Several recent studies have suggested that PcG proteins, in particular EZH2 and Bmi-1 are overexpressed in human cancers. Recent elegant studies have clearly shown that oncogenic transformation of human cells is a multi-step process (48
). It is very likely that overexpression of EZH2 or Bmi-1 alone is not sufficient to cause transformation of human cells. To gain an insight into breast cancer progression, here we examined the transformation potential of Bmi-1 oncoprotein in immortalized HMECs. Although immortalized HMECs that we studied lack p16INK4A
and p14-ARF, Bmi-1 expression still provides an oncogenic signal in these cells by the activation of PI3KAKT pathway (25
). However, the oncogenic signal provided by Bmi-1 alone does not appear to be sufficient to cause transformation of HMECs, despite these cells being immortal and lacking p16INK4A
). This observation underscores the stringency of transformation in HMECs. Nonetheless, Bmi-1 overexpression is frequently observed in invasive breast tumors (8
), suggesting the involvement of additional oncogenic events during breast cancer progression in such tumors.
To understand the genetic basis of these presumptive additional oncogenic events, we overexpressed a constitutively active mutant G12V of H-Ras (33
) in Bmi-1 overexpressing MCF10A cells. G12V mutant of H-Ras promotes proliferation and oncogenesis via activation of mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK and the phosphoinositide 3-kinase (PI-3K)/AKT pathways. However, the activation of these pathways and their outcome is cell-type specific. For example, in primary cells, activation of these pathways lead to induction of OIS, while in immortalized cells with compromised p53-p21 and/or p16INK4A
pathways, H-Ras G12V may promote proliferation. Our reasoning behind using H-Ras G12V in these assays was based on its relevance to breast cancer, and its reported use in oncogenic assays involving HMECs (32
). Although, the direct mutational activation of H-Ras is rare in breast cancer, its hyperactivation by persistent growth factor signaling caused by EGFR and HER2/neu overexpression occurs in a proportion of breast cancers (49
OIS caused by G12V mutant of H-Ras may require both functional p16INK4A
and p53. In MCF10A cells, which have functional p53, we initially noticed the appearance of a heterogeneous culture with approximately 40−50% cells exhibiting senescent morphology upon H-Ras overexpression. Consistent with partial senescence induction, our Western blot data also indicated upregulation of p53 protein in these cells. Senescence acts as a strong barrier to oncogenesis (20
), hence the initial induction of senescence in a proportion of MCF10A cells by H-Ras indicates an anti-oncogenic response. As expected, these early passage cells were not transformed by soft-agar and Matrigel assays. However, late passage culture, which were much more homogenous and did not contain cells with senescent morphology, displayed transformed phenotype in Matrigel and soft-agar assays. Ras and Ki-67 co-staining data also suggest that early passage culture of MCF10A-H-Ras are more heterogeneous in terms of Ras expression, while the late passage culture of these cells are homogenous in terms of Ras expression. Importantly, only low Ras expressing cells tend to be Ki-67 positive suggesting that low Ras permits proliferation, while high Ras blocks proliferation, possibly via OIS. This differential effect of Ras on proliferation explains the emergence of low Ras expressing culture at late passages.
The H-Ras overexpression in Bmi-1 overexpressing MCF10A cells caused senescence only in a minority of cells. When compared to H-Ras overexpressing culture, homogenous culture with proliferating cells appeared much more rapidly from MCF10A-Bmi-1+H-Ras cultures. These data indicate that to some extent, Bmi-1 can overcome H-Ras induced OIS, even in p16INK4A negative cells, presumably via p16INK4a/ARF-independent targets of Bmi-1. The homogenous culture that rapidly emerged from Bmi-1+H-Ras expressing cells continued to express high Ras. Most cells in this culture were Ki-67 positive despite expressing high Ras, suggesting that in the presence of Bmi-1, cells can tolerate high Ras, and thus there is no selection for cells expressing low Ras.
Analysis of Ras effector pathways suggest that activation of PI3K-AKT pathway may also play a role in Bmi-1 and H-Ras induced transformation of HMECs. Although, after EGF stimulation, compared to control MCF10A cells, early passage H-Ras expressing cells exhibited higher phospho-AKT, EGF starved MCF10A-H-Ras had no phospho-AKT, while Bmi-1 and Bmi-1+H-Ras expressing MCF10A cells contained significant level of phospho-AKT even under growth factor starved conditions. On the other hand, early passage MCF10A-H-Ras and MCF10A-Bmi-1+H-Ras cells exhibited constitutively high ERK kinase activity, which was inducible but not constitutive in control MCF10A and MCF10A-Bmi-1 cells. Collectively, our data suggest that Bmi-1 induces AKT activity, which is further augmented by H-Ras co-overexpression, and that H-Ras constitutively activates ERK kinases in MCF10A cells.
