The presence of cancer stem cells (CSCs) is one of the primary causes of recurring therapy resistance because they have two main capacities: self-renewal and avoiding apoptotic pathways. Despite their relevance, no full bibliometric analysis has yet been done in this topic. The goal of this work is to use bibliometric analysis to map the fundamental and emergent areas in therapeutics targeting colon cancer stem cells. To perform bibliometric analysis on colon cancer stem cells (CCSCs) literature, spanning roughly the last 40 years, in order to establish a firm base for future projections by emphasizing the findings of the most notable research. All information pertinent to CCSCs was accessed from Web of Science Core Collection database. In order to identify and analyze the research hotspots and trends related to this topic, Biblioshiny (RStudio) and VOSviewer were utilized to ascertain the countries/regions, institutions, journals, authors, references, and keywords involved. The targeted time span covered 1735 research-, and review articles. The most frequent keywords were "colorectal cancer," "cancer stem cells," and "colon cancer," while the most trending keywords in the last few years were "protein stability," "spheroid formation," "ubiquitination," "exosomes," "patient-derived organoids," and "gut microbiota." Over the past 40 years, there has been a significant advancement in researchers' understanding of colon cancer stem cells. In addition, the cluster map of co-cited literature showed that colon cancer stem cell research has emerged as a research hotspot. It was also anticipated that the main focus of the future efforts appears to involve clinical applications of cell-targeted colon cancer therapy. These results provide researchers with a comprehensive understanding of this field and provide insightful ideas for further research.

In multicellular organisms, Wnt proteins govern stem and progenitor cell renewal and differentiation to regulate embryonic development, adult tissue homeostasis and tissue regeneration. Defects in canonical Wnt signalling, which is transduced intracellularly by β-catenin, have been associated with developmental disorders, degenerative diseases and cancers. Although a simple model describing Wnt-β-catenin signalling is widely used to introduce this pathway and has largely remained unchanged over the past 30 years, in this Review we discuss recent studies that have provided important new insights into the mechanisms of Wnt production, receptor activation and intracellular signalling that advance our understanding of the molecular mechanisms that underlie this important cell-cell communication system. In addition, we review the recent development of molecules capable of activating the Wnt-β-catenin pathway with selectivity in vitro and in vivo that is enabling new lines of study to pave the way for the development of Wnt therapies for the treatment of human diseases.

RSPO2 (R-spondin 2) is a key regulator of the Wnt/β-catenin signaling pathway, involved in embryogenesis, tissue homeostasis, and cancer progression. Despite its therapeutic potential, effective agents targeting RSPO2 remain elusive. To address the unmet need for RSPO2-targeted therapies, we aimed to develop high-affinity nanobodies via phage display and prokaryotic expression, characterizing their binding specificity and functional blockade of RSPO2-LGR4 interactions. This study provides foundational insights into nanobody-mediated inhibition of Wnt signaling, supporting future therapeutic strategies against RSPO2-driven pathologies.

Recombinant RSPO2 proteins were constructed and purified using PCR-based recombination. Camels (Camelus bactrianus) were immunized with RSPO2, and phage display was employed to screen nanobody libraries. High-affinity nanobodies were cloned, expressed, purified, and assessed for specificity and binding affinity using biolayer interferometry and protein blotting. Functional validation was performed using TOPFLASH assays to evaluate their impact on Wnt/β-catenin signaling.

Nanobodies with high specificity and nanomolar-range affinity constants (KDs) for RSPO2 were identified. The nanobody effectively inhibited RSPO2-induced Wnt/β-catenin signaling in human renal epithelial cells.

The development of RSPO2-targeting nanobodies offers new prospects for treating RSPO2-related diseases. The nanobody serve as valuable tools for functional research and hold potential as diagnostic and therapeutic agents for RSPO2-driven conditions.

Granulosa cells (GCs) are important drives of the reproductive process, not only the supporting cells for nutrition, but also cells with endocrine functions. Their differentiation and development parallel the entire menstruation period and even during pregnancy, making it tightly linked to the fate of the follicle. To elucidate the underlying mechanism is of great significance for related researches. The life course of GCs is briefly divided into five stages, from epithelial cells to pre-granulosa cells, GCs, mural and cumulus cells, lutein cells, and eventually disappear. A wide variety of genes and transcription factors participate in the regulation of different stages, and more importantly, various hormones secreted by the pituitary gland and GCs themselves play a leading role. These endogenous and exogenous signalling molecules interact to form a cross-linked communication network, promoting the development of GCs. Together with oocytes, theca cells and other functional cells in the ovary, GCs drive one of the most vital biological processes in women.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a transcriptional target gene of the Wnt signalling pathway, is overexpressed in multiple cancers, including colorectal cancer (CRC), hepatocellular carcinoma (HCC) and pre-B acute lymphoblastic leukaemia (pre-B ALL) and has emerged as a promising therapeutic target. Here, we reflect on the bottom-up development of a novel α-LGR5 therapeutic antibody we have recently reported, into a palette of LGR5-targeting immunotherapeutic modalities: antibody-drug conjugates (ADCs), bispecific T cell engagers (bispecific engagers), and chimeric antigen receptor (CAR) T cells. The α-LGR5 antibody is highly specific and accurately detects LGR5 protein expression levels, enabling its use as a prognostic biomarker for identifying LGR5+ tumour types. Preclinical studies road-testing the various α-LGR5-based modalities established potent and safe elimination of LGR5-expressing cancer cells in vitro and efficacy in a mouse model of human cancer in vivo. In this review, we discuss the utility of our antibody as the building block for a novel set of immunotherapeutics and highlight the importance of matching specific α-LGR5-based therapeutic modalities to individual tumour type and patient characteristics.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma and is characterized by a complex molecular landscape driven by genetic and epigenetic alternations. Among the crucial signaling pathways implicated in ccRCC, the Wnt/β-catenin pathway plays a significant role in tumor progression and prognosis. This review delves into the molecular basis of ccRCC, highlighting the genetic and epigenetic modifications that contribute to its pathogenesis. We explore the significance of the Wnt/β-catenin pathway, focusing on its role in disease development, particularly the nuclear transport of β-catenin and its activation and downstream effects. Furthermore, we examine the role of antagonist genes in regulating this pathway within the context of ccRCC, providing insights into potential therapeutic targets. Dysregulation of this pathway, which is characterized by abnormal activation and nuclear translocation of β-catenin, plays a significant role in promoting tumor growth and metastasis. We explore the intricate molecular aspects of ccRCC, with a particular emphasis on this topic, underscoring the role of the pathway and emphasizing the importance and relevance of antagonist genes. Understanding the intricate interplay between these molecular mechanisms is crucial for developing innovative strategies to improve ccRCC treatment and patient outcomes.

