IBC is a unique and highly aggressive form of locally advanced breast cancer with distinct clinical presentation. We hypothesized that upregulated expression of RhoC, as reported by others to be characteristic of IBC, contributes to the unusual pathological presentation of IBC. For the first time, we have compared the actin architecture, invasive, and adhesive properties of the IBC cell line SUM 149 with a cell line reported to express less RhoC mRNA compared to SUM 149 but share a deletion in LIBC [5
]. Using a commercially available specific antibody to RhoC, we report that RhoC is not overexpressed at the protein level by the IBC cell line SUM 149. Interestingly, we confirmed overexpression of RhoA by utilizing an anti-RhoA specific antibody. However, post-transcriptional regulation of RhoC expression may account for the observed discrepancy. It is possible that our results do not agree with the reported mRNA expression due to specificity problems with the commercially developed antibodies. Furthermore, we demonstrate that, compared to SUM 102, SUM 149 is less invasive and migratory, and displays impaired adhesion to basal lamina components but strong adhesion to connective tissue proteins.
The role of the Rho protein in cancer cell invasion is somewhat controversial. RhoA is known to be involved in cell contractility, both in the formation of bundled actin fibers and through the activation of Rho kinase and subsequent activation of myosin light chain [30
]. Such contractile cells have previously been shown to be less motile [31
]. However, Rho overexpression has been documented in various human cancers such as bladder and ovarian, and correlates with lymph node invasion, metastasis, and poor patient prognosis [32
]. Overexpression of RhoC by human mammary epithelial cells increased invasion, motility, and anchorage independent growth, similar to SUM 149 [9
]. Expression of dominant negative Rho T19N has been demonstrated to block melanoma cell invasion [34
]. Some investigators report that Rho overexpression has little impact on invasion and cell motility, while others demonstrate a positive correlation between Rho expression and cell migration capacity [35
]. Rho is required for cell body contraction and tail retraction during directed cell motility, while active Rac and Cdc42 are required for lamellipodia and filopodia extension at the leading edge [30
]. Thus, invasive potential is considered to be a balance between Rac, Cdc42, and Rho activities. Overexpression or activation of one of these Rho GTPases will shift this balance and result in a cellular phenotype dominated by the actin structure promoted by the activated Rho GTPase [38
]. SUM 149 may display reduced invasion and migration in vitro
compared to SUM 102 due to the overexpression of RhoA alone, thus masking the motile effects of Rac and Cdc42.
Another aspect that makes IBC so remarkable is that this form of aggressive breast cancer maintains strong E-cadherin expression [11
]. Typically, loss of E-cadherin expression correlates with progression to metastatic disease since cancer cells must break inter-cell adhesions before attaining a motile phenotype [29
]. Here, we demonstrate that the SUM 149 model of IBC maintains strong E-cadherin expression in culture, as seen in other IBC xenograft models and IBC pathological specimens. Previous reports indicate the E-cadherin axis is also complete and functional [11
]. IBC histology reveals an extensive invasion of E-cadherin positive tumor cell emboli within the dermal lymphatics [11
]. The expression of E-cadherin may be critical for invasion in that IBC is thought by some to be passively disseminated, an invasion mechanism that necessitates cell-cell attachment [12
]. In this scenario, tumor cells maintain strong cell-cell connections and enter circulation via vasculogenesis around a tumor cell embolus. Others hold that E-cadherin expression varies with the malignant stage of the disease, and is lost during invasion but reestablished once tumor cells invade the vasculature [15
]. The finding reported here, in which the IBC cell line SUM 149 was less invasive and adhesive in vitro
compared to the reportedly less aggressive breast cancer cell line SUM 102, seems to support an alternative mode for IBC dissemination from classic actin cytoskeleton- mediated cell motility.
The high expression levels of both E-cadherin and RhoA by SUM 149 may contribute to the uniquely invasive phenotype of IBC. However, signaling via E-cadherin to Rho is unclear with E-cadherin-mediated Rho activation and inhibition reported in a cell line specific manner [39
]. Dominant negative RhoA expression in EL and nEαCL cells has been reported to reduce E-cadherin activity [40
]. During the embryonic development of stratified epithelium, it was found that α-catenin, Rho, and Rho kinase were vital for coordinated tissue movement. In this sense, cells maintain tissue architecture via cadherin binding but move as a unit through actin reorganization mediated by Rho and its downstream effector Rho kinase [41
]. A parallel argument could be made for the dissemination of IBC, in which tightly bound tumor cells move as a coordinated front. This possibility was tested in a wound healing assay, in which we found that the SUM 149 cells do not polarize or move into the wound after 7.5 hours, suggesting that this form of invasion is not the mechanism by which IBC is dissemination.