DKK1 Expression Research Articles

HDAC1 regulates inflammation and osteogenic differentiation of ankylosing spondylitis fibroblasts through the Wnt-Smad signaling pathway

Ankylosing spondylitis (AS) is a refractory autoimmune disease, whose typical pathology is the development of inflammation to ossification and ankylosis. Histone deacetylase 1 (HDAC1) is considered to be a key factor involved in inflammatory gene transduction, but its role in AS remains unclear. The purpose of this study was to explore the role and possible mechanism of HDAC1 in AS based on the Wnt-Smad pathway. Fibroblasts were isolated from hip synovial tissues of AS patients, adeno-associated virus (AAV) was used to regulate the expression of HDAC1, DKK-1 and SIS3 was used to inhibit Wnt and Smad, respectively. The expressions of Wnt-Smad pathway-related proteins were analyzed by WB, and the TRP ion channel proteins were analyzed by immunofluorescence and WB. The proliferation of AS fibroblasts was detected by CCK-8, the expression of inflammatory cytokines was detected by ELISA, and the effects of HDAC1 on osteogenic differentiation of AS fibroblasts were investigated by alkaline phosphatase (ALP) activity, intracellular calcium concentration, mineralization and osteogenic proteins expressions. Results showed that HDAC1 significantly affected the protein expressions of the Wnt-Smad pathway in AS fibroblasts, and Wnt inhibitor DKK-1 and Smad3 inhibitor SIS3 could significantly reverse the effect of HDAC1 on the Wnt-Smad pathway. In addition, HDAC1 significantly activated the TRP ion channel and promoted the proliferation, inflammatory response and osteogenic differentiation of AS fibroblasts. DKK-1 or SIS3 treatment significantly inhibit the effect of HDAC-1 on AS fibroblasts, suggesting that the Wnt-Smad pathway is involved in the regulation of AS by HDAC1. In conclusion, HDAC1 promotes the proliferation, inflammatory response and osteogenic differentiation of AS fibroblasts through the Wnt-Smad pathway.

HCV Core protein represses DKK3 expression via epigenetic silencing and activates the Wnt/β-catenin signaling pathway during the progression of HCC.

The Wnt/β-catenin signaling pathway is frequently activated in hepatocellular carcinoma (HCC). A number of studies have focused on the aberrant hypermethylation of the DKK family proteins and its role in regulating the activation of specific signaling pathways. However, the exact way by which DKK regulates the signaling pathway caused by Core protein of HCV has not been reported. In the present study, we evaluated the expression level of DKK and its aberrant promoter methylation to investigate the involvement of epigenetic regulation in hepatoma cell lines. The transcription and protein expression of DKK1 was significantly increased, whereas the transcription and protein expression levels of DKK2, DKK3, and DKK4 were significantly decreased following overexpression of Core protein. Pyrosequencing indicated that hypermethylation of DKK3 was increased. This was associated with increased expression of Dnmt1. The investigation of the molecular mechanism indicated that HCV Core protein interacted with Dnmt1, which combined with the promoter of DKK3, leading to methylation of DKK3. Functional studies indicated that Core protein promoted the growth, migration and invasion of cancer cells. However, upregulation of the expression of DKK3 and/or the knockdown of the expression of Dnmt1 inhibited the growth, migration and invasion of cancer cells. Taken together, the data indicated that epigenetic silencing of DKK3 caused by Dnmt1 activated the Wnt/β-catenin pathway in HCV Core-mediated HCC. Therefore, DKK3 may be a potential diagnostic and therapeutic target for HCC.

DKK-1 Is Underexpressed in Mesenchymal Stem Cells from Patients with Ankylosing Spondylitis and Further Downregulated by IL-17

