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Tumor metastasis is the leading cause of cancer-associated mortality. Unfortunately, the underlying mechanism of metastasis is poorly understood. Expression of legumain (LGMN), an endo-lysosomal cysteine protease, positively correlates with breast cancer metastatic progression and poor prognosis. Here, we report that LGMN is secreted in the zymogen form by motile breast cancer cells. Through binding to cell surface integrin αvβ3 via an RGD motif, the autocrine pro-LGMN activates FAK-Src-RhoA signaling in cancer cells and promotes cancer cell migration and invasion independent of LGMN protease activity. Either silencing LGMN expression or mutationally abolishing pro-LGMN‒αvβ3 interaction significantly inhibits cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Finally, we developed a monoclonal antibody against LGMN RGD motif, which blocks pro-LGMN‒αvβ3 binding, and effectively suppresses cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Thus, disruption of pro-LGMN‒integrin αvβ3 interaction may be a potentially promising strategy for treating breast cancer metastasis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Intestinal stem cell (ISC) differentiation is regulated precisely by a niche in the crypt, where lymphocytes may interact with stem and transient amplifying (TA) cells. However, whether and how lymphocyte-stem/TA cell contact affects ISC differentiation is largely unknown. Here, we uncover a novel role of T cell-stem/TA cell contact in ISC fate decisions. We show that intestinal lymphocyte depletion results in skewed ISC differentiation in mice, which can be rescued by T cell transfer. Mechanistically, integrin αEβ7 expressed on T cells binds to E-cadherin on ISCs and TA cells, triggering E-cadherin endocytosis and the consequent Wnt and Notch signaling alterations. Blocking αEβ7-E-cadherin adhesion suppresses Wnt signaling and promotes Notch signaling in ISCs and TA cells, leading to defective ISC differentiation. Thus, αEβ7 + T cells regulate ISC differentiation at single-cell level through cell-cell contact-mediated αEβ7-E-cadherin adhesion signaling, highlighting a critical role of the T cell-stem/TA cell contact in maintaining intestinal homeostasis., (© 2021. The Author(s), under exclusive licence to Center for Excellence in Molecular Cell Science, CAS.)

Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells 1,2 . However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported 3-6 , are strategically localized at the bases of colon crypts, and function to maintain LGR5 + intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)-MAP3K2-ERK5-KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine.

Breast cancer is a heterogeneous disease. In particular, triple-negative breast cancer (TNBC) comprises various molecular subgroups with unclear identities and currently has few targeted treatment options. Our previous study identified protein C receptor (Procr) as a surface marker on mammary stem cells (MaSCs) located in the basal layer of the normal mammary gland. Given the possible connection of TNBC with basal layer stem cells, we conducted comparative analyses of Procr in breast cancers of mouse and human origin. In mouse mammary tumors, we showed that Procr + cells are enriched for cancer stem cells (CSCs) in Wnt1 basal-like tumors, but not in Brca1 basal-like tumors or PyVT luminal tumors. In human cancers, PROCR was robustly expressed in half of TNBC cases. Experiments with patient-derived xenografts (PDXs) revealed that PROCR marks CSCs in this discrete subgroup (referred to as PROCR + TNBC). Interfering with the function of PROCR using an inhibitory nanobody reduced the CSC numbers, arrested tumor growth and prevented rapid tumor recurrence. Our data suggest a key role of MaSC in breast tumorigenesis. Moreover, our work indicates that PROCR can be used as a biomarker to stratify TNBC into clinically relevant subgroups and may provide a novel targeted treatment strategy for this clinically important tumor subtype.

In the mammary gland, it is widely believed that the luminal cells are unipotent after birth, contributing only to the luminal compartment in normal development. Here, by lineage tracing, we uncovered an unexpected potential of luminal cells that can give rise to basal cells during pregnancy. These luminal-derived basal cells (LdBCs) persisted through mammary regression and generated more progeny in successive rounds of pregnancies. LdBCs express basal markers as well as estrogen receptor α (ERα). In ovariectomized (OVX) mice, stimulation with estrogen and progesterone promoted the formation of LdBCs. In serial transplantation assays, LdBCs were able to reconstitute new mammary glands in a hormone-dependent manner. Transcriptome analysis and genetic experiments suggest that Wnt/β-catenin signaling is essential for the formation and maintenance of LdBCs. Our data uncover an unexpected bi-potency of luminal cells in a physiological context. The discovery of ERα + basal cells, which can respond to hormones and are endowed with stem cell-like regenerative capacity in parous mammary gland, provides new insights into the association of hormones and breast cancer.

Wnt/β-catenin signaling is instrumental for the development of mammary gland and the properties of mammary stem cells (MaSCs). The Wnt signaling downstream effectors that engage in regulating MaSCs have not been extensively studied. Here, we report that Neuropilin-1 (Nrp1) expression is induced by Wnt/β-catenin signaling in MaSCs, and its function is critical for the activity of MaSCs. Nrp1 is particularly expressed in MaSCs that are marked by the expression of Protein C Receptor (Procr). Knockdown of Nrp1 by shRNA diminishes MaSCs' in vitro colony formation and in vivo mammary gland reconstitution ability. Similar results are seen when antagonizing Nrp1 using a dominant negative peptide. In genetic experiments, deletion of Nrp1 results in delay of mammary development. In addition, knockdown of Nrp1 inhibits MMTV-Wnt1 tumor growth in xenograft. Our data demonstrate that Nrp1 is critical for mammary development and tumorigenesis, revealing new insights into MaSC regulation and targeting stem cells in treatment of breast cancer.

Vascular growth and remodeling are dependent on the generation of new endothelial cells from stem cells and the involvement of perivascular cells to maintain vessel integrity and function. The existence and cellular identity of vascular endothelial stem cells (VESCs) remain unclear. The perivascular pericytes in adult tissues are thought to arise from the recruitment and differentiation of mesenchymal progenitors during early development. In this study, we identified Protein C receptor-expressing (Procr + ) endothelial cells as VESCs in multiple tissues. Procr + VESCs exhibit robust clonogenicity in culture, high vessel reconstitution efficiency in transplantation, long-term clonal expansion in lineage tracing, and EndMT characteristics. Moreover, Procr + VESCs are bipotent, giving rise to de novo formation of endothelial cells and pericytes. This represents a novel origin of pericytes in adult angiogenesis, reshaping our understanding of blood vessel development and homeostatic process. Our study may also provide a more precise therapeutic target to inhibit pathological angiogenesis and tumor growth.

The mammary gland is composed of multiple types of epithelial cells, which are generated by mammary stem cells (MaSCs) residing at the top of the hierarchy. However, the existence of these multipotent MaSCs remains controversial and the nature of such cells is unknown. Here we demonstrate that protein C receptor (Procr), a novel Wnt target in the mammary gland, marks a unique population of multipotent mouse MaSCs. Procr-positive cells localize to the basal layer, exhibit epithelial-to-mesenchymal transition characteristics, and express low levels of basal keratins. Procr-expressing cells have a high regenerative capacity in transplantation assays and differentiate into all lineages of the mammary epithelium by lineage tracing. These results define a novel multipotent mammary stem cell population that could be important in the initiation of breast cancer.