Complex and variable regulation of ΔNp63 and TAp63 by TGFß has implications for the dynamics of squamous cell epithelial to mesenchymal transition.

TGFß has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFß signaling, with contradictory effects reported. We investigated the effects of TGFß on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFß signaling pathways. TGFß selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFß receptor inhibition, indicating activation of canonical TGFß pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFß in squamous cells that depend on the extent of canonical TGFß pathway aberrations. The interplay between TGFß and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy.

Patients with oral tongue squamous cell carcinoma and co­existing diabetes exhibit lower recurrence rates and improved survival: Implications for treatment.

Locoregional recurrences and distant metastases are major problems for patients with squamous cell carcinoma of the head and neck (SCCHN). Because SCCHN is a heterogeneous group of tumours with varying characteristics, the present study concentrated on the subgroup of squamous cell carcinoma of the oral tongue (SCCOT) to investigate the use of machine learning approaches to predict the risk of recurrence from routine clinical data available at diagnosis. The approach also identified the most important parameters that identify and classify recurrence risk. A total of 66 patients with SCCOT were included. Clinical data available at diagnosis were analysed using statistical analysis and machine learning approaches. Tumour recurrence was associated with T stage (P=0.001), radiological neck metastasis (P=0.010) and diabetes (P=0.003). A machine learning model based on the random forest algorithm and with attendant explainability was used. Whilst patients with diabetes were overrepresented in the SCCOT cohort, diabetics had lower recurrence rates (P=0.015 after adjusting for age and other clinical features) and an improved 2-year survival (P=0.025) compared with non-diabetics. Clinical, radiological and histological data available at diagnosis were used to establish a prognostic model for patients with SCCOT. Using machine learning to predict recurrence produced a classification model with 71.2% accuracy. Notably, one of the findings of the feature importance rankings of the model was that diabetics exhibited less recurrence and improved survival compared with non-diabetics, even after accounting for the independent prognostic variables of tumour size and patient age at diagnosis. These data imply that the therapeutic manipulation of glucose levels used to treat diabetes may be useful for patients with SCCOT regardless of their diabetic status. Further studies are warranted to investigate the impact of diabetes in other SCCHN subtypes.

ΔNp63 is regulated by insulin/IGF-1 signaling in normal basal/progenitor mammary cells and in luminal-type breast cancer cells.

Breast cancers are a heterogeneous group of tumors classified according to their histological growth patterns and receptor expression characteristics. Intratumor heterogeneity also exists, with subpopulations of cells with different phenotypes found in individual cancers, including cells with stem or progenitor cell properties. At least two types of breast cancer stem cells (CSCs) exist, the epithelial and the basal/mesenchymal subtypes, although how these phenotypes are controlled is unknown. ΔNp63 is a basal cell marker and regulator of stem/progenitor cell activities in the normal mammary gland and is expressed in the basal-like CSC subpopulation in some estrogen receptor-positive (ER+) and/or human epidermal growth factor receptor 2-positive (HER2+) breast adenocarcinomas. Whilst p63 is known to directly impart CSC properties in luminal breast cancer cells, how p63 is regulated and induced in these cells is unknown. We initially confirmed the existence of a small subpopulation of ΔNp63+ cells in lymph node metastases of ER+ human ductal adenocarcinomas, indicating together with previous reports that ΔNp63+ tumor cells are present in approximately 40% of these metastases. Notably, ΔNp63+ cells show a preferential location at the edge of tumor areas, suggesting possible regulation of ΔNp63 by the tumor microenvironment. Subsequently, we showed that the high levels of ΔNp63 in basal non-transformed MCF-10A mammary epithelial cells rely on insulin in their culture medium, whilst ΔNp63 levels are increased in MCF-7 ER+ luminal-type breast cancer cells treated with insulin or insulin-like growth factor 1 (IGF-1). Mechanistically, small molecule inhibitors and siRNA gene knockdown demonstrated that induction of ΔNp63 by IGF-1 requires PI3K, ERK1/2, and p38 MAPK activation, and acts through FOXO transcriptional inactivation. We also show that metformin inhibits ΔNp63 induction. These data reveal an IGF-mediated mechanism to control basal-type breast CSCs, with therapeutic implications to modify intratumor breast cancer cell heterogeneity and plasticity.

Recent Advances in Pathology: the 2023 Annual Review Issue of The Journal of Pathology.

