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The immunosuppressive microenvironment in pancreatic ductal adenocarcinoma (PDAC) prevents tumor control and strategies to restore anti-cancer immunity (i.e. by increasing CD8 T-cell activity) have had limited success. Here, we demonstrate how inducing localized physical damage using ionizing radiation (IR) unmasks the benefit of immunotherapy by increasing tissue-resident natural killer (trNK) cells that support CD8 T activity. Our data confirms that targeting mouse orthotopic PDAC tumors with IR together with CCR5 inhibition and PD1 blockade reduces E-cadherin positive tumor cells by recruiting a hypoactive NKG2D -ve NK population, phenotypically reminiscent of trNK cells, that supports CD8 T-cell involvement. We show an equivalent population in human single-cell RNA sequencing (scRNA-seq) PDAC cohorts that represents immunomodulatory trNK cells that could similarly support CD8 T-cell levels in a cDC1-dependent manner. Importantly, a trNK signature associates with survival in PDAC and other solid malignancies revealing a potential beneficial role for trNK in improving adaptive anti-tumor responses and supporting CCR5 inhibitor (CCR5i)/αPD1 and IR-induced damage as a novel therapeutic approach., Competing Interests: SG, CD, GV, SH, SL, HF, EA, SS, RB, MM, SM, JM, KJ, EN No competing interests declared, (© 2024, Go, Demetriou, Valenzano et al.)

Background: RASopathies, which include neurofibromatosis type 1 (NF1), are defined by Ras/mitogen-activated protein kinase (Ras/MAPK) pathway activation. They represent a group of clinically related disorders often characterised by multiple Café au Lait Macules (CALMs)., Objectives: To determine, using in depth transcriptomic analysis of NF1 melanocytes from CALM and unaffected skin, (1) the gene(s) responsible for melanocyte proliferation and migration, and (2) the activated signalling pathway(s) in NF1 melanoma., Methods: Classical NF1 ( n  = 2, who develop tumours) and 3bp deletion NF1 (p. Met992del, who do not develop tumours) ( n  = 3) patients underwent skin biopsies from CALM and unaffected skin. Melanocytes were isolated and propagated, with five replicates from each tissue sample. DNA and RNA were extracted for mutational analysis and transcriptomic profiling with six replicates per sample. Mechanistic determination was undertaken using melanocyte and melanoma cell lines., Results: All CALMs in NF1 were associated with biallelic NF1 loss, resulting in amplification of Ras/MAPK and Wnt pathway signalling. CALMs were also associated with reduced SERPINF1 gene expression (and pigment epithelium-derived factor (PEDF) levels, the reciprocal protein), a known downstream target of the master regulator of melanocyte differentiation microphthalmia-associated transcription factor (MITF), leading to increased melanocyte proliferation, migration and invasion. In classical NF1 and melanoma, but not 3bp deletion NF1, there was also activation of the PI3K/AKT pathway. Pigment epithelium-derived factor was found to reduce cell proliferation and invasion of NF1 melanoma., Conclusions: Melanocyte proliferation and migration leading to CALMs in NF1 arises from biallelic NF1 loss, resulting in RAS/MAPK pathway activation, and reduced expression of the tumour suppressor PEDF. Activation of the PI3K/AKT pathway in classical NF1 and NF1 melanoma may facilitate tumour growth., Competing Interests: The authors state no conflict of interest., (© 2024 The Authors. Skin Health and Disease published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.)

The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor-initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells to suppress MAPK pathway signaling that in turn blocked indirect (IFN-γ release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ-regulated gene transcription and led to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL, which resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell-specific immune checkpoint, which is responsible for NK cell-associated poor outcomes in many cancers.

