Wnt Pathway Components Research Articles

Impaired LEF1 Activation Accelerates iPSC-Derived Keratinocytes Differentiation in Hutchinson-Gilford Progeria Syndrome.

Hutchinson–Gilford progeria syndrome (HGPS) is a detrimental premature aging disease caused by a point mutation in the human LMNA gene. This mutation results in the abnormal accumulation of a truncated pre-lamin A protein called progerin. Among the drastically accelerated signs of aging in HGPS patients, severe skin phenotypes such as alopecia and sclerotic skins always develop with the disease progression. Here, we studied the HGPS molecular mechanisms focusing on early skin development by differentiating patient-derived induced pluripotent stem cells (iPSCs) to a keratinocyte lineage. Interestingly, HGPS iPSCs showed an accelerated commitment to the keratinocyte lineage than the normal control. To study potential signaling pathways that accelerated skin development in HGPS, we investigated the WNT pathway components during HGPS iPSCs-keratinocytes induction. Surprisingly, despite the unaffected β-catenin activity, the expression of a critical WNT transcription factor LEF1 was diminished from an early stage in HGPS iPSCs-keratinocytes differentiation. A chromatin immunoprecipitation (ChIP) experiment further revealed strong bindings of LEF1 to the early-stage epithelial developmental markers K8 and K18 and that the LEF1 silencing by siRNA down-regulates the K8/K18 transcription. During the iPSCs-keratinocytes differentiation, correction of HGPS mutation by Adenine base editing (ABE), while in a partial level, rescued the phenotypes for accelerated keratinocyte lineage-commitment. ABE also reduced the cell death in HGPS iPSCs-derived keratinocytes. These findings brought new insight into the molecular basis and therapeutic application for the skin abnormalities in HGPS.

Increase in ADAR1p110 activates the canonical Wnt signaling pathway associated with aggressive phenotype in triple negative breast cancer cells

Triple-negative breast cancer (TNBC) represents a challenge in the search for new therapeutic targets. TNBCs are aggressive and generate resistance to chemotherapy. Tumors of TNBC patients with poor prognosis present a high level of adenosine deaminase acting on RNA1 (ADAR1). We explore the connection of ADAR1 with the canonical Wnt signaling pathway and the effect of modulation of its expression in TNBC. Expression data from cell line sequencing (DepMap) and TCGA samples were downloaded and analyzed. We lentivirally generated an MDA-MB-231 breast cancer cell line that overexpress (OE) ADAR1p110 or an ADAR knockdown. Abundance of different proteins related to Wnt/β-catenin pathway and activity of nuclear β-catenin were analyzed by Western blot and luciferase TOP/FOP reporter assay, respectively. Cell invasion was analyzed by matrigel assay. In mice, we study the behavior of tumors generated from ADAR1p110 (OE) cells and tumor vascularization immunostaining were analyzed. ADAR1 connects to the canonical Wnt pathway in TNBC. ADAR1p110 overexpression decreased GSK-3β, while increasing active β-catenin. It also increased the activity of nuclear β-catenin and increased its target levels. ADAR1 knockdown has the opposite effect. MDA-MB-231 ADAR1 (OE) cells showed increased capacity of invasion. Subsequently, we observed that tumors derived from ADAR1p110 (OE) cells showed increased invasion towards the epithelium, and increased levels of Survivin and CD-31 expressed in vascular endothelial cells. These results indicate that ADAR1 overexpression alters the expression of some key components of the canonical Wnt pathway, favoring invasion and neovascularization, possibly through activation of the β-catenin, which suggests an unknown role of ADAR1p110 in aggressiveness of TNBC tumors.

Dickkopf proteins in pathological inflammatory diseases.