The 2023 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 12 invited reviews on topics of current interest in pathology. This year, our subjects include immuno-oncology and computational pathology approaches for diagnostic and research applications in human disease. Reviews on the tissue microenvironment include the effects of apoptotic cell-derived exosomes, how understanding the tumour microenvironment predicts prognosis, and the growing appreciation of the diverse functions of fibroblast subtypes in health and disease. We also include up-to-date reviews of modern aspects of the molecular basis of malignancies, and our final review covers new knowledge of vascular and lymphatic regeneration in cardiac disease. All of the reviews contained in this issue are written by expert groups of authors selected to discuss the recent progress in their particular fields and all articles are freely available online (https://pathsocjournals.onlinelibrary.wiley.com/journal/10969896). © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Early detection of squamous cell carcinoma of the oral tongue using multidimensional plasma protein analysis and interpretable machine learning.

BACKGROUND: Interpretable machine learning (ML) for early detection of cancer has the potential to improve risk assessment and early intervention. METHODS: Data from 261 proteins related to inflammation and/or tumor processes in 123 blood samples collected from healthy persons, but of whom a sub-group later developed squamous cell carcinoma of the oral tongue (SCCOT), were analyzed. Samples from people who developed SCCOT within less than 5 years were classified as tumor-to-be and all other samples as tumor-free. The optimal ML algorithm for feature selection was identified and feature importance computed by the SHapley Additive exPlanations (SHAP) method. Five popular ML algorithms (AdaBoost, Artificial neural networks [ANNs], Decision Tree [DT], eXtreme Gradient Boosting [XGBoost], and Support Vector Machine [SVM]) were applied to establish prediction models, and decisions of the optimal models were interpreted by SHAP. RESULTS: Using the 22 selected features, the SVM prediction model showed the best performance (sensitivity = 0.867, specificity = 0.859, balanced accuracy = 0.863, area under the receiver operating characteristic curve [ROC-AUC] = 0.924). SHAP analysis revealed that the 22 features rendered varying person-specific impacts on model decision and the top three contributors to prediction were Interleukin 10 (IL10), TNF Receptor Associated Factor 2 (TRAF2), and Kallikrein Related Peptidase 12 (KLK12). CONCLUSION: Using multidimensional plasma protein analysis and interpretable ML, we outline a systematic approach for early detection of SCCOT before the appearance of clinical signs.

Evidence for etiologic field changes in tongue distant from tumor in patients with squamous cell carcinoma of the oral tongue.

Oral cancer is a paradigm of Slaughter's concept of field cancerization, where tumors are thought to originate within an area of cells containing genetic alterations that predispose to cancer development. The field size is unclear but may represent a large area of tissue, and the origin of mutations is also unclear. Here, we analyzed whole exome and transcriptome features in contralateral tumor-distal tongue (i.e. distant from the tumor, not tumor-adjacent) and corresponding tumor tissues of 15 patients with squamous cell carcinoma of the oral tongue. The number of point mutations ranged from 41 to 237 in tumors and from one to 78 in tumor-distal samples. Tumor-distal samples showed mainly clock-like (associated with aging) or tobacco smoking mutational signatures. Tumors additionally showed mutations that associate with cytidine deaminase AID/APOBEC enzyme activities or a UV-like signature. Importantly, no point mutations were shared between a tumor and the matched tumor-distal sample in any patient. TP53 was the most frequently mutated gene in tumors (67%), whereas a TP53 mutation was detected in only one tumor-distal sample, and this mutation was not shared with the matched tumor. Arm-level copy number variation (CNV) was found in 12 tumors, with loss of chromosome (Chr) 8p or gain of 8q being the most frequent events. Two tumor-distal samples showed a gain of Chr8, which was associated with increased expression of Chr8-located genes in these samples, although gene ontology did not show a role for these genes in oncogenic processes. In situ hybridization revealed a mixed pattern of Chr8 gain and neutral copy number in both tumor cells and adjacent nontumor epithelium in one patient. We conclude that distant field cancerization exists but does not present as tumor-related mutational events. The data are compatible with etiologic field effects, rather than classical monoclonal field cancerization theory. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Antigen peptide transporters are upregulated in squamous cell carcinoma of the oral tongue and show sex-specific associations with survival.

