Niche inflammatory signals control oscillating mammary regeneration and protect stem cells from cytotoxic stress.

Stem cells are known for their resilience and enhanced activity post-stress. The mammary gland undergoes frequent remodeling and is subjected to recurring stress during the estrus cycle, but it remains unclear how mammary stem cells (MaSCs) respond to the stress and contribute to regeneration. We discovered that cytotoxic stress-induced activation of CD11c+ ductal macrophages aids stem cell survival and prevents differentiation. These macrophages boost Procr+ MaSC activity through IL1ß-IL1R1-NF-κB signaling during the estrus cycle in an oscillating manner. Deleting IL1R1 in MaSCs results in stem cell loss and skewed luminal differentiation. Moreover, under cytotoxic stress from the chemotherapy agent paclitaxel, ductal macrophages secrete higher IL1ß levels, promoting MaSC survival and preventing differentiation. Inhibiting IL1R1 sensitizes MaSCs to paclitaxel. Our findings reveal a recurring inflammatory process that regulates regeneration, providing insights into stress-induced inflammation and its impact on stem cell survival, potentially affecting cancer therapy efficacy.

LRP12 is an endogenous transmembrane inactivator of α4 integrins.

Dynamic regulation of integrin activation and inactivation is critical for precisely controlled cell adhesion and migration in physiological and pathological processes. The molecular basis for integrin activation has been intensively studied; however, the understanding of integrin inactivation is still limited. Here, we identify LRP12 as an endogenous transmembrane inhibitor for α4 integrin activation. The LRP12 cytoplasmic domain directly binds to the integrin α4 cytoplasmic tail and inhibits talin binding to the ß subunit, thus keeping integrin inactive. In migrating cells, LRP12-α4 interaction induces nascent adhesion (NA) turnover at the leading-edge protrusion. Knockdown of LRP12 leads to increased NAs and enhanced cell migration. Consistently, LRP12-deficient T cells show an enhanced homing capability in mice and lead to aggravated chronic colitis in a T cell-transfer colitis model. Altogether, LRP12 is a transmembrane inactivator for integrins that inhibits α4 integrin activation and controls cell migration by maintaining balanced NA dynamics.

Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet-Induced Adiposity.

Recent genetic evidence has linked WNT downstream mutations to fat distribution. However, the roles of WNTs in human obesity remain unclear. Here, the authors screen all Wnt-related paracrine factors in 1994 obese cases and 2161 controls using whole-exome sequencing (WES) and identify that 12 obese patients harbor the same mutations in RSPO1 (p.R219W/Q) predisposing to human obesity. RSPO1 is predominantly expressed in visceral fat, primarily in the fibroblast cluster, and is increased with adiposity. Mice overexpressing human RSPO1 in adipose tissues develop obesity under a high-fat diet (HFD) due to reduced brown/beige fat thermogenesis. In contrast, Rspo1 ablation resists HFD-induced adiposity by increasing thermogenesis. Mechanistically, RSPO1 overexpression or administration significantly inhibits adipocyte mitochondrial respiration and thermogenesis via LGR4-Wnt/ß-catenin signaling pathway. Importantly, humanized knockin mice carrying the hotspot mutation (p.R219W) display suppressed thermogenesis and recapitulate the adiposity feature of obese carriers. The mutation disrupts RSPO1's electrostatic interaction with the extracellular matrix, leading to excessive RSPO1 release that activates LGR4-Wnt/ß-catenin signaling and attenuates thermogenic capacity in differentiated beige adipocytes. Therefore, these findings identify that gain-of-function mutations and excessive expression of RSPO1, acting as a paracrine Wnt activator, suppress fat thermogenesis and contribute to obesity in humans.

Activation of Wnt/ß-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry.