The human body encounters various challenges. Tissue repair and regeneration processes are augmented after tissue injury to reinstate tissue homeostasis. The Wnt pathway plays a crucial role in tissue repair since it induces target genes required for cell proliferation and differentiation. Since tissue injury causes inflammatory immune responses, it has become increasingly clear that the Wnt ligands can function as immunomodulators while critical for tissue homeostasis. The Wnt pathway and Wnt ligands have been studied extensively in cancer biology and developmental biology. While the Wnt ligands are being studied actively, how the Wnt antagonists and their regulatory mechanisms can modulate immune responses during chronic pathological inflammation remain elusive. This review summarizes DKK family proteins as immunomodulators, aiming to provide an overarching picture for tissue injury and repair. To this end, we first review the Wnt pathway components and DKK family proteins. Next, we will review DKK family proteins (DKK1, 2, and 3) as a new class of immunomodulatory protein in cancer and other chronic inflammatory diseases. Taken together, DKK family proteins and their immunomodulatory functions in chronic inflammatory disorders provide novel insights to understand immune diseases and make them attractive molecular targets for therapeutic intervention.

Comprehensive analysis of oncogenic fusions in mismatch repair deficient colorectal carcinomas by sequential DNA and RNA next generation sequencing

BackgroundColorectal carcinoma (CRC) harboring oncogenic fusions has been reported to be highly enriched in mismatch repair deficient (dMMR) tumors with MLH1 hypermethylation (MLH1me+) and wild-type BRAF and RAS. In this study, dMMR CRCs were screened for oncogene fusions using sequential DNA and RNA next generation sequencing (NGS).ResultsComprehensive analysis of fusion variants, genetic profiles and clinicopathological features in fusion-positive dMMR CRCs was performed. Among 193 consecutive dMMR CRCs, 39 cases were identified as MLH1me+BRAF/RAS wild-type. Eighteen fusion-positive cases were detected by DNA NGS, all of which were MLH1me+ and BRAF/RAS wild-type. RNA NGS was sequentially conducted in the remaining 21 MLH1me+BRAF/RAS wild-type cases lacking oncogenic fusions by DNA NGS, and revealed four additional fusions, increasing the proportion of fusion-positive tumors from 46% (18/39) to 56% (22/39) in MLH1me+BRAF/RAS wild-type dMMR cases. All 22 fusions were found to involve RTK-RAS pathway. Most fusions affected targetable receptor tyrosine kinases, including NTRK1(9/22, 41%), NTRK3(5/22, 23%), ALK(3/22, 14%), RET(2/22, 9%) and MET(1/22, 5%), whilst only two fusions affected mitogen-activated protein kinase cascade components BRAF and MAPK1, respectively. RNF43 was identified as the most frequently mutated genes, followed by APC, TGFBR2, ATM, BRCA2 and FBXW7. The vast majority (19/22, 86%) displayed alterations in key WNT pathway components, whereas none harbored additional mutations in RTK-RAS pathway. In addition, fusion-positive tumors were typically diagnosed in elder patients and predominantly right-sided, and showed a significantly higher preponderance of hepatic flexure localization (P < 0.001) and poor differentiation (P = 0.019), compared to fusion-negative MLH1me+ CRCs.ConclusionsWe proved that sequential DNA and RNA NGS was highly effective for fusion detection in dMMR CRCs, and proposed an optimized practical fusion screening strategy. We further revealed that dMMR CRCs harboring oncogenic fusion was a genetically and clinicopathologically distinctive subgroup, and justified more precise molecular subtyping for personalized therapy.

The m6 A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons.

Messenger RNA m6A modification is shown to regulate local translation in axons. However, how the m6A codes in axonal mRNAs are read and decoded by the m6A reader proteins is still unknown. Here, it is found that the m6A readers YTHDF1 and YTHDF2 are both expressed in cerebellar granule cells (GCs) and their axons. Knockdown (KD) of YTHDF1 or YTHDF2 significantly increases GC axon growth rates in vitro. By integrating anti‐YTHDF1&2 RIP‐Seq with the quantitative proteomic analysis or RNA‐seq after KD of YTHDF1 or YTHDF2, a group of transcripts which may mediate the regulation of GC axon growth by YTHDFs is identified. Among them, Dvl1 and Wnt5a, encoding the key components of Wnt pathway, are further found to be locally translated in axons, which are controlled by YTHDF1 and YTHDF2, respectively. Specific ablation of Ythdf1 or Ythdf2 in GCs increases parallel fiber growth, promotes synapse formation in cerebellum in vivo, and improves motor coordination ability. Together, this study identifies a mechanism by which the m6A readers YTHDF1 and YTHDF2 work synergistically on the Wnt5a pathway through regulating local translation in GC axons to control cerebellar parallel fiber development.

