Transcriptomic and Proteomic Profiles for Elucidating Cisplatin Resistance in Head-and-Neck Squamous Cell Carcinoma.

To identify the novel genes involved in chemoresistance in head and neck squamous cell carcinoma (HNSCC), we explored the expression profiles of the following cisplatin (CDDP) resistant (R) versus parental (sensitive) cell lines by RNA-sequencing (RNA-seq): JHU029, HTB-43 and CCL-138. Using the parental condition as a control, 30 upregulated and 85 downregulated genes were identified for JHU029-R cells; 263 upregulated and 392 downregulated genes for HTB-43-R cells, and 154 upregulated and 68 downregulated genes for CCL-138-R cells. Moreover, we crossed-checked the RNA-seq results with the proteomic profiles of HTB-43-R (versus HTB-43) and CCL-138-R (versus CCL-138) cell lines. For the HTB-43-R cells, 21 upregulated and 72 downregulated targets overlapped between the proteomic and transcriptomic data; whereas in CCL-138-R cells, four upregulated and three downregulated targets matched. Following an extensive literature search, six genes from the RNA-seq (CLDN1, MAGEB2, CD24, CEACAM6, IL1B and ISG15) and six genes from the RNA-seq and proteomics crossover (AKR1C3, TNFAIP2, RAB7A, LGALS3BP, PSCA and SSRP1) were selected to be studied by qRT-PCR in 11 HNSCC patients: six resistant and five sensitive to conventional therapy. Interestingly, the high MAGEB2 expression was associated with resistant tumours and is revealed as a novel target to sensitise resistant cells to therapy in HNSCC patients.

TSPAN1, a novel tetraspanin member highly involved in carcinogenesis and chemoresistance.

The tetraspanin (TSPAN) family constitutes a poorly explored family of membrane receptors involved in various physiological processes, with relevant roles in anchoring multiple proteins, acting as scaffolding proteins, and cell signaling. Recent studies have increasingly demonstrated the involvement of TSPANs in cancer. In particular, tetraspanin 1 (also known as TSPAN1, NET-1, TM4C, C4.8 or GEF) has been implicated in cell survival, proliferation and invasion. Recently, our laboratory revealed a key role of TSPAN1 in the acquired resistance of tumor cells to conventional chemotherapy (e.g., cisplatin). In this review, we summarize and discuss the latest research on the physiological mechanisms of TSPANs in cancer and, in particular, on TSPAN1 regulating resistance to chemotherapy. A model of TSPAN1 action is proposed, and the potential of targeting TSPAN1 in anticancer therapeutic strategies is discussed.

Targeting Sphingolipids for Cancer Therapy.

Sphingolipids are an extensive class of lipids with different functions in the cell, ranging from proliferation to cell death. Sphingolipids are modified in multiple cancers and are responsible for tumor proliferation, progression, and metastasis. Several inhibitors or activators of sphingolipid signaling, such as fenretinide, safingol, ABC294640, ceramide nanoliposomes (CNLs), SKI-II, α-galactosylceramide, fingolimod, and sonepcizumab, have been described. The objective of this review was to analyze the results from preclinical and clinical trials of these drugs for the treatment of cancer. Sphingolipid-targeting drugs have been tested alone or in combination with chemotherapy, exhibiting antitumor activity alone and in synergism with chemotherapy in vitro and in vivo. As a consequence of treatments, the most frequent mechanism of cell death is apoptosis, followed by autophagy. Aslthough all these drugs have produced good results in preclinical studies of multiple cancers, the outcomes of clinical trials have not been similar. The most effective drugs are fenretinide and α-galactosylceramide (α-GalCer). In contrast, minor adverse effects restricted to a few subjects and hepatic toxicity have been observed in clinical trials of ABC294640 and safingol, respectively. In the case of CNLs, SKI-II, fingolimod and sonepcizumab there are some limitations and absence of enough clinical studies to demonstrate a benefit. The effectiveness or lack of a major therapeutic effect of sphingolipid modulation by some drugs as a cancer therapy and other aspects related to their mechanism of action are discussed in this review.

Characterization of genetically modified mice for phosphoglycerate mutase, a vitally-essential enzyme in glycolysis.

Glycolytic metabolism is closely involved in physiological homeostasis and pathophysiological states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been reported to exert certain physiological role in vitro, whereas its impact on glucose metabolism in vivo remains unclear. Here, we report the characterization of Pgam1 knockout mice. We observed that homozygous knockout mice of Pgam1 were embryonic lethal. Although we previously reported that both PGAM-1 and -2 affect global glycolytic profile of cancers in vitro, in vivo glucose parameters were less affected both in the heterozygous knockout of Pgam1 and in Pgam2 transgenic mice. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before.