Dickkopf-1 (Dkk-1) is a key regulator of bone remodeling in spondyloarthropathies. Nevertheless, data regarding its expression in cells of pathophysiologic relevance, such as mesenchymal stem cells (MSCs), are lacking. Herein, we aimed to address DKK1 gene expression and Wnt pathway activation in MSCs from patients with ankylosing spondylitis (AS) and explore the effect of IL-17 on MSCs with respect to DKK-1 expression and Wnt pathway activation. Primary MSCs were isolated from the bone marrow of the femoral head of two patients with AS and two healthy controls undergoing orthopedic surgery. MSCs were cultured for 7 days in expansion medium and for 21 days in osteogenic medium in the presence or absence of IL-17A. Gene expression of DKK-1 and osteoblastic markers was determined by RT-PCR. Alkaline phosphatase activity, alizarin red and Van Kossa staining were used to assess osteoblastic function and mineralization capacity. DKK-1 was significantly downregulated in MSCs and osteoblasts from patients with AS compared to controls. Moreover, MSCs and osteoblasts from AS patients displayed increased Wnt pathway activation and enhanced osteoblastic activity, as indicated by increased expression of osteoblast marker genes and alkaline phosphatase activity. IL-17 downregulated DKK-1 expression and increased osteoblastic activity and mineralization capacity. DKK-1 is underexpressed in MSCs from AS patients compared to controls, whereas IL-17 has an inhibitory effect on DKK-1 expression and stimulates osteoblastic function. These data may have pathogenetic and clinical implications in AS.

Abstract LB100: Dickkopf-1 induces breast cancer progression by limiting T cell infiltration into the tumor microenvironment

Abstract Activation of Wnt/β-catenin signaling pathway in the tumor cells has been shown to promote cell proliferation and correlate with cancer progression. Therefore, dickkopf-1 (Dkk1), a soluble Wnt/β-catenin inhibitor, has been considered as a tumor suppressor. However, contradicting evidence demonstrated that high levels of circulating Dkk1 correlate with poor prognosis in various cancers. Although Dkk1 is best known to modulate bone remodeling, we recently reported that Dkk1 supports the growth of subcutaneous B16 melanomas by creating an immune-suppressive environment. However, the detailed mechanism by which Dkk1 alters anti-tumor immune responses remains to be elucidated. In this study, we used mice orthotopically injected with hormone-resistant luminal B, ER+ PyMT, EO771 and triple-negative 4T1 cell lines. We find that Dkk1 is upregulated in all models and its neutralization reduces tumor growth. To our surprise, Dkk1 was barely expressed by each tumor lines tested. However, we found elevated expression of Dkk1 in the bone by the osteoblasts (OBs) and in the breast tumor stroma by cancer-associated fibroblasts (CAFs), although at a much lower level compared to the OBs. To address the role of bone-derived versus tumor stroma-derived Dkk1 during tumor progression, we generated mouse models specifically deleting Dkk1 from OBs (OsxCre;Dkk1fl/fl), and from CAFs (Fsp1Cre;Dkk1fl/fl and aSMACreERT2;Dkk1fl/fl). To our surprise, all our mouse models reduced orthotopic PyMT tumor growth significantly, suggesting that Dkk1 could act both systemically and locally to modulate tumor progression. Interestingly, however, deletion of Dkk1 in the bone or in the CAFs did not appear to change the immune composition in the bone marrow, spleen, or at the tumor site. Next, we orthotopically injected PyMT cells into the severely immune-compromised NSG mice treated with anti-Dkk1 or IgG. Strikingly, Dkk1 neutralization did not reduce tumor growth in NSG mice, suggesting that Dkk1 modulates breast cancer growth by altering anti-tumor immune responses. Similar to mice with deletion of Dkk1 in the bone or in the CAFs, Dkk1 neutralization did not alter the percentage of the myeloid and lymphoid cell subsets or the activation status of T cells in bone marrow, spleen, or tumor site compared to IgG control mice. However, by IHC we observed that tumors from anti-Dkk1-treated mice or with deletion of Dkk1 in the CAFs had increased numbers of CD4+ and CD8+ T cells infiltrating the center of tumor mass while these immune populations remained at the edges of the tumors in control mice. In summary, we provide evidence that Dkk1 exerts local and systemic effects to promote tumor progression by modulating the infiltration of immune cells into the TME. Citation Format: Seunghyun Lee, Biancamaria Ricci, Roberta Faccio. Dickkopf-1 induces breast cancer progression by limiting T cell infiltration into the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB100.

CCN1 interacts with integrins to regulate intestinal stem cell proliferation and differentiation

Intestinal stem cells (ISCs) at the crypt base contribute to intestinal homeostasis through a balance between self-renewal and differentiation. However, the molecular mechanisms regulating this homeostatic balance remain elusive. Here we show that the matricellular protein CCN1/CYR61 coordinately regulates ISC proliferation and differentiation through distinct pathways emanating from CCN1 interaction with integrins αvβ3/αvβ5. Mice that delete Ccn1 in Lgr5 + ISCs or express mutant CCN1 unable to bind integrins αvβ3/αvβ5 exhibited exuberant ISC expansion and enhanced differentiation into secretory cells at the expense of absorptive enterocytes in the small intestine, leading to nutrient malabsorption. Analysis of crypt organoids revealed that through integrins αvβ3/αvβ5, CCN1 induces NF-κB-dependent Jag1 expression to regulate Notch activation for differentiation and promotes Src-mediated YAP activation and Dkk1 expression to control Wnt signaling for proliferation. Moreover, CCN1 and YAP amplify the activities of each other in a regulatory loop. These findings establish CCN1 as a niche factor in the intestinal crypts, providing insights into how matrix signaling exerts overarching control of ISC homeostasis.

