MiR-151a: a robust endogenous control for normalizing small extracellular vesicle cargo in human cancer.

Small extracellular vesicles (sEVs) in the blood of cancer patients contain higher amounts of tumor markers than those identified as free-circulating. miRNAs have significant biomedical relevance due to their high stability and feasible detection. However, there is no reliable endogenous control available to measure sEVs-miRNA content, impairing the acquisition of standardized consistent measurements in cancer liquid biopsy. In this study, we identified three miRNAs from a panel of nine potential normalizers that emerged from a comprehensive analysis comparing the sEV-miRNA profile of six lung and ovarian human cancer cell lines in the absence of or under different conditions. Their relevance as normalizers was tested in 26 additional human cancer cell lines from nine different tumor types undergoing chemotherapy or radiotherapy treatment. The validation cohorts were comprised of 242 prospective plasma and ascitic fluid samples from three different human tumor types. Variability and normalization properties were tested in comparison to miR-16, the most used control to normalize free-circulating miRNAs in plasma. Our results indicate that miR-151a is consistently represented in small extracellular vesicles with minimal variability compared to miR-16, providing a novel normalizer to measure small extracellular vesicle miRNA content that will benefit liquid biopsy in cancer patients.

Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function.

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

Prognosis Value of Immunoregulatory Molecules in Oral Cancer Microenvironment: An Immunohistochemical Study.

OBJECTIVES: To evaluate the relationship of the immune-checkpoint PD-1/PD-L1 with the clinical evolution of OSCC; to assess survival in OSCC based on the characteristics of TME and histologic risk score; to evaluate the clinical and histopathological relationship of OSCC with immunological TME. MATERIAL AND METHODS: A retrospective study was carried out on 65 samples from patients with OSCC on the floor of the mouth or tongue. Clinicopathological variables and the expression of the biomarkers PD-1, PD-L1, FoxP3, CD4, CD8, CSF1R, and p16 were recorded. The relationship of the clinical and histological variables with the expression of the biomarkers and survival was studied. RESULTS: The univariate and multivariate analysis indicated that positive PD-1 expression was an independent protective factor for survival (overall, disease-free, disease-specific survival) and that high PD-L1 also improved survival. Poorly differentiated histological grades and metastasis were associated with a worse prognosis. CONCLUSIONS: PD-1 is a protective survival factor that is maintained independently of PD-L1 expression. High values of PD-L1 expression also improve survival. Higher expression of PD-1 is observed in smaller tumors, and higher expression of PD-L1 is more likely in women. No relationship between the tumor microenvironment and histologic risk score was found to influence the survival patterns studied in the OSCC. There is no evidence of a relationship between the histopathological features and the studied markers, although the positive PD-1 and PD-L1 cases have a lower risk of a high WPOI score, and positive PD-1 expression was associated with a lower DOI.

Comparison of Colorectal Cancer Stem Cells and Oxaliplatin-Resistant Cells Unveils Functional Similarities.

Colorectal cancer is the second most common cancer in women, the third in men, and an important cause of cancer-related mortality. Recurrence and the development of chemotherapy resistance are major hindrances for patients' treatment. The presence of cancer stem cells with chemotherapy resistance able to generate proliferating tumor cells contributes to tumor recurrence and resistance. In addition, tumor cells can develop chemoresistance through adaptation mechanisms. In this article, cancer stem cells were isolated from HT29 and SW620 colorectal cancer cell lines. Oxaliplatin resistance was induced by a single drug treatment simulating the usual guidelines of patient treatment. A comparison of these two populations showed similarities since cancer stem cells presented increased oxaliplatin resistance, and resistant cells contained an increased number of cancer stem cells. Cancer stem cells isolated from resistant cells showed increased oxaliplatin resistance. Cell invasion capacity and epithelial-mesenchymal transition were increased both in cancer stem cells and oxaliplatin-resistant cells. mRNA expression analysis showed that both cell types shared a significant proportion of commonly regulated genes. In summary, the data presented indicate that colorectal cancer stem cells and oxaliplatin-resistant cells are highly related cell populations that might have interesting implications in the development of tumor recurrence and resistance to chemotherapy.

Mechanisms of Cisplatin Resistance in HPV Negative Head and Neck Squamous Cell Carcinomas.