Cold-inducible RNA binding protein promotes breast cancer cell malignancy by regulating Cystatin C levels.

Cold-inducible RNA binding protein (CIRBP) is a stress-responsive protein that promotes cancer development and inflammation. Critical to most CIRBP functions is its capacity to bind and posttranscriptionally modulate mRNA. However, a transcriptome-wide analysis of CIRBP mRNA targets in cancer has not yet been performed. Here, we use an ex vivo breast cancer model to identify CIRBP targets and mechanisms. We find that CIRBP transcript levels correlate with breast cancer subtype and are an indicator of luminal A/B prognosis. Accordingly, overexpression of CIRBP in nontumoral MCF-10A cells promotes cell growth and clonogenicity, while depletion of CIRBP from luminal A MCF-7 cells has opposite effects. We use RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) to identify a set of 204 high confident CIRBP targets in MCF-7 cells. About 10% of these showed complementary changes after CIRBP manipulation in MCF-10A and MCF-7 cells, and were highly interconnected with known breast cancer genes. To test the potential of CIRBP-mediated regulation of these targets in breast cancer development, we focused on Cystatin C (CST3), one of the most highly interconnected genes, encoding a protein that displays tumor suppressive capacities. CST3 depletion restored the effects of CIRBP depletion in MCF-7 cells, indicating that CIRBP functions, at least in part, by down-regulating CST3 levels. Our data provide a resource of CIRBP targets in breast cancer, and identify CST3 as a novel downstream mediator of CIRBP function.

Five microRNAs in Serum Are Able to Differentiate Breast Cancer Patients From Healthy Individuals.

Breast cancer is the cancer with the most incidence and mortality in women. microRNAs are emerging as novel prognosis/diagnostic tools. Our aim was to identify a serum microRNA signature useful to predict cancer development. We focused on studying the expression levels of 30 microRNAs in the serum of 96 breast cancer patients vs. 92 control individuals. Bioinformatic studies provide a microRNA signature, designated as a predictor, based on the expression levels of five microRNAs. Then, we tested the predictor in a group of 60 randomly chosen women. Lastly, a proteomic study unveiled the overexpression and downregulation of proteins differently expressed in the serum of breast cancer patients vs. that of control individuals. Twenty-six microRNAs differentiate cancer tissue from healthy tissue, and 16 microRNAs differentiate the serum of cancer patients from that of the control group. The tissue expression of miR-99a, miR-497, miR-362, and miR-1274, and the serum levels of miR-141 correlated with patient survival. Moreover, the predictor consisting of miR-125b, miR-29c, miR-16, miR-1260, and miR-451 was able to differentiate breast cancer patients from controls. The predictor was validated in 20 new cases of breast cancer patients and tested in 60 volunteer women, assigning 11 out of 60 women to the cancer group. An association of low levels of miR-16 with a high content of CD44 protein in serum was found. Circulating microRNAs in serum can represent biomarkers for cancer prediction. Their clinical relevance and the potential use of the predictor here described are discussed.

Autophagy Takes Center Stage as a Possible Cancer Hallmark.

Cancer remains one of the leading causes of death worldwide, despite significant advances in cancer research and improvements in anticancer therapies. One of the major obstacles to curing cancer is the difficulty of achieving the complete annihilation of resistant cancer cells. The resistance of cancer cells may not only be due to intrinsic factors or factors acquired during the evolution of the tumor but may also be caused by chemotherapeutic treatment failure. Conversely, autophagy is a conserved cellular process in which intracellular components, such as damaged organelles, aggregated or misfolded proteins and macromolecules, are degraded or recycled to maintain cellular homeostasis. Importantly, autophagy is an essential mechanism that plays a key role in tumor initiation and progression. Depending on the cellular context and microenvironmental conditions, autophagy acts as a double-edged sword, playing a role in inducing apoptosis or promoting cell survival. In this review, we propose several scenarios in which autophagy could contribute to cell survival or cell death. Moreover, a special focus on novel promising targets and therapeutic strategies based on autophagic resistant cells is presented.

TSPAN1: A Novel Protein Involved in Head and Neck Squamous Cell Carcinoma Chemoresistance.