The Wnt signaling pathway is critically involved in orchestrating cellular functions such as proliferation, migration, survival, and cell fate determination during development. Given its pivotal role in cellular communication, aberrant Wnt signaling has been extensively linked to the pathogenesis of various diseases. This review offers an in-depth analysis of the Wnt pathway, detailing its signal transduction mechanisms and principal components. Furthermore, the complex network of interactions between Wnt cascades and other key signaling pathways, such as Notch, Hedgehog, TGF-β, FGF, and NF-κB, is explored. Genetic mutations affecting the Wnt pathway play a pivotal role in disease progression, with particular emphasis on Wnt signaling's involvement in cancer stem cell biology and the tumor microenvironment. Additionally, this review underscores the diverse mechanisms through which Wnt signaling contributes to diseases such as cardiovascular conditions, neurodegenerative disorders, metabolic syndromes, autoimmune diseases, and cancer. Finally, a comprehensive overview of the therapeutic progress targeting Wnt signaling was given, and the latest progress in disease treatment targeting key components of the Wnt signaling pathway was summarized in detail, including Wnt ligands/receptors, β-catenin destruction complexes, and β-catenin/TCF transcription complexes. The development of small molecule inhibitors, monoclonal antibodies, and combination therapy strategies was emphasized, while the current potential therapeutic challenges were summarized. This aims to enhance the current understanding of this key pathway.

Sex determination and differentiation are fundamental processes that are not only essential for fertility but also influence the development of many other organs, and hence, are important for species diversity and survival. In mammals, sex is determined by the inheritance of an X or a Y chromosome from the father. The Y chromosome harbours the testis-determining gene SRY, and it has long been thought that its absence is sufficient for ovarian development. Consequently, the ovarian pathway has been treated as a default pathway, in the sense that ovaries do not have or need a female-determining factor. Recently, a female-determining factor has been identified in mouse as the master regulator of ovarian development. Interestingly, this scenario was predicted as early as 1983. In this Review, we discuss the model predicted in 1983, how the mechanisms and genes currently known to be important for sex determination and differentiation in mammals have changed or supported this model, and finally, reflect on what these findings might mean for sex determination in other vertebrates.

Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), a member of the G-protein-coupled receptor (GPCR) family, has been implicated in various regulatory functions across multiple differentiation stages and numerous target sites in bone diseases. Therefore, LGR4 is a potential regulator of nuclear factor-κB ligand (RANKL) during osteoclast differentiation. However, a comprehensive investigation of its functions and applications in bone immunology is lacking. This review discusses the molecular characteristics, signaling pathways, and role of LGR4 in osteoimmunology, with a particular focus on its interactions with RANKL during osteoclast differentiation, while identifying gaps that warrant further research.

Emerging evidence underscored the significance of leucine-rich repeat-containing G protein-coupled receptor (LGR) 4 in endocrine and metabolic disorders. Despite this, its role in LGR4 in hepatic glucose metabolism remains poorly understood. In this study we set out to test whether LGR4 regulates glucose production in liver through a specific signaling pathway.

Hepatic glucose production and gluconeogenic gene expressions were detected after silence of LGR4 in three obese mice models. Then, whole-body LGR4-deficient (LGR4 KO) mice, liver-specific LGR4 knockout (LGR4LKO) mice, and liver-specific LGR4 overexpression (LGR4LOV) mice were generated, in which we analyzed the effects of LGR4 on hepatic glucose metabolism upon HFD feeding, among which live imaging and quantitative analysis of hepatic phosphoenolpyruvate carboxykinase (PEPCK)-luciferase activity were conducted.

LGR4 expression was significantly upregulated in the liver of three obese mouse models, and presented dynamic expression patterns in response to nutritional fluxes. We utilized global and liver-specific LGR4 knockouts (LGR4LKO), along with adenoviral-mediated LGR4 knockdown in mice, to show improved glucose tolerance and decreased hepatic gluconeogenesis. Specifically, the expression of rate-limiting gluconeogenic enzymes, PEPCK was significantly downregulated. Conversely, mouse model with adenovirus-mediated LGR4 overexpression (LGR4LOV) exhibited elevated gluconeogenesis and PEPCK expression and reversed the suppression observed in LGR4 knockout models. Notably, neither RANKL nor PKA signaling pathways, which were reported to take part in LGR4's function, were involved in the process of LGR4 regulating PEPCK. Instead, TopFlash reporter system and inhibitors application suggested that LGR4's influence on hepatic gluconeogenesis operates through the canonical Wnt/β-catenin/TCF7L2 signaling pathway.

Overall, these findings underscore a novel mechanism by which LGR4 regulates hepatic gluconeogenesis, presenting a potential therapeutic target for diabetes management.

