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.

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.

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.

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.

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.

A Regulation Loop between YAP and NR4A1 Balances Cell Proliferation and Apoptosis.

The Hippo signaling pathway maintains organ size and tissue homeostasis via orchestration of cell proliferation and apoptosis. How this pathway triggers cell apoptosis remains largely unexplored. Here, we identify NR4A1 as a target of the Hippo pathway that mediates the pro-apoptotic and anti-tumor effects of the Hippo pathway whereby YAP regulates the transcription, phosphorylation, and mitochondrial localization of NR4A1. NR4A1, in turn, functions as a feedback inhibitor of YAP to promote its degradation, thereby inhibiting the function of YAP during liver regeneration and tumorigenesis. Our studies elucidate a regulatory loop between NR4A1 and YAP to coordinate Hippo signaling activity during liver regeneration and tumorigenesis and highlight NR4A1 as a marker of Hippo signaling, as well as a therapeutic target for hepatocellular carcinoma.

A novel function of R-spondin1 in regulating estrogen receptor expression independent of Wnt/ß-catenin signaling.

R-spondin1 (Rspo1) has been featured as a Wnt agonist, serving as a potent niche factor for stem cells in many tissues. Here we unveil a novel role of Rspo1 in promoting estrogen receptor alpha (Esr1) expression, hence regulating the output of steroid hormone signaling in the mouse mammary gland. This action of Rspo1 relies on the receptor Lgr4 and intracellular cAMP-PKA signaling, yet is independent of Wnt/ß-catenin signaling. These mechanisms were reinforced by genetic evidence. Luminal cells-specific knockout of Rspo1 results in decreased Esr1 expression and reduced mammary side branches. In contrast, luminal cells-specific knockout of Wnt4, while attenuating basal cell Wnt/ß-catenin signaling activities, enhances Esr1 expression. Our data reveal a novel Wnt-independent role of Rspo1, in which Rspo1 acts as a bona fide GPCR activator eliciting intracellular cAMP signaling. The identification of Rspo1-ERα signaling axis may have a broad implication in estrogen-associated diseases.

Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr+ Progenitors.

It has generally proven challenging to produce functional ß cells in vitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing ∼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. ß cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.

Amphiregulin mediates the hormonal regulation on Rspondin-1 expression in the mammary gland.

The steroid hormones are instrumental for the growth of mammary epithelial cells. Our previous study indicates that hormones regulate the expression of Rspondin-1 (Rspo1). Yet, the regulatory mechanism remains unknown. In the current study, we identify Amphiregulin (Areg) as a novel upstream regulator of Rspo1 expression mediating the hormonal influence. In response to hormonal signaling, Areg emanating from estrogen receptor (ER)-positive luminal cells, induce the expression of Rspo1 in ER-negative luminal cells. The paracrine action of Areg on Rspo1 expression is dependent on Egfr. Our data reveal a novel Estrogen-Areg-Rspo1 regulatory axis in the mammary gland, providing new evidence for the orchestrated action of systemic hormones and local growth factors.

Procr-expressing progenitor cells are responsible for murine ovulatory rupture repair of ovarian surface epithelium.

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and repair. The OSE replenishing mechanism post ovulation remains unclear. Here we report that the expression of Protein C Receptor (Procr) marks a progenitor population in adult mice that is responsible for OSE repair post ovulation. Procr+ cells are the major cell source for OSE repair. The mechanism facilitating the rapid re-epithelialization is through the immediate expansion of Procr+ cells upon OSE rupture. Targeted ablation of Procr+ cells impedes the repairing process. Moreover, Procr+ cells displayed robust colony-formation capacity in culture, which we harnessed and established a long-term culture and expansion system of OSE cells. Finally, we show that Procr+ cells and previously reported Lgr5+ cells have distinct lineage tracing behavior in OSE homeostasis. Our study suggests that Procr marks progenitor cells that are critical for OSE ovulatory rupture and homeostasis, providing insight into how adult stem cells respond upon injury.

Protein C receptor is a therapeutic stem cell target in a distinct group of breast cancers.

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.

Glucocorticoid Receptor Signaling Activates TEAD4 to Promote Breast Cancer Progression.

The Hippo pathway plays a critical role in cell growth and tumorigenesis. The activity of TEA domain transcription factor 4 (TEAD4) determines the output of Hippo signaling; however, the regulation and function of TEAD4 has not been explored extensively. Here, we identified glucocorticoids (GC) as novel activators of TEAD4. GC treatment facilitated glucocorticoid receptor (GR)-dependent nuclear accumulation and transcriptional activation of TEAD4. TEAD4 positively correlated with GR expression in human breast cancer, and high expression of TEAD4 predicted poor survival of patients with breast cancer. Mechanistically, GC activation promoted GR interaction with TEAD4, forming a complex that was recruited to the TEAD4 promoter to boost its own expression. Functionally, the activation of TEAD4 by GC promoted breast cancer stem cells maintenance, cell survival, metastasis, and chemoresistance both in vitro and in vivo. Pharmacologic inhibition of TEAD4 inhibited GC-induced breast cancer chemoresistance. In conclusion, our study reveals a novel regulation and functional role of TEAD4 in breast cancer and proposes a potential new strategy for breast cancer therapy. SIGNIFICANCE: This study provides new insight into the role of glucocorticoid signaling in breast cancer, with potential for clinical translation.

Endothelial cell clonal expansion in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is a neurovascular familial or sporadic disease that is characterised by capillary-venous cavernomas, and is due to loss-of-function mutations to any one of three CCM genes. Familial CCM follows a two-hit mechanism similar to that of tumour suppressor genes, while in sporadic cavernomas only a small fraction of endothelial cells shows mutated CCM genes. We reported that in mouse models and in human patients, endothelial cells lining the lesions have different features from the surrounding endothelium, as they express mesenchymal/stem-cell markers. Here we show that cavernomas originate from clonal expansion of few Ccm3-null endothelial cells that express mesenchymal/stem-cell markers. These cells then attract surrounding wild-type endothelial cells, inducing them to express mesenchymal/stem-cell markers and to contribute to cavernoma growth. These characteristics of Ccm3-null cells are reminiscent of the tumour-initiating cells that are responsible for tumour growth. Our data support the concept that CCM has benign tumour characteristics.

Lung regeneration by multipotent stem cells residing at the bronchioalveolar-duct junction.

Characterizing the stem cells responsible for lung repair and regeneration is important for the treatment of pulmonary diseases. Recently, a unique cell population located at the bronchioalveolar-duct junctions has been proposed to comprise endogenous stem cells for lung regeneration. However, the role of bronchioalveolar stem cells (BASCs) in vivo remains debated, and the contribution of such cells to lung regeneration is not known. Here we generated a genetic lineage-tracing system that uses dual recombinases (Cre and Dre) to specifically track BASCs in vivo. Fate-mapping and clonal analysis showed that BASCs became activated and responded distinctly to different lung injuries, and differentiated into multiple cell lineages including club cells, ciliated cells, and alveolar type 1 and type 2 cells for lung regeneration. This study provides in vivo genetic evidence that BASCs are bona fide lung epithelial stem cells with deployment of multipotency and self-renewal during lung repair and regeneration.

Hormones induce the formation of luminal-derived basal cells in the mammary gland.

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/ß-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.