We identified the ubiquitin-conjugating enzyme E2-EPF in DNA microarray and EST database-mining studies as a gene overexpressed in breast tumors relative to normal tissues. Our study demonstrated that the E2-EPF protein is more highly expressed in the majority of breast infiltrating ductal carcinomas than in normal tissues and is regulated during the HeLa cell cycle, achieving the highest levels during mitosis. In tumors, E2-EPF mRNA expression correlated with genes involved in mitotic surveillance, but RNAi-mediated knockdown of the E2-EPF protein did not alter cell cycle distribution or affect the proliferation of HeLa cervical cancer cells or MDA-231 and MDA-MB-453 breast cancer cells. Instead, E2-EPF protein knockdown sensitized HeLa cells to the antiproliferative effects of the topo II inhibitors etoposide and doxorubicin and resulted in elevated topo IIα protein levels.
Our observation that ER-
breast tumors are more likely to express the E2-EPF protein and have detectable E2-EPF expression in a greater percentage of cells than ER+
tumors is consistent with the presence of E2-EPF in the metasignature for undifferentiated cancer [1
] because ER-
tumors are typically poorly differentiated and are of higher mitotic grade than ER+
]. Yet, RNAi-mediated reduction of E2-EPF protein levels to < 15% of control levels in HeLa cells or in ER-
MDA-231 and MDA-MB-453 breast cancer cells did not affect their proliferation. Our results differ from those of Jung et al. [4
], who demonstrated that adenovirus-encoded E2-EPF siRNA that downregulated E2-EPF protein levels inhibited the proliferation of C8161 melanoma and VHL-expressing 786-O renal carcinoma cells by ~ 1.5-fold to 2-fold 4 days after seeding (approximately two population doublings) and more markedly suppressed their in vivo
tumor growth. It is possible that the 80% to 90% reduction in E2-EPF levels achieved in our study is not of sufficient magnitude or duration to impact the proliferation of the cancer cells analyzed or that only certain cancers are dependent on E2-EPF for their growth. In this regard, it is noteworthy that VHL expression was critical for tumor growth inhibition following adenoviral E2-EPF siRNA delivery in Jung et al. [4
], as growth of parental 786-O tumors was not affected by E2-EPF downmodulation. Because the cell lines studied here express a wild-type functional VHL protein [17
], the lack of an E2-EPF requirement for cell proliferation suggests that the presence of a functional VHL-HIF-1α pathway is not, by itself, responsible for determining E2-EPF dependency.
In our survey of multiple cancer microarray studies using the ONCOMINE database, we found that E2-EPF expression was most frequently correlated with genes such as CCNB2, CENPA, TOP2A, CDC20
, and TPX2
, which are all implicated in cell cycle progression through G2
/M phase. Their coexpression with E2-EPF in tumors may indicate that E2-EPF is expressed in a proliferating tumor cell population identified by the expression of these G2
/M genes. The relatively weak correlation of E2-EPF with these G2
/M genes, compared with genes such as TOP2A, CDC20, UBE2C
, or CKS2
, is consistent with the fact that reduction of E2-EPF levels following siRNA treatment had no effect on cell proliferation, but may also suggest that E2-EPF is important for proliferation in only a subset of cancers or may function in a particular tumor microenvironment. Our analysis explored E2-EPF activity in two ER-
breast cancer cell lines that may represent different types of breast cancer. MDA-MB-453 is classified as an ERBB2-amplified luminal epithelial cell cancer, whereas MDA-231 is a highly invasive cell line with mesenchymal cell characteristics [18
]. Evaluation of additional cell lines representative of other breast cancers, as well as different conditions of in vitro
culture (e.g., extracellular matrix, cellular stress), for effects of E2-EPF depletion may therefore be warranted.
The cell cycle-regulated expression of E2-EPF and its coexpression in tumors with genes implicated in chromosome decatenation (i.e., TOP2A
), spindle assembly (TPX2
), and mitotic checkpoint function (BUB1B, BUB1
, and TTK
) prompted us to ask whether E2-EPF might have a function in cancer cells that might be revealed only under conditions of cellular stress, modeled by treating cells with agents that induce a G2
checkpoint or a mitotic checkpoint. We discovered that HeLa cells treated with E2-EPF siRNA were approximately two-fold more sensitive to the topo II inhibitors etoposide and doxorubicin than were cells transfected with negative control siRNA. This degree of sensitization is similar to that seen with agents, such as the poly(ADP-Ribose) polymerase inhibitor AG14361, that enhance the potency of topo I inhibitors [19
]. Importantly, no sensitization was found for the topo I inhibitor camptothecin, which also induces DNA damage, or for the microtubule-stabilizing drug Taxol. Lack of sensitization to camptothecin suggests that sensitization to topo II inhibitors by E2-EPF depletion was not due to a reduced ability of the cells to sense double-stranded DNA breaks, to trigger the S phase and/or G2
DNA damage checkpoints, or to repair damaged DNA. A possible explanation for the increased antiproliferative effect of topo II inhibitors following E2-EPF knockdown is that topo IIα protein levels were increased. Because topo II inhibitors form tertiary complexes with the enzyme and DNA to enhance strand breakage [11
], higher levels of topo II can lead to greater drug sensitivity. Indeed, enhanced sensitivity to topo II inhibitors has been shown in tumors with elevated levels of topo II [13,14,20
]. Alternatively, E2-EPF may decrease drug sensitivity by playing a role in the turnover of topo II inhibitor-induced topo II-DNA complexes, thereby enabling repair of DNA damage. Topo IIβ-DNA covalent complexes have been shown to be degraded by the 26S
proteasome following ubiquitinylation [21
]. In our study, E2-EPF knockdown resulted in an increase in topo IIα, but not topo IIβ, levels in exponentially growing cells that were not treated with drugs. Further studies will be needed to address the effect of topo II inhibitor treatment on topo II protein levels under conditions of E2-EPF depletion, as well as to determine whether ubiquitinylation of topo IIα is affected by knockdown or overexpression of E2-EPF.
Proteasomal activity has also been shown to be important for the regulation of topo IIα protein degradation at the M/G1
transition of the cell cycle [22
]. The specific E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases responsible for topo II turnover have not yet been identified; our E2-EPF depletion, as well as cell cycle studies demonstrating peak levels of E2-EPF during mitosis, suggests but does not prove that E2-EPF is one of the regulators of this process. It is possible that the frequent coexpression of E2-EPF and TOP2A
reflects a tumor-specific mechanism that generates adequate levels of E2-EPF to ensure appropriate regulation of topo IIα concentrations and activity.
In summary, we have shown that the E2-EPF ubiquitin-conjugating enzyme is overexpressed in the majority of breast cancers, but elevated levels of E2-EPF are not required for cell proliferation under standard culture conditions, as suggested by RNAi-mediated depletion studies with the tested cancer cell lines. However, sensitivity to topo II inhibitors was enhanced in HeLa cells on treatment with E2-EPF-directed siRNA concomitant with an elevation in topo IIα protein levels. Studies that extend these observations to other cancer cells and explore the mechanism(s) by which E2-EPF modulates sensitivity to topo II inhibition are warranted to provide additional rationale for combination therapy with topo II and E2-EPF inhibitors.