Title Colon cancer cells adopt an invasive phenotype without mesenchymal transition in 3-D but not 2-D culture upon combined stimulation with EGF and crypt growth factors Permalink

Background: The intestinal crypt homeostasis is maintained by a combination of growth factors including Wnt, R-Spondin1, Noggin and the epidermal growth factor (EGF). In human colorectal cancer, the Wnt pathway is constitutively activated through genetic and epigenetic alterations in as many as 11 genes encoding components of this crypt stem-cell maintenance mechanism. Although the proliferation of colon cancer cells does not require Wnt, it is possible that colon cancer cells can still respond to the crypt growth factors in the colonic microenvironment. A number of studies have shown that epithelial cells behave differently in 3-D versus 2-D cultures. Because the 3-D conditions more closely mimic the in vivo environment, we examined the effects of Wnt and other crypt growth factors on colon cancer cell growth in 3-D culture. Methods: Colon cancer cells were grown in 3-D matrigel supplemented with different combinations of crypt growth factors and colonies were examined for morphology and pathways. Results: When colon cancer cells were cultured in 3-D with EGF, they grew as round spheroid colonies. However, colon cancer cells also grew as flat, disc-like colonies when cultured with EGF plus Wnt, R-Spondin1 and Noggin. Disc colonies were found to have comparable levels of E-cadherin as the spheroid colonies, but showed decreased E-cadherin at the cell-matrix contact sites. Disc colonies also elaborated F-actin rich protrusions (FRP) at the cell-matrix edge, reminiscent of an invasive phenotype but without the expression of vimentin. These E-cadherin and F-actin alterations were not induced by the four growth factors in 2-D culture. Formation of the disc colonies was inhibited by the knockdown of β-catenin and by protein kinase inhibitors such as gefitinib, imatinib and MK-2206. Furthermore, withdrawal of the crypt growth factors was able to revert the disc colonies to spheroid growth, showing that the invasive phenotype was reversible dependent on the availability of growth factors. Conclusions: These findings show that colon cancer cells remain responsive to the growth factors in the crypt microenvironment and can be induced to undergo morphological transformation in the more physiologically relevant 3-D culture. * Correspondence: jywang@ucsd.edu Moores UCSD Cancer Center, 3855 Health Sciences Drive, La Jolla, CA 92093-0820, USA Department of Medicine, Division of Hematology-Oncology, University of California, La Jolla, CA 92093, USA © 2013 Ludwig et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ludwig et al. BMC Cancer 2013, 13:221 Page 2 of 12 http://www.biomedcentral. com/1471-2407/13/221 Background Invasive growth is a critical step in the progression of tumorigenesis as it is what distinguishes a malignant from a benign tumor [1]. A tumor’s ability to disseminate, invade and migrate to distant tissues correlates with worse prognosis [2]. The edge of an invasive tumor is characterized by the loss of apico-basal polarity along with a loss of cell-cell junctions and decreased E-cadherin expression. The actin cytoskeleton is reorganized with the formation of F-actin rich protrusions (FRP) at the leading edge of an invasive tumor, where the cell changes from a cuboidal shape to a motile spindle shape [3]. The cell motility pathways such as those controlled by the integrin receptors, the focal adhesion kinase (FAK), the Rho and Rac family of small G-proteins, and the metalloproteases (MMPs) are also activated in the invasive tumor cells [4]. Histology of colon tumor samples has shown that some of these characteristics, i.e. change in shape and loss of E-cadherin, are found only at the leading edge of the tumor in cells that have direct contact with the ECM, while cells fully encased in the solid tumor maintain expression of E-cadherin [5]. It thus appears that the invasive phenotype might occur in individual cells responding to the external cues rather than the entire tumor mass undergoing global changes. The recent TCGA (The Cancer Genome Atlas) analysis of human colorectal cancer (CRC) has established that the Wnt and the TGF-β (BMP) pathways are consistently up or down regulated, respectively, by genetic and epigenetic mechanisms in 97% and 87% of CRC in the hypermutated group [6]. The Wnt pathway is also upregulated in 92% of CRC in the non-hypermutated group [6]. This finding is consistent with the fact that maintenance of the intestinal crypt stem cells requires full activation of the Wnt pathway and inactivation of the BMP pathway by the anti-BMP ligand Noggin [7]. In the intestinal crypt compartment, binding of locally produced Wnt and R-Spondin to their respective seven transmembraneserpetine receptors, Frizzled and Lgr4/5, leads to the assembly of a Wnt signaling complex involving the recruitment of another membrane receptor, LRP, and the stabilization of cytoplasmic β-catenin [8]. The accumulation of cytoplasmic β-catenin is a pre-request for its nuclear translocation, which is regulated by a variety of factors, as β-catenin itself does not contain any nuclear localization signals [9]. Nuclear β-catenin associates with the TCF-family of transcription factors to stimulate gene expression that promotes cell cycle progression and inhibits apoptosis [8]. In the normal regenerating intestinal tissue, Wnt and Noggin levels are high at the base of the crypt to stimulate proliferation and inhibit differentiation. The concentrations of these factors are reduced in the villi, where Wnt and Noggin levels are low and BMP levels are high, promoting differentiation [10]. With the constitutive activation of the Wnt and the receptor tyrosine kinase (RTK) pathways as well as the downregulation of the TGF-β pathway, colon cancer cells do not require this complement of factors to proliferate. In this study, we show that established colon cancer cells remain responsive to the stimulation of a complement of crypt growth factors to undergo a reversible and localized invasive phenotype but only in 3-D cultures. This invasive response requires activation of β-catenin and EGFR and can be inhibited by drugs that interfere with the function of downstream effectors such as ABL or AKT.