We have previously demonstrated that WISP3 is lost in 80% of IBC tumors and that it has tumor-suppressor functions in IBC [5,6
]. Studies on the SUM149 IBC cell line showed that restoration of WISP3 expression has potent growth- and angiogenesis-inhibitory functions in vitro
and in vivo
]. Restoration of WISP3 resulted in a significant decrease in anchorage-independent growth in soft agar and cellular proliferation, as well as a drastic decrease in the invasive capabilities of the SUM149 cells, which are highly invasive in their wild-type state. Furthermore, restoration of WISP3 expression in SUM149 cells resulted in a biologically relevant decrease in the level of angiogenic factors (VEGF, bFGF, and IL-6) in the conditioned media of the cells. In vivo
, restoration of WISP3 expression in SUM149 cells caused a drastic decrease in tumor volume and rate of tumor growth when injected in nude mice [6
]. Taken together, this body of work had strongly supported a tumor-suppressor role for WISP3 in mammary tumor progression. In the present study, we sought to discover the molecular mechanisms underlying the tumor-suppressor function of WISP3.
WISP3 belongs to the CCN family of proteins, which are highly conserved, putatively secreted proteins with important roles in development during chondrogenesis and skeletogenesis [7
]. The CCN proteins have been recently also implicated in carcinogenesis [7,22–26
]. It is not well understood, however, how the functions of the CCN proteins in development relate to their role in cancer. Moreover, their expression during tumorigenesis cannot be generalized across different tissue types. This may be due to tissue-specific functions of the CCN proteins, perhaps mediated by their multimodular structure and the presence of different affinities for binding partners and ligands in different tissues [7
]. The presence of different receptors and differential processing of the CCN proteins (e.g., cleavage by proteases) may account also for their diverse functions in different tissues. In this paper, we focused on determining whether WISP3 is secreted into the conditioned media and its relationship to IGF signaling pathways.
Analysis of the protein sequence of WISP3 revealed that it contains a signal peptide at the N-terminal region that may participate in the secretion of the protein into the extracellular media [7–9
]. Indeed, by Western blot, using two different specific antibodies, we were able to detect WISP3 protein in the conditioned media of SUM149 and HEK293 cells transfected with WISP3. Furthermore, consistent with these results, by immunohistochemical analysis, WISP3 protein was detected in the secretions accumulated in the lumens of ducts and lobules in normal breast tissues. The fact that WISP3 is secreted and present in the conditioned media (thereby alluding to its stability in solution) led us to the hypothesis that it may directly or indirectly regulate IGF signaling.
Although the signaling pathways that are required for the effects of IGF-I in breast cancer have not been completely elucidated, the contribution of IGF-I-induced IGF-IR activation appears to be critical in hormone-dependent and -independent breast cancer [27–30
]. IGF-I is locally released by breast cancer cells and stromal fibroblasts, and it is involved in autocrine and paracrine stimulation of the mammary epithelium [31
]. In breast cancer cells, when IGF-I binds IGF-IR, signaling occurs mainly through activation of IRS-1 and RAS-dependent phosphorylation of MAP kinase with subsequent activation of nuclear transcription factors [32,33
]. IGF-I signaling promotes cell growth, survival, and motility of breast cancer cells, as well as resistance to therapeutic interventions [10–12,14–18
]. We hypothesized that expression of WISP3 could result in a series of molecular events that leads to the modulation of IGF-IR activation and downstream signaling. Contributing to this hypothesis is the fact that we have shown that WISP3 is secreted into the media where it has the opportunity to directly or indirectly modulate the strength of IGF signaling. Indeed, in the presence of IGF-I, WISP3 containing conditioned media decreased IGF-IR phosphorylation and the phosphorylation of two main downstream IGF-IR signaling molecules, IRS1 and ERK-1/2. This inhibition was not evident under baseline conditions, without stimulation with IGF-I. Our experiments thus show that even relatively small concentrations of WISP3 secreted by WISP3-transfected cells are able to modulate, directly or indirectly, IGF-I signaling in the setting of IGF-I stimulation.
A major growth-regulatory IGF-IR downstream pathway that regulates breast cancer growth and survival converges on ERK-1/2 cascade [34
]. We observed a decrease in ERK-1/2 phosphorylation by addition of WISP3 containing conditioned media in the presence of IGF-I stimulation. ERK-1/2 influence chromatin remodeling and activation of gene expression, leading to enhanced cellular proliferation and decreased apoptosis [35–37
]. Specifically, ERK-1/2 have been shown to activate the transcription of key genes involved in cell cycle progression including cyclin D1 and cyclin E. We have shown previously that restoration of WISP3 expression in the highly malignant SUM149 IBC cell line markedly decreased the levels of cyclin E and PCNA, a reliable marker of cellular proliferation [6
The mechanism whereby WISP3 may modulate IGF-IR activation in the presence of IGF-I remains to be elucidated. WISP3 contains a highly conserved motif (GCGCCXXC) characteristic of IGFBPs, which may provide the proper protein folding to interact with IGF-like ligands, thereby enabling interference with IGF signaling. Although initial studies reported that two other CCN proteins, CTGF (CTGF) and Nov, specifically bind to IGF-I [38,39
], these results have not been subsequently built on and they remain to be duplicated by other investigators. Whether WISP3 physically binds to IGF-I warrants further investigation, in light of out data.
Another mechanism that may explain the modulation of IGF-IR phosphorylation by WISP3-containing conditioned media is the formation of a WISP3/IGF-I complex that may bind to the IGF-IR and occupy IGF binding sites, but the complex may be either inhibitory or may be only a weak agonist of the receptor. In another system [40
], this hypothesis is supported by recent data showing that IGF-I can still freely bind to the receptor even when complexed to a truncated N-terminal fragment of IGF-binding protein 5 (mini-IGFBP5); interestingly, the N-terminal portion of IGFBP5 has high homology to the N-terminal portion of WISP3. Mini-IGFBP5 binding to IGF-I resulted in incomplete inhibition of receptor binding [40
]. In a similar manner, a WISP3/IGF-I complex might still bind to the IGF-IR but exert only a weak agonist effect, effectively resulting in physiologic antagonism of IGF-I action under conditions of high IGF-I stimulation. WISP3-rich conditioned media was able to significantly decrease the proliferation rate of IBC cells. The fact that this effect was seen both in the presence and absence of IGF-I simulation suggests that, in addition to interfering with IGF-I signaling pathways, WISP3 may have IGF-independent functions in IBC. In sum, we show that WISP3 is a secreted protein that modulates IGF-I signaling pathways, leading to a decrease in the growth of IBC cells.