On examination of Ser-37 and Ser-15 phosphorylation of p53 in response to DNA damage, we found that Ser-37 phosphorylation of p53 is significantly low and not inducible in both late passage H-Ras, and Bmi-1+H-Ras expressing cells. However, PRAK expression is not affected in these cells. These data suggest that the kinase activity of PRAK responsible for Ser-37 phosphorylation is lost in H-Ras overexpressing MCF10A cells. In addition, these cells also had much lower induction of Ser-15 phosphorylated p53, suggesting a possible defect in other p53 activating kinases such as ATM. A detailed analysis of various p53 phosphorylating kinases in late passage MCF10A-H-Ras and MCF10A-Bmi-1+H-Ras remains to be elucidated. Nevertheless, our data clearly indicate that these late passage H-Ras and Bmi-1+H-Ras expressing cells have defects in p53 phosphorylating pathways, which results in attenuation of induction of p53 targets such as p21 and PUMA. This compromised induction of p53 targets may contribute to a transformed phenotype of MCF10A cells expressing Bmi-1 and H-Ras.
The differential behavior of early and late passage H-Ras overexpressing MCF10A cells with respect to the transformed phenotype explains the different results that are reported in the literature (36
). Our data suggest that in cases where H-Ras expressing MCF10A cells showed a transformed phenotype and gave rise to tumors in nude mice assays, late passage H-Ras expressing cells with defective p53 regulation may have been used. In other studies, where transformation of H-Ras expressing MCF10A cells was not reported, early passage H-Ras expressing MCF10A cells may have been used. Alternatively, the transforming potential of H-Ras cells could be correlated with the level of expression of H-Ras. In studies where H-Ras alone was reported to be transforming, the expression of H-Ras may be low, which permits proliferation and expression of other oncogenic functions of Ras. On the other hand, in cases where Ras was reported to be insufficient for transformation, the expression of Ras may be very high, which causes proliferation arrest and OIS. Neither of these possibilities is mutually exclusive and both possibilities are likely to contribute to transformation of HMECs by H-Ras. Recently it was shown that low levels of K-Ras induce proliferation and mammary epithelial cell hyperplasias, while high expression of K-Ras induces proliferation arrest and OIS in dox-inducible K-Ras
transgenic mice (51
). In this report, it was also shown that inactivation of p53 permits transformation of mammary epithelial cells and tumor formation by high expression of Ras (51
). Our in vitro data are consistent with this report.
The results of histopathology, including special stains and immunohistochemistry, confirm that the MCF10A+H-Ras tumors are composed of multiple different populations of varying phenotypes (smooth muscle, hemangiomatous and mast cells), suggesting that these populations may be in part an in-vivo response to the xenografted tumor population, rather than original components of the neoplastic population that have undergone dedifferentiation and redifferentiation along multiple lines. The MCF10A-Bmi-1+H-Ras tumors on the other hand, represent a pure population of highly atypical, poorly-differentiated, and infiltrative spindle cells consistent with a mesenchymal phenotype. Although the α-SMA immunohistochemistry was negative in these tumors, the Masson's Trichrome stain along with positive immunohistochemistry for vimentin would suggest that these cells may represent a myoepithelial phenotype consistent with EMT.
The striking differences seen in tumor morphology both grossly and histologically are reflected in the survival data of these animals over time. For example, due to the hemorrhagic nature of their tumors, many animals injected with MCF10A+H-Ras cells were removed from study prematurely due to morbidity as a result of anemia, rather than tumor burden. On the other hand, animals receiving MCF10A-Bmi-1+H-ras cells generally survived until they became moribund due to tumor burden. These data would suggest that expression of Bmi-1 concurrently with H-Ras promotes a more poorly differentiated tumor phenotype that is directly responsible for clinical morbidity due to tumor burden in comparison to H-Ras alone, in which morbidity is primarily due to anemia.
Our preliminary data also suggest that tumors formed by MCF10A-Bmi-1+H-Ras (LP) cells are much more metastatic and invasive compared to tumors formed by MCF10A-H-Ras (LP) cells. Although, MCF10A-Bmi-1+H-Ras(LP) and MCF10A-H-Ras cells give rise to histologically distinct type of tumors, biochemically, these cells show only minor differences in regulation of growth regulatory pathways. The only significant difference between these two cell lines is that H-Ras (LP) cells expressed higher levels of BCL2 oncoprotein, which may contribute to the oncogenicity of these cells. The other significant difference between MCF10A-Bmi-1+H-Ras (LP) and MCF10A-H-Ras cells appears to be that MCF10A-Bmi-1+H-Ras (LP) cells can proliferate despite high Ras. The biochemical basis for proliferation of MCF10A-Bmi-1+H-Ras (LP) cells despite high Ras remains to be elucidated. In any case, we did not observe tumor formation by MCF10A-Bmi-1 (LP) cells suggesting the involvement of additional oncogenic events such as downregulation of p53, overexpression of CDK4 and cyclin D1, and upregulation of AKT and ERK activities in the transformation of HMECs and breast cancer progression. Our data also indicate that Bmi-1 may cooperate with Ras in transformation by simply allowing high Ras expressing cells to proliferate. The additional oncogenic events then may be largely contributed to by H-Ras in the experiments described here. It remains to be determined which of these oncogenic lesions, together with Bmi-1, are sufficient to transform HMECs and form tumors in vivo.