The establishment of a functional vasculature requires endothelial cells to enter quiescence during the completion of development, otherwise pathological overgrowth occurs. How such a transition is regulated remains unclear. Here, we uncover a role of Zeb1 in defining vascular quiescence entry. During quiescence acquisition, Zeb1 increases along with the progressive decline of endothelial progenitors' activities, with Zeb1 loss resulting in endothelial overgrowth and vascular deformities. RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) analyses reveal that Zeb1 represses Wif1, thereby activating Wnt/ß-catenin signaling. Knockdown of Wif1 rescues the overgrowth induced by Zeb1 deletion. Importantly, local administration of surrogate Wnt molecules in the retina ameliorates the overgrowth defects of Zeb1 mutants. These findings show a mechanism by which Zeb1 induces quiescence of endothelial progenitors during the establishing of vascular homeostasis, providing molecular insight into the inherited neovascular pathologies associated with human ZEB1 mutations, suggesting pharmacological activation of Wnt/ß-catenin signaling as a potential therapeutical approach.

CDK14 inhibition reduces mammary stem cell activity and suppresses triple negative breast cancer progression.

The Wnt/ß-catenin signaling pathway plays an important role in regulating mammary organogenesis and oncogenesis. However, therapeutic methods targeting the Wnt pathway against breast cancer have been limited. To address this challenge, we investigate the function of cyclin-dependent kinase 14 (CDK14), a member of the Wnt signaling pathway, in mammary development and breast cancer progression. We show that CDK14 is expressed in the mammary basal layer and elevated in triple negative breast cancer (TNBC). CDK14 knockdown reduces the colony-formation ability and regeneration capacity of mammary basal cells and inhibits the progression of murine MMTV-Wnt-1 basal-like mammary tumor. CDK14 knockdown or pharmacological inhibition by FMF-04-159-2 suppresses the progression and metastasis of TNBC. Mechanistically, CDK14 inhibition inhibits mammary regeneration and TNBC progression by attenuating Wnt/ß-catenin signaling. These findings highlight the significance of CDK14 in mammary development and TNBC progression, shedding light on CDK14 as a promising therapeutic target for TNBC.

Autocrine pro-legumain promotes breast cancer metastasis via binding to integrin αvß3.

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 αvß3 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‒αvß3 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‒αvß3 binding, and effectively suppresses cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Thus, disruption of pro-LGMN‒integrin αvß3 interaction may be a potentially promising strategy for treating breast cancer metastasis.

Isolation of mouse pancreatic islet Procr+ progenitors and long-term expansion of islet organoids in vitro.

Insulin production is required for glucose homeostasis. Pancreatic islet ß cells are the only cells that produce insulin in humans; however, generation of functional ß cells in vitro from embryonic or adult tissues has been challenging. Here, we describe isolation of pancreatic islet progenitors from adult mice, which enables the efficient generation and long-term expansion of functional islet organoids in vitro. This protocol starts with purification of protein C receptor (Procr)-expressing islet progenitors. Coculture with endothelial cells generates islet organoids in vitro that can be expanded by passage. Functional maturation is achieved as a consequence of a prolonged culture period and cyclic glucose stimulation. Primary islet organoids form in 7-10 days. Subsequently, each passage takes 1 week, with the final maturation step requiring 3 weeks of additional culture. The resulting organoids are predominantly composed of ß cells but also contain small proportions of α, δ and pancreatic polypeptide cells. The organoids sense glucose and secrete insulin. This approach thus provides a strategy for ß cell generation in vitro and an organoid system to study islet regeneration and diseases.

Procr functions as a signaling receptor and is essential for the maintenance and self-renewal of mammary stem cells.

The protein C receptor (Procr) has been implicated as a stem cell surface marker in several tissues. It is unknown whether Procr acts as a functional signaling receptor in stem cells. Here, by conditional knockout in mammary stem cells (MaSCs), we demonstrate that Procr is essential for mammary gland development and homeostasis. Through proteomics profiling, we identify that, upon stimulation by the ligand protein C, Procr interacts with heat shock protein 90 (HSP90AA1) via its short cytoplasmic tail, recruiting Src and IGF1R to the complex at the plasma membrane. We show that Procr acts as a signaling receptor of protein C in regulation of MaSCs through HSP90, Src, and IGF1R in vitro. In vivo, IGF1R deletion in MaSCs displays similar phenotypes to Procr deletion. These findings illustrate the essential role of Procr signaling in MaSC maintenance, shedding light onto the molecular regulation by Procr in tissue stem cells.

