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1.
J Vis Exp ; (88)2014 Jun 11.
Article in English | MEDLINE | ID: mdl-24961804

ABSTRACT

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.


Subject(s)
Breast Neoplasms/pathology , Cell Culture Techniques/methods , Basement Membrane/pathology , Cell Line, Tumor , Female , Humans , Imaging, Three-Dimensional/methods , MCF-7 Cells , Neoplasm Invasiveness , Tumor Cells, Cultured
3.
J Cancer ; 4(8): 653-61, 2013 Sep 26.
Article in English | MEDLINE | ID: mdl-24155777

ABSTRACT

Kisspeptins (KP), peptide products of the kisspeptin-1 (KISS1) gene are the endogenous ligands for a G protein-coupled receptor (GPCR) - KP receptor (KISS1R). KISS1R couples to the Gαq/11 signaling pathway. KISS1 is a metastasis suppressor gene and the KP/KISS1R signaling has anti-metastatic and tumor-suppressant effects in numerous human cancers. On the other hand, recent studies indicate that KP/KISS1R pathway plays detrimental roles in breast cancer. In this review, we summarize recent developments in the understanding of the mechanisms regulating KP/KISS1R signaling in breast cancer metastasis.

4.
Endocrinology ; 154(6): 1999-2014, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23525242

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 α (ERα)-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 ERα-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 ERα-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 ERα-positive MCF7 and T47D breast cancer cells. This suggested that ERα 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 ERα 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 ERα status of mammary cells dictates whether KISS1R may be a novel clinical target for treating breast cancer metastasis.


Subject(s)
Cell Movement , Epithelial Cells/metabolism , Estrogen Receptor alpha/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line , Cell Line, Tumor , Chick Embryo , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Estrogen Receptor alpha/genetics , Female , Humans , Inositol Phosphates/metabolism , Kisspeptins/pharmacology , MCF-7 Cells , Mammary Glands, Human , Microscopy, Fluorescence , Neoplasm Invasiveness , RNA Interference , Receptors, G-Protein-Coupled/genetics , Receptors, Kisspeptin-1 , ras GTPase-Activating Proteins/genetics , ras GTPase-Activating Proteins/metabolism
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