Wnt pathway modulators in cancer therapeutics: An update on completed and ongoing clinical trials.

Wnt signaling plays an essential role in the initiation and progression of various types of cancer. Besides, the Wnt pathway components have been established as reliable biomarkers and potential targets for cancer therapy. Wnt signaling is categorized into canonical and noncanonical pathways. The canonical pathway is involved in cell survival, proliferation, differentiation and migration, while the noncanonical pathway regulates cell polarity and migration. Apart from its biological role in development and homeostasis, the Wnt pathway has been implicated in several pathological disorders, including cancer. As a result, inhibiting this pathway has been a focus of cancer research with multiple targetable candidates in development. In this review, our focus will be to summarize information about ongoing and completed clinical trials targeting various Wnt pathway components, along with describing current and emerging Wnt targeted therapies. In addition, we will discuss potential opportunities and associated challenges of inhibiting Wnt signaling for cancer therapy.

MRNA coexpression patterns of Wnt pathway components and their clinicopathological associations in breast and colorectal cancer

Aberrant Wnt signaling is implicated in carcinogenesis triggering efforts for the development of new therapeutic agents, many of which have entered clinical trials. We extend our previous analysis of WNT3, FZD7, LEF1 expression levels in breast and colorectal cancer including WNT2, FZD4 and β-catenin expression, in an effort to delineate their relative expression levels along with concurrent expression patterns and possible prognostic value. We analyzed 82 breast and 102 colorectal carcinomas for relative mRNA expression levels of the investigated genes by RT-PCR relative quantification with the ΔΔCt method. Statistical analysis was performed in order to determine associations of relative mRNA expression and linear correlations. β-catenin expression was determined by immunochemistry. Regarding breast carcinomas, decreased relative mRNA expression levels of WNT2, FZD4 were found frequently and WNT2 expression was correlated with ER/ PR status (p = 0.045/p = 0.028), whereas β-catenin with grade (p = 0.026). In colorectal carcinomas, increased relative mRNA expression levels of WNT2 and FZD4 were found in 59% and 32% of cases respectively, whereas β-catenin showed decreased mRNA expression levels in 57% of cases and a correlation with pN-category (p = 0.037). Linear correlations were observed between WNT2/FZD4 (R=0.542, p < 0.001), WNT2/β-catenin (R=0.254, p = 0.010), FZD4/β-catenin (R=0.406, p < 0.001) expression and a correlation between mRNA expression and membranous/cytoplasmic β-catenin emerged (p = 0.039/0.046). Our results suggest a possible clinical significance for Wnt pathway gene expression levels in both tumour types. The concurrent expression of the investigated genes as well as the different expression profiles, underlines the complexity of this pathway and the necessity of patient selection in order to maximize the efficacy of drugs targeting Wnt pathway.

A bibliometric analysis of Wnt signaling pathway: from the top-100 cited articles to emerging trends.

BackgroundWnt signaling pathway plays a vital role in the regulation of development. An increasing number of articles about Wnt pathway components have been published. By analyzing these studies’ characteristics and qualities, we aim to reveal the current research focus and emerging trends in Wnt signaling.MethodsThe databases of Web of Science Core Collection, BIOSIS Citation Index, MEDLINE, etc. were utilized to identify articles on May 23rd, 2020. Wnt signaling pathway-related articles were identified, the 100 most cited articles and articles in the last decade were selected and calculated for citations without self-citation. The subsequent analysis included citation density (citations/article age), time-related flux, authorship, institution, journal, geographic distribution, and theme.ResultsThese articles were published mainly from 2000 to 2009 (62%). Citations per article ranged from 599 to 3,780 with a median number of 880 times. Most studies (66%) came from the United States. Nusse Roel and Clevers Hans (15 and 13 papers) have contributed significantly to the field. The most highlighted study themes were cancer (15%), embryo development (14%), and cytoplasm signal transduction (11%). From 2011 to 2020, interest in emerging subtopics, including osteogenesis, immune, apoptosis, autophagy, microRNA, and cancer stem cell, are rising.ConclusionsCancer, embryo development, stem cell, and signal transduction process still play a major role in the field. With multiple emerging subtopics and investigation on an integrated view of the Wnt signal network, the association of Wnt with diseases was further revealed.

