Probes for INS

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4+ hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.

Increased testing for human papillomavirus (HPV) in oropharyngeal carcinomas has broadened the range of HPV-associated malignancies identified at this site. While HPV-related oropharyngeal non-keratinizing squamous cell carcinomas (SCC) are known to have a better prognosis than their non-HPV counterparts, HPV positivity may not alter the aggressive nature of HPV-associated small cell neuroendocrine carcinomas (SCNEC). We report a unique case of a mixed non-keratinizing type HPV-associated tonsillar SCC with SCNEC differentiation, and provide a comparison with the rare reported cases of such mixed carcinomas in the literature. Our patient is only the second such case positive for HPV genotype 18 and the only case in which this HPV-related mixed tonsillar tumor occurred in a patient with small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL). The case discussion supports the concept that HPV positivity does not confer a better prognosis in such mixed non-keratinizing type SCC with SCNEC. Our report also alerts pathologists to the need to evaluate for the possibility of a coexisting neuroendocrine component when oropharyngeal squamous cell carcinoma (OPSCC) is diagnosed, as its presence will affect the patients’ clinical management and prognosis

Tracheobronchomalacia (TBM) is a common congenital disorder in which the cartilaginous rings of the trachea are weakened or missing. Despite the high prevalence and clinical issues associated with TBM, the etiology is largely unknown. Our previous studies demonstrated that Wntless (Wls) and its associated Wnt pathways are critical for patterning of the upper airways. Deletion of Wls in respiratory endoderm caused TBM and ectopic trachealis muscle. To understand mechanisms by which Wls mediates tracheal patterning, we performed RNA sequencing in prechondrogenic tracheal tissue of Wlsf/f;ShhCre/wt embryos. Chondrogenic Bmp4, and Sox9 were decreased, while expression of myogenic genes was increased. We identified Notum, a deacylase that inactivates Wnt ligands, as a target of Wls induced Wnt signaling. Notum's mesenchymal ventral expression in prechondrogenic trachea overlaps with expression of Axin2, a Wnt/β-catenin target and inhibitor. Notum is induced by Wnt/β-catenin in developing trachea. Deletion of Notum activated mesenchymal Wnt/β-catenin and caused tracheal mispatternning of trachealis muscle and cartilage as well as tracheal stenosis. Notum is required for tracheal morphogenesis, influencing mesenchymal condensations critical for patterning of tracheal cartilage and muscle. We propose that Notum influences mesenchymal cell differentiation by generating a barrier for Wnt ligands produced and secreted by airway epithelial cells to attenuate Wnt signaling.

Human papillomavirus (HPV)-driven cutaneous squamous cell carcinoma (cSCC) is the most common cancer in immunosuppressed patients. Despite indications suggesting that HPV promotes genomic instability during cSCC development, the molecular pathways underpinning HPV-driven cSCC development remain unknown. We compared the transcriptome of HPV-driven mouse cSCC with normal skin and observed higher amounts of transcripts for Porcupine and WNT ligands in cSCC, suggesting a role for WNT signaling in cSCC progression. We confirmed increased Porcupine expression in human cSCC samples. Blocking the secretion of WNT ligands by the Porcupine inhibitor LGK974 significantly diminished initiation and progression of HPV-driven cSCC. Administration of LGK974 to mice with established cSCC resulted in differentiation of cancer cells and significant reduction of the cancer stem cell compartment. Thus, WNT/β-catenin signaling is essential for HPV-driven cSCC initiation and progression as well as for maintaining the cancer stem cell niche. Interference with WNT secretion may thus represent a promising approach for therapeutic intervention.

Basal cell carcinoma (BCC) is the most frequent cancer in humans and results from constitutive activation of the Hedgehog pathway1. Several Smoothened inhibitors are used to treat Hedgehog-mediated malignancies, including BCC and medulloblastoma2. Vismodegib, a Smoothened inhibitor, leads to BCC shrinkage in the majority of patients with BCC3, but the mechanism by which it mediates BCC regression is unknown. Here we used two genetically engineered mouse models of BCC4 to investigate the mechanisms by which inhibition of Smoothened mediates tumour regression. We found that vismodegib mediates BCC regression by inhibiting a hair follicle-like fate and promoting the differentiation of tumour cells. However, a small population of tumour cells persists and is responsible for tumour relapse following treatment discontinuation, mimicking the situation found in humans5. In both mouse and human BCC, this persisting, slow-cycling tumour population expresses LGR5 and is characterized by active Wnt signalling. Combining Lgr5 lineage ablation or inhibition of Wnt signalling with vismodegib treatment leads to eradication of BCC. Our results show that vismodegib induces tumour regression by promoting tumour differentiation, and demonstrates that the synergy between Wnt and Smoothened inhibitors is a clinically relevant strategy for overcoming tumour relapse in BCC.