Transporter associated with antigen processing 1 (TAP1) and TAP2 serve pivotal roles in adaptive immunity. Tumor cells often show reduced antigen presentation on their surface as one mechanism to escape immune recognition. Whether downregulation of TAPs is a common mechanism of tumor immune evasion in squamous cell carcinoma of the oral tongue (SCCOT) is unclear. In the present study, samples from 78 patients with SCCOT and 17 patients with benign hyperplastic tongue lesions were analyzed for TAP1 and TAP2 expression by immunohistochemistry. The percentage of positive cells and staining intensity were scored. Associations with clinicopathological variables and survival outcome were also investigated. The results demonstrated that TAP1 and TAP2 levels were highly associated with each other in individual samples and were upregulated in SCCOT compared with benign lesions (P<0.001). The proportion of TAP1- or TAP2-positive tumor cells was >80% in all but two of the tumors, whereas 25.6 and 23.0% of the tumors showed weak intensity of TAP1 and TAP2, respectively. There were no significant associations with clinicopathological variables or survival outcomes between TAP-intermediate/strong and TAP-weak tumors. However, in patients <70 years old and with early stage SCCOT, male patients had better outcomes than female patients (log-rank P<0.05), and the best outcome was observed in male patients with intermediate/strong TAP expression. In conclusion, loss of TAP was not a frequent event in SCCOT and stronger TAP expression in male patients was associated with improved survival, providing further evidence for sex-specific immune modulation in cancer.

DNA Demethylation Switches Oncogenic ΔNp63 to Tumor Suppressive TAp63 in Squamous Cell Carcinoma.

The TP63 gene encodes two major protein variants; TAp63 contains a p53-like transcription domain and consequently has tumor suppressor activities whereas ΔNp63 lacks this domain and acts as an oncogene. The two variants show distinct expression patterns in normal tissues and tumors, with lymphocytes and lymphomas/leukemias expressing TAp63, and basal epithelial cells and some carcinomas expressing high levels of ΔNp63, most notably squamous cell carcinomas (SCC). Whilst the transcriptional functions of TAp63 and ΔNp63 isoforms are known, the mechanisms involved in their regulation are poorly understood. Using squamous epithelial cells that contain high levels of ΔNp63 and low/undetectable TAp63, the DNA demethylating agent decitabine (5-aza-2'-deoxycytidine, 5-dAza) caused a dose-dependent increase in TAp63, with a simultaneous reduction in ΔNp63, indicating DNA methylation-dependent regulation at the isoform-specific promoters. The basal cytokeratin KRT5, a direct ΔNp63 transcriptional target, was also reduced, confirming functional alteration of p63 activity after DNA demethylation. We also showed high level methylation of three CpG sites in the TAP63 promoter in these cells, which was reduced by decitabine. DNMT1 depletion using inducible shRNAs partially replicated these effects, including an increase in the ratio of TAP63:ΔNP63 mRNAs, a reduction in ΔNp63 protein and reduced KRT5 mRNA levels. Finally, high DNA methylation levels were found at the TAP63 promoter in clinical SCC samples and matched normal tissues. We conclude that DNA methylation at the TAP63 promoter normally silences transcription in squamous epithelial cells, indicating DNA methylation as a therapeutic approach to induce this tumor suppressor in cancer. That decitabine simultaneously reduced the oncogenic activity of ΔNp63 provides a "double whammy" for SCC and other p63-positive carcinomas. Whilst a variety of mechanisms may be involved in producing the opposite effects of DNA demethylation on TAp63 and ΔNp63, we propose an "either or" mechanism in which TAP63 transcription physically interferes with the ability to initiate transcription from the downstream ΔNP63 promoter on the same DNA strand. This mechanism can explain the observed inverse expression of p63 isoforms in normal cells and cancer.

Stat3 Tyrosine 705 and Serine 727 Phosphorylation Associate With Clinicopathological Characteristics and Distinct Tumor Cell Phenotypes in Triple-Negative Breast Cancer.