Canonical Wnt Pathway Is Activated in Myeloid Leukemia Stem Cells Via Hematopoietic Cell Kinase That Is Phosphorylated By an Autocrine TIM-3/Galectin-9 Signaling

(Introduction)Activation of β-catenin is critical for self-renewal of cancer stem cells (CSCs). A number of recurrent mutations related to the aberrant accumulation and activation of β-catenin have been identified in CSCs in various types of cancer, but such somatic mutations are barely found in leukemic stem cells (LSCs) in acute myeloid leukemia (AML). We have recently identified the autocrine loop consisted of LSC-specific surface molecule T-cell immunoglobulin mucin-3 (TIM-3) and its ligand galectin-9 (Gal-9) in human myeloid malignancies. When Gal-9 binds to TIM-3, TIM-3 signaling induces accumulation of β-catenin in nucleus (Kikushige et al, Cell Stem Cell, 2015). In this study, we sought to identify the molecular mechanism of aberrant β-catenin activation in LSCs of AML.(Results)To test whether TIM-3 signaling can activate the canonical Wnt pathway, we evaluated the activation of LDL receptor-related protein 6 (LRP6), a key component of canonical Wnt pathway. Immunoprecipitation assay by using anti-LRP6 antibody revealed that Gal-9 stimulation promoted the formation of the “LRP6-signalosome”, the protein complex consisted of Wnt components including LRP6, Axin and Dvl. Gal-9 stimulation also induced phosphorylation of LRP6at several sites including Thr1479 and subsequent β-catenin accumulation in primary TIM-3+ AML cells. On the other hand, this effect on LRP6 induced by Gal-9 stimulation was completely canceled in the presence of an anti-TIM-3 antibody (F38-2E2), which can block the TIM-3/Gal-9 interaction. Since the Wnt ligand is not involved in this assay system, the TIM-3/Gal-9 interaction should directly activate the canonical Wnt pathway, not through the previously identified Wnt signaling machinery.Gal-9 stimulation induced phosphorylation of cytoplasmic tail of TIM-3, which induces recruitment of several Src family kinases (SFKs). We found that Gal-9 ligation to TIM-3 activated several SFKs including hematopoietic cell kinase (HCK) by phosphorylation of the SH1 domain (Tyr410 in HCK) in TIM-3+ AML cells. Furthermore, an inhibitor specific for HCK, but not inhibitors effective for other SFKs, inhibited LRP6 phosphorylation induced by TIM-3/Gal-9 signaling, indicating that HCK should be a critical mediator for TIM-3 signaling in human LSCs.Next, we focused on p120-catenin because this molecule is known as not only a substrate for SFKs but also an indispensable molecule for the initial step of the LRP6-signalosome formation. We found that the phosphorylation of p120-catenin at Tyr228 was induced in response to Gal-9 stimulation. HCK inhibitors efficiently blocked the Tyr228 phosphorylation and subsequent activation of canonical β-catenin pathway. These results suggested that HCK activation induced by TIM-3/Gal-9 ligation could activate canonical β-catenin pathway through phosphorylation of p120-catenin.(Conclusion)In myeloid leukemia, TIM-3/Gal-9 signaling activates HCK, which induces p120-catenin phosphorylation, eventually acitavate the canonical β-catenin pathway, and these processes should be independent of Wnt ligands. Since TIM-3/Gal-9 autocrine loop signaling is constitutively active in AML, this novel machinery should contribute to the maintenance of AML LSCs through accumulation of β-catenin. Our study suggest that TIM-3 and Gal-9 as well as HCK should be important therapeutic targets in AML LCSs. DisclosuresNo relevant conflicts of interest to declare.

Expression of Wnt and TGF-Beta Pathway Components during Whole-Body Regeneration from Cell Aggregates in Demosponge Halisarca dujardinii.

