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EMT-associated factors promote invasive properties of uveal melanoma cells.

Asnaghi L, Gezgin G, Tripathy A, Handa JT, Merbs SL, van der Velden PA, Jager MJ, Harbour JW, Eberhart CG - Mol. Vis. (2015)

Bottom Line: The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells.Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%.The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, MD.

ABSTRACT

Purpose: Transcription factors regulating the epithelial-to-mesenchymal transition (EMT) program contribute to carcinogenesis and metastasis in many tumors, including cutaneous melanoma. However, little is known about the role of EMT factors in the growth and metastatic dissemination of uveal melanoma cells. Here, we analyzed the expression and functions of the EMT factors ZEB1, Twist1, and Snail1 in uveal melanoma cell lines and primary tumors.

Methods: ZEB1, Twist1, and Snail1 mRNA levels were measured using qPCR in five uveal melanoma cell lines and in 30 primary tumors. Gene expression was used to determine class 1 and class 2 signatures in the primary tumors. Short hairpin RNA was used to downregulate the expressions of the EMT factors; then, growth and transwell invasion assays were performed.

Results: ZEB1, Twist1, and Snail1 were expressed in all five uveal melanoma lines, with ZEB1 having the highest protein levels. ZEB1 mRNA was significantly elevated in highly metastatic class 2 primary tumors for which survival data were not available, whereas a high gene expression of Twist1 was associated with a worse prognosis in a separate tumor cohort analyzed by expression profiling. The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells. Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%. The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth.

Conclusions: The downregulation of ZEB1, Twist1, and Snail1 reduces the invasive properties of uveal melanoma cells, and the elevated mRNA levels of ZEB1 and Twist1 are associated with a more aggressive clinical phenotype in uveal melanoma samples. Therefore, these factors could represent new therapeutic targets in patients with ocular melanoma.

No MeSH data available.


Related in: MedlinePlus

Reduction in Twist1 decreases both growth and invasion in uveal melanoma cells. A: Twist1 mRNA levels were determined by qPCR in Mel290 and OMM1 cells transduced with Twist1 shRNA. Data shown are mean ± SD; *p = 0.01; ***p = 0.0001 versus sh-Scramble. B: An MTS assay shows a reduction in cell growth when the Twist1 expression was reduced in both cell lines, as compared to scramble shRNA; ***p = 0.0002 versus sh-Scramble. C: A transwell invasion assay reveals a significant reduction in the invasive capacity of Mel290 and OMM1 cells upon the downregulation of Twist1, compared to vector control. Microphotographs in the right panels show the invading cells on the lower surface of the Matrigel-coated filter after overnight incubation; ***p = 0.0001 versus sh-Scramble.
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f4: Reduction in Twist1 decreases both growth and invasion in uveal melanoma cells. A: Twist1 mRNA levels were determined by qPCR in Mel290 and OMM1 cells transduced with Twist1 shRNA. Data shown are mean ± SD; *p = 0.01; ***p = 0.0001 versus sh-Scramble. B: An MTS assay shows a reduction in cell growth when the Twist1 expression was reduced in both cell lines, as compared to scramble shRNA; ***p = 0.0002 versus sh-Scramble. C: A transwell invasion assay reveals a significant reduction in the invasive capacity of Mel290 and OMM1 cells upon the downregulation of Twist1, compared to vector control. Microphotographs in the right panels show the invading cells on the lower surface of the Matrigel-coated filter after overnight incubation; ***p = 0.0001 versus sh-Scramble.

Mentions: To assess the effects of Twist1 in uveal melanoma, we suppressed its expression using two separate shRNA constructs targeting Twist1 mRNA, both of which reduced the mRNA levels by 60%–70% in Mel290 and OMM1 cells, as compared to scramble shRNA (Figure 4A). This reduction correlated with a significant inhibition of growth in both cell lines (Figure 4B), as found by MTS assay. However, a transwell invasion assay showed that Twist1 inhibition produced a stronger effect on cellular invasion, which was inhibited by approximately 80%–90% in Mel290 and 60%–75% in OMM1 cells (Figure 4C).


EMT-associated factors promote invasive properties of uveal melanoma cells.