Sensitization of resistant cells and cancer stem cells (CSCs) represents a major challenge in cancer therapy. A proteomic study revealed tetraspanin-1 (TSPAN1) as a protein involved in acquisition of cisplatin (CDDP) resistance (Data are available via ProteomeXchange with identifier PXD020159). TSPAN1 was found to increase in CDDP-resistant cells, CSCs and biopsies from head and neck squamous cell carcinoma (HNSCC) patients. TSPAN1 depletion in parental and CDDP-resistant HNSCC cells reduced cell proliferation, induced apoptosis, decreased autophagy, sensitized to chemotherapeutic agents and inhibited several signaling cascades, with phospho-SRC inhibition being a major common target. Moreover, TSPAN1 depletion in vivo decreased the size and proliferation of parental and CDDP-resistant tumors and reduced metastatic spreading. Notably, CDDP-resistant tumors showed epithelial-mesenchymal transition (EMT) features that disappeared upon TSPAN1 inhibition, suggesting a link of TSPAN1 with EMT and metastasis. Immunohistochemical analysis of HNSCC specimens further revealed that TSPAN1 expression was correlated with phospho-SRC (pSRC), and inversely with E-cadherin, thus reinforcing TSPAN1 association with EMT. Overall, TSPAN1 emerges as a novel oncogenic protein and a promising target for HNSCC therapy.

Phosphoglycerate Mutase Cooperates with Chk1 Kinase to Regulate Glycolysis.

Dysregulated glycolysis, including the cancerous Warburg effect, is closely involved in pathological mechanisms of diseased states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been known to exert certain physiological impact in vitro, whereas its regulatory role on glycolysis remains unclear. Here, we identified that PGAM plays a key role in regulating glycolysis in cancer cells but not in standard cells. Cancer-prone phenotype by PGAM overexpression in vivo was associated with upregulated glycolytic features. PGAM interacts and cooperates with Chk1 to regulate the enhanced glycolysis in cancer cells, especially under oncogenic Ras expressing conditions. Genetic or chemical interference of the PGAM-Chk1 interaction, with intact PGAM activity, abrogated the maintenance of cancerous enhanced glycolysis. Thus, the nonenzymatic function of PGAM is essential for the Warburg effect that accompanies cancerous proliferation.

Tumor Profiling at the Service of Cancer Therapy.

Cancer treatment options have evolved significantly in the past few years. From the initial surgical procedures, to the latest next-generation technologies, we are now in the position to analyze and understand tumors in a one-by-one basis and use that to our advantage to provide with individualized treatment options that may increase patient survival. In this review, we will focus on how tumor profiling has evolved over the past decades to deliver more efficient and personalized treatment options, and how novel technologies can help us envisage the future of precision oncology toward a better management and, ultimately, increased survival.

Autophagy inhibition as a promising therapeutic target for laryngeal cancer.

To identify the putative relevance of autophagy in laryngeal cancer, we performed an immunohistochemistry study to analyze the expression of the proteins involved in this process, namely, LC3, ATG5 and p62/SQSTM1. Additionally, Prostate tumor-overexpressed gene 1 protein (PTOV1) was included due to its potential relevance in laryngeal cancer. Moreover, as cancer resistance might involve autophagy in some circumstances, we studied the intrinsic drug resistance capacity of primary tumor cultures derived from 13 laryngeal cancer biopsies and their expression levels of LC3, ATG5, p62 and PTOV1. Overall, our results suggest that (i) cytoplasmic p62 and PTOV1 can be considered prognostic markers in laryngeal cancer, (ii) the acquisition of resistance seems to be related to PTOV1 and autophagy-related protein overexpression, (iii) by increasing autophagy, PTOV1 might contribute to resistance in this model and (iv) the expression of autophagy-related proteins could classify a subgroup of laryngeal cancer patients who will benefit from a therapy based upon autophagy inhibition. Our study suggests that autophagy inhibition with hydroxychloroquine could be a promising strategy for laryngeal cancer patients, particularly those patients with high resistance to the CDDP treatment that in addition have autophagy upregulation.

Common Metabolic Pathways Implicated in Resistance to Chemotherapy Point to a Key Mitochondrial Role in Breast Cancer.

