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. 2004;6(1):R110-5.
doi: 10.1186/bcr755.

WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer

Celina G Kleer  1 , Yanhong Zhang, Quintin Pan, Gary Gallagher, Mei Wu, Zhi-Fen Wu, Sofia D Merajver
Affiliations

WISP3 and RhoC guanosine triphosphatase cooperate in the development of inflammatory breast cancer

Celina G Kleer et al. Breast Cancer Res. 2004.

Abstract

Background: Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer. We found concordant and consistent alterations of two genes in 90% of IBC tumors when compared to stage matched, non-IBC tumors: overexpression of RhoC GTPase and loss of WISP3. Further work revealed that RhoC is a transforming oncogene for HME cells. Despite the aggressiveness of the RhoC-driven phenotype, it does not quantitatively reach that of the true IBC tumors. We have demonstrated that WISP3 has tumor growth and angiogenesis inhibitory functions in IBC. We hypothesized that RhoC and WISP3 cooperate in the development of IBC.

Methods: Using an antisense approach, we blocked WISP3 expression in HME cells (HME/AS WISP3). Cellular proliferation and anchorage independent growth were determined using the MTT assay and the anchorage independent growth in soft agar assay. VEGF was measured in the conditioned media of the HME/ AS WISP3 by ELISA.

Results: Antisense inhibition of WISP3 expression in HME cells increased the levels of RhoC mRNA and increased cellular proliferation, anchorage independent growth and secretion of VEGF in these cells. Conversely, restoration of WISP3 expression in the highly malignant IBC cell line SUM149 was able to decrease the expression of RhoC protein.

Conclusion: WISP3 modulates RhoC expression in HME cells and in the IBC cell line SUM149, and provide further evidence in support that these two genes act in concert to give rise to the highly aggressive IBC phenotype. We propose a model of this interaction as a starting point for further investigations.

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Figures

Figure 1
Figure 1
Inhibition of WISP3 in human mammary epithelial (HME) cells results in an increase in RhoC transcript levels. Reverse transcriptase polymerase chain reaction was conducted on vector and HME cells that have inhibition of WISP3 expression using full-length WISP3 antisense mRNA. HME/AS WISP3 cells showed increased levels of RhoC transcript in comparison with controls.
Figure 2
Figure 2
Inhibition of WISP3 induces anchorage-independent growth, proliferation and secretion of vascular endothelial growth factor (VEGF) in human mammary epithelial (HME) cells. (a) Inhibition of WISP3 expression in HME cells. HME cells greatly increased the number of colonies formed in soft agar in comparison with empty vector control (HME/Flag; t-test, P < 0.05 for both clones). (b) Effect of inhibition of WISP3 expression on the proliferation of HME cells was studied with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The stable HME/AS WISP3 cells have a significant increase in the proliferation rate in comparison with the empty vector control. Results are expressed as means ± SEM of three independent experiments. In all, 3000 cells were assessed in each plate (t-test, P < 0.05). (c) Increase in VEGF measured by enzyme-linked immunosorbent assay, as a result of inhibition of WISP3 expression in HME cells. Results are expressed as means ± SEM; t-test, P < 0.05 for all clones.
Figure 3
Figure 3
Restoration of WISP3 expression in SUM149 inflammatory breast cancer cells decreases RhoC protein expression. Western immunoblot of cell culture of SUM 149 cells, empty vector control (SUM149/Flag), and two WISP3-expressing clones with antibodies against RhoC and actin. Gels were scanned and pixel intensity values were obtained. Values for RhoC were corrected for loading by dividing the RhoC pixel intensity by the actin pixel intensity.
Figure 4
Figure 4
RhoC overexpression in MCF10A cells results in a decrease in WISP3 mRNA level. Reverse transcriptase polymerase chain reaction was conducted on vector and MCF10A cells stably overexpressing RhoC. RhoC-overexpressing cells had decreased levels of WISP3 mRNA.
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