Head and neck squamous cell carcinomas (HNSCCs) are the eighth most common cancers worldwide. While promising new therapies are emerging, cisplatin-based chemotherapy remains the gold standard for advanced HNSCCs, although most of the patients relapse due to the development of resistance. This review aims to condense the different mechanisms involved in the development of cisplatin resistance in HNSCCs and highlight future perspectives intended to overcome its related complications. Classical resistance mechanisms include drug import and export, DNA repair and oxidative stress control. Emerging research identified the prevalence of these mechanisms in populations of cancer stem cells (CSC), which are the cells mainly contributing to cisplatin resistance. The use of old and new CSC markers has enabled the identification of the characteristics within HNSCC CSCs predisposing them to treatment resistance, such as cell quiescence, increased self-renewal capacity, low reactive oxygen species levels or the acquisition of epithelial to mesenchymal transcriptional programs. In the present review, we will discuss how cell intrinsic and extrinsic cues alter the phenotype of CSCs and how they influence resistance to cisplatin treatment. In addition, we will assess how the stromal composition and the tumor microenvironment affect drug resistance and the acquisition of CSCs' characteristics through a complex interplay between extracellular matrix content as well as immune and non-immune cell characteristics. Finally, we will describe how alterations in epigenetic modifiers or other signaling pathways can alter tumor behavior and cell plasticity to induce chemotherapy resistance. The data generated in recent years open up a wide range of promising strategies to optimize cisplatin therapy, with the potential to personalize HNSCC patient treatment strategies.

Intermittent Hypoxia Mediates Paraspeckle Protein-1 Upregulation in Sleep Apnea.

As some evidence suggests that hypoxia might be an inducer of nuclear paraspeckle formation, we explore whether intermittent hypoxia (IH)-mediated paraspeckle protein-1 (PSPC1) overexpression might contribute to the activation of tumor growth factor (TGF)ß-SMAD pathway in patients with obstructive sleep apnea (OSA). This activation would promote changes in intracellular signaling that would explain the increased cancer aggressiveness reported in these patients. Here, we show that patients with OSA exhibit elevated PSPC1 levels both in plasma and in monocytes. Our data suggest that PSPC1 is ultimately delivered to the plasma through its cleavage from OSA monocytes by matrix metalloproteinase-2 (MMP2). In addition, IH promotes PSPC1, TGFß, and MMP2 expression in monocytes through the hypoxia-inducible factor. Lastly, both PSPC1 and TGFß induce increased expression of genes that drive the epithelial-to-mesenchymal transition. Our study details the mechanism by which hypoxemia upmodulates the extracellular release of PSPC1 by means of MMP2, such that plasma PSPC1 together with TGFß activation signaling further promotes tumor metastasis and supports cancer aggressiveness in patients with OSA.

Tissue-resident macrophages provide a pro-tumorigenic niche to early NSCLC cells.

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis3-5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions1. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial-mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8+ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions.

A Novel Role for the Tumor Suppressor Gene ITF2 in Tumorigenesis and Chemotherapy Response.

Despite often leading to platinum resistance, platinum-based chemotherapy continues to be the standard treatment for many epithelial tumors. In this study we analyzed and validated the cytogenetic alterations that arise after treatment in four lung and ovarian paired cisplatin-sensitive/resistant cell lines by 1-million microarray-based comparative genomic hybridization (array-CGH) and qRT-PCR methodologies. RNA-sequencing, functional transfection assays, and gene-pathway activity analysis were used to identify genes with a potential role in the development of this malignancy. The results were further explored in 55 lung and ovarian primary tumors and control samples, and in two extensive in silico databases. Long-term cell exposure to platinum induces the frequent deletion of ITF2 gene. Its expression re-sensitized tumor cells to platinum and recovered the levels of Wnt/ß-catenin transcriptional activity. ITF2 expression was also frequently downregulated in epithelial tumors, predicting a worse overall survival. We also identified an inverse correlation between ITF2 and HOXD9 expression, revealing that Non-small cell lung cancer (NSCLC) patients with lower expression of HOXD9 had a better overall survival rate. We defined the implication of ITF2 as a molecular mechanism behind the development of cisplatin resistance probably through the activation of the Wnt-signaling pathway. This data highlights the possible role of ITF2 and HOXD9 as novel therapeutic targets for platinum resistant tumors.

KLF4 as a rheostat of osteolysis and osteogenesis in prostate tumors in the bone.

