Interaction of the tumor suppressor SMAD4 and WNT signaling in progression to oral squamous cell carcinoma.

SMAD4 is a tumor suppressor mutated or silenced in multiple cancers, including oral cavity squamous cell carcinoma (OSCC). Human clinical samples and cell lines, mouse models and organoid culture were used to investigate the role that SMAD4 plays in progression from benign disease to invasive OSCC. Human OSCC lost detectable SMAD4 protein within tumor epithelium in 24% of cases, and this loss correlated with worse progression-free survival independent of other major clinical and pathological features. A mouse model engineered for KrasG12D expression in the adult oral epithelium induced benign papillomas, however the combination of KrasG12D with loss of epithelial Smad4 expression resulted in rapid development of invasive carcinoma with features of human OSCC. Examination of regulatory pathways in 3D organoid cultures of SMAD4+ and SMAD4- mouse tumors with Kras mutation found that either loss of SMAD4 or inhibition of TGFß signaling upregulated the WNT pathway and altered the extracellular matrix. The gene signature of the mouse tumor organoids lacking SMAD4 was highly similar to the gene signature of human head and neck squamous cell carcinoma. In summary, this work has uncovered novel mechanisms by which SMAD4 acts as a tumor suppressor in OSCC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Wnt/ß-catenin signaling is a therapeutic target in anaplastic thyroid carcinoma.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy that has consistently shown Wnt/ß-catenin (canonical) signaling activation in various study populations. There are currently no targetable treatments for BRAF-wildtype ATC and a lack of effective treatment for BRAFV600EATC. Our aim is to identify whether Wnt inhibitors could be potential therapeutic agents for ATC patients with limited treatment options. METHODS: In this Institutional Review Board-approved study, we utilize a cohort of 32 ATCs and 20 non-neoplastic multinodular goiters (MNG). We also use 4 ATC spheroid cell lines (THJ-16T, THJ-21T, THJ-29T, and THJ-11T) and two primary patient-derived ATC organoid cultures (VWL-T5 and VWL-T60). Finally, we use a murine xenograft mouse model of ATC for in vivo treatment studies. RESULTS: Using a large patient cohort, we demonstrate that this near-universal Wnt signaling activation is associated with ligand expression- rather than being mutationally-driven. We show that pyrvinium pamoate, a potent Wnt inhibitor, exhibits in vitro efficacy against both ATC cell lines and primary patient-derived ATC organoids VWL-T5 (p < 0.05) and VWL-T60 (p < 0.01) Finally, using a murine xenograft model of ATC, we show that pyrvinium significantly delays the growth of ATC tumors in THJ-16T (p < 0.005) and THJ-21T (p < 0.001). CONCLUSIONS: We tested Wnt inhibitor treatment, both in vitro and in vivo, as a potential novel therapy for this highly lethal disease. Future large-scale studies utilizing multiple Wnt inhibitors will lay the foundation for the development of these novel therapies for patients with ATC.

Case Report of a Child with Colocolic Intussusception with a Primary Lead Point.

Intussusception is the telescoping of bowel into an adjacent segment of bowel and has an associated risk for bowel ischemia and perforation. The classic triad of abdominal pain, blood in stool, and an abdominal mass is present in less than 40% of pediatric cases and is less common in older children.1 Ultrasound has a high sensitivity and specificity for the diagnosis of intussusception, and once diagnosed, treatment modalities include reduction by either ultrasound or fluoroscopic guided air or hydrostatic enema. The risk of recurrence after successful reduction occurs in up to 12% of pediatric patients and occurs more frequently in older children and children with a pathologic lead point.2 We present a case of a 6-year-old child with colocolic intussusception that was successfully reduced and recurred within five days due to a large colonic polyp. Topics: Intussusception, lead point, pediatrics.

Molecular signature incorporating the immune microenvironment enhances thyroid cancer outcome prediction.

Genomic and transcriptomic analysis has furthered our understanding of many tumors. Yet, thyroid cancer management is largely guided by staging and histology, with few molecular prognostic and treatment biomarkers. Here, we utilize a large cohort of 251 patients with 312 samples from two tertiary medical centers and perform DNA/RNA sequencing, spatial transcriptomics, and multiplex immunofluorescence to identify biomarkers of aggressive thyroid malignancy. We identify high-risk mutations and discover a unique molecular signature of aggressive disease, the Molecular Aggression and Prediction (MAP) score, which provides improved prognostication over high-risk mutations alone. The MAP score is enriched for genes involved in epithelial de-differentiation, cellular division, and the tumor microenvironment. The MAP score also identifies aggressive tumors with lymphocyte-rich stroma that may benefit from immunotherapy. Future clinical profiling of the stromal microenvironment of thyroid cancer could improve prognostication, inform immunotherapy, and support development of novel therapeutics for thyroid cancer and other stroma-rich tumors.

