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Snail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here an EMT-independent action of Snail1 on tumor invasion, as it is required for the activation of cancer-associated fibroblasts (CAF). Snail1 expression in fibroblasts requires signals derived from tumor cells, such as TGFβ; reciprocally, in fibroblasts, Snail1 organizes a complex program that stimulates invasion of epithelial cells independent of the expression of Snail1 in these cells. Epithelial cell invasion is stimulated by the secretion by fibroblast of diffusible signaling molecules, such as prostaglandin E2. The capability of human or murine CAFs to promote tumor invasion is dependent on Snail1 expression. Inducible Snail1 depletion in mice decreases the invasion of breast tumors; moreover, epithelial tumor cells coxenografted with Snail1-depleted fibroblasts originated tumors with lower invasion than those transplanted with control fibroblasts. Therefore, these results demonstrate that the role of Snail1 in tumor invasion is not limited to EMT, but it is also dependent on its activity in stromal fibroblasts, where it orchestrates the cross-talk with epithelial tumor cells. Cancer Res; 76(21); 6205–17. ©2016 AACR.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Invasion of colon and breast cancer cell lines (S1); Mesenchymal Stem Cells enhance tumor invasion through Collagen or Matrigel matrices (S2); Invasion of HT-29 M6 is preceded by that of MSCs (S3); Generation of MSCs and MEFs depleted in Snail1 (S4); Generation of HT-29 M6 Snail1 CRISPR KO cells (S5); Genes differently regulated by HT-29 M6 in MSCs wild-type and Snail1 KO (S6); Prostaglandin E2 acts as a chemoattractant (S7); Inhibition of PGE2 signaling prevents HT-29 M6 invasion (S8); PGE2 stimulates HT-29 M6 invasion (S9); Snail1 expression in MSCs is required for the MSCs stimulation of invasion of different epithelial cell lines (S10); Expression of EP4 PGE2 receptor in epithelial cell lines (S11); Isolation of murine and human CAFs with different Snail1 levels (S12); Identification of ePyMT metastases in the lung (S13); Celecoxib does not modify tumor size in ePyMT-MSCs co-xenografts (S14).

Tumors are complex tissues composed of transformed epithelial cells as well as cancer-activated fibroblasts (CAF) that facilitate epithelial tumor cell invasion. We show here that CAFs and other mesenchymal cells rely much more on glutamine than epithelial tumor cells; consequently, they are more sensitive to inhibition of glutaminase. Glutamine dependence drove CAF migration toward this amino acid when cultured in low glutamine conditions. CAFs also invaded a Matrigel matrix following a glutamine concentration gradient and enhanced the invasion of tumor cells when both cells were cocultured. Accordingly, glutamine directed invasion of xenografted tumors in immunocompromised mice. Stimulation of glutamine-driven epithelial tumor invasion by fibroblasts required previous CAF activation, which involved the TGFβ/Snail1 signaling axis. CAFs moving toward Gln presented a polarized Akt2 distribution that was modulated by the Gln-dependent activity of TRAF6 and p62 in the migrating front, and depletion of these proteins prevented Akt2 polarization and Gln-driven CAF invasion. Our results demonstrate that glutamine deprivation promotes CAF migration and invasion, which in turn facilitates the movement of tumor epithelial cells toward nutrient-rich territories. These results provide a novel molecular mechanism for how metabolic stress enhances invasion and metastasis.Significance:Cancer-associated fibroblasts migrate and invade toward free glutamine and facilitate invasion of tumor epithelial cells, accounting for their movement away from the hostile conditions of the tumor towards nutrient-rich adjacent tissues.

Video showing migration of activated CAFs toward the Gln-rich compartment when cultured in Gln-high conditions (2 mM)

Supplementary Information containing supplementary methods, eight supplementary figures with legends and one supplementary table.

Video showing migration of activated CAFs toward the Gln-rich compartment when cultured in a Gln-concentration gradient (0.2-2 mM)

Legend for Supplementary Figures S1-S14.

Video S4. MDA-MB231 cells on 3D-ECM generated by TGFβ-treated KO MEFs.

Video S6. Ela-Myc cells on 3D-ECM generated by TGFβ-treated Control MEFs.

Supplemental Methods

Supplementary Figure S1. TGF beta remodels the ECM generated by mesenchymal stem and 1BR3G cells in a Snail1-dependent manner. Supplementary Figure S2. Cell Commitment is Modulated by Fibroblast-derived Matrices in a Snail1-dependent Manner. Supplementary Figure S3. TGF beta controls focal contacts in a Snail1-dependent manner. Supplementary Figure S4. TGF beta regulates N-cadherin intercellular contacts in a Snail1-dependent manner. Supplementary Figure S5. Alignment of MEFs bo-cultured with CAFs. Supplementary Table S1. Baseline Characteristics According to Snail1 Expression in the Stroma Supplementary Table S2. Overall Survival Analysis in Patients with Snail1 Expression in Stroma Supplementary Table S3. Antibodies Used for Protein Analysis

Video S5. Ela-Myc cells on 3D-ECM generated by Control MEFs.

Video S7. Ela-Myc cells on 3D-ECM generated by KO MEFs.

Video S1. MDA-MB231 cells on 3D-ECM generated by Control MEFs.

Video S3. MDA-MB231 cells on 3D-ECM generated by KO MEFs.

Video S2. MDA-MB231 cells on 3D-ECM generated by TGFβ-treated Control MEFs.

Video S8. Ela-Myc cells on 3D-ECM generated by TGFβ-treated KO MEFs.

Crosstalk between tumor and stromal cells in the tumor microenvironment alter its properties in ways that facilitate the invasive behavior of tumor cells. Here, we demonstrate that cancer-associated fibroblasts (CAF) increase the stiffness of the extracellular matrix (ECM) and promote anisotropic fiber orientation, two mechanical signals generated through a Snail1/RhoA/αSMA–dependent mechanism that sustains oriented tumor cell migration and invasiveness. Snail1-depleted CAF failed to acquire myofibroblastic traits in response to TGFβ, including RhoA activation, αSMA-positive stress fibers, increased fibronectin fibrillogenesis, and production of a stiff ECM with oriented fibers. Snail1 expression in human tumor–derived CAF was associated with an ability to organize the ECM. In coculture, a relatively smaller number of Snail1-expressing CAF were capable of imposing an anisotropic ECM architecture, compared with nonactivated fibroblasts. Pathologically, human breast cancers with Snail1+ CAF tended to exhibit desmoplastic areas with anisotropic fibers, lymph node involvement, and poorer outcomes. Snail1 involvement in driving an ordered ECM was further confirmed in wound-healing experiments in mice, with Snail1 depletion preventing the anisotropic organization of granulation tissue and delaying wound healing. Overall, our results showed that inhibiting Snail1 function in CAF could prevent tumor-driven ECM reorganization and cancer invasion. Cancer Res; 75(2); 284–95. ©2014 AACR.