Cancer cells are known to reprogram their metabolism to adapt to adverse conditions dictated by tumor growth and microenvironment. A subtype of cancer cells with stem-like properties, known as cancer stem cells (CSC), is thought to be responsible for tumor recurrence. In this study, we demonstrated that CSC and chemoresistant cells derived from triple negative breast cancer cells display an enrichment of up- and downregulated proteins from metabolic pathways that suggests their dependence on mitochondria for survival. Here, we selected antibiotics, in particular - linezolid, inhibiting translation of mitoribosomes and inducing mitochondrial dysfunction. We provided the first in vivo evidence demonstrating that linezolid suppressed tumor growth rate, accompanied by increased autophagy. In addition, our results revealed that bactericidal antibiotics used in combination with autophagy blocker decrease tumor growth. This study puts mitochondria in a spotlight for cancer therapy and places antibiotics as effective agents for eliminating CSC and resistant cells.

Otologic, audiometric and speech findings in patients undergoing surgery for cleft palate.

BACKGROUND: Although considerable progress has been made in the last 30 years in the treatment of cleft palate (CP), a multidisciplinary approach combining examinations by a paediatrician, maxillofacial surgeon, otolaryngologist and speech and language pathologist followed by surgical operation is still required. In this work, we performed an observational cross-sectional study to determine whether the CP grade or number of ventilation tubes received was associated with tympanic membrane abnormalities, hearing loss or speech outcomes. METHODS: Otologic, audiometric, tympanometric and speech evaluations were performed in a cohort of 121 patients (children > 6 years) who underwent an operation for CP at the Vall d'Hebron Hospital, Barcelona from 2000 to 2014. RESULTS: The most and least frequent CP types evaluated according to the Veau grade were type III (55.37%) and I (8.26%), respectively. A normal appearance of the membrane was observed in 58% individuals, of whom 55% never underwent ventilation ear tube insertion. No statistically significant associations were identified between the CP type and number of surgeries for insertion of tubes (p = 0.820). The degree of hearing loss (p = 0.616), maximum impedance (p = 0.800) and tympanic membrane abnormalities indicative of chronic otitis media (COM) (p = 0.505) among examined patients revealed no statistically significant association with the grade of CP. However, an association was identified between hypernasality and the grade of CP (p = 0.053), COM (p = 0.000), hearing loss (p = 0.000) and number of inserted ventilation tubes. CONCLUSION: Although the placement of tympanic ventilation tubes has been accompanied by an increased rate of COM, it is still important to assess whether this is a result of the number of ventilation tubes inserted or it is intrinsic to the natural history of middle ear inflammatory disease of such patients. Our results do not support improvements in speech, hearing, or tympanic membrane abnormalities with more aggressive management of COM with tympanostomy tubes.

Expression patterns and bioinformatic analysis of miR-1260a and miR-1274a in Prostate Cancer Tunisian patients.

Currently, microRNAs (miRs) represent great biomarkers in cancer due to their stability and their potential role in diagnosis, prognosis and therapy. This study aims to evaluate the expression levels of miRs-1260 and -1274a in prostate cancer (PC) samples and to identify their eventual targets by using bioinformatic analysis. In this project, we evaluated the expression status of miRs-1260 and -1274a in 86 PC patients and 19 controls by using real-time quantitative PCR and 2-ΔΔCt method. Moreover, we retrieved validated and predicted targets of miRs from several datasets by using the "multiMir" R/Bioconductor package. We have found that miRs-1260 and -1274a were over-expressed in PC patients compared to controls (p < 1 × 10-5). Moreover ROC curve for miRs-1260 and 1274a showed a good performance to distinguish between controls group and PC samples with an area under the ROC curve of 0.897 and 0.784 respectively. However, no significant association could be shown between these two miRs and clinical parameters such as PSA levels, Gleason score, tumor stage, D'Amico classification, lymph node metastasis statues, tumor recurrence, metastasis status and progression after a minimum of 5 years follow-up. Finally, a bioinformatic analysis revealed the association between these two miRs and several targets implicated in prostate cancer initiation pathways.

The interplay between autophagy and tumorigenesis: exploiting autophagy as a means of anticancer therapy.

In wild-type cells, autophagy represents a tumour-suppressor mechanism, and dysfunction of the autophagy machinery increases genomic instability, DNA damage, oxidative stress and stem/progenitor expansion, which are events associated with cancer onset. Autophagy occurs at a basal level in all cells depending on cell type and cellular microenvironment. However, the role of autophagy in cancer is diverse and can promote different outcomes even in a single tumour. For example, in hypoxic tumour regions, autophagy emerges as a protective mechanism and allows cancer cell survival. By contrast, in cancer cells surrounding the tumour mass, the induction of autophagy by radio- or chemotherapy promotes cell death and significantly reduces the tumour mass. Importantly, inhibition of autophagy compromises tumorigenesis by mechanisms that are not entirely understood. The aim of this review is to explain the apparently contradictory role of autophagy as a mechanism that both promotes and inhibits tumorigenesis using different models. The induction/inhibition of autophagy as a mechanism for cancer treatment is also discussed.