Cell-autonomous Wnt signaling has well-characterized functions in controlling stem cell activity, including in the prostate. While niche cells secrete Wnt ligands, the effects of Wnt signaling in niche cells per se are less understood. Here, we show that stromal cells in the proximal prostatic duct near the urethra, a mouse prostate stem cell niche, not only produce multiple Wnt ligands but also exhibit strong Wnt/β-catenin activity. The non-canonical Wnt ligand Wnt5a, secreted by proximal stromal cells, directly inhibits proliefration of prostate epithelial stem or progenitor cells whereas stromal cell-autonomous canonical Wnt/β-catenin signaling indirectly suppresses prostate stem or progenitor activity via the transforming growth factor β (TGFβ) pathway. Collectively, these pathways restrain the proliferative potential of epithelial cells in the proximal prostatic ducts. Human prostate likewise exhibits spatially restricted distribution of stromal Wnt/β-catenin activity, suggesting a conserved mechanism for tissue patterning. Thus, this study shows how distinct stromal signaling mechanisms within the prostate cooperate to regulate tissue homeostasis.

Dysregulated expression of oncogenic types of E6 and E7 is necessary for human papillomavirus (HPV)-driven carcinogenesis. An HPV E6/E7 mRNA in situ hybridization (ISH) assay covering 18 common high-risk types ("HR-RISH," aka HR-HPV RNA18 ISH) has not been extensively studied in the anogenital tract or validated on automated technology. We herein compare HR-RISH to DNA polymerase chain reaction (PCR), p16 immunohistochemistry, and a previously available HPV DNA ISH assay in HPV-related anogenital and head and neck (H&N) neoplasia. A total of 102 squamous intraepithelial lesions (16 CIN1, 25 CIN3, 3 AIN1, 12 AIN3, 9 VIN3)/invasive squamous cell carcinomas (17 cervical, 2 anal, 18 H&N) as well as 10 normal and 15 reactive cervix samples were collected. HR-RISH, DNA ISH, and p16 immunohistochemistry were performed on whole formalin-fixed, paraffin-embedded sections. RNA ISH for 6 low-risk HPV types (LR-RISH) was also performed. RNA and DNA ISH assays used automated systems. HR-HPV PCR was performed on morphology-directed formalin-fixed, paraffin-embedded punches. HR-RISH was ≥97% sensitive for PCR+ and p16+ neoplasia, as well as morphologically defined anogenital high grade squamous intraepithelial lesion/invasive squamous cell carcinoma. HR-RISH was also positive in 78% of anogenital low grade squamous intraepithelial lesion, including 81% of CIN1. Furthermore, a subset of PCR-negative/invalid and p16-negative lesions was positive for HR-RISH. Only 1 problematic reactive cervix sample and no normal cervix samples stained. These results demonstrate that HR-RISH is a robust method for the detection of HR-HPV-related neoplasia and provides insight into HPV pathobiology. Performance meets or exceeds that of existing assays in anogenital and H&N lesions and may play a role in resolving diagnostically challenging CIN1 versus reactive cases.

Cervical low-grade squamous intraepithelial lesions (LSIL) (aka cervical intraepithelial neoplasia, grade 1 [CIN1]) can present considerable diagnostic challenges and are associated with poor interobserver reproducibility and overdiagnosis. Furthermore, ancillary studies such as p16 immunohistochemistry have shown little utility in resolving the LSIL versus negative/reactive differential. Human papillomavirus (HPV) RNA in situ hybridization (ISH) has shown promise as a diagnostic aid in this setting, but has not been studied in a large case series. We herein investigate high-risk and low-risk HPV RNA ISH in 126 cervical biopsies originally diagnosed as LSIL/CIN1 and compare HPV RNA ISH results to expert-adjudicated morphologic diagnosis to assess whether this assay can help routine cases attain the existing "gold standard" of morphologic consensus diagnosis. We also assess whether this criterion standard can be further improved by integration of HPV RNA ISH results. A consensus diagnosis of intraepithelial lesion (CIN1) was confirmed in 61% of cases, whereas 57% were HPV RNA. HPV-RNA positivity was 84% sensitive and 86% specific for an expert-adjudicated diagnosis of CIN1. Conversely, consensus diagnosis was 90% sensitive and 78% specific for the presence of HPV RNA. Integrating RNA ISH into morphologic review led to further reclassification of 10% of cases, resulting in 95% sensitivity and 98% specificity of HPV RNA ISH for a CIN1 diagnosis and 98% sensitivity and 92% specificity of CIN1 for the presence of HPV RNA. These findings suggest that judicious use of HPV RNA ISH can improve the accuracy of LSIL/CIN1 diagnosis for morphologically ambiguous cases.