Liver repair and regeneration are crucial physiological responses to hepatic injury and are orchestrated through intricate cellular and molecular networks. This review systematically delineates advancements in the field, emphasizing the essential roles played by diverse liver cell types. Their coordinated actions, supported by complex crosstalk within the liver microenvironment, are pivotal to enhancing regenerative outcomes. Recent molecular investigations have elucidated key signaling pathways involved in liver injury and regeneration. Viewed through the lens of metabolic reprogramming, these pathways highlight how shifts in glucose, lipid, and amino acid metabolism support the cellular functions essential for liver repair and regeneration. An analysis of regenerative variability across pathological states reveals how disease conditions influence these dynamics, guiding the development of novel therapeutic strategies and advanced techniques to enhance liver repair and regeneration. Bridging laboratory findings with practical applications, recent clinical trials highlight the potential of optimizing liver regeneration strategies. These trials offer valuable insights into the effectiveness of novel therapies and underscore significant progress in translational research. In conclusion, this review intricately links molecular insights to therapeutic frontiers, systematically charting the trajectory from fundamental physiological mechanisms to innovative clinical applications in liver repair and regeneration.

Breast cancer stem cells (BCSC) are a subpopulation responsible for cancer resistance and relapse. The receptor activator of nuclear factor kappa-Β ligand (RANKL) is a cytokine capable of activating RANK and LGR4 receptors. RANKL/RANK signaling maintains the self-renewal of BCSCs, however, the effect of RANKL via LGR4 remains unclear. Evidence from osteoclasts suggests that RANKL/LGR4 axis disrupts RANK signaling, leading to opposing cellular responses. Anti-RANKL inhibitors are potential agents for eradicating CSCs, but their effect on RANKL/LGR4 signal has not been demonstrated.

This project aimed to elucidate the role of RANKL in regulating stemness depending on the expression of its receptors.

We use in vitro and in vivo approaches to evaluate the effects of RANKL inhibition in stemness in low or high-LGR4 expressing cells. Furthermore, we analyze the effects of RANKL stimulation on the stemness of LGR4 or RANK overexpressing cells. Additionally, we evaluated the impact of RANKL/LGR4 signaling in the activity of Wnt/β-catenin and NF-κB signaling pathways.

Our findings indicated that elevated RANKL expression is related to a favorable prognosis in patients with high LGR4 levels. Furthermore, RANKL inhibition decreased BCSC properties in LGR4-low cell lines, while it promoted migration in LGR4-high cells. Additionally, the RANKL/RANK axis activated NF-κB signaling and enhanced BCSCs in RANK-overexpressing cells. In contrast, in LGR4-overexpressing cells, RANKL failed to activate NF-κB but instead inhibited the Wnt/β-catenin pathway, leading to a reduction in BCSCs.

Our findings suggest that RANKL exerts different responses according to the expression of its receptors.

In the dentate gyrus of the adult hippocampus, neurogenesis from neural stem cells (NSCs) is regulated by Wnt signals from the local microenvironment. The Wnt/β-catenin pathway is active in NSCs, where it regulates proliferation and fate commitment, and subsequently its activity is strongly attenuated. The mechanisms controlling Wnt activity are poorly understood. In stem cells from adult peripheral tissues, secreted R-spondin proteins (RSPO1-4) interact with LGR4-6 receptors and control Wnt signaling strength. Here, we found that RSPO1-3 and LGR4-6 are expressed in the adult dentate gyrus and in cultured NSCs isolated from the adult mouse hippocampus. LGR4-5 expression decreased in cultured NSCs upon differentiation, concomitantly with the reported decrease in Wnt activity. Treatment with RSPO1-3 increased NSC proliferation and the expression of Cyclin D1 but did not induce the expression of Axin2 or RNF43, 2 well-described Wnt target genes. However, RSPOs enhanced the effect of Wnt3a on Axin2 and RNF43 expression as well as on Wnt/β-catenin reporter activity, indicating that they can potentiate Wnt activity in NSCs. Moreover, RSPO1-3 was found to be expressed by cultured dentate gyrus astrocytes, a crucial component of the neurogenic niche. In co-culture experiments, the astrocyte-induced proliferation of NSCs was prevented by RSPO2 knockdown in astrocytes and LGR5 knockdown in hippocampal NSCs. Additionally, RSPO2 knockdown in the adult mouse dentate gyrus reduced proliferation of neural stem and progenitor cells in vivo. Altogether, our results indicate that RSPO/LGR signaling is present in the dentate gyrus and plays a crucial role in regulating neural precursor cell proliferation.

Cancer stem cells play an important role in tumor progression and chemotherapy resistance. Leucine-rich G repeat-containing protein-coupled receptor 5 (LGR5) has been identified as a cancer stem cell marker in several cancer types. LGR5 is involved in cancer development and progression via several pathways including WNT/β-catenin signaling pathway. LGR5 plays a role in tumor progression by promoting cancer cell migration, invasion, metastasis, and angiogenesis in many cancers including colorectal, brain, gastric, and ovarian cancer. This review summarises the current knowledge on the expression and functional role of LGR5 in cancers, the molecular mechanisms regulated by LGR5, and the relationship between LGR5 and chemotherapy resistance. The review also includes highlights potential strategies to inhibit LGR5 expression and function. The majority of functional studies have shown that LGR5 plays an important role in promoting cancer progression, metastasis and chemotherapy resistance however, in some contexts LGR5 can also activate tumor-suppressive pathways and LGR5 negative cells can also promote cancer progression. The review highlights that targeting LGR5 is a promising anti-cancer treatment but the functional effect of LGR5 on tumor cells is complex may be dependent on cancer type, tumor microenvironment and cross-talk with other molecules in the LGR5 signaling pathway.