The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6.

The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. We demonstrate that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/ß-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient.

Experimental Insights into the Interplay between Histone Modifiers and p53 in Regulating Gene Expression.

Chromatin structure plays a fundamental role in regulating gene expression, with histone modifiers shaping the structure of chromatin by adding or removing chemical changes to histone proteins. The p53 transcription factor controls gene expression, binds target genes, and regulates their activity. While p53 has been extensively studied in cancer research, specifically in relation to fundamental cellular processes, including gene transcription, apoptosis, and cell cycle progression, its association with histone modifiers has received limited attention. This review explores the interplay between histone modifiers and p53 in regulating gene expression. We discuss how histone modifications can influence how p53 binds to target genes and how this interplay can be disrupted in cancer cells. This review provides insights into the complex mechanisms underlying gene regulation and their implications for potential cancer therapy.

SELENOP modifies sporadic colorectal carcinogenesis and WNT signaling activity through LRP5/6 interactions.

Although selenium deficiency correlates with colorectal cancer (CRC) risk, the roles of the selenium-rich antioxidant selenoprotein P (SELENOP) in CRC remain unclear. In this study, we defined SELENOP's contributions to sporadic CRC. In human single-cell cRNA-Seq (scRNA-Seq) data sets, we discovered that SELENOP expression rose as normal colon stem cells transformed into adenomas that progressed into carcinomas. We next examined the effects of Selenop KO in a mouse adenoma model that involved conditional, intestinal epithelium-specific deletion of the tumor suppressor adenomatous polyposis coli (Apc) and found that Selenop KO decreased colon tumor incidence and size. We mechanistically interrogated SELENOP-driven phenotypes in tumor organoids as well as in CRC and noncancer cell lines. Selenop-KO tumor organoids demonstrated defects in organoid formation and decreases in WNT target gene expression, which could be reversed by SELENOP restoration. Moreover, SELENOP increased canonical WNT signaling activity in noncancer and CRC cell lines. In defining the mechanism of action of SELENOP, we mapped protein-protein interactions between SELENOP and the WNT coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6). Last, we confirmed that SELENOP-LRP5/6 interactions contributed to the effects of SELENOP on WNT activity. Overall, our results position SELENOP as a modulator of the WNT signaling pathway in sporadic CRC.

Efficacy and safety of tirzepatide for treatment of overweight or obesity. A systematic review and meta-analysis.

BACKGROUND: Recent studies suggest that tirzepatide, a dual glucose-dependent insulinotropic-peptide (GIP) and glucagon-like peptide-1 receptor agonist (GLP-1 RA), has significant weight loss effects. This systematic review and meta-analysis aims to assess the efficacy and safety of tirzepatide for weight loss in patients with overweight or obesity. METHODS: Medline, Embase and Cochrane CENTRAL were searched for randomized controlled trials (RCTs) on tirzepatide's weight loss efficacy for these patients. A single arm meta-analysis of proportions estimated primary outcomes, ≥5%, ≥10%, and ≥15% weight loss, and adverse events (AEs); while meta-analysis of means estimated secondary outcomes. Comparative meta-analysis was conducted between tirzepatide and control arms where mean differences and odds ratios were estimated for continuous and dichotomous outcomes respectively. RESULTS: RCTs included in this study revealed that among 5800 patients, 78.22% (95% CI: 72.15% to 83.73%), 55.60% (95% CI: 46.54% to 64.47%), 32.28% (95% CI: 23.17% to 42.12%) achieved ≥5%, ≥10%, and ≥15% weight loss, respectively. Tirzepatide 5 mg demonstrated weight loss superiority relative to placebo (MD: -12.47 kg, 95% CI: -13.94 kg to -11.00 kg) and semaglutide (n = 1409, MD: -1.90 kg, 95% CI: -2.97 kg to -0.83 kg) with dose-dependent increase for 10 mg and 15 mg doses. The comparison between tirzepatide and semaglutide was examined in the SURPASS-2 trial that was included in this systematic review. For AEs, there was increase odds of experiencing gastrointestinal AEs with tirzepatide compared to placebo, but no significant difference with semaglutide. CONCLUSION: Tirzepatide has significant potential as a weight loss drug in patients with overweight and obesity, with little increase in AEs compared to other weight loss drugs. With its ability to concurrently target multiple aspects of metabolic syndrome, it should be considered as the next helm of weight loss therapies.

Wnt Signaling in the Phenotype and Function of Tumor-Associated Macrophages.

