KISS1R Induces Invasiveness of Estrogen Receptor-Negative Human Mammary Epithelial and Breast Cancer Cells

Abstract

Kisspeptins (KPs), peptide products of the KISS1 metastasis-suppressor gene, are endogenous ligands for a G protein-coupled receptor (KISS1R). KISS1 acts as a metastasis suppressor in numerous human cancers. However, recent studies have demonstrated that an increase in KISS1 and KISS1R expression in patient breast tumors correlates with higher tumor grade and metastatic potential. We have shown that KP-10 stimulates invasion of estrogen receptor ␣ (ER␣)-negative MDA-MB-231 breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR). Here, we report that either KP-10 treatment of ER␣-negative nonmalignant mammary epithelial MCF10A cells or expression of KISS1R in MCF10A cells induced a mesenchymal phenotype and stimulated invasiveness. Similarly, exogenous expression of KISS1R in ER␣-negative SKBR3 breast cancer cells was sufficient to trigger invasion and induced extravasation in vivo. In contrast, KP-10 failed to transactivate EGFR or stimulate invasiveness in the ER␣-positive MCF7 and T47D breast cancer cells. This suggested that ER␣ negatively regulates KISS1R-dependent breast cancer cell migration, invasion, and EGFR transactivation. In support of this, we found that these KP-10-induced effects were ablated upon exogenous expression of ER␣ in the MDA-MB-231 cells, by down-regulating KISS1R expression. Lastly, we have identified IQGAP1, an actin cytoskeletal binding protein as a novel binding partner of KISS1R, and have shown that KISS1R regulates EGFR transactivation in breast cancer cells in an IQGAP1-dependent manner. Overall, our data strongly suggest that the ER␣ status of mammary cells dictates whether KISS1R may be a novel clinical target for treating breast cancer metastasis. (Endocrinology

Stage of Breast Cancer Progression Influences Cellular Response to Activation of the WNT/Planar Cell Polarity Pathway

“…In order to assess cell invasiveness in vivo, we used a chick embryo chorioallantoic membrane vein injection assay for cell extravasation33343536. In this assay, tumor cells are injected intravenously into ex ovo chicken embryos.…”

“…The chorioallantoic membrane (CAM) of the chicken embryo is a well-established model for quantifying extravasation rates by IV-injected tumor cells33343536. Immediately after IV-injection, ZsGreen positive 21T cells arrest within the intravascular portion of the CAM as identified by lectin-rhodamine injected IV.…”

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

“…Alternately, the capacity of breast cancer cells to form invasive protrusions can be quantified upon genetically modifying the cells to express a potential oncogene or reduced gene expression using RNA interference (such as shRNA) 18,24,25 .…”

“…By utilizing immunofluorescence staining, the expression of these molecules can be visually detected within the 3D culture. In Figure 3E, we show representative invasive (stellate) colonies of the MDA-MB-231 cells displaying a loss of membrane integrity and diffuse localization of the basement membrane protein laminin V 24 . In stark contrast to what is observed in the breast cancer cells, laminin V was localized to an intact basement membrane layer enclosing the mammary acini of untreated non-malignant MCF10A cells (Figure 3E) These innovative 3D culture models allow researchers to study phenotypic changes that are not possible when studying cells in monolayer, using traditional transwell chamber invasion assays.…”

“…To conclude, the variety of possible applications makes the use of 3D cultures a dynamic assay that can be used with a wide spectrum of cells, whether pathological and/or normal in origin allowing for the evaluation of changes in cellular morphology and invasion in fixed samples or in real-time 18,19,24 .…”