Signal transducer and activator of transcription 3 (Stat3) is responsible for many aspects of normal development and contributes to the development and progression of cancer through regulating epithelial cell identity and cancer stem cells. In breast cancer, Stat3 is associated with triple-negative breast cancers (TNBC) and its function has been related to the activation of p63, itself a marker of basal-like TNBC and a master regulator of stem cell activities. Stat3 activation is controlled by dual phosphorylation at tyrosine 705 (pTyr705) and serine 727 (pSer727), although it is unclear whether these have equivalent effects, and whether they are related or independent events. To address these issues, we investigated Stat3 phosphorylation at the two sites by immunohistochemistry in 173 patients with TNBC. Stat3 phosphorylation was assessed by automated quantitative measurements of digitized scanned images and classified into four categories based on histoscore. The results were analyzed for associations with multiple markers of tumor phenotype, proliferation, BRCA status, and clinicopathological characteristics. We show that the levels of pTyr705- and pSer727-Stat3 were independent in 34% of tumors. High pTyr705-Stat3 levels were associated with the luminal differentiation markers ERß/AR and MUC1, whereas tumors with high levels of pSer727-Stat3 were more likely to be positive for the basal marker CK5/6, but were independent of p63 and were EGFR negative. Combined high pSer727- and low Tyr705-Stat3 phosphorylation associated with basal-like cancer. Although high Stat3 phosphorylation levels were associated with less aggressive tumor characteristics, they did not associate with improved survival, indicating that Stat3 phosphorylation is an unfavorable indicator for tumors with an otherwise good prognosis according to clinicopathological characteristics. These findings also show that pTyr705-Stat3 and pSer727-Stat3 associate with specific breast tumor phenotypes, implying that they exert distinct functional activities in breast cancer.

Reiterative modeling of combined transcriptomic and proteomic features refines and improves the prediction of early recurrence in squamous cell carcinoma of head and neck.

BACKGROUND: Patients with squamous cell carcinoma of the head and neck (SCCHN) have a high-risk of recurrence. We aimed to develop machine learning methods to identify transcriptomic and proteomic features that provide accurate classification models for predicting risk of early recurrence in SCCHN patients. METHODS: Clinical, genomic, transcriptomic and proteomic features distinguishing recurrence risk were examined in SCCHN patients from The Cancer Genome Atlas (TCGA). Recurrence within one year after treatment was classified as high-risk and no recurrence as low-risk. RESULTS: No significant differences in individual clinicopathological characteristics, mutation profiles or mRNA expression patterns were seen between the groups using conventional statistical analysis. Using the machine learning algorithm, extreme gradient boosting (XGBoost), ten proteins (RAD50, 4E-BP1, MYH11, MAP2K1, BECN1, NF2, RAB25, ERRFI1, KDR, SERPINE1) and five mRNAs (PLAUR, DKK1, AXIN2, ANG and VEGFA) made the greatest contribution to classification. These features were used to build improved models in XGBoost, achieving the best discrimination performance when combining transcriptomic and proteomic data, providing an accuracy of 0.939 and an Area Under the ROC Curve (AUC) of 0.951. CONCLUSIONS: This study highlights machine learning to identify transcriptomic and proteomic factors that play important roles in predicting risk of recurrence in patients with SCCHN and to develop such models by iterative cycles to enhance their accuracy, thereby aiding the introduction of personalized treatment regimens.

Recent Advances in Pathology: the 2022 Annual Review Issue of The Journal of Pathology.

The 2022 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 15 invited reviews on research areas of growing importance in pathology. This year, the articles include those that focus on digital pathology, employing modern imaging techniques and software to enable improved diagnostic and research applications to study human diseases. This subject area includes the ability to identify specific genetic alterations through the morphological changes they induce, as well as integrating digital and computational pathology with 'omics technologies. Other reviews in this issue include an updated evaluation of mutational patterns (mutation signatures) in cancer, the applications of lineage tracing in human tissues, and single cell sequencing technologies to uncover tumour evolution and tumour heterogeneity. The tissue microenvironment is covered in reviews specifically dealing with proteolytic control of epidermal differentiation, cancer-associated fibroblasts, field cancerisation, and host factors that determine tumour immunity. All of the reviews contained in this issue are the work of invited experts selected to discuss the considerable recent progress in their respective fields and are freely available online (https://onlinelibrary.wiley.com/journal/10969896). © 2022 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Identifying pathways regulating the oncogenic p53 family member ΔNp63 provides therapeutic avenues for squamous cell carcinoma.

