Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells

Abstract

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial-mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three-dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/β-catenin pathway, or short hairpin RNA targeting β-catenin expression demonstrated that the invasive activity was not mediated by β-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the β-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures.

Therapeutic Targeting of Tumor-Derived R-Spondin Attenuates β-Catenin Signaling and Tumorigenesis in Multiple Cancer Types

“…The identification of Rspo2 and Rspo3 as sites of integration for MMTV-induced mammary tumors in mice supported a role as breast cancer oncogenes (26)(27)(28). Ectopic expression of Rspo2 was shown to increase tumorigenic and invasive properties of mouse breast cell lines (25). Genomic rearrangements and transcriptional activation in human tumors that result in elevated RSPO expression have been identified, suggesting that RSPO may be functionally important for tumor development (29)(30)(31)(32).…”

“…In most cases, RSPOs function by enhancing Wnt/b-catenin signaling. However, RSPO2 or 3 were also shown to signal through cascades that are separate from the canonical pathway (5,8,19,(23)(24)(25).…”

The wnt/β‐catenin signaling pathway: A potential therapeutic target in the treatment of triple negative breast cancer

“…Furthermore, it has been recently demonstrated that mouse mammary epithelial cells expressing Wnt1 and Rspo2 independently or together can develop into tumors in nude mice. In addition, Rspo2-derived tumors exhibit a strong epithelial mesenchymal transformation (EMT) phenotype and are highly metastatic to the lung as well as the spleen [Klauzinska et al, 2011]. These studies suggest a potential positive contribution of Rspos to mammary gland tumorigenesis.…”

Breast cancer lung metastasis: Molecular biology and therapeutic implications

“…60 Other studies suggested that Wnt/b-catenin can also regulate breast cancer cell proliferation. 61,62 The proteins of the Wnt family are functionally separated into two classes: those activating the canonical Wnt/b-catenin pathway and those activating the planar cell polarity and Wnt/calcium pathways, which do not involve b-catenin. 63,64 Recently, it was reported that the inhibitor of Wnt Dickkopf-related protein 1 (DKK1) suppresses macrophage and neutrophil recruitment in breast cancer lung metastases in part by antagonizing cancer cell non-canonical Wnt-JNK signaling.…”