Tumor-associated macrophages (TAM) play an important role in supporting tumor growth and suppressing antitumor immune responses, and TAM infiltration has been associated with poor patient prognosis in various cancers. TAMs can be classified as pro-inflammatory, M1-like, or anti-inflammatory, M2-like. While multiple factors within the tumor microenvironment affect the recruitment, polarization, and functions of TAMs, accumulating evidence suggests that Wnt signaling represents an important, targetable driver of an immunosuppressive, M2-like TAM phenotype. TAM production of Wnt ligands mediates TAM-tumor cross-talk to support cancer cell proliferation, invasion, and metastasis. Targeting TAM polarization and the protumorigenic functions of TAMs through inhibitors of Wnt signaling may prove a beneficial treatment strategy in cancers where macrophages are prevalent in the microenvironment.

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon.

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN-CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.

Utilizing Three-Dimensional Culture Methods to Improve High-Throughput Drug Screening in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) is the most aggressive endocrine neoplasm, with a median survival of just four to six months post-diagnosis. Even with surgical and chemotherapeutic interventions, the five-year survival rate is less than 5%. Although combination dabrafenib/trametinib therapy was recently approved for treatment of the ~25% of ATCs harboring BRAFV600E mutations, there are no approved, effective treatments for BRAF-wildtype disease. Herein, we perform a screen of 1525 drugs and evaluate therapeutic candidates using monolayer cell lines and four corresponding spheroid models of anaplastic thyroid carcinoma. We utilize three-dimensional culture methods, as they have been shown to more accurately recapitulate tumor responses in vivo. These three-dimensional cultures include four distinct ATC spheroid lines representing unique morphology and mutational drivers to provide drug prioritization that will be more readily translatable to the clinic. Using this screen, we identify three exceptionally potent compounds (bortezomib, cabazitaxel, and YM155) that have established safety profiles and could potentially be moved into clinical trial for the treatment of anaplastic thyroid carcinoma, a disease with few treatment options.

DDR1 contributes to kidney inflammation and fibrosis by promoting the phosphorylation of BCR and STAT3.

Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen, contributes to chronic kidney disease. However, its role in acute kidney injury and subsequent development of kidney fibrosis is not clear. Thus, we performed a model of severe ischemia/reperfusion-induced acute kidney injury that progressed to kidney fibrosis in WT and Ddr1-null mice. We showed that Ddr1-null mice had reduced acute tubular injury, inflammation, and tubulointerstitial fibrosis with overall decreased renal monocyte chemoattractant protein (MCP-1) levels and STAT3 activation. We identified breakpoint cluster region (BCR) protein as a phosphorylated target of DDR1 that controls MCP-1 production in renal proximal tubule epithelial cells. DDR1-induced BCR phosphorylation or BCR downregulation increased MCP-1 secretion, suggesting that BCR negatively regulates the levels of MCP-1. Mechanistically, phosphorylation or downregulation of BCR increased ß-catenin activity and in turn MCP-1 production. Finally, we showed that DDR1-mediated STAT3 activation was required to stimulate the secretion of TGF-ß. Thus, DDR1 contributes to acute and chronic kidney injury by regulating BCR and STAT3 phosphorylation and in turn the production of MCP-1 and TGF-ß. These findings identify DDR1 an attractive therapeutic target for ameliorating both proinflammatory and profibrotic signaling in kidney disease.

The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling.

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.

Immunofluorescent staining of cancer spheroids and fine-needle aspiration-derived organoids.

Our organoid generation technique has allowed for the development of downstream organoid applications. Here, we detail an accessible, straightforward protocol for immunofluorescent staining and imaging of thyroid cancer organoids, particularly those with tumor de-differentiation. Immunofluorescence is a powerful tool to help understand the localization of cell types within organoids and determine the interactions between those cells. As organoids have been shown to recapitulate patient tumor morphology, immunofluorescent staining and imaging of organoids allows for enhanced understanding of near in vivo structures. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020) and Vilgelm et al. (2020).

Obtaining patient-derived cancer organoid cultures via fine-needle aspiration.

Patient-derived tumor organoid cultures are an essential and innovative methodology for translational research. However, current techniques to establish these cultures are cumbersome, expensive, and often require irreplaceable clinical tissue from surgery or core biopsies. Fine-needle aspiration (FNA) provides a minimally invasive biopsy technique commonly performed in clinical settings. Here, we provide a protocol for FNA. We have found that FNA provides a cost-effective, rapid, and streamlined method for tissue acquisition for cancer organoid culture. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020) and Vilgelm et al. (2020).