Antibody-drug conjugates (ADCs) have emerged as a major modality of targeted cancer therapy, yet no ADC has been approved specifically for colorectal cancer (CRC). LGR4/5/6 (leucine-rich repeat containing, G protein-coupled receptor 4, 5, 6) are three related receptors that are expressed at high levels together or alternately in nearly all cases of CRC. ADCs targeting LGR5 have been shown to have robust anti-tumor potency, but not all CRC cells express LGR5 and LGR5-positive tumor cells may lose LGR5 expression due to cancer cell plasticity. R-spondin 4 (RSPO4) is a natural protein ligand of LGR4/5/6 with high affinity for all three receptors. We fused a mutant form of RSPO4 furin domain that retains high affinity binding to LGR4/5/6 to human IgG1 Fc to create a peptibody designated R462. Conjugation of R462 with a camptothecin analog designated CPT2 at eight drugs per peptibody led to the generation of R462-CPT2 that showed highly potent cytotoxic activity in vitro in CRC cell lines expressing any of LG4/5/6. In cell line xenograft and PDX models of CRC, R462-CPT2 demonstrated robust anti-tumor effect. Importantly, R462-CPT2 showed no major adverse effect at therapeutically effective dose levels. These results strongly support the use of RSPO ligand drug-conjugates that target LGR4/5/6 simultaneously for the treatment of CRC.

WNT signalling pathway dysregulation is often a critical early component in colorectal neoplasia, particularly the chromosomal instability pathway. Using two WNT reporters, LGR5 and AXIN2, we sought to assess whether these polyps demonstrate predictable expression patterns and if these patterns show diagnostic value.

We evaluated 23 adenomas (TA), 23 sessile serrated lesions (SSLs), 14 SSL with dysplasia and 38 traditional serrated adenomas (TSA). Chromogenic in situ hybridisation stains (ISH) for LGR5 and AXIN2 were performed. Reactivity was defined as strong, intermediate or weak. Upper third crypt reactivity was defined as full-thickness staining. Accentuation within ectopic crypts (ECF) was recorded.

TAs (91%) showed strong reactivity and full-thickness staining with LGR5. TSAs showed full-thickness and weak to intermediate LGR5 reactivity (79%) and ECF with LGR5 accentuation was exclusively seen in TSA. SSL showed weak LGR5 reactivity confined to the basal crypt region (100%). SSL with dysplasia also showed weak or intermediate (100%) LGR5 reactivity, but the reactivity pattern was full thickness (88%). AXIN2 expression parallels LGR5 expression (Pearson coefficient=0.63) regarding signal intensity for the examined polyp groups.

Qualitative and quantitative differences in AXIN2 and LGR5 expression assist in the diagnosis of SSL with dysplasia.

Cancer represents an intricate and heterogeneous ailment that evolves from a multitude of epigenetic and genetic variations that disrupt normal cellular function. The WNT/β-catenin pathway is essential in maintaining the balance between cell renewal and differentiation in various tissues. Abnormal activation of this pathway can lead to uncontrolled cell growth and initiate cancer across a variety of tissues such as the colon, skin, liver, and ovary. It enhances characteristics that lead to cancer progression, including angiogenesis, invasion and metastasis. Processes like autophagy and apoptosis which regulate cell death and play a crucial role in maintaining cellular equilibrium are also intimately linked with WNT/ β-catenin pathway. Thus, targeting WNT pathway has become a key strategy in developing antitumor therapies. Employing small molecule inhibitors has emerged as a targeted therapy to improve the clinical outcome compared to conventional cancer treatments. Many strategies using small molecule inhibitors for modulating the WNT/β-catenin pathway, such as hindering WNT ligands' secretion or interaction, disrupting receptor complex, and blocking the nuclear translocation of β-catenin have been investigated. These interventions have shown promise in both preclinical and clinical settings. This review provides a comprehensive understanding of the role of WNT/β-catenin signalling pathway's role in cancer, emphasizing its regulation of autophagy and apoptosis. Our goal is to highlight the potential of specific small molecule inhibitors targeting this pathway, fostering the development of novel, tailored cancer treatments.

WNT/β-catenin signaling plays key roles in development and cancer1,2. ZNRF3/RNF43 modulates Frizzleds through ubiquitination, dampening WNT/β-catenin signaling. Conversely, RSPO1-4 binding to LGR4-6 and ZNRF3/RNF43 enhances WNT/β-catenin signaling3-5. Here, we elucidate the overall landscape of architectures in multiple LGR4, RSPO2, and ZNRF3 assemblies, showcasing varying stoichiometries and arrangements. These structures reveal that LGR4 and RSPO2 capture distinct states of ZNRF3. The intrinsic heterogeneity of the LGR4-RSPO2-ZNRF3 assembly is influenced by LGR4 content. Particularly, in the assembly complex with a 2:2:2 ratio, two LGR4 protomers induce and stabilize the inactive state of ZNRF3, characterized by a wide inward-open conformation of two transmembrane helices (TM helices). This specific assembly promotes a stable complex, facilitating LGR4-induced endocytosis of ZNRF3. In contrast, the active dimeric ZNRF3, bound by a single LGR4, adopts a coiled-coil TM helices conformation and dimerization of RING domains. Our findings unveil how LGR4 content mediates diverse assemblies, leading to conformational rearrangements in ZNRF3 to regulate WNT/β-catenin signaling, and provide a structural foundation for drug development targeting Wnt-driven cancers.