The phenomenon of whole-body regeneration means rebuilding of the whole body of an animal from a small fragment or even a group of cells. In this process, the old axial relationships are often lost, and new ones are established. An amazing model for studying this process is sponges, some of which are able to regenerate into a definitive organism after dissociation into cells. We hypothesized that during the development of cell aggregates, primmorphs, new axes are established due to the activation of the Wnt and TGF-beta signaling pathways. Using in silico analysis, RNA-seq, and whole-mount in situ hybridization, we identified the participants in these signaling pathways and determined the spatiotemporal changes in their expression in demosponge Halisarca dujardinii. It was shown that Wnt and TGF-beta ligands are differentially expressed during primmorph development, and transcripts of several genes are localized at the poles of primmorphs, in the form of a gradient. We suppose that the Wnt and TGF-beta signaling cascades are involved in the initial axial patterning of the sponge body, which develops from cells after dissociation.

Wnt- and glutamate-receptors orchestrate stem cell dynamics and asymmetric cell division.

The Wnt-pathway is part of a signalling network that regulates many aspects of cell biology. Recently, we discovered crosstalk between AMPA/Kainate-type ionotropic glutamate receptors (iGluRs) and the Wnt-pathway during the initial Wnt3a-interaction at the cytonemes of mouse embryonic stem cells (ESCs). Here, we demonstrate that this crosstalk persists throughout the Wnt3a-response in ESCs. Both AMPA and Kainate receptors regulate early Wnt3a-recruitment, dynamics on the cell membrane, and orientation of the spindle towards a Wnt3a-source at mitosis. AMPA receptors specifically are required for segregating cell fate components during Wnt3a-mediated asymmetric cell division (ACD). Using Wnt-pathway component knockout lines, we determine that Wnt co-receptor Lrp6 has particular functionality over Lrp5 in cytoneme formation, and in facilitating ACD. Both Lrp5 and 6, alongside pathway effector β-catenin act in concert to mediate the positioning of the dynamic interaction with, and spindle orientation to, a localised Wnt3a-source. Wnt-iGluR crosstalk may prove pervasive throughout embryonic and adult stem cell signalling.

Profiling genomic characteristics related to immunotherapy for gastric cancer with amplified MET.

e16066 Background: Gastric cancer (GCA) is one of the most deadly cancers all over the world, with an estimate of 26,560 new cases and 11,180 deaths in United States according to the cancer statistics of 2021. As immunotherapy has become available for GCA, it is necessary to distinguish patients who might be resistant to it. Methods: We identified 41 patients with amplified MET from a large Chinese cohort including 934 GCA (4.4% out of them) according to the ratio of purity adjusted copy number of MET and chromosome 7. Then, we comprehensively profiled genomic characteristics related to immunotherapy for GCA with amplified MET and compared them with other GCA. Results: First, mutated RNF43 and AXIN1 were much more prevalent in GCA with amplified MET. These two genes are components of Wnt pathway, whose aberrance is associated with resistance to immunotherapy. Second, carrier ratios of amplified CDK6 and CCND1 were much higher in GCA with amplified MET. These two genes play important roles in cell cycle pathway, while amplification of CCND1 has been recognized as a negatively predictive biomarker for immunotherapy. Third, we calculated mutational load of immunotherapy-related positively (mutated ATM, BRCA1, BRCA2, CHEK2, ERCC2, ERCC4, FANCA, KRAS, MLH1, MSH2, MSH6, NRAS, PALB2, PBRM1, PMS2, POLD1, POLE, RAD50 and TP53; deleted POLE) and negatively (mutated B2M, CTNNB1, DNMT3A, EGFR, JAK1, JAK2 and STK11; amplified CCND1, EGFR, FGF19, FGF3, FGF4, MDM2 and MDM4; deleted B2M, JAK2, PTEN and STK11) predictive biomarkers for GCA with amplified MET. We defined the mutational load as mean carrier ratio of point mutations or copy number variations (CNVs) appeared in a gene set. As is expected, the mutational load of point mutations in 19 positively predictive biomarkers in GCA with amplified MET was much lower than that in other GCA, whereas the mutational load of CNV in 11 negatively predictive biomarkers in GCA with amplified MET was much higher than that in other GCA. Finally, we found GCA with amplified MET possessed lower tumor mutational burden (TMB) and microsatellite instability (MSI), depleted somatic signature of defective DNA mismatch repair and higher copy number instability (CNI). Conclusions: Genomic characteristics of GCA with amplified MET are associated with adverse response to immunotherapy and thus it should be carefully evaluated when these patients are treated with immunotherapy.