We previously showed that KLF4, a gene highly expressed in murine prostate stem cells, blocks the progression of indolent intraepithelial prostatic lesions into aggressive and rapidly growing tumors. Here, we show that the anti-tumorigenic effect of KLF4 extends to PC3 human prostate cancer cells growing in the bone. We compared KLF4 null cells with cells transduced with a DOX-inducible KLF4 expression system, and find KLF4 function inhibits PC3 growth in monolayer and soft agar cultures. Furthermore, KLF4 null cells proliferate rapidly, forming large, invasive, and osteolytic tumors when injected into mouse femurs, whereas KLF4 re-expression immediately after their intra-femoral inoculation blocks tumor development and preserves a normal bone architecture. KLF4 re-expression in established KLF4 null bone tumors inhibits their osteolytic effects, preventing bone fractures and inducing an osteogenic response with new bone formation. In addition to these profound biological changes, KLF4 also induces a transcriptional shift from an osteolytic program in KLF4 null cells to an osteogenic program. Importantly, bioinformatic analysis shows that genes regulated by KLF4 overlap significantly with those expressed in metastatic prostate cancer patients and in three individual cohorts with bone metastases, strengthening the clinical relevance of the findings in our xenograft model.

De Novo PITX1 Expression Controls Bi-Stable Transcriptional Circuits to Govern Self-Renewal and Differentiation in Squamous Cell Carcinoma.

Basal tumor propagating cells (TPCs) control squamous cell carcinoma (SCC) growth by self-renewing and differentiating into supra-basal SCC cells, which lack proliferative potential. While transcription factors such as SOX2 and KLF4 can drive these behaviors, their molecular roles and regulatory interactions with each other have remained elusive. Here, we show that PITX1 is specifically expressed in TPCs, where it co-localizes with SOX2 and TRP63 and determines cell fate in mouse and human SCC. Combining gene targeting with chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analyses reveals that PITX1 cooperates with SOX2 and TRP63 to sustain an SCC-specific transcriptional feed-forward circuit that maintains TPC-renewal, while inhibiting KLF4 expression and preventing KLF4-dependent differentiation. Conversely, KLF4 represses PITX1, SOX2, and TRP63 expression to prevent TPC expansion. This bi-stable, multi-input network reveals a molecular framework that explains self-renewal, aberrant differentiation, and SCC growth in mice and humans, providing clues for developing differentiation-inducing therapeutic strategies.

KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer.

There is a considerable need to identify those individuals with prostate cancer who have indolent disease. We propose that genes that control adult stem cell homeostasis in organs with slow turnover, such as the prostate, control cancer fate. One such gene, KLF4, overexpressed in murine prostate stem cells, regulates their homeostasis, blocks malignant transformation, and controls the self-renewal of tumor-initiating cells. KLF4 loss induces the molecular features of aggressive cancer and converts PIN lesions to invasive sarcomatoid carcinomas; its re-expression in vivo reverses this process. Bioinformatic analysis links these changes to human cancer. KLF4 and its downstream targets make up a gene signature that identifies indolent tumors and predicts recurrence-free survival. This approach may improve prognosis and identify therapeutic targets for advanced cancer.

TGF-ß-Induced Quiescence Mediates Chemoresistance of Tumor-Propagating Cells in Squamous Cell Carcinoma.

Squamous cell carcinomas (SCCs) are heterogeneous tumors sustained by tumor-propagating cancer cells (TPCs). SCCs frequently resist chemotherapy through still unknown mechanisms. Here, we combine H2B-GFP-based pulse-chasing with cell-surface markers to distinguish quiescent from proliferative TPCs within SCCs. We find that quiescent TPCs resist DNA damage and exhibit increased tumorigenic potential in response to chemotherapy, whereas proliferative TPCs undergo apoptosis. Quiescence is regulated by TGF-ß/SMAD signaling, which directly regulates cell-cycle gene transcription to control a reversible G1 cell-cycle arrest, independent of p21CIP function. Indeed, genetic or pharmacological TGF-ß inhibition increases the susceptibility of TPCs to chemotherapy because it prevents entry into a quiescent state. These findings provide direct evidence that TPCs can reversibly enter a quiescent, chemoresistant state and thereby underscore the need for combinatorial approaches to improve treatment of chemotherapy-resistant SCCs.

ß-catenin signaling is required for RAS-driven thyroid cancer through PI3K activation.