LncRNAs UC.145 and PRKG1-AS1 Determine the Functional Output of DKK1 in Regulating the Wnt Signaling Pathway in Gastric Cancer

Simple SummaryThe long non-coding RNA (lncRNA) UC.145, which is conserved in gastric cancer tissue, is presented as a potential novel biomarker for the diagnosis, prognosis, and prediction of gastric cancer. In this study, we analyzed and correlated UC.145 expression with that of DKK1, an inhibitor of the canonical Wnt pathway involved in various cancers. Furthermore, we provide evidence that UC.145 regulates the expression of DKK1 by directly targeting another lncRNA, PRKG1-AS1. Regulation of the three genes was closely associated with the overall survival of patients with gastric cancer.DKK1 inhibits the canonical Wnt signaling pathway that is known to be involved in various cancers. However, whether DKK1 acts as an oncogene or tumor suppressor gene remains controversial. Furthermore, the DKK1-regulating mechanism in gastric cancer has not yet been defined. The aim of this study was to explore whether the ultraconserved region UC.145 regulates epigenetic changes in DKK1 expression in gastric cancer. Microarray analysis revealed that UC.145 exhibited the highest binding affinity to EZH2, a histone methyltransferase. The effects of UC.145 inactivation were assessed in gastric cancer cell lines using siRNA. The results indicated that UC.145 triggers DKK1 methylation via interaction with EZH2 and is involved in the canonical Wnt signaling pathway. Additionally, interaction between UC.145 and another long non-coding RNA adjacent to DKK1, PRKG1-AS1, induced a synergistic effect on Wnt signaling. The regulation of these three genes was closely associated with patient overall survival. Inactivation of UC.145 induced apoptosis and inhibited the growth and migratory, invasive, and colony-forming abilities of gastric cancer cells. The study findings provide insights into Wnt signaling in gastric cancer and support UC.145 as a potential novel predictive biomarker for the disease.

Apoptotic extracellular vesicles are metabolized regulators nurturing the skin and hair.

Over 300 billion of cells die every day in the human body, producing a large number of endogenous apoptotic extracellular vesicles (apoEVs). Also, allogenic stem cell transplantation, a commonly used therapeutic approach in current clinical practice, generates exogenous apoEVs. It is well known that phagocytic cells engulf and digest apoEVs to maintain the body's homeostasis. In this study, we show that a fraction of exogenous apoEVs is metabolized in the integumentary skin and hair follicles. Mechanistically, apoEVs activate the Wnt/β-catenin pathway to facilitate their metabolism in a wave-like pattern. The migration of apoEVs is enhanced by treadmill exercise and inhibited by tail suspension, which is associated with the mechanical force-regulated expression of DKK1 in circulation. Furthermore, we show that exogenous apoEVs promote wound healing and hair growth via activation of Wnt/β-catenin pathway in skin and hair follicle mesenchymal stem cells. This study reveals a previously unrecognized metabolic pathway of apoEVs and opens a new avenue for exploring apoEV-based therapy for skin and hair disorders.

MO415: Kidney Expression of Canonical WNT Inhibitors and Β-Catenin in Experimental Models of Arterial Hypertension and Nephrectomy Induced CKD

