Probes for INS

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.

Abstract

HPV-related and HPV-unrelated oropharyngeal squamous cell carcinomas are two distinct entities according to the Union for International Cancer Control, with a better prognosis conferred to HPV-related oropharyngeal squamous cell carcinomas. However, variable clinical outcomes are observed among patients with p16 positive oropharyngeal squamous cell carcinoma, which is a surrogate marker of HPV infection. We aimed to investigate the prognostic value of RNA CISH against E6 and E7 transcripts (HPV RNA CISH) to predict such variability. We retrospectively included 50 histologically confirmed p16 positive oropharyngeal squamous cell carcinomas (p16 positive immunostaining was defined by a strong staining in 70% or more of tumor cells). HPV RNA CISH staining was assessed semi-quantitatively to define two scores: RNA CISH “low” and RNA CISH “high”. Negative HPV RNA CISH cases were scored as RNA CISH “low”. This series contained 29 RNA CISH low cases (58%) and 21 RNA CISH high cases (42%). Clinical and pathologic baseline characteristics were similar between the two groups. RNA CISH high staining was associated with a better overall survival in both univariate and multivariate analyses (p = 0.033 and p = 0.042, respectively). Other recorded parameters had no prognostic value. In conclusion, HPV RNA CISH might be an independent prognostic marker in p16 positive oropharyngeal squamous cell carcinomas and might help guide therapeutics.

Abstract

Background

Accurate identification of HPV-driven oropharyngeal cancer (OPC) is a major issue and none of the current diagnostic approaches is ideal. An in situ hybridization (ISH) assay that detects high-risk HPV E6/E7 mRNA, called the RNAscope HPV-test, has been recently developed. Studies have suggested that this assay may become a standard to define HPV-status.

Methods

To further assess this test, we compared its performance against the strategies that are used in routine clinical practice: p16 immunohistochemistry (IHC) as a single test and algorithms combining p16-IHC with HPV-DNA identification by PCR (algorithm-1) or ISH (algorithm-2).

Results

105 OPC specimens were analyzed. The prevalence of HPV-positive samples varied considerably: 67% for p16-IHC, 54% for algorithm-1, 61% for algorithm-2 and 59% for the RNAscope HPV-test. Discrepancies between the RNAscope HPV-test and p16-IHC, algorithm-1 and 2 were noted in respectively 13.3%, 13.1%, and 8.6%.

The 4 diagnostic strategies were able to identify 2 groups with different prognosis according to HPV-status, as expected. However, the greater survival differential was observed with the RNAscope HPV-test [HR: 0.19, 95% confidence interval (CI), 0.07–0.51, p = 0.001] closely followed by algorithm-1 (HR: 0.23, 95% CI, 0.08–0.66, p = 0.006) and algorithm-2 (HR: 0.26, 95% CI, 0.1–0.65, p = 0.004). In contrast, a weaker association was found when p16-IHC was used as a single test (HR: 0.33, 95% CI, 0.13–0.81, p = 0.02).

Conclusions

Our findings suggest that the RNAscope HPV-test and p16-based algorithms perform better that p16 alone to identify OPC that are truly driven by HPV-infection. The RNAscope HPV-test has the advantage of being a single test.

Abstract

BACKGROUND:

There is increasing evidence that high-risk human papillomavirus plays significant role in oropharyngeal cancer; however, there is lack of knowledge on the interplay between the virus and its downstream related molecules and their possible prognostic values. The objectives of the study are to better understand the interplay of the HR-HPV and its associated downstream molecules and to evaluate potential biomarkers for patient outcomes.

METHODS:

We conducted a retrospective study with available formalin-fixed, paraffin-embedded tissue from 244 oropharyngeal cancer patients that received curative radiotherapy or concurrent chemoradiotherapy from 2000 to 2008. In addition to chart review, we performed HPV DNA and RNA in situ hybridization and immunohistochemistry for p53, the retinoblastoma protein, p16, and cyclin D1 analysis. Cox-proportional hazard and Kaplan-Meier survival analysis were used to determine the prognostic markers for clinical outcomes.

