A YAP-centered mechanotransduction loop drives collective breast cancer cell invasion.

Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion.

R-spondin-3 promotes proliferation and invasion of breast cancer cells independently of Wnt signaling.

We recently identified R-spondin-3 (RSPO3) as a novel driver of breast cancer associating with reduced patient survival, expanding its clinical value as potential therapeutic target that had been recognized mostly for colorectal cancer so far. (Pre)clinical studies exploring RSPO3 targeting in colorectal cancer approach this indirectly with Wnt inhibitors, or directly with anti-RSPO3 antibodies. Here, we address the clinical relevance of RSPO3 in breast cancer and provide insight in the oncogenic activities of RSPO3. Utilizing the RSPO3 breast cancer mouse model, we show that RSPO3 drives the aberrant expansion of luminal progenitor cells expressing cancer stem cell marker CD61, inducing proliferative, poorly differentiated and invasive tumors. Complementary studies with tumor organoids and human breast cancer cell lines demonstrate that RSPO3 consistently promotes the proliferation and invasion of breast cancer cells. Importantly, RSPO3 exerts these oncogenic effects independently of Wnt signaling, rejecting the therapeutic value of Wnt inhibitors in RSPO3-driven breast cancer. Instead, direct RSPO3 targeting effectively inhibited RSPO3-driven growth of breast cancer cells. Conclusively, our data indicate that RSPO3 exerts unfavorable oncogenic effects in breast cancer, enhancing proliferation and malignancy in a Wnt-independent fashion, proposing RSPO3 itself as a valuable therapeutic target in breast cancer.

Spatial collagen stiffening promotes collective breast cancer cell invasion by reinforcing extracellular matrix alignment.

The tumor micro-environment often contains stiff and irregular-bundled collagen fibers that are used by tumor cells to disseminate. It is still unclear how and to what extent, extracellular matrix (ECM) stiffness versus ECM bundle size and alignment dictate cancer cell invasion. Here, we have uncoupled Collagen-I bundling from stiffness by introducing inter-collagen crosslinks, combined with temperature induced aggregation of collagen bundling. Using organotypic models from mouse invasive ductal and invasive lobular breast cancers, we show that increased collagen bundling in 3D induces a generic increase in breast cancer invasion that is independent of migration mode. However, systemic collagen stiffening using advanced glycation end product (AGE) crosslinking prevents collective invasion, while leaving single cell invasion unaffected. Collective invasion into collagen matrices by ductal breast cancer cells depends on Lysyl oxidase-like 3 (Loxl3), a factor produced by tumor cells that reinforces local collagen stiffness. Finally, we present clinical evidence that collectively invading cancer cells at the invasive front of ductal breast carcinoma upregulate LOXL3. By uncoupling the mechanical, chemical, and structural cues that control invasion of breast cancer in three dimensions, our data reveal that spatial control over stiffness and bundling underlie collective dissemination of ductal-type breast cancers.