Expression and effect of inhibition of the ubiquitin-conjugating enzyme E2C on esophageal adenocarcinoma

Abstract

Ubiquitin-dependent proteolysis of cyclins plays a critical role in cell cycle progression and tumorigenesis. We examined the expression of ubiquitin-conjugating enzyme E2C (UBE2C) during progression from Barrett's metaplasia to esophageal adenocarcinoma (EA) and the effects of targeting this enzyme on EA-derived cell lines. Using oligonucleotide microarrays UBE2C expression was elevated in 73% (11 of 15) of EAs relative to Barrett's metaplasia. Tissue microarray showed elevated UBE2C in 70% (7 of 10) of dysplastic samples and in 87% (58 of 67) of tumors relative to metaplastic samples. Transfection of dominant-negative UBE2C into Seg-1 cells decreased proliferation (P = .04) and increased mitotic arrest compared to vector controls (63.5% vs 6.8%; P < .001). Transfection of UBE2C small interfering RNA also caused inhibiton of cell proliferation and distortion of the cell cycle, with maximal increase of G(2) cells (155% of mock cells) at 72 hours and of S-phase cells (308% of mock cells) at 24 hours. Treatment of Seg-1 cells with the proteasome inhibitor MG-262 (1 nM-1 microM) showed decreased proliferation (P = .02). EA-derived cells expressing UBE2C are sensitive to treatment with MG-262 and to silencing of UBE2C, suggesting that patients with EAs overexpressing UBE2C may benefit from agents targeting this ubiquitin-conjugating enzyme.

Figure 1

Oligonucleotide microarray analysis of 46 esophageal samples revealed at least a two-fold overexpression (Y axis) of UBE2C in 11 of 15 (73%) cases of esophageal adenocarcinomas relative to Barrett's metaplasia. BM, Barrett's metaplasia; LGD, low-grade dysplasia; HGD, high-grade dysplasia; EA, esophageal adenocarcinoma. Numbers on X axis labels represent tumor ID.

Figure 2

TMA immunohistochemistry showing (A) intense nuclear staining (arrow) in tumor D48-T, (B) intense staining (arrow) in esophageal adenocarcinoma R35-T, (C) moderate staining (arrow) in Barrett's dysplasia sample P60-BD, and (D) no staining in Barrett's metaplasia sample I93-B. Original magnification, x200. Sections were counterstained with hematoxylin.

Figure 3

(A) Western blot analysis shows expression of UBE2C (∼ 19 kDa) in esophageal adenocarcinomas S96-T, M48-T, K32T, and P28-T. The Barrett's metaplasia sample M48-B and the normal esophageal sample M48-N express small amounts of UBE2C, whereas there was no UBE2C protein expressed in either the Barrett's dysplasia sample S96-BD or the normal esophageal samples A54-N and D67-N. (B) Western blot analysis of nine esophageal cell lines shows expression of UBE2C (∼ 19 kDa) in the esophageal adenocarcinoma cell lines BA, OE33, Flo-1, Seg-1, and Bic-1, whereas L20-T and H80-T showed smaller amounts of UBE2C protein. The squamous cell line Het-1A and the Barrett's metaplasia-derived S95-B also expressed UBE2C protein. β-Actin expression was used as loading control.

Figure 4

(A) Seg-1 cells, which overexpressed UBE2C, showed a significant decrease in cell proliferation after transfection with the dominant-negative UBE2C when compared to control cultures using the MTT assay. All experiments were repeated in triplicate. (B) Although transfection efficiency was not significantly different between vector controls and dominant-negative UBE2C plasmids in Seg-1 cells, the percentage of cells in the mitotic phase was significantly higher after transfection with the dominant-negative UBE2C. All experiments were repeated in triplicate. (C) Seg-1 cells showed a significant decrease in cell proliferation after treatment with the proteasome inhibitor MG-262 when compared to control cultures using the MTT assay. All experiments were repeated in triplicate (*P < .05, **P < .005).

Figure 5

UBE2C silencing with gene-specific siRNA treatment in Seg-1 cells. (A) Real-time quantitative RT-PCR indicated a potent knockdown of UBE2C mRNA expression, with four to five threshold cycles (C_t) between mock control and siRNA-mediated UBE2C-silencing transfectants. (B) A > 95% reduction of UBE2C mRNA expression was detected at 24 and 48 hours and in both 1.5 x 10³ and 2.5 x 10³ cells/well transfectants. (C) Western blot analysis of Seg-1 cells treated with UBE2C-specific siRNA at various time periods. The lack of expression in the treated samples indicates complete abrogation of the UBE2C protein.

Figure 6

WST-1 cell proliferation assay. The reduction of cell proliferation in Seg-1 cells treated with siRNA against UBE2C demonstrated a maximal decrease of 44.3% compared to mock control cells. Cell proliferation was decreased in 22.4% and 31.0% of mock controls after the 24-hour and 72-hour treatments, respectively, of siRNA-mediated UBE2C silencing. There was no difference in cell proliferation observed between treated cells and mock cells after 96 hours.

Figure 7

Flow cytometry of cells labeled with PI. (A) Change in cell cycle distribution over time was observed for Seg-1 cells following treatment with siRNA against UBE2C. Maximal G2 cell accumulation was observed 72 hours after treatment with siRNA (155.4% increase compared to mock cells and/or a normal range of cell cycle distribution). Shown also in this figure is that S-phase cell number increased significantly 24 hours after siRNA transfection. S-phase cells were also increased in 72-hour transfectants treated with an additional siRNA transfection at the 48-hour time point (24 hours before harvest of 72-hour transfectants). (B) Cell cycle distributions over time after siRNA treatment against UBE2C.