RASSF1A promoter methylation has been correlated with tumor dedifferentiation and aggressive oncogenic behavior. Nevertheless, the underlying mechanism of RASSF1A-dependent tumor dedifferentiation remains elusive. Here, we show that RASSF1A directly uncouples the NOTCH-HES1 axis, a key suppressor of differentiation. Interestingly, the crosstalk of RASSF1A with HES1 occurs independently from the signaling route connecting RASSF1A with the Hippo pathway. At the molecular level, we demonstrate that RASSF1A acts as a scaffold essential for the SUMO-targeted E3 ligase SNURF/RNF4 to target HES1 for degradation. The reciprocal relationship between RASSF1A and HES1 is evident across a wide range of human tumors, highlighting the clinical significance of the identified pathway. We show that HES1 upregulation in a RASSF1A-depleted environment renders cells non-responsive to the downstream effects of γ-secretase inhibitors (GSIs) which restrict signaling at the level of the NOTCH receptor. Taken together, we report a mechanism through which RASSF1A exerts autonomous regulation of the critical Notch effector HES1, thus classifying RASSF1A expression as an integral determinant of the clinical effectiveness of Notch inhibitors., (© 2021 The Authors.)

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by advanced disease stage at presentation, aggressive disease biology, and resistance to therapy, resulting in an extremely poor 5-year survival rate of <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression, but exactly why is unclear and is hindered by the technical limitations of analyzing clinical samples., Methods: We performed genome-wide epigenetic mapping of DNA modifications 5-methylcytosine and 5-hydroxymethylcytosine (5hmc) using oxidative bisulfite sequencing from formalin-embedded sections. We identified overlap with transcriptional signatures in formalin-fixed, paraffin-embedded tissue from resected patients, via bioinformatics using iCluster and mutational profiling and confirmed them in vivo., Results: We found that aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci, including GATA6, which promote differentiated classical pancreatic subtypes. We showed that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5-methylcytosine hydroxylase TET2. Furthermore, we found that SMAD4 directly supports TET2 levels in classical pancreatic tumors, and loss of SMAD4 expression was associated with reduced 5hmc, GATA6, and squamous-like tumors. Importantly, enhancing TET2 stability using metformin and vitamin C/ascorbic acid restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo., Conclusions: We identified epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data showed that restoring epigenetic control increases biomarkers of classical pancreatic tumors that are associated with improved therapeutic responses and survival., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Identification of human basal cell carcinoma (BCC) cancer stem cells and cellular hierarchy inherently implies the presence of differentiation. By conventional histological analysis, BCC demonstrates tumour nodules that appear relatively homogeneous., Aim: As BCCs arise from hair follicle (HF) keratinocytes, we sought to define the pattern of HF differentiation., Methods: BCC, squamous cell carcinoma (SCC) and normal skin tissues were analysed using a microarray chip. The expression of individual keratins, regulatory pathways and proliferative states were analysed using reverse transcription-PCR and immunofluorescence microscopy., Results: Microarray analysis of BCC, SCC and normal hair-bearing skin revealed that BCCs express a wide range of HF genes, including HF- specific keratins. BCC demonstrated outer (KRT5, KRT514, KRT516, KRT517 and KRT519) and inner (KRT25, KRT27, KRT28, KRT32, KRT35, KRT71, KRT75 and KRT85) root sheath differentiation, but not hair shaft differentiation. As in the HF, differentiation-specific keratins in BCC keratinocytes correlated with a reduced proliferative index and regulatory pathway activation despite the oncogenic drive towards tumour growth. Our findings show the close correlation between HF and BCC keratinocyte differentiation., Conclusion: This work has defined the differentiation pattern within BCCs, enabling development of targeted therapies that promote differentiation and result in BCC cancer stem cell exhaustion., (© 2019 British Association of Dermatologists.)

Background: Consistent with cancer stem cell driven pattern of growth, human basal cell carcinomas (BCCs) demonstrate differentiation along hair follicle (HF) lineages., Aim: To define the pattern of differentiation and therapeutic targets that promote BCC differentiation and therefore BCC cancer stem cell exhaustion., Methods: An alkaline phosphatase substrate kit was used to determine dermal papilla cells within the BCC stroma. Autonomous HF cycle-dependent gene expression was identified by analysis of the human homologues of a murine gene set (total 2289 genes) that is differentially expressed in hair cycle phases. The findings were validated by quantitative real-time PCR and immunofluorescence, as well as in vitro transforming growth factor (TGF)-β2 stimulation of BCC cancer stem cell colonies., Results: As in the HF, keratin expression in the inner root sheath and matrix in BCC correlated with proliferative index and was tightly regulated, despite the absence of dermal papilla cells. Cross-species microarray analysis comparing human BCC and murine synchronous HF growth cycle datasets revealed 74% concordance with telogen differentiation compared with anagen (23%, P < 0.01) and catagen (49%; P < 0.01). Incomplete anagen differentiation within BCC was characterized by reduced expression of the anagen master regulator DLX3 (-5.5-fold), and increased expression of telogen-associated genes: AEBP1 (2.2-fold), DEFB8 (35.3-fold), MMP3 (106.0-fold) and MMP12 (12.9-fold). Restoration of dermal papilla signals by in vitro addition of TGF-β2 enhanced anagen differentiation., Conclusion: Our findings show that BCC cells differentiate along HF lineages and may be susceptible to exogenous HF cycle modulators., (© 2019 British Association of Dermatologists.)