Mutations in ß-catenin are traditionally described as late events in thyroid cancer progression. However, the functional implications of ß-catenin dysregulation in the context of tumor initiating events remain unclear. The aim of this work was to investigate whether the two main oncogenic drivers in thyroid cancer, RAS and BRAF, could activate the Wnt/ß-catenin pathway. Expression of HRASV12 but not BRAFV600E in thyroid cells induced ß-catenin nuclear localization, increased ß-catenin-dependent transcriptional activity and inhibited GSK3ß. In a panel of human thyroid cancer cell lines representative of the main genetic events in thyroid cancer, ß-catenin activation was highly dependent on PI3K/AKT activity through its phosphorylation at S552, but not on MAPK. Silencing of ß-catenin expression in cell lines led to a dramatic reduction in proliferation due to an induction of senescence, which was concordant with a reduction in tumor size in nude mice. Moreover, ß-catenin silencing suppressed the expression of EMT-related genes and reduced the invasive capacity of the tumor cells. In conclusion, this work demonstrates that RAS-driven tumors induce PI3K/AKT-dependent ß-catenin activation.

Selenium Increases Thyroid-Stimulating Hormone-Induced Sodium/Iodide Symporter Expression Through Thioredoxin/Apurinic/Apyrimidinic Endonuclease 1-Dependent Regulation of Paired Box 8 Binding Activity.

AIMS: The sodium-iodide symporter (NIS) mediates the uptake of I(-) by the thyroid follicular cell and is essential for thyroid hormone biosynthesis. Nis expression is stimulated by thyroid-stimulating hormone (TSH) and also requires paired box 8 (Pax8) to bind to its promoter. Pax8 binding activity depends on its redox state by a mechanism involving thioredoxin/thioredoxin reductase-1 (Txn/TxnRd1) reduction of apurinic/apyrimidinic endonuclease 1 (Ape1). In this study, we investigate the role of Se in Nis expression. RESULTS: Selenium increases TSH-induced Nis expression and activity in rat thyroid cells. The stimulatory effect of Se occurs at the transcriptional level and is only observed for Nis promoters containing a Pax8 binding site in the Nis upstream enhancer, suggesting that Pax8 is involved in this effect. In fact, Se increases Pax8 expression and its DNA-binding capacity, and in Pax8-silenced rat thyroid cells, Nis is not Se responsive. By inhibiting Ape1 and TxnRd1 functions, we found that both enzymes are crucial for TSH and TSH plus Se stimulation of Pax8 activity and mediate the Nis response to Se treatment. INNOVATION: We describe that Se increases Nis expression and activity. We demonstrate that this effect is dependent on the redox functions of Ape1 and Txn/TxnRd1 through control of the DNA binding activity of Pax8. CONCLUSION: Nis expression is controlled by Txn/Ape1 through a TSH/Se-dependent mechanism. These findings open a new field of study regarding the regulation of Nis activity in thyroid cells. Antioxid. Redox Signal. 24, 855-866.

The miR-146b-3p/PAX8/NIS Regulatory Circuit Modulates the Differentiation Phenotype and Function of Thyroid Cells during Carcinogenesis.

The presence of differentiated thyroid cells in thyroid cancer is critical for the antitumor response to radioactive iodide treatment, and loss of the differentiated phenotype is a key hallmark of iodide-refractory metastatic disease. The role of microRNAs (miRNA) in fine-tuning gene expression has become a major regulatory mechanism by which developmental and pathologic processes occur. In this study, we performed next-generation sequencing and expression analysis of eight papillary thyroid carcinomas (PTC) to comprehensively characterize miRNAs involved in loss of differentiation. We found that only a small set of abundant miRNAs is differentially expressed between PTC tissue and normal tissue from the same patient. In addition, we integrated computational prediction of potential targets and mRNA sequencing and identified a master miRNA regulatory network involved in essential biologic processes such as thyroid differentiation. Both mature products of mir-146b (miR-146b-5p and -3p) were among the most abundantly expressed miRNAs in tumors. Specifically, we found that miR-146b-3p binds to the 3'-untranslated region of PAX8 and sodium/iodide symporter (NIS), leading to impaired protein translation and a subsequent reduction in iodide uptake. Furthermore, our findings show that miR-146b and PAX8 regulate each other and share common target genes, thus highlighting a novel regulatory circuit that governs the differentiated phenotype of PTC. In conclusion, our study has uncovered the existence of a miR-146b-3p/PAX8/NIS regulatory circuit that may be exploited therapeutically to modulate thyroid cell differentiation and iodide uptake for improved treatment of advanced thyroid cancer.

