Our previous work showed that the overexpression of RhoC GTPase and the loss of WISP3 expression are alterations that occur concordantly, more often in IBC than in slow-growing locally advanced breast cancers. WISP3 loss was found in concert with RhoC GTPase overexpression in 90% of archival patient samples of IBC, but rarely in stage-matched non-IBC tumors. Our laboratory further demonstrated that RhoC GTPase is a transforming oncogene for HME cells and that WISP3 has tumor inhibitory functions in IBC. However, neither alteration occurring in isolation seems to be sufficient to develop the full-blown, highly malignant IBC phenotype. Here we postulate that dysregulation of WISP3 might upregulate RhoC GTPase and thus enhance the aggressiveness of the phenotype that results when these two alterations are present.
We have shown that overexpression of RhoC GTPase in immortalized HME cells produced a striking tumorigenic effect that, for the most part, recapitulates the phenotype of the SUM149 IBC cell line. HME cells stably transfected with RhoC exhibited greatly increased growth under anchorage-independent conditions [15
]. HME cells overexpressing RhoC produced up to 100-fold more colonies than the controls, about 60% of the level of colony formation of the SUM149 IBC cell line. RhoC overexpression induced motility and invasion in HME cells, and markedly induced the production of angiogenic mediators including VEGF [17
]. The HME/RhoC transfectants formed tumors when injected into the mammary fat pad of athymic nude mice [15
Importantly, restoration of WISP3 in SUM149 cells ameliorated these features of the malignant phenotype. The SUM149/WISP3+
cells exhibited decreased growth in vitro
and in vivo
in comparison with SUM149 cells transfected with the empty vector. The invasiveness of SUM149 cells was greatly decreased by restoring WISP3 expression. We also found that WISP3 markedly decreased the concentration of angiogenic mediators in the conditioned medium, especially VEGF, basic fibroblast growth factor, and interleukin-6 [9
]. Given the high specificity of WISP3 and RhoC alterations in IBC and their interrelated functions in tumorigenesis, we propose that they cooperate in the development of IBC.
Using an antisense approach, inhibition of WISP3 expression in HME cells resulted in a threefold increase of RhoC GTPase transcript levels. The HME/AS WISP3 cells also exhibited increased cellular proliferation and anchorage-independent growth in soft agar. The HME/AS WISP3 cells produced significantly more colonies in soft agar in comparison with the control cells, an average of 58% of the level of colonies formed by the SUM149 IBC cells. HME/AS WISP3 cells also exhibited decreased production of VEGF in the conditioned medium.
The relationship between RhoC and WISP3 expression seems to be reciprocal. Restoration of WISP3 expression in SUM149 cells, which have lost WISP3 in the wild-type state, induced a 1.5-fold decrease in RhoC GTPase expression. These results are intriguing because changes in expression in Rho proteins by subtle factors such as 1.5–1.8 can be sufficient to modulate cellular behavior. Overexpression of RhoC in spontaneously immortalized HME cells, MCF10A, resulted in a 2.5-fold decrease in WISP3 mRNA expression.
In summary, overexpression of RhoC GTPase and loss of WISP3 are key genetic alterations in the development of IBC, and they have complementary functions. RhoC GTPase has a primary a role in motility, invasion, and angiogenesis [15
]. WISP3 has a pivotal role in tumor growth, invasion, and angiogenesis [9
]. Here we have further strengthened the evidence that these genes cooperate in the development of IBC, because WISP3 expression modulates the expression of RhoC GTPase and its functions.