (p73ΔEx2/3) overexpression is a key event of melanoma progression, which independently from early driver mutations induces a mesenchymal phenotype switch via EMT activation [12], as well as pluripotency and stemness-like characteristics [13]. In the context of these studies, we further observed that when DNp73-overexpressing melanoma cells were injected into mice, they developed depigmented tumors. This co-existence of aggressive tumor features together with depigmentation in the same tumor intringued us to address pigmentation relative to invasion and shed light on a potential connection between EMT and tyrosinase in the context of aggressive melanomas. Herein, we show that DNp73 (p73ΔEx2/3) enhances tyrosinase ER-arrest and posttranslational degradation in an IGF1R-dependent manner. Loss of active tyrosinase supports EMT, leading to depigmented, invasive and melanoblast-like cells.