Asnaghi L, Gezgin G, Tripathy A, Handa JT, Merbs SL, van der Velden PA, Jager MJ, Harbour JW, Eberhart CG - Mol. Vis. (2015)

Reduction in Twist1 decreases both growth and invasion in uveal melanoma cells. A: Twist1 mRNA levels were determined by qPCR in Mel290 and OMM1 cells transduced with Twist1 shRNA. Data shown are mean ± SD; *p = 0.01; ***p = 0.0001 versus sh-Scramble. B: An MTS assay shows a reduction in cell growth when the Twist1 expression was reduced in both cell lines, as compared to scramble shRNA; ***p = 0.0002 versus sh-Scramble. C: A transwell invasion assay reveals a significant reduction in the invasive capacity of Mel290 and OMM1 cells upon the downregulation of Twist1, compared to vector control. Microphotographs in the right panels show the invading cells on the lower surface of the Matrigel-coated filter after overnight incubation; ***p = 0.0001 versus sh-Scramble.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548792&req=5

f4: Reduction in Twist1 decreases both growth and invasion in uveal melanoma cells. A: Twist1 mRNA levels were determined by qPCR in Mel290 and OMM1 cells transduced with Twist1 shRNA. Data shown are mean ± SD; *p = 0.01; ***p = 0.0001 versus sh-Scramble. B: An MTS assay shows a reduction in cell growth when the Twist1 expression was reduced in both cell lines, as compared to scramble shRNA; ***p = 0.0002 versus sh-Scramble. C: A transwell invasion assay reveals a significant reduction in the invasive capacity of Mel290 and OMM1 cells upon the downregulation of Twist1, compared to vector control. Microphotographs in the right panels show the invading cells on the lower surface of the Matrigel-coated filter after overnight incubation; ***p = 0.0001 versus sh-Scramble.
Mentions: To assess the effects of Twist1 in uveal melanoma, we suppressed its expression using two separate shRNA constructs targeting Twist1 mRNA, both of which reduced the mRNA levels by 60%–70% in Mel290 and OMM1 cells, as compared to scramble shRNA (Figure 4A). This reduction correlated with a significant inhibition of growth in both cell lines (Figure 4B), as found by MTS assay. However, a transwell invasion assay showed that Twist1 inhibition produced a stronger effect on cellular invasion, which was inhibited by approximately 80%–90% in Mel290 and 60%–75% in OMM1 cells (Figure 4C).

Bottom Line: The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells.Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%.The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, MD.

ABSTRACT

Purpose: Transcription factors regulating the epithelial-to-mesenchymal transition (EMT) program contribute to carcinogenesis and metastasis in many tumors, including cutaneous melanoma. However, little is known about the role of EMT factors in the growth and metastatic dissemination of uveal melanoma cells. Here, we analyzed the expression and functions of the EMT factors ZEB1, Twist1, and Snail1 in uveal melanoma cell lines and primary tumors.

Methods: ZEB1, Twist1, and Snail1 mRNA levels were measured using qPCR in five uveal melanoma cell lines and in 30 primary tumors. Gene expression was used to determine class 1 and class 2 signatures in the primary tumors. Short hairpin RNA was used to downregulate the expressions of the EMT factors; then, growth and transwell invasion assays were performed.

Results: ZEB1, Twist1, and Snail1 were expressed in all five uveal melanoma lines, with ZEB1 having the highest protein levels. ZEB1 mRNA was significantly elevated in highly metastatic class 2 primary tumors for which survival data were not available, whereas a high gene expression of Twist1 was associated with a worse prognosis in a separate tumor cohort analyzed by expression profiling. The genetic downregulation of ZEB1 in OCM1, OMM1, and 92.1 resulted in a more than 50% reduction in invasion, but only suppressed growth in OMM1 cells. Suppression of Twist1 in Mel290 and OMM1 reduced growth and invasion by more than 50%. The downregulation of Snail1 in the 92.1 cell line reduced invasion by 50%, but did not interfere with growth.

Conclusions: The downregulation of ZEB1, Twist1, and Snail1 reduces the invasive properties of uveal melanoma cells, and the elevated mRNA levels of ZEB1 and Twist1 are associated with a more aggressive clinical phenotype in uveal melanoma samples. Therefore, these factors could represent new therapeutic targets in patients with ocular melanoma.

No MeSH data available.


Related in: MedlinePlus