Abstract BACKGROUND AND AIMS The canonical Wnt (cWnt) is known to be involved in tubular recovery after kidney injury and fibrosis, while arterial hypertension (AH) promotes cWnt activity. The aim of the study was to investigate the kidney expression of β-catenin and cWnt inhibitors in AH and chronic kidney disease (CKD) models. METHOD AH-CKD was performed by 3/4 nephrectomy (NE) in spontaneously hypertensive rats (SHR) with 2 or 6 months of experimental exposure (Table). Sham-operated (SO) SHRs and Wistar Kyoto rats (WKY) served as controls, and 6 mo exposure SO-SHRs were used as AH-induced CKD. Serum creatinine (Cr), renal sclerostin (Sost) and dickkopf-1 (Dkk1) gene expression were measured. The following parameters of kidney morphology were analyzed and calculated quantitatively: the areas of tubular dystrophy (TD) (PAS), renal fibrosis (RF) (Masson's trichrome), β-catenin (BC), sclerostin (SOST) and dickkopf-1 (DKK1) IHC-staining. RESULTS The experimental models were corresponded to early (SO6, NE2) and mild (NE6) CKD (Table). In all groups, BC membrane IHC staining in epithelial cells and nuclear expression in podocytes were determined, SOST and DKK1 cytoplasmic IHC staining in tubular epithelium were detected (Figure). The tubular and interstitial lesions in NE (Table) were accompanied by cytoplasmic BC localization in epithelial and interstitial cells (Figure) with a decrease in dickkopf-1 gene (Table) and protein (Figure) expression, increase in Sost gene expression and SOST IHC staining of tubular intracellular vesicles (Figure). Long-term exposure to hypertension (SO6) resulted in the renal function decline and RF comparable to NE2 (Table), while TD and levels of Wnt inhibitors and BC IHC staining area did not differ significantly vs SO2, WKY (Table). In the pooled analysis, the RF directly correlated with the BC renal expression (r = 0.76, P = 0.005) and negatively with Dkk1 expression (r = −0.45, P = 0.028); the TD negatively correlated with Sost expression (r = −0.80, P = 0.002). CONCLUSION Tubular and interstitial damage in NE and AH-induced CKD is associated with BC up-regulation. The decrease in dickkopf1 and BC cytoplasmic accumulation may be required for renal fibrosis. The sclerostin expression is likely to be essential for the pathogenesis of TD.

Involvement of RUVBL1 in WNT/β-Catenin Signaling in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of head and neck squamous cell carcinoma (HNSCC), but the causes and molecular mechanisms remain unclear. The wingless-integrated/β-catenin (WNT/β-catenin) signaling pathway plays a vital role in cancer cell proliferation, differentiation, and metastasis, including OSCC. To screen potential β-catenin-associated genes involved in OSCC, the intersection of these genes in the STRING and IMEx databases was assessed using differential expression genes (DEG) from public microarrays, and 22 were further selected to construct a β-catenin-protein interaction network. The top 14 hub genes (node degree > 10) within the network were selected. Pearson's correlation analysis showed that β-catenin expression correlated positively with the expression of 11 genes, including AR, BIRC5, CDK6, DKK1, GSK3B, MET, MITF, PARD3, RUVBL1, SLC9A3R1, and SMAD7. A heat map of overall hub gene survival was created, and elevated expression of DKK1 and RUVBL1 was associated with poor survival using the Mantel-Cox test. To identify the function of RUVBL1, colony formation assay, transwell assay, and western blotting revealed that knock-down of RUVBL1 by siRNA decreased H157 and Cal-27 cell proliferation and metastasis by inhibiting β-catenin signaling. These findings suggest that RUVBL1 may serve as a diagnostic and prognostic biomarker for OSCC, as well as a therapeutic target, and may help to uncover additional molecular mechanisms of β-catenin-driven OSCC tumorigenesis.

The Apoptotic Resistance of BRCA1-Deficient Ovarian Cancer Cells is Mediated by cAMP.

Breast cancer type 1 susceptibility protein (BRCA1) is essential for homologous recombination repair of DNA double-strand breaks. Loss of BRCA1 is lethal to embryos due to extreme genomic instability and the activation of p53-dependent apoptosis. However, the apoptosis is resisted in BRCA1-deficient cancer cells even though their p53 is proficient. In this study, by analysis of transcriptome data of ovarian cancer patients bearing BRCA1 defects in TCGA database, we found that cAMP signaling pathway was significantly activated. Experimentally, we found that BRCA1 deficiency caused an increased expression of ADRB1, a transmembrane receptor that can promote the generation of cAMP. The elevated cAMP not only inhibited DNA damage-induced apoptosis through abrogating p53 accumulation, but also suppressed the proliferation of cytotoxic T lymphocytes by enhancing the expression of immunosuppressive factors DKK1. Inhibition of ADRB1 effectively killed cancer cells by abolishing the apoptotic resistance. These findings uncover a novel mechanism of apoptotic resistance in BRCA1-deficient ovarian cancer cells and point to a potentially new strategy for treating BRCA1-mutated tumors.