RESULTS:

Patients averaged 57.3±9.4 year-old and were mostly males (76.2%) and ever-smokers (76.2%). All patients received curative radiotherapy and 44.3% received concurrent chemoradiotherapy. We detected the human papillomavirus in 77.9% of study patients. Ever-smokers, more advanced tumor stage, and receiving radiotherapy only had poorer 5-year overall survival, disease-specific survival, and loco-regional recurrence. Cases with positive human papillomavirus and p53 overexpression had poorer disease-specific survival. Cases without human papillomavirus, but cyclin D1 overexpression, was associated with poorer 5-year overall survival.

CONCLUSIONS:

Our data suggests that additional p53 and cyclin D1 testing may benefit oropharyngeal cancer patients with known human papillomavirus status.

The aim of this study is comparing two in situ hybridization (ISH) detection methods for human papilloma virus (HPV) 16 E6/E7 mRNA, i.e. the RNAscope™ 2.0 High Definition (HD) and the upgraded RNAscope™ 2.5 HD version. The RNAscope™ 2.5 HD has recently replaced the RNAscope™ 2.0 HD detection kit. Therefore, this investigation starts from the need to analytically validate the new mRNA ISH assay and, possibly, to refine the current algorithm for HPV detection in oropharyngeal squamous cell carcinoma (OSCC) with the final goal to apply it to daily laboratory practice. The study was based on HPV status and on generated data, interpreted by a scoring algorithm. The results highlighted that the compared RNAscope HPV tests had a good level of interchangeability and enabled to identify OSCC that are truly driven by high risk-HPV infection. This was also supported by the comparison of the RNAscope HPV test with HPV E6/E7 mRNA real time reverse transcriptase-polymerase chain reaction (RT-PCR), in a fraction of cases where material for HPV E6/E7 mRNA real time RT-PCR was available. Furthermore, the algorithm that associates p16 immunohistochemistry (IHC) with the identification of HPV mRNA by RNAscope was more effective than the one that associated p16 IHC with the identification of HPV DNA by ISH.

The heterogeneity of cellular states in cancer has been linked to drug resistance, cancer progression and the presence of cancer cells with properties of normal tissue stem cells. Secreted Wnt signals maintain stem cells in various epithelial tissues, including in lung development and regeneration. Here we show that mouse and human lung adenocarcinomas display hierarchical features with two distinct subpopulations, one with high Wnt signalling activity and another forming a niche that provides the Wnt ligand. The Wnt responder cells showed increased tumour propagation ability, suggesting that these cells have features of normal tissue stem cells. Genetic perturbation of Wnt production or signalling suppressed tumour progression. Small-molecule inhibitors targeting essential posttranslational modification of Wnt reduced tumour growth and markedly decreased the proliferative potential of lung cancer cells, leading to improved survival of tumour-bearing mice. These results indicate that strategies for disrupting pathways that maintain stem-like and niche cell phenotypes can translate into effective anti-cancer therapies.

Chronic itch is a highly debilitating condition affecting about 10% of the general population. The relay of itch signals is under tight control by inhibitory circuits of the spinal dorsal horn, which may offer a hitherto unexploited therapeutic opportunity. Here, we found that specific pharmacological targeting of inhibitory α2 and α3GABAA receptors reduces acute histaminergic and non-histaminergic itch in mice. Systemic treatment with an α2/α3GABAA receptor selective modulator alleviates also chronic itch in a mouse model of atopic dermatitis and in dogs sensitized to house dust mites, without inducing sedation, motor dysfunction, or loss of antipruritic activity after prolonged treatment. Transsynaptic circuit tracing, immunofluorescence, and electrophysiological experiments identify spinal α2 and α3GABAA receptors as likely molecular targets underlying the antipruritic effect. Our results indicate that drugs targeting α2 and α3GABAA receptors are well-suited to alleviate itch, including non-histaminergic chronic itch for which currently no approved treatment exists.

Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/β-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike β-catenin gain-of-function models, which induce high Wnt/β-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/β-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing β-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/β-catenin activation, which is regulated by ZNRF3.