BACKGROUND: ΔNp63 overexpression is a common event in squamous cell carcinoma (SCC) that contributes to tumorigenesis, making ΔNp63 a potential target for therapy. METHODS: We created inducible TP63-shRNA cells to study the effects of p63-depletion in SCC cell lines and non-malignant HaCaT keratinocytes. DNA damaging agents, growth factors, signaling pathway inhibitors, histone deacetylase inhibitors, and metabolism-modifying drugs were also investigated for their ability to influence ΔNp63 protein and mRNA levels. RESULTS: HaCaT keratinocytes, FaDu and SCC-25 cells express high levels of ΔNp63. HaCaT and FaDu inducible TP63-shRNA cells showed reduced proliferation after p63 depletion, with greater effects on FaDu than HaCaT cells, compatible with oncogene addiction in SCC. Genotoxic insults and histone deacetylase inhibitors variably reduced ΔNp63 levels in keratinocytes and SCC cells. Growth factors that regulate proliferation/survival of squamous cells (IGF-1, EGF, amphiregulin, KGF, and HGF) and PI3K, mTOR, MAPK/ERK or EGFR inhibitors showed lesser and inconsistent effects, with dual inhibition of PI3K and mTOR or EGFR inhibition selectively reducing ΔNp63 levels in HaCaT cells. In contrast, the antihyperlipidemic drug lovastatin selectively increased ΔNp63 in HaCaT cells. CONCLUSIONS: These data confirm that ΔNp63-positive SCC cells require p63 for continued growth and provide proof of concept that p63 reduction is a therapeutic option for these tumors. Investigations of ΔNp63 regulation identified agent-specific and cell-specific pathways. In particular, dual inhibition of the PI3K and mTOR pathways reduced ΔNp63 more effectively than single pathway inhibition, and broad-spectrum histone deacetylase inhibitors showed a time-dependent biphasic response, with high level downregulation at the transcriptional level within 24 h. In addition to furthering our understanding of ΔNp63 regulation in squamous cells, these data identify novel drug combinations that may be useful for p63-based therapy of SCC.

Variation in Plasma Levels of TRAF2 Protein During Development of Squamous Cell Carcinoma of the Oral Tongue.

As early detection is crucial for improvement of cancer prognosis, we searched for biomarkers in plasma from individuals who later developed squamous cell carcinoma of the oral tongue (SCCOT) as well as in patients with an already established SCCOT. Levels of 261 proteins related to inflammation and/or tumor processes were measured using the proximity extension assay (PEA) in 179 plasma samples (42 collected before diagnosis of SCCOT with 81 matched controls; 28 collected at diagnosis of SCCOT with 28 matched controls). Statistical modeling tools principal component analysis (PCA) and orthogonal partial least square - discriminant analysis (OPLS-DA) were applied to provide insights into separations between groups. PCA models failed to achieve group separation of SCCOT patients from controls based on protein levels in samples taken prior to diagnosis or at the time of diagnosis. For pre-diagnostic samples and their controls, no significant OPLS-DA model was identified. Potentials for separating pre-diagnostic samples collected up to five years before diagnosis (n = 15) from matched controls (n = 28) were seen in four proteins. For diagnostic samples and controls, the OPLS-DA model indicated that 21 proteins were important for group separation. TNF receptor associated factor 2 (TRAF2), decreased in pre-diagnostic plasma (< 5 years) but increased at diagnosis, was the only protein showing altered levels before and at diagnosis of SCCOT (p-value < 0.05). Taken together, changes in plasma protein profiles at diagnosis were evident, but not reliably detectable in pre-diagnostic samples taken before clinical signs of tumor development. Variation in protein levels during cancer development poses a challenge for the identification of biomarkers that could predict SCCOT development.

Resistance mechanisms to inhibitors of p53-MDM2 interactions in cancer therapy: can we overcome them?

Since the discovery of the first MDM2 inhibitors, we have gained deeper insights into the cellular roles of MDM2 and p53. In this review, we focus on MDM2 inhibitors that bind to the p53-binding domain of MDM2 and aim to disrupt the binding of MDM2 to p53. We describe the basic mechanism of action of these MDM2 inhibitors, such as nutlin-3a, summarise the determinants of sensitivity to MDM2 inhibition from p53-dependent and p53-independent points of view and discuss the problems with innate and acquired resistance to MDM2 inhibition. Despite progress in MDM2 inhibitor design and ongoing clinical trials, their broad use in cancer treatment is not fulfilling expectations in heterogenous human cancers. We assess the MDM2 inhibitor types in clinical trials and provide an overview of possible sources of resistance to MDM2 inhibition, underlining the need for patient stratification based on these aspects to gain better clinical responses, including the use of combination therapies for personalised medicine.