Dickkopf1 (Dkk1) Alleviates Vascular Calcification by Regulating the Degradation of Phospholipase D1 (PLD1).

Vascular calcification (VC) is a significant risk factor for cardiovascular mortality and morbidity in patients with atherosclerosis (AS), chronic kidney disease, and diabetes. Dickkopf1 (Dkk1) is a multifunctional secreted glycoprotein that has been explored as a novel potential antitumor target. Recently, Dkk1 was shown to be closely associated with AS development. However, the role of Dkk1 in VC remains elusive. In this study, we explored the role and molecular mechanisms of Dkk1 in VC based on a smooth muscle-specific Dkk1-knockout (Dkk1SMKO) mouse model. Our data indicated that Dkk1 expression was decreased under calcifying conditions and that Dkk1 overexpression alleviated high phosphate-induced vascular calcification. In vivo, smooth muscle Dkk1-specific knockout aggravated vascular calcification in mice. However, phospholipase D1 (PLD1) overexpression partially weakened the protective effect of Dkk1 against vascular calcification. Mechanistically, Dkk1 slowed vascular calcification by promoting the degradation of PLD1 via the regulating autophagosome formation and maturation. In conclusion, we found that Dkk1 could alleviate vascular calcification by regulating the degradation of PLD1.

Correction: Study on the Action Mechanism of Dkk-1, TGF-β1 and TNF-α Expression Levels in Dupuytren's Contracture.

Correction: Study on the Action Mechanism of Dkk-1, TGF-β1 and TNF-α Expression Levels in Dupuytren's Contracture.

MiR-148a-3p facilitates osteogenic differentiation of fibroblasts in ankylosing spondylitis by activating the Wnt pathway and targeting DKK1.

Ankylosing spondylitis (AS) is a chronic inflammatory form of arthritis. MicroRNAs (miRNAs) have been identified to serve as therapeutic targets in various inflammatory diseases. The aim of the present study was to determine the functional mechanism of miR-148a-3p on AS. Specimens were collected from AS patients and non-AS patients. Fibroblasts were delivered with the aid of miR-148a-3p inhibitor. Cell staining was performed to observe the morphological changes, calcified nodules, and mineralization degree. The binding sites of miR-148a-3p and DKK1 were predicted on the Starbase website and subsequently verified by means of dual-luciferase reporter assay. AS fibroblasts with silenced miR-148a-3p were transfected with si-DKK1. Levels of RUNX2 and Osteocalcin, DKK1 and Wnt1 protein and phosphorylation level of β-catenin were detected by means of western blot analysis. Results of the present study denoted that AS upregulated miR-148a-3p in fibroblasts to exacerbate osteogenic differentiation, resulting in increased calcified nodules and mineralization degree. Silencing miR-148a-3p could reverse the upregulation of RUNX2 and Osteocalcin in AS fibroblasts and reduce the calcified nodules and mineralization degree. miR-148a-3p targeted DKK1. DKK1 knockdown averted the effect of silencing miR-148a-3p in AS fibroblasts. In addition, silencing miR-148a-3p reversed the upregulation of Wnt1 and β-catenin proteins in AS fibroblasts. To conclude, miR-148a-3p exacerbated the osteogenic differentiation of AS fibroblasts by inhibiting DKK1 expression and activating the Wnt pathway.

Morpholino-driven blockade of Dkk-1 in osteosarcoma inhibits bone damage and tumour expansion by multiple mechanisms

BackgroundOsteosarcoma (OS) is the most common primary bone malignancy. Chemotherapy plays an essential role in OS treatment, potentially doubling 5-year event-free survival if tumour necrosis can be stimulated. The canonical Wnt inhibitor Dickkopf-1 (Dkk-1) enhances OS survival in part through upregulation of aldehyde-dehydrogenase-1A1 which neutralises reactive oxygen species originating from nutritional stress and chemotherapeutic challenge.MethodsA vivo morpholino (DkkMo) was employed to block the expression of Dkk-1 in OS cells. Cell mitosis, gene expression and bone destruction were measured in vitro and in vivo in the presence and absence of doxorubicin (DRB).ResultsDkkMo reduced the expression of Dkk-1 and Aldh1a1, reduced expansion of OS tumours, preserved bone volume and architecture and stimulated tumour necrosis. This was observed in the presence or absence of DRB.ConclusionThese results indicate that administration of DkkMo with or without chemotherapeutics can substantially improve OS outcome with respect to tumour expansion and osteolytic corruption of bone in experimental OS model.