SOX2 is a cancer-specific regulator of tumour initiating potential in cutaneous squamous cell carcinoma.

Although the principles that balance stem cell self-renewal and differentiation in normal tissue homeostasis are beginning to emerge, it is still unclear whether cancer cells with tumour initiating potential are similarly governed, or whether they have acquired distinct mechanisms to sustain self-renewal and long-term tumour growth. Here we show that the transcription factor Sox2, which is not expressed in normal skin epithelium and is dispensable for epidermal homeostasis, marks tumour initiating cells (TICs) in cutaneous squamous cell carcinomas (SCCs). We demonstrate that Sox2 is required for SCC growth in mouse and human, where it enhances Nrp1/Vegf signalling to promote the expansion of TICs along the tumour-stroma interface. Our findings suggest that distinct transcriptional programmes govern self-renewal and long-term growth of TICs and normal skin epithelial stem and progenitor cells. These programmes present promising diagnostic markers and targets for cancer-specific therapies.

Wnt-independent role of ß-catenin in thyroid cell proliferation and differentiation.

The Wnt/ß-catenin pathway has been associated with thyroid cell growth and tumorigenesis. However, little is known regarding its involvement in the response to the key regulators of thyroid cell proliferation and differentiation. Here we show that TSH and IGF-1 increase ß-catenin nuclear accumulation and its transcriptional activity in differentiated thyroid cells. This effect takes place in a Wnt-independent manner because TSH and IGF-1, through the activation of protein kinase A and protein kinase B/Akt, phosphorylate ß-catenin at S552 and S675, which results in ß-catenin release from E-cadherin at the adherens junctions. Nuclear ß-catenin regulates thyroid cell proliferation, because its silencing or the overexpression of a dominant-negative form of T-cell factor 4 resulted in reduced levels of cyclin D1 and DNA synthesis. Furthermore, the ß-catenin silencing markedly reduced the expression of Pax8, the main transcription factor involved in epithelial thyroid cell differentiation. Finally, we observed that ß-catenin physically interacts with the transcription factor Pax8, increasing its transcriptional activity on the sodium iodide symporter (NIS) gene, a critical gene required for thyroid cell physiology. Taken together, our findings show that ß-catenin plays a not yet described role in thyroid function including a functional interaction with Pax8.

An epistatic interaction between the PAX8 and STK17B genes in papillary thyroid cancer susceptibility.

Papillary Thyroid Cancer (PTC) is a heterogeneous and complex disease; susceptibility to PTC is influenced by the joint effects of multiple common, low-penetrance genes, although relatively few have been identified to date. Here we applied a rigorous combined approach to assess both the individual and epistatic contributions of genetic factors to PTC susceptibility, based on one of the largest series of thyroid cancer cases described to date. In addition to identifying the involvement of TSHR variation in classic PTC, our pioneer study of epistasis revealed a significant interaction between variants in STK17B and PAX8. The interaction was detected by MD-MBR (p = 0.00010) and confirmed by other methods, and then replicated in a second independent series of patients (MD-MBR p = 0.017). Furthermore, we demonstrated an inverse correlation between expression of PAX8 and STK17B in a set of cell lines derived from human thyroid carcinomas. Overall, our work sheds additional light on the genetic basis of thyroid cancer susceptibility, and suggests a new direction for the exploration of the inherited genetic contribution to disease using association studies.

Role of the wnt pathway in thyroid cancer.

Aberrant activation of Wnt signaling is involved in the development of several epithelial tumors. Wnt signaling includes two major types of pathways: (i) the canonical or Wnt/ß-catenin pathway; and (ii) the non-canonical pathways, which do not involve ß-catenin stabilization. Among these pathways, the Wnt/ß-catenin pathway has received most attention during the past years for its critical role in cancer. A number of publications emphasize the role of the Wnt/ß-catenin pathway in thyroid cancer. This pathway plays a crucial role in development and epithelial renewal, and components such as ß-catenin and Axin are often mutated in thyroid cancer. Although it is accepted that altered Wnt signaling is a late event in thyroid cell transformation that affects anaplastic thyroid tumors, recent data suggest that it is also altered in papillary thyroid carcinoma (PTC) with RET/PTC mutations. Therefore, the purpose of this review is to summarize the main relevant data of Wnt signaling in thyroid cancer, with special emphasis on the Wnt/ß-catenin pathway.