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20 p.-8 fig.-1 tab., Endoglin (CD105) is an auxiliary receptor of transforming growth factor (TGF)-β family members that is expressed in human melanomas. It is heterogeneously expressed by primary and metastatic melanoma cells, and endoglin targeting as a therapeutic strategy for melanoma tumors is currently been explored. However, its involvement in tumor development and malignancy is not fully understood. Here, we find that endoglin expression correlates with malignancy of primary melanomas and cultured melanoma cell lines. Next, we have analyzed the effect of ectopic endoglin expression on two miRNAs (hsa-mir-214 and hsa-mir-370), both involved in melanoma tumor progression and endoglin regulation. We show that compared with control cells, overexpression of endoglin in the WM-164 melanoma cell line induces; (i) a significant increase of hsa-mir-214 levels in small extracellular vesicles (EVs) as well as an increased trend in cells; and (ii) significantly lower levels of hsa-mir-370 in the EVs fractions, whereas no significant differences were found in cells. As hsa-mir-214 and hsa-mir-370 are not just involved in melanoma tumor progression, but they can also target endoglin-expressing endothelial cells in the tumor vasculature, these results suggest a complex and differential regulatory mechanism involving the intracellular and extracellular signaling of hsa-mir-214 and hsa-mir-370 in melanoma development and progression., This work was supported by grants from Ministerio de Ciencia, Innovación y Universidades (SAF2013-43421-R to CB), Consejo Superior de Investigaciones Científicas (201920E022 to CB), Fondo de Investigación Sanitaria (FIS) de la Seguridad Social (PI-20/01553 to JLR-P), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER;ISCIII-CB06/07/0038 to CB and contract CNV-234-PRF-360 to LR-L) of Spain. CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds. The CNIO, certified as a Severo Ochoa Excellence Centre, is supported by the Spanish government through the ISCIII.

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ERlo/Notchhi feed-forward loop responsible for the generation of CD133hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6–pStat3 pathway promoted the biogenesis of onco-miR-221hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSCs. Conversely, in models of CD133neg, HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927–41. ©2017 AACR.

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INTRODUCTION Analgesics are widely used, but evidence regarding whether their use increases the risk of inflammatory bowel disease (IBD) flares or complications is unclear. Therefore, self-medication with analgesics in IBD is usually not recommended. The aim of this study was to explore the prevalence of self-medication with analgesics in a cohort of ulcerative colitis (UC) patients and to identify reasons and factors associated with self-medication. METHODS This cross-sectional study included consecutive unselected adult patients with UC. Participants were asked to complete an anonymous web-based survey with multiple-choice questions and closed responses. No clinical data were collected. RESULTS A total of 546 patients (61.2% women, mean age 39.9 years) completed the survey. The prevalence of self-medication with analgesics was 49.8% (272/546). Paracetamol (45.2%) and metamizole (21.2%) were the most frequently used drugs; frequencies of self-medication were

PurposeMalignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive softtissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGF-β coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs.Experimental DesignENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathways and in vivo MPNST growth and metastasis were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models.ResultsENG expression was found to be upregulated in both human MPNST tumor tissues and plasma circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST tumor growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Importantly, combination of anti-ENG therapy with MEK inhibition reduced more effectively tumor cell growth and angiogenesis.ConclusionsOur data unveil a tumor-promoting function of ENG in MPNSTs and support the combined use of anti-ENG antibodies and MEK inhibitors as a novel potential combination to control MPNSTs growth and metastasis.Statement of translational relevanceMalignant peripheral nerve sheath tumors (MPNSTs) present a poor clinical outcome due to tumor aggressiveness and the absence of effective treatments that underline the need for identifying novel therapeutic approaches. Here, we show that ENG is upregulated in human MPNSTs and we uncover an important role for this coreceptor in MPNST. ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST tumor growth and metastasis in xenograft models, supporting a novel use of anti-ENG therapies for MPNST treatment. Currently, MEK inhibitors are used in the clinic to shrink peripheral nerve sheath tumors (e.g. plexiform neurofibromas). Importantly, we demonstrate that combination of anti ENG antibodies with MEK inhibitors efficiently blocked MPNST growth and metastasis. Our findings provide a rationale for combining anti-ENG and MEK inhibitors as a new strategy for MPNST management.

BACKGROUND Few studies have examined self-medication with corticosteroids among patients with ulcerative colitis (UC). AIMS To assess the frequency of self-medication with oral corticosteroids in UC patients, and associated factors and reasons. METHODS An anonymous, voluntary, web-based survey was administered to adults with UC recruited via a Spanish patient association (ACCU) and hospital gastroenterology departments. Information was provided by patients; no clinical data were collected. Descriptive statistics and comparisons of frequencies are displayed. RESULTS Among 546 respondents (mean age 39.9 years, median duration of UC since diagnosis 7 years,) 36 (6.6%) reported self-medication with oral corticosteroids during the past year (once: 23 patients; 2-3 times: 10 patients; >3 times: 3 patients). Self-medication was more common among patients managed in general gastroenterology vs. inflammatory bowel disease clinics [23 (9.0%) vs. 11 (2.9%), P = 0.019], patients with no regular follow-up [4 (22.2%) vs. 32 (6.1%), P = 0.026] and patients with more flares (P < 0.001). Patients who stored steroids from previous flares (17.9% vs. 6.0%, P < 0.001) or who lived with a partner taking steroids (9.3% vs. 1.1%, P = 0.038) were more likely to self-medicate than other patients. Common reasons for self-medicating included the need for quick symptom relief (55.6%), fear of worsening (47.2%) and difficulty in getting an appointment (25.0%). Only seven patients (19.4%) informed their physician when they started self-medicating and only four (11.1%) declared they would not start corticosteroids again. CONCLUSION Self-medication with oral corticosteroids is not a common practice among patients with UC in Spain, but several areas of improvement exist.

