Heme Oxygenase-1 Overexpression Promotes Uveal Melanoma Progression and Is Associated with Poor Clinical Outcomes

Abstract

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. To date, the main strategies to counteract its progression consist of focal radiation on the tumor site and ocular enucleation. Furthermore, many UM patients develop liver metastasis within 10 years following diagnosis, eventually resulting in a poorer prognosis for those patients. Dissecting the molecular mechanism involved in UM progression may lead to identify novel prognostic markers with significative clinical applications. The aim of the present study was to evaluate the role of Heme Oxygenase 1 (HO-1) in regulating UM progression. UM cell lines (92.1) were treated with Hemin (CONC e time), a strong inducer of HO-1, and VP13/47, a selective inhibitor of its enzymatic activity. Interestingly, our results showed an enhanced 92.1 cellular proliferation and wound healing ability following an HO-1 increase, overall unveiling the role played by this protein in tumor progression. Similar results were obtained following treatment with two different CO releasing molecules (CORM-3 and CORM-A1). These results were further confirmed in a clinical setting using our UM cohort. Our results demonstrated an increased median HO-1 expression in metastasizing UM when compared to nonmetastasizing patients. Overall, our results showed that HO-1 derived CO plays a major role in UM progression and HO-1 protein expression may serve as a potential prognostic and therapeutical factor in UM patients.

Keywords: cancer; carbon monoxide; heme oxygenase; proliferation; uveal melanoma.

Figure 1

Hemin, CORM-A1, and CORM-3 trigger HO-1 accumulation. (A) Hemin (10 μM) increases HO-1 mRNA levels. qPCR analysis showing the increase in HO-1 mRNA levels upon hemin supplementation 3, 6, and 12 h post-treatment. (B) CORM-A1 (25 μM) induces HO-1 expression. HO-1 increases its expression 6, 12, and 24 h post-treatment. (C) CORM-3 (25 μM) leads to HO-1 overexpression. HO-1 expression 3, 6, and 12 h post-treatment increases post CORM-3 treatment. (D) Hemin, CORM-A1, and CORM-3 increase HO-1 protein levels. HO-1 accumulates when 92.1 are treated with different concentrations of Hemin, CORM-A1, and CORM-3. Each result represents the average of four different replicas (mean ± SD). p values < 0.05 were statistically significant (** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. 0 h and untreated).

Figure 2

Hemin, CORM-A1, and CORM-3 increase 92.1 proliferation and number of colonies. (A) Hemin (10 μM), (B) CORM-A1 (25 μM), and (C) CORM-3 (25 μM) enhance UM proliferation. xCELLigence assay monitoring real-time 92.1 cell proliferation upon Hemin, CORM-A1, and CORM-3 supplementation, showing an enhanced cellular proliferation already 16, 24, and 20 h post-treatment, respectively Interestingly, VP 13/47 addition (50 μM) disrupts hemin-, CORM-A1-, and CORM-3-, induced proliferation. Each line is expressing the average of four different experiments (mean ± SD). p values < 0.05 were statistically significant. (D) Clonogenic assay on 92.1 supplemented with Hemin, CORM-A1, and CORM-3. Colony forming ability of 92.1 cells was assayed by Clonogenic assay. (E) Hemin (10 μM), (F) CORM-A1 (25 μM), and (G) CORM-3 (25 μM) enhance 92.1 number of colonies. Clonogenic assay quantification unveiled an increased 92.1 number of colonies following Hemin, CORM-A1, and CORM-3 treatment for 9 days. Of note, VP 13/47 administration (50 μM) disrupted the 92.1 colony forming ability. Each result is representative of four different replicas (mean ± SD). p values < 0.05 were statistically significant (*** p < 0.001; **** p < 0.0001 vs. untreated).

Figure 3

Hemin and CORMs increase 92.1 wound healing ability. (A) 92.1 supplemented with Hemin (10 μM), CORM-A1 (25 μM), and CORM-3 (25 μM) increase their wound closure speed. The differences are already visible 24 h post-treatment. (B) VP 13/47 (50 μM) impairs Hemin- and CORMs- induced 92,1 wound closures. The opening of the wound is drastically affected by VP 13/47 supplementation, eventually hampering hemin, CORM-A1, and CORM-3-induced closure. Quantification of the percentage of wideness resulting from (C) Hemin (10 μM), (D) CORM-A1 (25 μM), and (E) CORM-3 (25 μM) supplementation. The three molecules, once administered, increase 92.1 wound closure. Their effect is disrupted by VP 13/47 (50 μM) addition. Each result is representative of four different replicas (mean ± SD). p values < 0.05 were statistically significant **** p < 0.0001 vs. untreated. ^#### p < 0.0001 vs. (C) Hemin, (D) CORM-A1, and (E) CORM-3).

Figure 4

CORMs (24 μM for 24 h) supplementation increases 92.1 mitochondrial fitness. (A–K) qPCR analysis unveils an increased expression of genes involved in UM mitochondrial fitness. (A) PGC1α, (B) SIRT1, (C) TFAM, (D) FIS1, (E) OPA1, (F) COX II, (G) COX IV, (H) CytB (I) ND4, (L) Ndufa6, and (M) ATP syn show an increase in expression upon CORM-A1 and CORM-3 supplementation. Each result is representative of four different replicas (mean ± SD). p values < 0.05 were statistically significant (* p< 0.05; ** p<0.01; *** p < 0.001; **** p < 0.0001 vs. untreated).

Figure 5

O-1 accumulation correlates with higher-risk UM. (A) Immunohistochemical expression of HO-1 anticorrelates with p16 expression in an epithelioid cell-type UM. HO-1 expression increases with the progression of this tumor, as showed by the anticorrelation with the tumor suppressor protein p16. (immunoperoxidase; original magnification 200×). (B) Kaplan–Meier survival curves in UM patients. Disease-free survival decreases when HO-1 is accumulating.