Selective YAP activation in Procr cells is essential for ovarian stem/progenitor expansion and epithelium repair.

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and OSE stem cells rapidly generate new cells for the repair. How the stem cell activation is triggered by the rupture and promptly turns on proliferation is unclear. Our previous study has identified that Protein C Receptor (Procr) marks OSE progenitors. In this study, we observed decreased adherent junction and selective activation of YAP signaling in Procr progenitors at OSE rupture site. OSE repair is impeded upon deletion of Yap1 in these progenitors. Interestingly, Procr+ progenitors show lower expression of Vgll4, an antagonist of YAP signaling. Overexpression of Vgll4 in Procr+ cells hampers OSE repair and progenitor proliferation, indicating that selective low Vgll4 expression in Procr+ progenitors is critical for OSE repair. In addition, YAP activation promotes transcription of the OSE stemness gene Procr. The combination of increased cell division and Procr expression leads to expansion of Procr+ progenitors surrounding the rupture site. These results illustrate a YAP-dependent mechanism by which the stem/progenitor cells recognize the murine ovulatory rupture, and rapidly multiply their numbers, highlighting a YAP-induced stem cell expansion strategy.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Organoids.

Organoids have attracted increasing attention because they are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration, and repair in human tissues. Organoids can also be used in diagnostics, disease modeling, drug discovery, and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumors. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer serves to highlight the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community is also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and key priorities in organoid engineering for the coming years.

Recent advances in tissue stem cells.

Stem cells are undifferentiated cells capable of self-renewal and differentiation, giving rise to specialized functional cells. Stem cells are of pivotal importance for organ and tissue development, homeostasis, and injury and disease repair. Tissue-specific stem cells are a rare population residing in specific tissues and present powerful potential for regeneration when required. They are usually named based on the resident tissue, such as hematopoietic stem cells and germline stem cells. This review discusses the recent advances in stem cells of various tissues, including neural stem cells, muscle stem cells, liver progenitors, pancreatic islet stem/progenitor cells, intestinal stem cells, and prostate stem cells, and the future perspectives for tissue stem cell research.

Integrin αEß7+ T cells direct intestinal stem cell fate decisions via adhesion signaling.

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 αEß7 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 αEß7-E-cadherin adhesion suppresses Wnt signaling and promotes Notch signaling in ISCs and TA cells, leading to defective ISC differentiation. Thus, αEß7+ T cells regulate ISC differentiation at single-cell level through cell-cell contact-mediated αEß7-E-cadherin adhesion signaling, highlighting a critical role of the T cell-stem/TA cell contact in maintaining intestinal homeostasis.

Lentiviral CRISPR-guided RNA library screening identified Adam17 as an upstream negative regulator of Procr in mammary epithelium.

BACKGROUND: Protein C receptor (Procr) has recently been shown to mark resident adult stem cells in the mammary gland, vascular system, and pancreatic islets. More so, high Procr expression was also detected and used as indicator for subsets of triple-negative breast cancers (TNBCs). Previous study has revealed Procr as a target of Wnt/ß-catenin signaling; however, direct upstream regulatory mechanism of Procr remains unknown. To comprehend the molecular role of Procr during physiology and pathology, elucidating the upstream effectors of Procr is necessary. Here, we provide a system for screening negative regulators of Procr, which could be adapted for broad molecular analysis on membrane proteins. RESULTS: We established a screening system which combines CRISPR-Cas9 guided gene disruption with fluorescence activated cell sorting technique (FACS). CommaDß (murine epithelial cells line) was used for the initial Procr upstream effector screening using lentiviral CRISPR-gRNA library. Shortlisted genes were further validated through individual lentiviral gRNA infection followed by Procr expression evaluation. Adam17 was identified as a specific negative inhibitor of Procr expression. In addition, MDA-MB-231 cells and Hs578T cells (human breast cancer cell lines) were used to verify the conserved regulation of ADAM17 over PROCR expression. CONCLUSION: We established an efficient CRISPR-Cas9/FACS screening system, which identifies the regulators of membrane proteins. Through this system, we identified Adam17 as the negative regulator of Procr membrane expression both in mammary epithelial cells and breast cancer cells.