High Levels of Low-Density Lipoproteins Correlate with Improved Survival in Patients with Squamous Cell Carcinoma of the Head and Neck.

Circulating lipoproteins as risk factors or prognostic indicators for various cancers have been investigated previously; however, no clear consensus has been reached. In this study, we aimed at evaluating the impact of serum lipoproteins on the prognosis of patients with squamous cell carcinoma of the head and neck (SCCHN). Levels of total cholesterol, low-density lipoproteins (LDL), high-density lipoproteins (HDL), triglycerides and lipoprotein(a) were measured in serum samples from 106 patients and 28 healthy controls. We found that HDL was the only lipoprotein exhibiting a significant difference in concentration between healthy controls and patients (p = 0.012). Kaplan-Meier survival curves indicated that patients with high levels of total cholesterol or LDL had better overall survival than patients with normal levels (p = 0.028 and p = 0.007, respectively). Looking at patients without lipid medication (n = 89) and adjusting for the effects of TNM stage and weight change, multivariate Cox regression models indicated that LDL was an independent prognostic factor for both overall (p = 0.005) and disease-free survival (p = 0.013). In summary, our study revealed that high LDL level is beneficial for survival outcome in patients with SCCHN. Use of cholesterol-lowering medicines for prevention or management of SCCHN needs to be evaluated carefully.

Recent Advances in Pathology: the 2021 Annual Review Issue of The Journal of Pathology.

The 2021 Annual Review Issue of The Journal of Pathology contains 14 invited reviews on current research areas of particular importance in pathology. The subjects included here reflect the broad range of interests covered by the journal, including both basic and applied research fields but always with the aim of improving our understanding of human disease. This year, our reviews encompass the huge impact of the COVID-19 pandemic, the development and application of biomarkers for immune checkpoint inhibitors, recent advances in multiplexing antigen/nucleic acid detection in situ, the use of genomics to aid drug discovery, organoid methodologies in research, the microbiome in cancer, the role of macrophage-stroma interactions in fibrosis, and TGF-ß as a driver of fibrosis in multiple pathologies. Other reviews revisit the p53 field and its lack of clinical impact to date, dissect the genetics of mitochondrial diseases, summarise the cells of origin and genetics of sarcomagenesis, provide new data on the role of TRIM28 in tumour predisposition, review our current understanding of cancer stem cell niches, and the function and regulation of p63. The reviews are authored by experts in their field from academia and industry, and provide comprehensive updates of the chosen areas, in which there has been considerable recent progress. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Low potential of circulating interleukin 1 receptor antagonist as a prediction marker for squamous cell carcinoma of the head and neck.

BACKGROUND: Circulating markers are attractive molecules for prognosis and management of cancer that allow sequential monitoring of patients during and after treatment. Based on previous protein profiling data, circulating interleukin 1 receptor antagonist (IL-1Ra) was evaluated as a potential diagnostic and prognostic marker for squamous cell carcinomas of the head and neck (SCCHN). In this study, we aimed at confirming the clinical relevance of plasma IL-1Ra in SCCHN and exploring its potential as a prediction marker for SCCHN. METHODS: Plasma from 87 patients with SCCHN, control plasma from 28 healthy individuals and pre-diagnostic plasma from 44 patients with squamous cell carcinoma of the oral tongue (SCCOT) and 88 matched controls were analysed with IL-1Ra electrochemiluminescence immunoassays from mesoscale diagnostics. RESULTS: Plasma IL-1Ra was found to be up-regulated in patients with oral tongue, gingiva and base of tongue tumours compared to healthy individuals (p < 0.01). IL-1Ra levels positively correlated with tumour size (p < 0.01) and body mass index (p = 0.013). Comparing pre-diagnostic plasma to the matched controls, similar IL1-Ra levels were seen (p = 0.05). CONCLUSION: The anti-inflammatory cytokine IL-1Ra could be a diagnostic marker for SCCHN, whereas its potential as a cancer prediction marker was not supported by our data.

The foggy world(s) of p63 isoform regulation in normal cells and cancer.