Leucine-rich repeat-containing G-protein-coupled receptors regulate stem cell activity and tissue homeostasis within female reproductive organs, primarily through their interaction with the Wnt/β-catenin signaling pathway. LGR4-6 are increasingly recognized for their roles in organ development, regeneration, and cancer. This review aims to provide a comprehensive overview of the roles of LGR4-6 in female reproductive organs, highlighting their significance in normal physiology and disease states, specifically in the context of ovarian cancer. LGR4 is essential for the proper development of the female reproductive system; its deficiency leads to significant reproductive abnormalities, including delayed menarche and follicle development issues. LGR5 is a well-established marker of stem cells in the ovary and fallopian tubes. It has been implicated in the pathogenesis of high-grade serous ovarian cancer. LGR6, while less studied, shares functional similarities with LGR5 and can maintain stemness. It contributes to chemoresistance in ovarian cancer. LGR6 is a marker for fallopian tube stem cells and is involved in stem cell maintenance and differentiation. LGR4-6 regulate the pathophysiology of female reproductive tissues. LGR4-6 are promising therapeutic targets for treating reproductive cancers and other related disorders. Molecular mechanisms underlying the functions of LGR4-6 should be studied.

ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the protein most negatively correlated with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptors, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.

The regulator of the canonical Wnt pathway, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), is expressed in the stem cell compartment of several tissues and overexpressed in different human carcinomas. The isoform of the stem cell marker LGR5, named LGR5Δ5 and first described by our group, is associated with prognosis and metastasis in oral squamous cell carcinoma (OSCC) and soft tissue sarcoma (STS). In a proof-of-principle analysis, the function of LGR5Δ5 was investigated in HEK293T cells, a model cell line of the Wnt pathway, compared to full-length LGR5 (FL) expression. The CRISPR/CAS knockout of LGR5 and LGR4 (thereby avoiding the side effects of LGR4) resulted in a loss of Wnt activity that cannot be restored by LGR5Δ5 but by LGR5FL rescue. The ability to migrate was not affected by LGR5Δ5, but was reduced by LGR5FL overexpression. The CRISPR/CAS of LGR4 and 5 induced radiosensitization, which was enhanced by the overexpression of LGR5FL or LGR5Δ5. RNA sequencing analysis revealed a significant increase in the ligand R-spondin 1 (RSPO1) level by LGR5Δ5. Furthermore, LGR5Δ5 appears to be involved in the regulation of genes related to the cytoskeleton, extracellular matrix stiffness, and angiogenesis, while LGR5FL is associated with the regulation of collagens and histone proteins.

Prostate cancer poses a significant risk to the well-being and way of life of countless men, with an increased likelihood of relapse recorded following modern treatment. This highlights the need for innovative approaches, specifically targeting LGR5. This systematic review aims to establish a connection between LGR5 and the various signaling pathways involved in the progression of prostate cancer. LGR5, a gene targeted by Wnt signaling, encodes a receptor protein that serves as a prognostic biomarker for stem cells and indicates the presence of cancer stem cells in colorectal and gastrointestinal cancers. The functions of LGR5 include processes such as cell proliferation, differentiation, and signaling pathways. Any modifications to the LGR5 gene, whether caused by mutations or mechanical stimuli, can lead to the development of treatment-resistant stem cell cancers. This review examines the molecular mechanisms associated with LGR5 and emphasizes methodologies aimed at targeting LGR5 to enhance understanding and promote the development of LGR5-specific therapies.

Lgr5-positive cells located in the basal layer of crypts have self-regenerative and proliferative differentiation potentials of intestinal stem cells (ISCs), maintaining a balance of regeneration-repair in mucosal epithelium. However, the mechanisms of mucosal repair that are regulated by ISCs in ulcerative colitis (UC) remain unclear.

Colon tissues from patients with UC were collected to test β-catenin and Notch1 expression by using Western blot and quantitative real-time polymerase chain reaction (PCR). β-cateninfl/fl mice, β-cateninTg mice, and Dll1tm1 Gos mice were used to cross with Lgr5-EGFP-IRES-creERT2 mice to generate mice of different genotypes, altering the activation of Wnt/β-catenin and Dll1-mediated Notch signaling in ISCs in vivo. Dextran sulfate sodium (DSS) was used to induce a colitis mice model. Intestinal organoids were isolated and cultured to observe the proliferation and differentiation levels of ISCs.

β-catenin and Notch1 expression were significantly increased in the inflamed colon tissues from patients with UC. Wnt/β-catenin activation and Dll1-mediated Notch pathway inhibition in Lgr5-positive stem cells promoted the expressions of E-cadherin, CK20, and CHGA in colonic organoids and epithelium, implying the promotion of colonic epithelial integrity. Activation of Wnt/β-catenin and suppression of Dll1-mediated Notch pathway in Lgr5-positive ISCs alleviated the DSS-induced intestinal mucosal inflammation in mice.

Lgr5-positive ISCs are characterized by self-renewal and high dividend potential, which play an important role in the injury and repair of intestinal mucosa. More importantly, the Wnt/β-catenin signaling pathway cooperates with the Notch signaling pathway to maintain the function of the Lgr5-positive ISCs.

Cancer Stem Cells (CSCs) are highly tumorigenic, chemoresistant, and immune evasive. They emerge as a central driver that gives rise to the bulk of tumoral mass, modifies the tumor microenvironment (TME), and exploits it, leading to poor clinical outcomes for patients with cancer. The existence of CSCs thus accounts for the failure of conventional therapies and immune surveillance. Identifying CSCs in solid tumors remains a significant challenge in modern oncology, with the use of cell surface markers being the primary strategy for studying, isolating, and enriching these cells. In this review, we explore CSC markers, focusing on the underlying signaling pathways that drive CSC self-renewal, which simultaneously makes them intrinsically chemoresistant and immune system evaders. We comprehensively discuss the autonomous and non-autonomous functions of CSCs, with particular emphasis on their interactions with the tumor microenvironment, especially immune cells. This reciprocal network enhances CSCs malignancy while compromising the surrounding niche, ultimately defining therapeutic vulnerabilities associated with each CSC marker. The most common CSCs surface markers addressed in this review-CD44, CD133, ICAM1/CD54, and LGR5-provide insights into the interplay between chemoresistance and immune evasion, two critically important phenomena in disease eradication. This new perspective on the state-of-the-art of CSCs will undoubtedly open new avenues for therapy.