Endothelial Wnts control mammary epithelial patterning via fibroblast signaling.

Endothelial and fibroblast niches are crucial for epithelial organs. How these heterotypic cells interact is of great interest. In this study, we reveal an axis of signaling in which fibroblasts relay Wnt signals from the endothelial niche to organize epithelial patterning. We generate an Axin2-membrane GFP (mGFP) reporter mouse and observe robust Wnt/ß-catenin signaling activities in fibroblasts surrounding the mammary epithelium. To enable cell-type-specific gene manipulation in vitro, we establish an organoid system via coculture of endothelial cells (ECs), fibroblasts, and mammary epithelial cells. Deletion of ß-catenin in fibroblasts impedes epithelium branching, and ECs are responsible for the activation of Wnt/ß-catenin signaling in fibroblasts. In vivo, EC deletion of Wntless inhibits Wnt/ß-catenin signaling activity in fibroblasts, rendering a reduction in epithelial branches. These findings highlight the significance of the endothelial niche in tissue patterning, shedding light on the interactive mechanisms in which distinct niche components orchestrate epithelial organogenesis and tissue homeostasis.

Procr-expressing granulosa cells are highly proliferative and are important for follicle development.

Granulosa cells (GCs) play a critical role in folliculogenesis. It remains unclear how GCs expand during follicle development and whether there is a subpopulation of cells that is responsible for GCs growth. Here, we observed that a small population of GCs expressed stem cell surface marker Procr (Protein C receptor). Procr GCs displayed higher proliferation ability and lower levels of hormone receptors compared with Procr- GCs. Knockdown of Procr inhibited proliferation. Lineage tracing experiments demonstrated that they contribute to increasing numbers of GCs during folliculogenesis. Targeted ablation of Procr+ cells disrupted ovarian follicle development, leading to phenotypes of polycystic ovary syndrome. Our findings suggest that Procr-expressing GCs are endowed with high proliferative capacity that is critical for follicle development.

MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche.

Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. 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 reported3-6, 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.

SHANK2 is a frequently amplified oncogene with evolutionarily conserved roles in regulating Hippo signaling.

Dysfunction of the Hippo pathway enables cells to evade contact inhibition and provides advantages for cancerous overgrowth. However, for a significant portion of human cancer, how Hippo signaling is perturbed remains unknown. To answer this question, we performed a genome-wide screening for genes that affect the Hippo pathway in Drosophila and cross-referenced the hit genes with human cancer genome. In our screen, Prosap was identified as a novel regulator of the Hippo pathway that potently affects tissue growth. Interestingly, a mammalian homolog of Prosap, SHANK2, is the most frequently amplified gene on 11q13, a major tumor amplicon in human cancer. Gene amplification profile in this 11q13 amplicon clearly indicates selective pressure for SHANK2 amplification. More importantly, across the human cancer genome, SHANK2 is the most frequently amplified gene that is not located within the Myc amplicon. Further studies in multiple human cell lines confirmed that SHANK2 overexpression causes deregulation of Hippo signaling through competitive binding for a LATS1 activator, and as a potential oncogene, SHANK2 promotes cellular transformation and tumor formation in vivo. In cancer cell lines with deregulated Hippo pathway, depletion of SHANK2 restores Hippo signaling and ceases cellular proliferation. Taken together, these results suggest that SHANK2 is an evolutionarily conserved Hippo pathway regulator, commonly amplified in human cancer and potently promotes cancer. Our study for the first time illustrated oncogenic function of SHANK2, one of the most frequently amplified gene in human cancer. Furthermore, given that in normal adult tissues, SHANK2's expression is largely restricted to the nervous system, SHANK2 may represent an interesting target for anticancer therapy.