CircCELF1 Inhibits Myocardial Fibrosis by Regulating the Expression of DKK2 Through FTO/m6A and miR-636.

The aim of this study is to explore the role of circCELF1/miR-636/DKK2 pathway in myocardial fibrosis (MF). RT-qPCR and western blot were used to detect the expression of circCELF1, miR-636, and DKK2 in activated cardiac fibroblasts (CFs) and the hearts of acute myocardial infarction (AMI) mice. The m6A level of DKK2 was detected by RIP and RT-qPCR. The regulation of circCELF1/miR-636/DKK2 pathway on CF viability, activation, apoptosis, and migration was verified by CCK-8, western blot, flow cytometry, and Transwell. Ang II induced downregulation of circCELF1 expression, while circCELF1 enhanced the expression of DKK2 by adsorbing miR-636. circCELF1 also reduced DKK2 m6A level by upregulating FTO expression, thereby inhibiting the binding of miR-636 to DKK2 and promoting DKK2 expression. Ang II promoted CF viability, activation, and migration through the circCELF1/miR-636/DKK2 pathway. Both miR-636 inhibitors and DKK2 effectively reduced MF and improved cardiac function in AMI mice.

MSX1 Drives Tooth Morphogenesis Through Controlling Wnt Signaling Activity

Tooth agenesis is a common structural birth defect in humans that results from failure of morphogenesis during early tooth development. The homeobox transcription factor Msx1 and the canonical Wnt signaling pathway are essential for “bud to cap” morphogenesis and are causal factors for tooth agenesis. Our recent study suggested that Msx1 regulates Wnt signaling during early tooth development by suppressing the expression of Dkk2 and Sfrp2 in the tooth bud mesenchyme, and it demonstrated partial rescue of Msx1-deficient molar teeth by a combination of DKK inhibition and genetic inactivation of SFRPs. In this study, we found that Sostdc1/Wise, another secreted Wnt antagonist, is involved in regulating the odontogenic pathway downstream of Msx1. Whereas Sostdc1 expression in the developing tooth germ was not increased in Msx1−/− embryos, genetic inactivation of Sostdc1 rescued maxillary molar, but not mandibular molar, morphogenesis in Msx1−/− mice with full penetrance. Since the Msx1−/−;Sostdc1−/− embryos exhibited ectopic Dkk2 expression in the developing dental mesenchyme, similar to Msx1−/− embryos, we generated and analyzed tooth development in Msx1−/−;Dkk2−/− double and Msx1−/−;Dkk2−/−;Sostdc1−/− triple mutant mice. The Msx1−/−;Dkk2−/− double mutants showed rescued maxillary molar morphogenesis at high penetrance, with a small percentage also exhibiting mandibular molars that transitioned to the cap stage. Furthermore, tooth development was rescued in the maxillary and mandibular molars, with full penetrance, in the Msx1−/−;Dkk2−/−;Sostdc1−/− mice. Together, these data reveal 1) that a key role of Msx1 in driving tooth development through the bud-to-cap transition is to control the expression of Dkk2 and 2) that modulation of Wnt signaling activity by Dkk2 and Sostdc1 plays a crucial role in the Msx1-dependent odontogenic pathway during early tooth morphogenesis.

DKN-01 and tislelizumab ± chemotherapy as a first-line (1L) and second-line (2L) investigational therapy in advanced gastroesophageal adenocarcinoma (GEA): DisTinGuish Trial.