Chemokine Receptor 2-targeted Molecular Imaging in Pulmonary Fibrosis. A Clinical Trial.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive inflammatory lung disease without effective molecular markers of disease activity or treatment responses. Monocyte and interstitial macrophages that express the C-C motif CCR2 (chemokine receptor 2) are active in IPF and central to fibrosis.Objectives: To phenotype patients with IPF for potential targeted therapy, we developed 64Cu-DOTA-ECL1i, a radiotracer to noninvasively track CCR2+ monocytes and macrophages using positron emission tomography (PET).Methods: CCR2+ cells were investigated in mice with bleomycin- or radiation-induced fibrosis and in human subjects with IPF. The CCR2+ cell populations were localized relative to fibrotic regions in lung tissue and characterized using immunolocalization, single-cell mass cytometry, and Ccr2 RNA in situ hybridization and then correlated with parallel quantitation of lung uptake by 64Cu-DOTA-ECL1i PET.Measurements and Main Results: Mouse models established that increased 64Cu-DOTA-ECL1i PET uptake in the lung correlates with CCR2+ cell infiltration associated with fibrosis (n = 72). As therapeutic models, the inhibition of fibrosis by IL-1ß blockade (n = 19) or antifibrotic pirfenidone (n = 18) reduced CCR2+ macrophage accumulation and uptake of the radiotracer in mouse lungs. In lung tissues from patients with IPF, CCR2+ cells concentrated in perifibrotic regions and correlated with radiotracer localization (n = 21). Human imaging revealed little lung uptake in healthy volunteers (n = 7), whereas subjects with IPF (n = 4) exhibited intensive signals in fibrotic zones.Conclusions: These findings support a role for imaging CCR2+ cells within the fibrogenic niche in IPF to provide a molecular target for personalized therapy and monitoring.Clinical trial registered with www.clinicaltrials.gov (NCT03492762).

High-throughput drug screening of fine-needle aspiration-derived cancer organoids.

Generation of fine-needle aspiration (FNA)-derived cancer organoids has allowed us to develop a number of downstream applications. In this protocol, we start with organoids cultured in a semi-solid format. We dissociate organoids into single cells and then plate in a 384-well format for high-throughput drug screening. While this method must be fine-tuned for each individual organoid culture, it offers a format well suited for rapidly screening medium-sized drug/compound libraries (500-5,000 molecules) and generating dose-response curves to measure relative efficacy. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020) and Vilgelm et al. (2020).

Fine-Needle Aspiration-Based Patient-Derived Cancer Organoids.

Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications.

Casein Kinase 1α as a Regulator of Wnt-Driven Cancer.

Wnt signaling regulates numerous cellular processes during embryonic development and adult tissue homeostasis. Underscoring this physiological importance, deregulation of the Wnt signaling pathway is associated with many disease states, including cancer. Here, we review pivotal regulatory events in the Wnt signaling pathway that drive cancer growth. We then discuss the roles of the established negative Wnt regulator, casein kinase 1α (CK1α), in Wnt signaling. Although the study of CK1α has been ongoing for several decades, the bulk of such research has focused on how it phosphorylates and regulates its various substrates. We focus here on what is known about the mechanisms controlling CK1α, including its putative regulatory proteins and alternative splicing variants. Finally, we describe the discovery and validation of a family of pharmacological CK1α activators capable of inhibiting Wnt pathway activity. One of the important advantages of CK1α activators, relative to other classes of Wnt inhibitors, is their reduced on-target toxicity, overcoming one of the major impediments to developing a clinically relevant Wnt inhibitor. Therefore, we also discuss mechanisms that regulate CK1α steady-state homeostasis, which may contribute to the deregulation of Wnt pathway activity in cancer and underlie the enhanced therapeutic index of CK1α activators.

Tubular ß-catenin and FoxO3 interactions protect in chronic kidney disease.

The Wnt/ß-catenin signaling pathway plays an important role in renal development and is reexpressed in the injured kidney and other organs. ß-Catenin signaling is protective in acute kidney injury (AKI) through actions on the proximal tubule, but the current dogma is that Wnt/ß-catenin signaling promotes fibrosis and development of chronic kidney disease (CKD). As the role of proximal tubular ß-catenin signaling in CKD remains unclear, we genetically stabilized (i.e., activated) ß-catenin specifically in murine proximal tubules. Mice with increased tubular ß-catenin signaling were protected in 2 murine models of AKI to CKD progression. Oxidative stress, a common feature of CKD, reduced the conventional T cell factor/lymphoid enhancer factor-dependent ß-catenin signaling and augmented FoxO3-dependent activity in proximal tubule cells in vitro and in vivo. The protective effect of proximal tubular ß-catenin in renal injury required the presence of FoxO3 in vivo. Furthermore, we identified cystathionine γ-lyase as a potentially novel transcriptional target of ß-catenin/FoxO3 interactions in the proximal tubule. Thus, our studies overturned the conventional dogma about ß-catenin signaling and CKD by showing a protective effect of proximal tubule ß-catenin in CKD and identified a potentially new transcriptional target of ß-catenin/FoxO3 signaling that has therapeutic potential for CKD.