Radiation-Induced DNA Damage in Uveal Melanoma Is Influenced by Dose Delivery and Chromosome 3 Status.

Purpose: The purpose of this study was to analyze the extent of DNA breaks in primary uveal melanoma (UM) with regard to radiotherapy dose delivery (single-dose versus fractionated) and monosomy 3 status. Methods: A total of 54 patients with UM were included. Stereotactic radiotherapy (SRT) was performed in 23 patients, with 8 undergoing single-dose SRT (sdSRT) treatment and 15 receiving fractionated SRT (fSRT). DNA breaks in the enucleated or endoresected tumors were visualized by a TUNEL assay and quantified by measuring the TUNEL-positive area. Protein expression was analyzed by immunohistochemistry. Co-detection of chromosome 3 with proteins was performed by immuno-fluorescent in situ hybridization. Results: The amount of DNA breaks in the total irradiated group was increased by 2.7-fold (P < 0.001) compared to non-irradiated tissue. Tumors treated with fSRT were affected more severely, showing 2.1-fold more DNA damage (P = 0.007) compared to the cases after single (high) dose irradiation (sdSRT). Monosomy 3 tumors showed less DNA breaks compared to disomy 3 samples (P = 0.004). The presence of metastases after radiotherapy correlated with monosomy 3 and less DNA breaks compared to patients with non-metastatic cancer in the combined group with fSRT and sdSRT (P < 0.05). Conclusions: Fractionated irradiation led to more DNA damage than single-dose treatment in primary UM. As tumors with monosomy 3 showed less DNA breaks than those with disomy 3, this may indicate that they are less radiosensitive, which may influence the efficacy of irradiation.

Quercetin Impairs the Growth of Uveal Melanoma Cells by Interfering with Glucose Uptake and Metabolism.

Monosomy 3 in uveal melanoma (UM) increases the risk of lethal metastases, mainly in the liver, which serves as the major site for the storage of excessive glucose and the metabolization of the dietary flavonoid quercetin. Although primary UMs with monosomy 3 exhibit a higher potential for basal glucose uptake, it remains unknown as to whether glycolytic capacity is altered in such tumors. Herein, we initially analyzed the expression of n = 151 genes involved in glycolysis and its interconnected branch, the "pentose phosphate pathway (PPP)", in the UM cohort of The Cancer Genome Atlas Study and validated the differentially expressed genes in two independent cohorts. We also evaluated the effects of quercetin on the growth, survival, and glucose metabolism of the UM cell line 92.1. The rate-limiting glycolytic enzyme PFKP was overexpressed whereas the ZBTB20 gene (locus: 3q13.31) was downregulated in the patients with metastases in all cohorts. Quercetin was able to impair proliferation, viability, glucose uptake, glycolysis, ATP synthesis, and PPP rate-limiting enzyme activity while increasing oxidative stress. UMs with monosomy 3 display a stronger potential to utilize glucose for the generation of energy and biomass. Quercetin can prevent the growth of UM cells by interfering with glucose metabolism.

Monosomy-3 Alters the Expression Profile of the Glucose Transporters GLUT1-3 in Uveal Melanoma.

Monosomy-3 in uveal melanoma (UM) cells increases the risk of fatal metastases. The gene encoding the low-affinity glucose transporter GLUT2 resides on chromosome 3q26.2. Here, we analyzed the expression of the glucose transporters GLUT1, GLUT2, and GLUT3 with regard to the histological and clinical factors by performing immunohistochemistry on the primary tumors of n = 33 UM patients. UMs with monosomy-3 exhibited a 57% lower immunoreactivity for GLUT2 and a 1.8×-fold higher ratio of GLUT1 to total GLUT1-3. The combined levels of GLUT1-3 proteins were reduced in the irradiated but not the non-irradiated tumors with monosomy-3. GLUT3 expression was stronger in the irradiated samples with disomy-3 versus monosomy-3, but the ratio of the GLUT3 isoform to total GLUT1-3 did not differ with regard to the monosomy-3 status in the irradiated or non-irradiated subgroups. Systemic metastases were associated with the presence of monosomy-3 in the primary and circulating tumor cells as well as a higher GLUT1 ratio. Upregulation of the high-affinity glucose transporter GLUT1 possibly as a compensation for the low-affinity isoform GLUT2 may be enhancing the basal glucose uptake in the UM cells with monosomy-3. Prevention of hyperglycemia might, therefore, be a valuable approach to delay the lethal UM metastases.

Metastasis of Uveal Melanoma with Monosomy-3 Is Associated with a Less Glycogenetic Gene Expression Profile and the Dysregulation of Glycogen Storage.

The prolonged storage of glucose as glycogen can promote the quiescence of tumor cells, whereas the accumulation of an aberrant form of glycogen without the primer protein glycogenin can induce the metabolic switch towards a glycolytic phenotype. Here, we analyzed the expression of n = 67 genes involved in glycogen metabolism on the uveal melanoma (UM) cohort of the Cancer Genome Atlas (TCGA) study and validated the differentially expressed genes in an independent cohort. We also evaluated the glycogen levels with regard to the prognostic factors via a differential periodic acid-Schiff (PAS) staining. UMs with monosomy-3 exhibited a less glycogenetic and more insulin-resistant gene expression profile, together with the reduction of glycogen levels, which were associated with the metastases. Expression of glycogenin-1 (Locus: 3q24) was lower in the monosomy-3 tumors, whereas the complementary isoform glycogenin-2 (Locus: Xp22.33) was upregulated in females. Remarkably, glycogen was more abundant in the monosomy-3 tumors of male versus female patients. We therefore provide the first evidence to the dysregulation of glycogen metabolism as a novel factor that may be aggravating the course of UM particularly in males.

Lower Levels of Adiponectin and Its Receptor Adipor1 in the Uveal Melanomas With Monosomy-3.

Purpose: Adiponectin is an insulin-sensitizing and anticarcinogenic hormone that is encoded by a gene on chromosome 3. Here, we analyzed the expression of adiponectin and its receptor Adipor1 in primary uveal melanoma (UM) with regard to the monosomy-3 status and clinical factors, as well as the physiological response of UM cells to adiponectin. Methods: Immunohistochemistry was performed on the primary UM of 34 patients. Circulating melanoma cells (CMC) were isolated by immunomagnetic enrichment. Monosomy-3 was evaluated by Immuno-FISH. Gene expression was analyzed using the RNAseq data of The Cancer Genome Atlas study. Cultures of choroidal melanocytes and UM were established from the samples of two patients. The proliferative potential of the UM cell lines Mel-270 and OMM-2.5 was determined by immunocytochemistry, immunoblotting, cell cycle analysis, nucleolar staining, and adenosine triphosphate (ATP) levels. Results: UM with monosomy-3 exhibited a lower immunoreactivity for adiponectin and Adipor1, which was associated with monosomy-3-positive CMC and the development of extraocular growth or metastases. Both proteins were more abundant in the irradiated tumors and present in the cultured cells. Gene expression profile indicated the impairment of adiponectin-mediated signaling in the monosomy-3 tumors. Adiponectin induced a significant decline in the ATP levels, Ki-67 expression, cells in the G2/M phase, and nucleolar integrity in UM cultures. Conclusions: Adiponectin deficiency appears to enhance the metastatic potential of the UM cells with monosomy-3 and the termination of tumor dormancy. Counteracting insulin resistance and improving the serum adiponectin levels might therefore be a valuable approach to prevent or delay the UM metastases.