292 Background: Despite recent approval of anti-PD-1 antibodies as 1L therapy in HER2(-) advanced GEA, benefit remains modest and limited largely to PD-L1(+) patients (pts), primarily those with combined positive scores (CPS) ≥5. Thus novel therapeutic approaches are needed for this pt population. DKN-01 is a targeted anti-DKK1 mAb which has demonstrated improved clinical outcomes in pts with elevated tumoral DKK1 expression, a subset of pts with more aggressive disease and shorter overall survival. Methods: DisTinGuish (NCT04363801) is a Phase 2a single arm 2-part trial; Part A investigated DKN-01 (D) + tislelizumab (TS) + CAPOX as 1L therapy for pts with advanced HER2(-) GEA regardless of DKK1 status; Part B investigated two dosing cohorts of D (300 mg and 600 mg) + TS as 2L therapy for DKK1-high advanced GEA pts. Primary objective was to examine safety and tolerability and secondary objectives evaluated multiple efficacy endpoints including overall response rate (ORR) in a modified intent to treat (mITT) population (>1 dose D). Results: Forty-nine pts enrolled between 01 Sept 2020 and 15 Sept 2021; 25 pts in Part A and 24 pts in Part B (D-300 mg). Key clinicopathologic features and efficacy outcomes are shown in Table. The most common D-related AEs were low grade (G1/2) fatigue, nausea, and diarrhea. Nine pts had D-related ≥G3 toxicities, elevated AST/ALT, elevated alkaline phosphatase, hypophosphatemia, hyponatremia, lymphopenia, neutropenia, diarrhea, vomiting, fatigue all occurring in 1 pt and pulmonary embolism in 2 pts (one G5 event). No new safety signals were observed in Part A or B1. Duration of response (DoR), median PFS and median OS have not been reached for Part A. Last pt enrolled in Part B1 on 15 Sept 2021. Conclusions: The combination of D/TS + CAPOX represents a well-tolerated, active 1L combination, particularly for DKK1-high patients consistent with the proposed mechanism of action. Activity appears to be independent of PD-L1 status. Part B1 is aligned with biomarker enrichment and efficacy and biomarker data will be presented along with updated Part A efficacy data. Clinical trial information: NCT04363801. [Table: see text]

Bone Involvement in Patients with Spondyloarthropathies.

Spondyloarthropathies (SpA) are common systemic inflammatory rheumatic diseases, in which, as in other rheumatic diseases, levels of markers of bone resorption are elevated, leading to bone loss and elevated risk of vertebral fractures. However, the diseases are also associated with new bone formation in the spine, the so-called syndesmophytes. We tried to unravel the pathogenesis of formation and growth of syndesmophytes and evaluated new diagnostic and treatment options. After a successful meeting of the Working Group on Rheumatic Diseases at the ECTS 2020, we (WL and CR) were excited about the quality of the speakers (CM, JH, AG, and GL) and their complimentary lectures. Given the relative lack of reviews on spondyloarthropathies and bone, we decided to work together on a comprehensive review that might be interesting for basic scientists and clinically relevant for clinicians. Radiographic progression in axSpA is linked to several risk factors, like male sex, smoking, HLA-B-27, increased levels of CRP, presence of syndesmophytes, and marked inflammation on MRI. The potential role of mechanical stress in the context of physically demanding jobs has been also suggested to promote structural damages. Different treatment options from NSAIDs to biologic agents like TNF inhibitors (TNFi) or IL-17inhibitors (IL-17i) result in a reduction of inflammation and symptoms. However, all these different treatment options failed to show clear and reproducible results on inhibition on syndesmophyte formation. The majority of data are available on TNFi, and some studies suggested an effect in subgroups of patients with ankylosing spondylitis. Less information is available on NSAIDs and IL-17i. Since IL-17i have been introduced quite recently, more studies are expected. IL-17 inhibitors (Il-17i) potently reduce signs and symptoms, but serum level of IL-17 is not elevated, therefore, IL-17 probably has mainly a local effect. The failure of anti-IL-23 in axSpA suggests that IL-17A production could be independent from IL-23. It may be upregulated by TNFα, resulting in lower expression of DKK1 and RANKL and an increase in osteogenesis. In active AS markers of bone resorption are increased, while bone formation markers can be increased or decreased. Bone Turnover markers and additional markers related to Wnt such as DKK1, sclerostin, and RANKL are valuable for elucidating bone metabolism on a group level and they are not (yet) able to predict individual patient outcomes. The gold standard for detection of structural lesions in clinical practice is the use of conventional radiographics. However, the resolution is low compared to the change over time and the interval for detecting changes are 2years or more. Modern techniques offer substantial advantages such as the early detection of bone marrow edema with MRI, the fivefold increased detection rate of new or growing syndesmophytes with low-dose CT, and the decrease in 18F-fluoride uptake during treatment with TNFα-inhibitors (TNFi) in a pilot study in 12 AS patients. Detection of bone involvement by new techniques, such as low-dose CT, MRI and 18-Fluoride PET-scans, and bone turnover markers, in combination with focusing on high-risk groups such as patients with early disease, elevated CRP, syndesmophytes at baseline, male patients and patients with HLA-B27 + are promising options for the near future. However, for optimal prevention of formation of syndesmophytes we need more detailed insight in the pathogenesis of bone formation in axSpA and probably more targeted therapies.

RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis.