The pancreas is a gland composed mainly by endocrine and exocrine cells, giving rise to three main tumour types. Pancreatic neuroendocrine tumour or PNET arise from the endocrine portion of the pancreas. On the contrary, pancreatic exocrine neoplasms include pancreatic ductal adenocarcinoma (PDAC) and acinar cell carcinoma. PDAC is the most common type of pancreatic cancer and one of the leading causes of cancer-related death. It has been shown that less than 3% of PDAC patients have an overall survival of up to 5 years in the U.K. This mainly arises since the majority of patients diagnosed with PDAC present with advanced unresectable disease, which is highly resistant to all forms of chemotherapy and radiotherapy. Activating mutations of an isoform of the RAS protein, KRAS, are found in almost all PDAC cases and occur during early stages of malignant transformation. KRAS mutations play a critical role as they are involved in both initiating and maintaining PDAC development. The interaction of RAS with GDP/GTP along with its recruitment to the membrane affects transduction of its activating signals to downstream effectors. In this review, we aim to summarise different mutations of RAS and their prevalence in pancreatic cancer along with other RAS-induced tumours. In addition, we briefly discuss the genetically engineered mouse models that have been developed to study KRAS-mutated adenocarcinomas in the pancreas. These provide an opportunity to also address the importance of targeting RAS for better treatment response in PDAC patients along with the challenges incurred herein., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Purpose: Multi-slice scanning in the abdomen and thorax of small animals is compromised by the effects of respiration unless imaging and respiration are synchronised. To avoid the signal modulations that result from respiration motion and a variable TR, blocks of fully relaxed slices are typically acquired during inter-breath periods, at the cost of scan efficiency. This paper reports a conceptually simple yet effective prospective gating acquisition mode for multi-slice scanning in free breathing small animals at any fixed TR of choice with reduced sensitivity to respiratory motion., Methods: Multi-slice scan modes have been implemented in which each slice has its own specific projection or phase encode loop index counter. When a breath is registered RF pulses continue to be applied but data are not acquired, and the corresponding counters remain fixed so that the data are acquired one TR later, providing it coincides with an inter-breath period. The approach is refined to reacquire the slice data that are acquired immediately before each breath is detected. Only the data with reduced motion artefact are used in image reconstruction. The efficacy of the method is demonstrated in the RARE scan mode which is well known to be particularly useful for tumour visualization., Results: Validation in mice with RARE demonstrates improved stability with respect to ungated scanning where signal averaging is often used to reduce artefacts. SNR enhancement maps demonstrate the improved efficiency of the proposed method that is equivalent to at least a 2.5 fold reduction in scan time with respect to ungated signal averaging. A steady-state magnetisation transfer contrast prepared gradient echo implementation is observed to highlight tumour structure. Supplementary simulations demonstrate that only small variations in respiration rate are required to enable efficient sampling with the proposed method., Conclusions: The proposed prospective gating acquisition scheme enables efficient multi-slice scanning in small animals at the optimum TR with reduced sensitivity to respiratory motion. The method is compatible with a wide range of complementary methods including non-Cartesian scan modes, partially parallel imaging, and compressed sensing. In particular, the proposed scheme reduces the need for continual close monitoring to effect operator intervention in response to respiratory rate changes, which is both difficult to maintain and precludes high throughput., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Small Ruminant Lentivirus (SRLV) subtype E1, also known as Roccaverano strain, is considered a low pathogenic virus on the basis of natural genetic deletions, in vitro properties and on-farm observations. In order to gain more knowledge on this atypical lentivirus we investigated the in vivo tropism of Roccaverano strain in both, experimentally and naturally infected goats. Antibody responses were monitored as well as tissue distribution and viral load, evaluated by real time PCR on single spliced (gag/env) and multiple spliced (rev) RNA targets respectively, that were compared to histopathological lesions. Lymph nodes, spleen, alveolar macrophages and mammary gland turned out to be the main tissue reservoirs of genotype E1-provirus. Moreover, mammary gland and/or mammary lymph nodes acted as active replication sites in dairy goats, supporting the lactogenic transmission of this virus. Notably, a direct association between viral load and concomitant infection or inflammatory processes was evident within organs such as spleen, lung and testis. Our results validate the low pathogenicity designation of SRLV genotype E1 in vivo, and confirm the monocyte-macrophage cell lineage as the main virus reservoir of this genotype. Accordingly, SRLV genotype E displays a tropism towards all tissues characterized by an abundant presence of these cells, either for their own anatomical structure or for an occasional infectious/inflammatory status., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Field cancerisation was originally described as a basis for multiple head and neck squamous cell carcinoma (HNSCC) and is a pre-malignant phenomenon that is frequently attributable to oncogenic human papillomavirus (HPV) infection. Our work on β-HPV-induced cutaneous squamous cell carcinomas identified a novel Lrig1+ hair follicle junctional zone keratinocyte stem cell population as the basis for field cancerisation. Herein, we describe the ability for HPV to infect adult tissue stem cells in order to establish persistent infection and induce their proliferation and displacement resulting in field cancerisation. By review of the HPV literature, we reveal how this mechanism is conserved as the basis of field cancerisation across many tissues. New insights have identified the capacity for HPV early region genes to dysregulate adult tissue stem cell self-renewal pathways ensuring that the expanded population preserve its stem cell characteristics beyond the stem cell niche. HPV-infected cells acquire additional transforming mutations that can give rise to intraepithelial neoplasia (IEN), from environmental factors such as sunlight or tobacco induced mutations in skin and oral cavity, respectively. With establishment of IEN, HPV viral replication is sacrificed with loss of the episome, and the tissue is predisposed to multiple cancer stem cell-driven carcinomas.