Primary Cilia Formation Does Not Rely on WNT/β-Catenin Signaling

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis.

Circulating Wnt Ligands Activate the Wnt Signaling Pathway in Mature Erythrocytes.

[Figure: see text].

Identification of novel human Wnt target genes using adult endodermal tissue-derived organoids

Canonical Wnt signaling plays a key role during organ development, homeostasis and regeneration and these processes are conserved between invertebrates and vertebrates. Mutations in Wnt pathway components are commonly found in various types of cancer. Upon activation of canonical Wnt signaling, β-catenin binds in the nucleus to members of the TCF-LEF family and activates the transcription of target genes. Multiple Wnt target genes, including Lgr5/LGR5 and Axin2/AXIN2, have been identified in mouse models and human cancer cell lines. Here we set out to identify the transcriptional targets of Wnt signaling in five human tissues using organoid technology. Organoids are derived from adult stem cells and recapitulate the functionality as well as the structure of the original tissue. Since the Wnt pathway is critical to maintain the organoids from the human intestine, colon, liver, pancreas and stomach, organoid technology allows us to assess Wnt target gene expression in a human wildtype situation. We performed bulk mRNA sequencing of organoids immediately after inhibition of Wnt pathway and identified 41 genes as commonly regulated genes in these tissues. We also identified large numbers of target genes specific to each tissue. One of the shared target genes is TEAD4, a transcription factor driving expression of YAP/TAZ signaling target genes. In addition to TEAD4, we identified a variety of genes which encode for proteins that are involved in Wnt-independent pathways, implicating the possibility of direct crosstalk between Wnt signaling and other pathways. Collectively, this study identified tissue-specific and common Wnt target gene signatures and provides evidence for a conserved role for these Wnt targets in different tissues.

Phase I dose-escalation study of MCLA-158, a first-in-class bispecific antibody targeting EGFR and LGR5, in metastatic colorectal cancer (CRC).

62 Background: The WNT and EGFR signaling pathways are oncogenic and mitogenic drivers in CRC and other cancers. MCLA-158 is an ADCC enhanced human IgG1 cLC bispecific antibody identified from functional screening of CRC patient (pt)-derived organoids (PDO). MCLA-158 exposure leads to EGFR degradation in LGR5+ cancer and exhibits potent growth inhibition of RASmut and wt CRC PDOs, while minimal inhibition is observed in non-tumoral PDOs. Blockade of metastasis initiation was seen in preclinical in vivo models. MCLA-158 also demonstrated tumor regression or stasis in esophageal squamous (6/6) and gastric adenocarcinoma (6/8) PDX models selected for LGR5 and EGFR expression. Methods: Metastatic CRC pts progressing after oxaliplatin, irinotecan and fluoropyrimidines, and EGFR monoclonal antibodies if RASwt, received MCLA-158 IV every 2 weeks (q2w; 4-week cycle) in a phase I study. Safety, efficacy (per RECIST 1.1), PK and ADA were assessed. WNT and EGFR pathway components were evaluated in tumor tissue. EGFR pathway mutations (ctDNA) and soluble EGFR (sEGFR) were assessed in blood. PDOs were derived from baseline biopsies and assessed for MCLA-158 responsiveness. Results: As of 7 September 2020, 33 pts were treated over 11 dose levels (5 to 1500 mg, flat dose). At enrollment, median age was 58 years (range 35-76), ECOG PS 0/1:22/11, and 15 pts were RASmut. Pts had a median of 3 metastatic sites (range 1-12) with a median of 4 prior lines of therapy (range 1-10). A median of 2 MCLA-158 cycles (range 1-6) were administered. No dose limiting toxicity occurred. Infusion-related reactions were the most common AEs (67% of pts), occurred mostly in cycle 1 (21/22 pts), and were manageable. Other common related AEs included rash acneiform (36%), diarrhea (15%), pyrexia (9%) and asthenia/fatigue (9%). Related AEs were mostly mild or moderate; G3 related AEs were IRRs (4 pts; 12%), diarrhea and hypophosphatemia (1 pt each). The RP2D (1500 mg) was selected based on safety, PK and receptor occupancy prediction. MCLA-158 exhibited target-mediated clearance with proportional PK from 750 mg (t1/2 ~80 h at 1500 mg). Low ADA titers were seen in 3/23 pts, with no effect on PK. Target saturation is predicted at steady state serum trough levels. No clinical responses were observed during dose escalation. No/low baseline tumor EGFR expression (0-1+ IHC) was observed in 17/30 pts and non-elevated sEGFR levels ( &lt; 4 ng/mL) in 18/22 pts. More than 1 activating mutation in EGFR signaling pathway genes were identified in ctDNA from 10/20 pts. 1/4 PDOs had &gt; 30% growth inhibition with MCLA-158 ex vivo. MCLA-158 activity on PDOs correlated negatively with the presence of EGFR pathway resistance-mutations. Conclusions: Dual EGFR/LRG5 blockade with MCLA-158 was well tolerated, and the RP2D was 1500 mg IV q2w. Enrollment of pts with gastric and other non-CRC cancers at the RP2D continues in the expansion phase. Clinical trial information: NCT03526835.