The human skeleton is a multifunctional organ made up of multiple cell types working in concert to maintain bone and mineral homeostasis and to perform critical mechanical and endocrine functions. From the beginning steps of chondrogenesis that prefigures most of the skeleton, to the rapid bone accrual during skeletal growth, followed by bone remodeling of the mature skeleton, cell differentiation is integral to skeletal health. While growth factors and nuclear proteins that influence skeletal cell differentiation have been extensively studied, the role of cellular metabolism is just beginning to be uncovered. Besides energy production, metabolic pathways have been shown to exert epigenetic regulation via key metabolites to influence cell fate in both cancerous and normal tissues. In this review, we will assess the role of growth factors and transcription factors in reprogramming cellular metabolism to meet the energetic and biosynthetic needs of chondrocytes, osteoblasts, or osteoclasts. We will also summarize the emerging evidence linking metabolic changes to epigenetic modifications during skeletal cell differentiation.

R-spondins (RSPOs) are a family of secreted proteins and stem cell growth factors that are potent co-activators of Wnt signaling. Recently, RSPO2 and RSPO3 were shown to be multifunctional, not only amplifying Wnt- but also binding BMP- and FGF receptors to downregulate signaling. The common mechanism underlying these diverse functions is that RSPO2 and RSPO3 act as "endocytosers" that link transmembrane proteins to ZNRF3/RNF43 E3 ligases and trigger target internalization. Thus, RSPOs are natural protein targeting chimeras for cell surface proteins. Conducting data mining and cell surface binding assays we report additional candidate RSPO targets, including SMO, PTC1,2, LGI1, ROBO4, and PTPR(F/S). We propose that there is an "R-spondin code" that imparts combinatorial signaling ON-OFF states of multiple growth factors. This code involves the modular RSPO domains, notably distinct motifs in the divergent RSPO-TSP1 domains to mediate target interaction and internalization. The RSPO code offers a novel framework for the understanding how diverse signaling pathways may be coordinately regulated in development and disease.

Tissue stem cells (TSCs), which reside in specialized tissues, constitute the major cell sources for tissue homeostasis and regeneration, and the contribution of transcriptional or epigenetic regulation of distinct biological processes in TSCs has been discussed in the past few decades. Meanwhile, ATP-dependent chromatin remodelers use the energy from ATP hydrolysis to remodel nucleosomes, thereby affecting chromatin dynamics and the regulation of gene expression programs in each cell type. However, the role of chromatin remodelers in tissue stem cell fate determination is less well understood. In this review, we systematically discuss recent advances in epigenetic control by chromatin remodelers of hematopoietic stem cells, intestinal epithelial stem cells, neural stem cells, and skin stem cells in their fate determination and highlight the importance of their essential role in tissue homeostasis, development, and regeneration. Moreover, the exploration of the molecular and cellular mechanisms of TSCs is crucial for advancing our understanding of tissue maintenance and for the discovery of novel therapeutic targets.

The gastrointestinal tract harbors diverse microorganisms in the lumen. Epithelial cells segregate the luminal microorganisms from immune cells in the lamina propria by constructing chemical and physical barriers through the production of various factors to prevent excessive immune responses against microbes. Therefore, perturbations of epithelial integrity are linked to the development of gastrointestinal disorders. Several mesenchymal stromal cell populations, including fibroblasts, myofibroblasts, pericytes, and myocytes, contribute to the establishment and maintenance of epithelial homeostasis in the gut through regulation of the self-renewal, proliferation, and differentiation of intestinal stem cells. Recent studies have revealed alterations in the composition of intestinal mesenchymal stromal cells in patients with inflammatory bowel disease and colorectal cancer. A better understanding of the interplay between mesenchymal stromal cells and epithelial cells associated with intestinal health and diseases will facilitate identification of novel biomarkers and therapeutic targets for gastrointestinal disorders. This review summarizes the key findings obtained to date on the mechanisms by which functionally distinct mesenchymal stromal cells regulate epithelial integrity in intestinal health and diseases at different developmental stages.

Mice hair follicles (HFs) are a valuable model for studying various aspects of hair biology, including morphogenesis, development, and regeneration due to their easily observable phenotype and genetic manipulability. The initiation and progression of hair follicle morphogenesis, as well as the hair follicle cycle, are regulated by various signaling pathways, of which the main role is played by the Wingless-type MMTV integration site family (Wnt) and the Bone Morphogenic Protein (BMP). During the hair follicle cycle, the BMP pathway maintains hair follicle stem cells (HFSCs) in a dormant state while the Wnt pathway activates them for hair growth. Given the pivotal role of the Wnt pathway in hair biology and HFSCs regulation, we investigated the influence of the Wnt modulator - R-spondin 3 (Rspo3), in these processes. For this purpose, we developed a transgenic mice model with the overexpression of Rspo3 (Rspo3GOF) in the whole ectoderm and its derivatives, starting from early morphogenesis. Rspo3GOF mice exhibited a distinct phenotype with sparse hair and visible bald areas, caused by reduced proliferation and increased apoptosis of hair matrix progenitor cells, which resulted in a premature anagen-to-catagen transition with a shortened growth phase and decreased overall length of all hair types. In addition, Rspo3GOF promoted induction of auchene and awl, canonical Wnt-dependent hair type during morphogenesis, but the overall hair amount remained reduced. We also discovered a delay in the pre-bulge formation during morphogenesis and prolonged immaturity of the HFSC population in the bulge region postnatally, which further impaired proper hair regeneration throughout the mice's lifespan. Our data supported that Rspo3 function observed in our model works in HFSCs' formation of pre-bulge during morphogenesis via enhancing activation of the canonical Wnt pathway, whereas in contrast, in the postnatal immature bulge, activation of canonical Wnt signaling was attenuated. In vitro studies on keratinocytes revealed changes in proliferation, migration, and colony formation, highlighting the inhibitory effect of constitutive overexpression of Rspo3 on these cellular processes. Our research provides novel insights into the role of Rspo3 in the regulation of hair morphogenesis and development, along with the formation and maturation of the HFSCs, which affect hair regeneration.