Many malignancies that occur in high excess in kidney transplant recipients (KTRs) are due to viruses that thrive in the setting of immunosuppression. Keratinocyte carcinoma (KC), the most frequently occurring cancer type in KTR, has been associated with skin infection by human papillomavirus (HPV) from the beta genus. In this report, we extend our previous investigation aimed at identifying the presence of active β-HPV infection in skin tumors from KTRs through detection of viral protein expression. Using a combination of antibodies raised against the E4 and L1 proteins of the β-genotypes, we were able to visualize infection in five tumors [one keratoacanthoma (KA), three actinic keratoses (AKs), and one seborrheic keratoses (SKs)] that were all removed from two patients who had been both transplanted twice, had developed multiple KCs, and presented with a long history of immunosuppression (>30 years). These infected tissues displayed intraepidermal hyperplasia and increased expression of the ΔNp63 protein, which extended into the upper epithelial layers. In addition, using a xenograft model system in nude mice displaying a humanized stromal bed in the site of grafting, we successfully engrafted three AKs, two of which were derived from the aforementioned KTRs and displayed β-HPV infection in the original tumor. Of note, one AK-derived xenograft, along with its ensuing lymph node metastasis, was diagnosed as squamous cell carcinoma (SCC). In the latter, both β-HPV infection and ΔNp63 expression were no longer detectable. Although the overall success rate of engrafting was very low, the results of this study show for the first time that β-HPV + and ΔNp63 + intraepidermal hyperplasia can indeed progress to an aggressive SCC able to metastasize. Consistent with a series of reports attributing a causative role of β-HPV at early stages of skin carcinogenesis through ΔNp63 induction and increased keratinocytes stemness, here we provide in vivo evidence that these events are also occurring in the affected skin of KTRs. Due to these β-HPV-driven molecular pathways, the nascent tumor cell is able to acquire a high enough number of carcinogenic insults that its proliferation and survival will eventually become independent of viral gene expression.