Reactive Oxygen Species-Mediated Autophagy Defines the Fate of Cancer Stem Cells.

Significance: A fraction of tumorigenic cells, also known as tumor initiating or cancer stem cells (CSCs), is thought to drive tumor growth, metastasis, and chemoresistance. However, little is known regarding mechanisms that convey relevant pathways contributing to their self-renewal, proliferation, and differentiation abilities. Recent Advances: Recent works on CSCs provide evidence on the role of redox disruption and regulation of autophagic flux. This has been linked to increased DNA repair capacity and chemoresistance. Critical Issues: The current review summarizes the most recent studies assessing the role of redox homeostasis, autophagy, and chemoresistance in CSCs, including some novel findings on microRNAs and their role in horizontal transfer within cancer cell populations. Future Directions: Rational anticancer therapy and prevention should rely on the fact that cancer is a redox disease with the CSCs being the apex modulated by redox-mediated autophagy. Antioxid. Redox Signal. 28, 1066-1079.

miR-99a reveals two novel oncogenic proteins E2F2 and EMR2 and represses stemness in lung cancer.

Lung cancer is one of the most aggressive tumours with very low life expectancy. Altered microRNA expression is found in human tumours because it is involved in tumour growth, progression and metastasis. In this study, we analysed microRNA expression in 47 lung cancer biopsies. Among the most downregulated microRNAs we focussed on the miR-99a characterisation. In vitro experiments showed that miR-99a expression decreases the proliferation of H1650, H1975 and H1299 lung cancer cells causing cell cycle arrest and apoptosis. We identified two novel proteins, E2F2 (E2F transcription factor 2) and EMR2 (EGF-like module-containing, mucin-like, hormone receptor-like 2), downregulated by miR-99a by its direct binding to their 3'-UTR. Moreover, miR-99a expression prevented cancer cell epithelial-to-mesenchymal transition (EMT) and repressed the tumourigenic potential of the cancer stem cell (CSC) population in both these cell lines and mice tumours originated from H1975 cells. The expression of E2F2 and EMR2 at protein level was studied in 119 lung cancer biopsies. E2F2 and EMR2 are preferentially expressed in adenocarcinomas subtypes versus other tumour types (squamous and others). Interestingly, the expression of E2F2 correlates with the presence of vimentin and both E2F2 and EMR2 correlate with the presence of ß-catenin. Moreover, miR-99a expression correlates inversely with E2F2 and directly with ß-catenin expression in lung cancer biopsies. In conclusion, miR-99a reveals two novel targets E2F2 and EMR2 that play a key role in lung tumourigenesis. By inhibiting E2F2 and EMR2, miR-99a represses in vivo the transition of epithelial cells through an EMT process concomitantly with the inhibition of stemness features and consequently decreasing the CSC population.

DNA Methylomes Reveal Biological Networks Involved in Human Eye Development, Functions and Associated Disorders.

This work provides a comprehensive CpG methylation landscape of the different layers of the human eye that unveils the gene networks associated with their biological functions and how these are disrupted in common visual disorders. Herein, we firstly determined the role of CpG methylation in the regulation of ocular tissue-specification and described hypermethylation of retinal transcription factors (i.e., PAX6, RAX, SIX6) in a tissue-dependent manner. Second, we have characterized the DNA methylome of visual disorders linked to internal and external environmental factors. Main conclusions allow certifying that crucial pathways related to Wnt-MAPK signaling pathways or neuroinflammation are epigenetically controlled in the fibrotic disorders involved in retinal detachment, but results also reinforced the contribution of neurovascularization (ETS1, HES5, PRDM16) in diabetic retinopathy. Finally, we had studied the methylome in the most frequent intraocular tumors in adults and children (uveal melanoma and retinoblastoma, respectively). We observed that hypermethylation of tumor suppressor genes is a frequent event in ocular tumors, but also unmethylation is associated with tumorogenesis. Interestingly, unmethylation of the proto-oncogen RAB31 was a predictor of metastasis risk in uveal melanoma. Loss of methylation of the oncogenic mir-17-92 cluster was detected in primary tissues but also in blood from patients.