The p53 family member p63 exists as two major protein variants (TAp63 and ΔNp63) with distinct expression patterns and functional properties. Whilst downstream target genes of p63 have been studied intensively, how p63 variants are themselves controlled has been relatively neglected. Here, we review advances in understanding ΔNp63 and TAp63 regulation, highlighting their distinct pathways. TAp63 has roles in senescence and metabolism, and in germ cell genome maintenance, where it is activated post-transcriptionally by phosphorylation cascades after DNA damage. The function and regulation of TAp63 in mesenchymal and haematopoietic cells is less clear but may involve epigenetic control through DNA methylation. ΔNp63 functions to maintain stem/progenitor cells in various epithelia and is overexpressed in squamous and certain other cancers. ΔNp63 is transcriptionally regulated through multiple enhancers in concert with chromatin modifying proteins. Many signalling pathways including growth factors, morphogens, inflammation, and the extracellular matrix influence ΔNp63 levels, with inconsistent results reported. There is also evidence for reciprocal regulation, including ΔNp63 activating its own transcription. ΔNp63 is downregulated during cell differentiation through transcriptional regulation, while post-transcriptional events cause proteasomal degradation. Throughout the review, we identify knowledge gaps and highlight discordances, providing potential explanations including cell-context and cell-matrix interactions. Identifying individual p63 variants has roles in differential diagnosis and prognosis, and understanding their regulation suggests clinically approved agents for targeting p63 that may be useful combination therapies for selected cancer patients. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

TAp63 and ΔNp63 (p40) in prostate adenocarcinomas: ΔNp63 associates with a basal-like cancer stem cell population but not with metastasis.

Like other malignancies, prostate tumors are thought to contain cancer stem-like cells (CSCs) that are responsible for growth, metastasis, and therapy resistance. ΔNp63 (also called p40) is a regulator of normal prostate stem/progenitor cell activities and a marker of normal basal epithelial cells. The levels of ΔNp63 are reduced in prostate adenocarcinomas, although there is also evidence that ΔNp63 is involved in CSC regulation and drives metastasis to the bone. We studied metastatic deposits of prostate cancers with isoform-specific ΔNp63 and TAp63 antibodies. We identified p63-positive cells in only 3 of 42 metastatic prostate tumors (7%), including 2/38 (5.3%) "usual-type" adenocarcinomas. ΔNp63 and TAp63 isoforms were present in the nuclei of a small subpopulation (< 1%) of tumor cells in these metastases. ΔNp63-positive cells showed a basal-like cell phenotype (cytokeratin 8- and androgen receptor-negative, high molecular weight cytokeratin- and cytokeratin 19-positive), distinct from the tumor bulk. TAp63-positive cells were similar but were sometimes cytokeratin 8-positive. A subset of ΔNp63-positive tumor cells were CD44-positive, a marker of "basal" CSCs but were not positive for the "epithelial" CSC marker ALDH1. TAp63 was not associated with either of these CSC markers. None of the tumors containing p63-positive cells showed evidence of bone metastasis, compared with 28% of the p63-negative tumors. These data show that both ΔNp63 and TAp63 are present in only a small proportion of prostate adenocarcinomas and do not associate with metastasis. The data suggest heterogeneity of CSCs in prostate cancer, similar to other cancer types.

Transfer-RNA-Derived Fragments Are Potential Prognostic Factors in Patients with Squamous Cell Carcinoma of the Head and Neck.

Transfer-RNA-derived fragments (tRFs) are a class of small non-coding RNAs that are functionally different from their parental transfer RNAs (tRNAs). tRFs can regulate gene expression by several mechanisms, and are involved in a variety of pathological processes. Here, we aimed at understanding the composition and abundance of tRFs in squamous cell carcinoma of the head and neck (SCCHN), and evaluated the potential of tRFs as prognostic markers in this cancer type. We obtained tRF expression data from The Cancer Genome Atlas (TCGA) HNSC cohort (523 patients) using MINTbase v2.0, and correlated to available TCGA clinical data. RNA-binding proteins were predicted according to the calculated Position Weight Matrix (PWM) score from the RNA-Binding Protein DataBase (RBPDB). A total of 10,158 tRFs were retrieved and a high diversity in expression levels was seen. Fifteen tRFs were found to be significantly associated with overall survival (Kaplan-Meier survival analysis, log rank test p-value < 0.01). The top prognostic marker, tRF-20-S998LO9D (p < 0.001), was further measured in tumor and tumor-free samples from 16 patients with squamous cell carcinoma of the oral tongue and 12 healthy controls, and was significantly upregulated in tumor compared to matched tumor-free tongue (p < 0.001). Results suggest that tRFs are useful prognostic markers in SCCHN.