Tumor progression is the evolution of already tumorigenic cells towards increasing malignancy [1, 2]. During tumor progression, further aberrant molecular events may occur in a specialized subset of low invasive cancer cells and ultimately promote these cells to acquire the ability of highly invasive growth . Identification of the molecules that predispose tumor cells to a more invasive phenotype is helpful for understanding the tumor metastasis process as well as providing potential therapeutic targets for tumor progression. In this study, we manipulated Id2 expression in poorly invasive MCF-7 and SKOV-3 cancer cells and determined the effects on cell proliferation, in vitro invasion and migration. Id2 could facilitate the invasive and migratory capabilities of MCF-7 and SKOV-3 cells in a dose-dependent manner. Importantly, structure and function analyses revealed that the HLH domain of Id2 is not required for its pro-invasive function. These observations suggest that the aberrant accumulation of Id2 in some specific non-aggressive epithelial tumor cells may be sufficient to convert them into more invasive cells.
Elevated levels of Id2 expression have been reported in carcinomas of breast, ovary, colon and prostate, in neural tumors, melanoma, Ewing's sarcoma and in hematological malignancies [13–15]. In some cases, high levels of Id2 expression are associated with disease severity and poor prognosis. However, significant contradictions exist in various types of human tumors. In addition, cell-based experiments indicated that Id2 has diverse and complex biological effects depending on cell lineage, differentiation state, and other contextual considerations . For example, down-regulation of Id2 expression in highly metastatic PC-3 human prostate cancer cells reduced their growth potential and invasiveness, which indicates the pro-proliferation and pro-invasion roles of Id2 in some epithelial cancer cells . Conversely, ectopic expression of Id2 in MDA-MB-231 and MDA-MB-436 human breast cancer cells did not significantly affect cell growth but markedly reduced the cells' invasive capacity [23, 35]. These controversial functional consequences of Id2 on tumor growth and invasion suggest the diverse nature of Id2 target signaling pathways in different cell contexts. Therefore, careful evaluation is required to unambiguously identify the tumor cell types or subtypes that may use Id2 to control their different phenotypes. Although MCF-7 is also a human breast cancer cell line, its genetic background and phenotypic characteristics differ greatly in MDA-MB-231 and MDA-MB-436 cells. Generally, the MCF-7 cell line is representative of ERα-positive and non-aggressive human breast cancers; however, MDA-MB-231 and MDA-MB-436 are representative of ERα-negative breast cancer cells. In addition, SKOV-3 is an ovarian carcinoma cell line with ERα-positive and non-aggressive phenotypes. In this study, overexpression of Id2 or either of its two mutants increased the invasive capacity of MCF-7 and SKOV-3 cells but did not alter the proliferation of either of these two cell lines. The functional role of Id2 in the cell invasion phenotype in MCF-7 and SKOV-3 cells is completely contrary to that previously observed in MDA-MB-231 and MDA-MB-436 cells. Considering the expression of ERα in MCF-7 and SKOV-3 cells, we postulated that the aberrant Id2 expression may play an important role in converting ERα-positive epithelial tumor cells into highly invasive cells. Although a high expression of Id2 in primary breast cancer cells was reported to confer favorable clinical outcome , our findings suggest that the analysis of Id2 expression in combination with ERα status may be better for prognostic reevaluation of breast cancer.
Our important finding is that the HLH domain of Id2 is not required for the pro-invasive activity of the protein and that the level of expression of an invasion-suppressing molecule, E-cadherin, is down-regulated by only the degradation-resistant Id2 form. These observations raise the possibility that different expression levels of Id2 can influence different gene expression through a heretofore unknown transcriptional activity of its non-HLH region. Consistent with this is our observation that the transactivation of E-cadherin promoter is significantly suppressed by the degradation-resistant full-length and HLH-deleted Id2 forms but not the wild-type form. Although MCF-7 and SKOV-3 cells are highly sensitive to changes in the levels of E-cadherin and will undergo enhanced invasion if E-cadherin is down-regulated, supplementation of E-cadherin in cells does not completely antagonize the invasion potential induced by Id2-DBM-δHLH. Overexpression of the wild-type Id2 in MCF-7 and SKOV-3 cells did not reduce E-cadherin expression but did indeed promote the invasion and migration of both cell lines. So, different mechanisms are involved in the invasion potential induced by different Id2 expression levels in cells.
Previous studies have linked Id and E-cadherin expression in some specific cell types. Id proteins have been shown to activate E-cadherin in normal epithelial cells by inhibiting E2A protein, which represses the most proximal E-cadherin promoter in these cells through interaction with the E-box elements [36, 37]. However, apparent contradiction exists in some specific cell types. For example, in uveal melanoma Mel202 and Mel290 cells, Id2 suppressed E-cadherin expression through inhibiting the transactivation of its proximal promoter by an unknown regulatory mechanism . In this study, Id2 suppressed E-cadherin expression in MCF-7 and SKOV-3 cells through a mechanism independent of its dimerization to bHLH factors when Id2 was accumulated to a high level in cells.
E-cadherin is often positive in ERα-positive cancer cells and often negative in ERα-negative tumor cells (such as MCF-7 versus MDA-MB-231 cells), and loss of E-cadherin may result in the more aggressive growth of ERα-positive cells by increasing the probability of invasion and metastasis . Epithelial cancers arise within an epithelium where cells are constrained by E-cadherin-mediated cell-cell interactions. Hence, in the early stages of these cancers, E-cadherin must be down-regulated to escape the local epithelial environment and invade local structures. In this paradigm, the aberrant accumulation of Id2 and the subsequent E-cadherin down-regulation as we described should provide a selective growth advantage in the tumor microenvironment by increasing the probability of invasion and metastasis.