While guided human cortical organoid (hCO) protocols reproducibly generate cortical cell types at one site, variability in hCO phenotypes across sites using a harmonized protocol has not yet been evaluated. To determine the cross-site reproducibility of hCO differentiation, three independent research groups assayed hCOs in multiple differentiation replicates from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol across 3 months. hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and expression of metabolism and cellular stress genes. Variability in hCO phenotypes correlated with stem cell gene expression prior to differentiation and technical factors associated with seeding, suggesting iPSC quality and treatment are important for differentiation outcomes. Cross-site reproducibility of hCO cell type proportions and organization encourages future prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.

Gastrointestinal cancers (GICs) are highly prevalent cancers that threaten human health worldwide. The Wnt/β-catenin signaling pathway has been reported to play a pivotal role in the carcinogenesis of GICs. Numerous interventions targeting the Wnt/β-catenin signaling in GICs are currently being tested in clinical trials with promising results. Unfortunately, there are no clinically approved drugs that effectively target this pathway. This comprehensive review aims to evaluate the impact of clinical therapies targeting the Wnt/β-catenin signaling pathway in GICs. By integrating data from bioinformatics databases and recent literature from the past five years, we examine the heterogeneous expression and regulatory mechanisms of Wnt/β-catenin pathway genes and proteins in GICs. Specifically, we focus on expression patterns, mutation frequencies, and clinical prognoses to understand their implications for treatment strategies. Additionally, we discuss recent clinical trial efforts targeting this pathway. Understanding the inhibitors currently under clinical investigation may help optimize foundational research and clinical strategies. We hope that elucidating the current status of precision therapeutic stratification for patients targeting the Wnt/β-catenin pathway will guide future innovations in precision medicine for GICs.

As a highly dynamic tissue, bone is continuously rebuilt throughout life. Both bone formation by osteoblasts and bone resorption by osteoclasts constitute bone reconstruction homeostasis. The equilibrium of bone homeostasis is governed by many complicated signaling pathways that weave together to form an intricate network. These pathways coordinate the meticulous processes of bone formation and resorption, ensuring the structural integrity and dynamic vitality of the skeletal system. Dysregulation of the bone homeostatic regulatory signaling network contributes to the development and progression of many skeletal diseases. Significantly, imbalanced bone homeostasis further disrupts the signaling network and triggers a cascade reaction that exacerbates disease progression and engenders a deleterious cycle. Here, we summarize the influence of signaling pathways on bone homeostasis, elucidating the interplay and crosstalk among them. Additionally, we review the mechanisms underpinning bone homeostatic imbalances across diverse disease landscapes, highlighting current and prospective therapeutic targets and clinical drugs. We hope that this review will contribute to a holistic understanding of the signaling pathways and molecular mechanisms sustaining bone homeostasis, which are promising to contribute to further research on bone homeostasis and shed light on the development of targeted drugs.

Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, affecting millions each year. It emerges from the colon or rectum, parts of the digestive system, and is closely linked to both genetic and environmental factors. In CRC, genetic mutations such as APC, KRAS, and TP53, along with epigenetic changes like DNA methylation and histone modifications, play crucial roles in tumor development and treatment responses. This paper delves into the complex biological underpinnings of CRC, highlighting the pivotal roles of genetic alterations, cell death pathways, and the intricate network of signaling interactions that contribute to the disease's progression. It explores the dysregulation of apoptosis, autophagy, and other cell death mechanisms, underscoring the aberrant activation of these pathways in CRC. Additionally, the paper examines how mutations in key molecular pathways, including Wnt, EGFR/MAPK, and PI3K, fuel CRC development, and how these alterations can serve as both diagnostic and prognostic markers. The dual function of autophagy in CRC, acting as a tumor suppressor or promoter depending on the context, is also scrutinized. Through a comprehensive analysis of cellular and molecular events, this research aims to deepen our understanding of CRC and pave the way for more effective diagnostics, prognostics, and therapeutic strategies.

Neuroblastoma (NB) is a cancer of the peripheral nervous system found in children under 15 years of age. It is the most frequently diagnosed cancer during infancy, accounting for ~12% of all cancer-related deaths in children. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a membrane receptor that is associated with the primary tumor formation and metastasis of cancers in the gastrointestinal system. Remarkably, high levels of LGR5 are found in NB tumor cells, and high LGR5 expression is strongly correlated with poor survival. Antibody-drug conjugates (ADCs) are monoclonal antibodies that are covalently linked to cell-killing cytotoxins to deliver the payloads into cancer cells. We generated an ADC with an anti-LGR5 antibody and pyrrolobenzodiazepine (PBD) dimer-based payload SG3199 using a chemoenzymatic conjugation method. The resulting anti-LGR5 ADC was able to inhibit the growth of NB cells expressing LGR5 with high potency and specificity. Importantly, the ADC was able to completely inhibit the growth of NB xenograft tumors in vivo at a clinically relevant dose for the PBD class of ADCs. The findings support the potential of targeting LGR5 using the PBD class of payload for the treatment of high-risk NBs.