β-Human papillomaviruses (HPVs) cause near ubiquitous latent skin infection within long-lived hair follicle (HF) keratinocyte stem cells. In patients with epidermodysplasia verruciformis, β-HPV viral replication is associated with skin keratosis and cutaneous squamous cell carcinoma. To determine the role of HF keratinocyte stem cells in β-HPV-induced skin carcinogenesis, we utilized a transgenic mouse model in which the keratin 14 promoter drives expression of the entire HPV8 early region (HPV8tg). HPV8tg mice developed thicker skin in comparison with wild-type littermates consistent with a hyperproliferative epidermis. HF keratinocyte proliferation was evident within the Lrig1+ keratinocyte stem cell population (69 vs. 55%, P < 0.01, n = 7), and not in the CD34+, LGR5+, and LGR6+ keratinocyte stem cell populations. This was associated with a 2.8-fold expansion in Lrig1+ keratinocytes and 3.8-fold increased colony-forming efficiency. Consistent with this, we observed nuclear p63 expression throughout this population and the HF infundibulum and adjoining interfollicular epidermis, associated with a switch from p63 transcriptional activation isoforms to ΔNp63 isoforms in HPV8tg skin. Epidermodysplasia verruciformis keratosis and in some cases actinic keratoses demonstrated similar histology associated with β-HPV reactivation and nuclear p63 expression within the HF infundibulum and perifollicular epidermis. These findings would suggest that β-HPV field cancerization arises from the HF junctional zone and predispose to squamous cell carcinoma., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Epidermodysplasia verruciformis (EV) is a rare, lifelong, autosomal recessive skin disease associated with an unusual susceptibility to infections with ubiquitous β-human papillomaviruses (β-HPVs), and in some cases also skin-tropic α genotypes. In this case report, HPV infection patterns were correlated with pathology and clinical manifestations of skin lesions from a patient with EV, without loss-of-function mutations in the EVER genes. HPV infection was investigated by both polymerase chain reaction (PCR) and laser capture microdissection (LCM) PCR, alongside immunofluorescence for the viral proteins E4 and L1. Analysis of eyebrow hair bulbs revealed multiple β-genus HPV infections, including HPV20 and HPV24, which were consistently found in all 11 skin lesions on the patient. Six lesions were also positive for the skin tropic α-genotype, HPV27. Clear-cut differences between two wart-like lesions, one caused by a skin-tropic α-genotype and the other by β-genotypes (as detected by LCM PCR) are shown, including the high cellular proliferation rate in β-HPV-induced lesions. Widespread expression of the early protein E4 was also evident in skin lesions positive for HPV20 by LCM PCR in both tumours and nearby intraepidermal proliferative areas. L1 expression was restricted to areas of intraepidermal proliferation showing productive infection. The patient's inability to control HPV infections is conclusive to the uncontrolled replication of few genotypes from both α and β genera, which cause proliferative lesions with clear-cut clinical and histological features., (© 2014 British Association of Dermatologists.)

The aim of this study was to determine whether detection of β-HPV gene products, as defined in epidermodysplasia verruciformis skin cancer, could also be observed in lesions from kidney transplant recipients alongside the viral DNA. A total of 111 samples, corresponding to 79 skin lesions abscised from 17 kidney transplant recipients, have been analyzed. The initial PCR analysis demonstrated that β-HPV-DNA was highly present in our tumor series (85%). Using a combination of antibodies raised against the E4 and L1 proteins of the β-genotypes, we were able to visualize productive infection in 4 out of 19 actinic keratoses, and in the pathological borders of 1 out of 14 squamous cell carcinomas and 1 out of 31 basal cell carcinomas. Increased expression of the cellular proliferation marker minichromosome maintenance protein 7 (MCM7), that extended into the upper epithelial layers, was a common feature of all the E4-positive areas, indicating that cells were driven into the cell cycle in areas of productive viral infections. Although the present study does not directly demonstrate a causal role of these viruses, the detection of E4 and L1 positivity in actinic keratosis and the adjacent pathological epithelium of skin cancer, clearly shows that β-HPV are actively replicating in the intraepidermal precursor lesions of kidney transplant recipients and can therefore cooperate with other carcinogenic agents, such as UVB, favoring skin cancer promotion.