Pathways involved in pony body size development

BackgroundThe mechanism of body growth in mammals is poorly understood. Here, we investigated the regulatory networks involved in body growth through transcriptomic analysis of pituitary and epiphyseal tissues of smaller sized Debao ponies and Mongolian horses at the juvenile and adult stages.ResultsWe found that growth hormone receptor (GHR) was expressed at low levels in long bones, although growth hormone (GH) was highly expressed in Debao ponies compared with Mongolian horses. Moreover, significant downregulated of the GHR pathway components m-RAS and ATF3 was found in juvenile ponies, which slowed the proliferation of bone osteocytes. However, WNT2 and PLCβ2 were obviously upregulated in juvenile Debao ponies, which led to premature mineralization of the bone extracellular matrix. Furthermore, we found that the WNT/Ca2+ pathway may be responsible for regulating body growth. GHR was demonstrated by q-PCR and Western blot analyses to be expressed at low levels in long bones of Debao ponies. Treatment with WNT antagonistI decreased the expression of WNT pathway components (P < 0.05) in vitro. Transduction of ATDC5 cells with a GHR-RNAi lentiviral vector decreased the expression of the GHR pathway components (P < 0.05). Additionally, the expression of the IGF-1 gene in the liver was lower in Debao ponies than in Mongolian horses at the juvenile and adult stages. Detection of plasma hormone concentrations showed that Debao ponies expressed higher levels of IGF-1 as juveniles and higher levels of GH as adults than Mongolian horses, indicating that the hormone regulation in Debao ponies differs from that in Mongolian horses.ConclusionOur work provides insights into the genetic regulation of short stature growth in mammals and can provide useful information for the development of therapeutic strategies for small size.

Nradd Acts as a Negative Feedback Regulator of Wnt/β-Catenin Signaling and Promotes Apoptosis.

Wnt/β-catenin signaling controls many biological processes for the generation and sustainability of proper tissue size, organization and function during development and homeostasis. Consequently, mutations in the Wnt pathway components and modulators cause diseases, including genetic disorders and cancers. Targeted treatment of pathway-associated diseases entails detailed understanding of the regulatory mechanisms that fine-tune Wnt signaling. Here, we identify the neurotrophin receptor-associated death domain (Nradd), a homolog of p75 neurotrophin receptor (p75NTR), as a negative regulator of Wnt/β-catenin signaling in zebrafish embryos and in mammalian cells. Nradd significantly suppresses Wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Nradd is localized at the plasma membrane, physically interacts with the Wnt receptor complex and enhances apoptosis in cooperation with Wnt/β-catenin signaling. Our functional analyses indicate that the N-glycosylated N-terminus and the death domain-containing C-terminus regions are necessary for both the inhibition of Wnt signaling and apoptosis. Finally, Nradd can induce apoptosis in mammalian cells. Thus, Nradd regulates cell death as a modifier of Wnt/β-catenin signaling during development.