Therapeutics targeting osteoclasts are commonly used treatments for bone metastasis; however, whether and how osteoclasts regulate premetastatic niche and bone tropism are largely unknown. In this study, we report that osteoclast precursors (OPs) can function as a premetastatic niche component that facilitates breast cancer (BCa) bone metastasis at early stages. At the molecular level, unbiased GPCR ligand/agonist screening in BCa cells suggested that R-spondin 2 (RSPO2) and RANKL, through interaction with their receptor LGR4, promoted osteoclastic premetastatic niche formation and enhanced BCa bone metastasis. This was achieved by RSPO2/RANKL-LGR4 signal modulating the WNT inhibitor DKK1 through Gαq and β-catenin signaling. DKK1 directly facilitated OP recruitment through suppression of its receptor LDL receptor–related protein 5 (LRP5) but not LRP6, upregulating Rnasek expression via inhibition of canonical WNT signaling. In clinical samples, RSPO2, LGR4, and DKK1 expression showed a positive correlation with BCa bone metastasis. Furthermore, soluble LGR4 extracellular domain (ECD) protein, acting as a decoy receptor for RSPO2 and RANKL, significantly alleviated bone metastasis and osteolytic lesions in a mouse bone metastasis model. These findings provide unique insights into the functional role of OPs as key components of the premetastatic niche for BCa bone metastasis and identify RSPO2/RANKL-LGR4 signaling as a promising target for inhibiting BCa bone metastasis.

Biological and molecular profile of fracture non-union tissue: A systematic review and an update on current insights.

Fracture non‐union represents a common complication, seen in 5%–10% of all acute fractures. Despite the enhancement in scientific understanding and treatment methods, rates of fracture non‐union remain largely unchanged over the years. This systematic review investigates the biological, molecular and genetic profiles of both (i) non‐union tissue and (ii) non–union‐related tissues, and the genetic predisposition to fracture non‐union. This is crucially important as it could facilitate earlier identification and targeted treatment of high‐risk patients, along with improving our understanding on pathophysiology of fracture non‐union. Since this is an update on our previous systematic review, we searched the literature indexed in PubMed Medline; Ovid Medline; Embase; Scopus; Google Scholar; and the Cochrane Library using Medical Subject Heading (MeSH) or Title/ words (non‐union(s), non‐union(s), human, tissue, bone morphogenic protein(s) (BMPs) and MSCs) from August 2014 (date of our previous publication) to 2 October 2021 for non‐union tissue studies, whereas no date restrictions imposed on non–union‐related tissue studies. Inclusion criteria of this systematic review are human studies investigating the characteristics and properties of non‐union tissue and non–union‐related tissues, available in full‐text English language. Limitations of this systematic review are exclusion of animal studies, the heterogeneity in the definition of non‐union and timing of tissue harvest seen in the included studies, and the search term MSC which may result in the exclusion of studies using historical terms such as ‘osteoprogenitors’ and ‘skeletal stem cells’. A total of 24 studies (non‐union tissue: n = 10; non–union‐related tissues: n = 14) met the inclusion criteria. Soft tissue interposition, bony sclerosis of fracture ends and complete obliteration of medullary canal are commonest macroscopic appearances of non‐unions. Non‐union tissue colour and surrounding fluid are two important characteristics that could be used clinically to distinguish between septic and aseptic non‐unions. Atrophic non‐unions had a predominance of endochondral bone formation and lower cellular density, when compared against hypertrophic non‐unions. Vascular tissues were present in both atrophic and hypertrophic non‐unions, with no difference in vessel density between the two. Studies have found non‐union tissue to contain biologically active MSCs with potential for osteoblastic, chondrogenic and adipogenic differentiation. Proliferative capacity of non‐union tissue MSCs was comparable to that of bone marrow MSCs. Rates of cell senescence of non‐union tissue remain inconclusive and require further investigation. There was a lower BMP expression in non‐union site and absent in the extracellular matrix, with no difference observed between atrophic and hypertrophic non‐unions. The reduced BMP‐7 gene expression and elevated levels of its inhibitors (Chordin, Noggin and Gremlin) could potentially explain impaired bone healing observed in non‐union MSCs. Expression of Dkk‐1 in osteogenic medium was higher in non‐union MSCs. Numerous genetic polymorphisms associated with fracture non‐union have been identified, with some involving the BMP and MMP pathways. Further research is required on determining the sensitivity and specificity of molecular and genetic profiling of relevant tissues as a potential screening biomarker for fracture non‐unions.