De novo hair follicle (HF) regeneration, achieved through the replenishment of the dermal papilla (DP), acknowledged as the principal orchestrator of the hair growth cycle, is emerging as a prospective therapeutic intervention for alopecia. Nonetheless, multiple attempts have shown that these cells lose key inductive properties when cultured in a two-dimensional (2D) monolayer, leading to precocious senescence engendered by oxidative stress and inflammatory processes. Consequently, the three-dimensional (3D) spheroid technique is presently widely employed for DP cell culture. Nevertheless, substantiating the regenerative potential of these cells within the hair follicle (HF) milieu remains a challenge. In this current study, we aim to find a new approach to activate the inductive properties of DP cells. This involves the application of hair-growth-stimulating agents that not only exhibit concurrent protective efficacy against the aging process but also induce HF regeneration. To achieve this objective, we initially synthesized a novel highly amphiphilic derivative derived from squalene (SQ), named triethylene glycol squalene (Tri-SQ). Squalene itself is a potent antioxidant and anti-inflammatory compound traditionally employed as a drug carrier for alopecia treatment. However, its application is limited due to its low solubility. Subsequently, we applied this newly synthesized derivative to DP cells. The data obtained demonstrated that the derivative exhibits robust antioxidant and anti-inflammatory activities while concurrently promoting the expression of genes associated with hair growth. Moreover, to further assess the hair regrowth inductive properties of DP cells, we cultured the cells and treated them with Tri-SQ within a 3D spheroid system. Subsequently, these treated cells were injected into the previously depilated dorsal area of six-week-old male C57BL/6 mice. Results revealed that 20 days postinjection, a complete regrowth of hair in the previously hairless area, particularly evident in the case of 3D spheroids treated with the derivative, was observed. Additionally, histological and molecular analyses demonstrated an upregulation of markers associated with hair growth and a concurrent decrease in aging hallmarks, specifically in the 3D spheroids treated with the compound. In summary, our approach, which involves the treatment of Tri-SQ combined with a 3D spheroid system, exhibited a notably robust stimulating effect. This effect was observed in the induction of inductive properties in DP cells, leading to HF regeneration, and concurrently, it demonstrated an inhibitory effect on cellular and follicular aging.

Wnts are secreted, lipid-modified proteins that bind to different receptors on the cell surface to activate canonical or non-canonical Wnt signaling pathways, which control various biological processes throughout embryonic development and adult life. Aberrant Wnt signaling pathway underlies a wide range of human disease pathogeneses. In this review, we provide an update of Wnt/β-catenin signaling components and mechanisms in bone formation, homeostasis, and diseases. The Wnt proteins, receptors, activators, inhibitors, and the crosstalk of Wnt signaling pathways with other signaling pathways are summarized and discussed. We mainly review Wnt signaling functions in bone formation, homeostasis, and related diseases, and summarize mouse models carrying genetic modifications of Wnt signaling components. Moreover, the therapeutic strategies for treating bone diseases by targeting Wnt signaling, including the extracellular molecules, cytosol components, and nuclear components of Wnt signaling are reviewed. In summary, this paper reviews our current understanding of the mechanisms by which Wnt signaling regulates bone formation, homeostasis, and the efforts targeting Wnt signaling for treating bone diseases. Finally, the paper evaluates the important questions in Wnt signaling to be further explored based on the progress of new biological analytical technologies.

Receptor tyrosine kinases (RTKs) control stem cell maintenance vs. differentiation decisions. Casitas B-lineage lymphoma (CBL) family ubiquitin ligases are negative regulators of RTKs, but their stem cell regulatory roles remain unclear. Here, we show that Lgr5+ intestinal stem cell (ISC)-specific inducible Cbl-knockout (KO) on a Cblb null mouse background (iDKO) induced rapid loss of the Lgr5 Hi ISCs with transient expansion of the Lgr5 Lo transit-amplifying population. LacZ-based lineage tracing revealed increased ISC commitment toward enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro, Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single-cell RNA sequencing in organoids identified Akt-mTOR (mammalian target of rapamycin) pathway hyperactivation upon iDKO, and pharmacological Akt-mTOR axis inhibition rescued the iDKO defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine-tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.

Selective and precise activation of signaling transduction cascades is key for cellular reprogramming and tissue regeneration. However, the development of small- or large-molecule agonists for many signaling pathways has remained elusive and is rate limiting to realize the full clinical potential of regenerative medicine. Focusing on the Wnt pathway, here we describe a series of disulfide-constrained peptides (DCPs) that promote Wnt signaling activity by modulating the cell surface levels of ZNRF3, an E3 ubiquitin ligase that controls the abundance of the Wnt receptor complex FZD/LRP at the plasma membrane. Mechanistically, monomeric DCPs induce ZNRF3 ubiquitination, leading to its cell surface clearance, ultimately resulting in FZD stabilization. Furthermore, we engineered multimeric DCPs that induce expansive growth of human intestinal organoids, revealing a dependence between valency and ZNRF3 clearance. Our work highlights a strategy for the development of potent, biologically active Wnt signaling pathway agonists via targeting of ZNRF3.

Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.

Neurodegenerative disorders represent a significant and growing health burden worldwide. Unfortunately, limited therapeutic options are currently available despite ongoing efforts. Over the past decades, research efforts have increasingly focused on understanding the molecular mechanisms underlying these devastating conditions. Orphan receptors, a class of receptors with no known endogenous ligands, emerge as promising druggable targets for diverse diseases. This review aims to direct attention to a subgroup of orphan GPCRs, in particular class A orphans that have roles in neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Multiple sclerosis. We highlight the diverse roles orphan receptors play in regulating critical cellular processes such as synaptic transmission, neuronal survival and neuro-inflammation. Moreover, we discuss the therapeutic potential of targeting orphan receptors for the treatment of neurodegenerative disorders, emphasizing recent advances in drug discovery and preclinical studies. Finally, we outline future directions and challenges in orphan receptor research.