Concomitant Up-Regulation of Hsa- Mir-374 and Down-Regulation of Its Targets, GSK-3β and APC, in Tissue Samples of Colorectal Cancer.

The WNT-pathway is involved in several cancers, including colorectal cancer (CRC). Many cell signaling components and pathways are controlled by microRNAs. The main purpose of the present study was to investigate the expression of hsa-miR-374, and its two target genes of the Wnt-pathway in CRC clinical samples. In this study, we predicted the miRNAs targeting key genes of WNT-pathway using bioinformatics algorithms. The expression levels of hsa-miR-374, APC and GSK-3β on 48 pairs of Formalin-Fixed Paraffin-Embedded (FFPE) CRC tumors and marginal-tumors were evaluated using real time-PCR. Additionally, the hsa-miR-374a-5p precursor sequence was amplified by whole-blood DNA as a template. This amplicon was cloned into pEGFP-c1 expression vector and transfected into SW742 cells. Aside from this, MTT assay was performed to evaluate the effect of miR-374 on cell viability. The bioinformatics analysis indicated that hsa-miR-374 binds to the regulatory region the key components of WNT-pathway, including APC and GSK-3β considering the recognition elements and mirSVR scores. Our results revealed significant down-regulation of GSK-3β (0.94 times, p= 0.0098) and APC (0.96 times, p= 0.03) and up-regulation of miR-374 (1.22 times, p= 0.0071) on tumor samples compared with their normal pairs. Meanwhile, the results of the over-expression of miR-374 showed down-regulation of APC and GSK-3β. MTT-assay also indicated that the miR-374 increased cell survival. The results of our study indicated a concomitant change in the expression of miR-374 and its two related target genes, in clinical samples of CRC. Hsa-miR-374 might be as a helpful biomarker or therapeutic target in CRC.

Angiogenesis-Related Functions of Wnt Signaling in Colorectal Carcinogenesis.

Simple SummaryAngiogenesis belongs to the most clinical characteristics of colorectal cancer (CRC) and is strongly linked to the activation of Wnt/β-catenin signaling. The most prominent factors stimulating constitutive activation of this pathway, and in consequence angiogenesis, are genetic alterations (mainly mutations) concerning APC and the β-catenin encoding gene (CTNNB1), detected in a large majority of CRC patients. Wnt/β-catenin signaling is involved in the basic types of vascularization (sprouting and nonsprouting angiogenesis), vasculogenic mimicry as well as the formation of mosaic vessels. The number of known Wnt/β-catenin signaling components and other pathways interacting with Wnt signaling, regulating angiogenesis, and enabling CRC progression continuously increases. This review summarizes the current knowledge about the role of the Wnt/Fzd/β-catenin signaling pathway in the process of CRC angiogenesis, aiming to improve the understanding of the mechanisms of metastasis as well as improvements in the management of this cancer.Aberrant activation of the Wnt/Fzd/β-catenin signaling pathway is one of the major molecular mechanisms of colorectal cancer (CRC) development and progression. On the other hand, one of the most common clinical CRC characteristics include high levels of angiogenesis, which is a key event in cancer cell dissemination and distant metastasis. The canonical Wnt/β-catenin downstream signaling regulates the most important pro-angiogenic molecules including vascular endothelial growth factor (VEGF) family members, matrix metalloproteinases (MMPs), and chemokines. Furthermore, mutations of the β-catenin gene associated with nuclear localization of the protein have been mainly detected in microsatellite unstable CRC. Elevated nuclear β-catenin increases the expression of many genes involved in tumor angiogenesis. Factors regulating angiogenesis with the participation of Wnt/β-catenin signaling include different groups of biologically active molecules including Wnt pathway components (e.g., Wnt2, DKK, BCL9 proteins), and non-Wnt pathway factors (e.g., chemoattractant cytokines, enzymatic proteins, and bioactive compounds of plants). Several lines of evidence argue for the use of angiogenesis inhibition in the treatment of CRC. In the context of this paper, components of the Wnt pathway are among the most promising targets for CRC therapy. This review summarizes the current knowledge about the role of the Wnt/Fzd/β-catenin signaling pathway in the process of CRC angiogenesis, aiming to improve the understanding of the mechanisms of metastasis as well as improvements in the management of this cancer.