Background: Despite the continuous adaptation of eHealth systems for patients with inflammatory bowel disease (IBD), a significant disconnection persists between users and developers. Since non-adherence remains high, it is necessary to better understand the patients&rsquo, perspective on telemonitoring for IBD. Accordingly, this study aimed to adapt the TECCU telemonitoring app to the preferences and needs of IBD patients. Methods: A qualitative study was carried out using successive focus groups of IBD patients. Meetings were audio-recorded and a thematic analysis was employed until data saturation was achieved. The first group included patients who had used the TECCU App in a pilot clinical trial, and subsequent meetings included patients with Crohn's disease and ulcerative colitis recruited from the Spanish Confederation of patient associations. The information collected at each meeting guided consecutive changes to the platform. Results: Data saturation was reached after three focus groups involving a total of 18 patients. Three main themes emerged: (1) platform usability, (2) the communication process, and (3) platform content. All participants indicated that TECCU is easy to use, permitting continuous and personalized feedback. According to patients&acute, perspectives, the platform was adapted to foster a flexible follow-up and shared decision-making using open and safe communication networks. Many participants appreciated the educational elements and, consequently, the app was connected to reliable and continuously updated webpages. Conclusions: IBD patients valued the usability and personalized monitoring offered by the TECCU App. Improvements in the messaging system and continuously updated educational content were introduced to address patients&acute, needs and favor their engagement.

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ERlo/Notchhi feed-forward loop responsible for the generation of CD133hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6–pStat3 pathway promoted the biogenesis of onco-miR-221hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSCs. Conversely, in models of CD133neg, HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927–41. ©2017 AACR.

Supplementation of micronutrients like folate is a double-edged sword in terms of their ambivalent role in cell metabolism. Although several epidemiological studies support a protective role of folate in carcinogenesis, there are also data arguing for an opposite effect. To address this issue in the context of human papillomavirus (HPV)-induced transformation, the molecular events of different folate availability on human keratinocytes immortalized by HPV16 E6 and E7 oncoproteins were examined. Several sublines were established: Control (4.5 µM folate), folate deficient (0.002 µM folate), and repleted cells (4.5 µM folate). Cells were analyzed in terms of oncogene expression, DNA damage and repair, karyotype changes, whole-genome sequencing, and transcriptomics. Here we show that folate depletion irreversibly induces DNA damage, impairment of DNA repair fidelity, and unique chromosomal alterations. Repleted cells additionally underwent growth advantage and enhanced clonogenicity, while the above mentioned impaired molecular properties became even more pronounced. Overall, it appears that a period of folate deficiency followed by repletion can shape immortalized cells toward an anomalous phenotype, thereby potentially contributing to carcinogenesis. These observations should elicit questions and inquiries for broader additional studies regarding folate fortification programs, especially in developing countries with micronutrient deficiencies and high HPV prevalence.

Innate immunity is the first line of host defense against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus 38 (HPV38) E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor Toll-like receptor 9 (TLR9) in human foreskin keratinocytes (HFK), a key mediator of the antiviral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA downregulation by promoting accumulation of ΔNp73α, an antagonist of p53 and p73. Inhibition of ΔNp73α expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of ΔNp73α in this event. Chromatin immunoprecipitation experiments showed that ΔNp73α is part of a negative transcriptional regulatory complex with IκB kinase beta (IKKβ) that binds to a NF-κB responsive element within the TLR9 promoter. In addition, the Polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex, leading to histone 3 trimethylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21 WAF1 and p27 Kip1 , decreased CDK2-associated kinase activity, and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can downregulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation. IMPORTANCE The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV38 downregulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated downregulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation.

The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNAhi EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS. In summary, we have demonstrated that the horizontal transfer of mtDNA from EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in OXPHOS-dependent breast cancer.

Folate is an essential water-soluble vitamin in food and nutrition supplements. As a one-carbon source, it is involved in many central regulatory processes, such as DNA, RNA, and protein methylation as well as DNA synthesis and repair. Deficiency in folate is considered to be associated with an increased incidence of several malignancies, including cervical cancer that is etiologically linked to an infection with “high-risk” human papilloma viruses (HPV). However, it is still not known how a recommended increase in dietary folate after its deprivation affects the physiological status of cells. To study the impact of folate depletion and its subsequent reconstitution in single cells, we used quantitative chromatin conformation measurements obtained by super-resolution fluorescence microscopy, i.e., single molecule localization microscopy (SMLM). As a read-out, we examined the levels and the (re)positioning of γ-H2AX tags and histone H3K9me3 heterochromatin tags after immunostaining in three-dimensional (3D)-conserved cell nuclei. As model, we used HPV16 positive immortalized human keratinocytes that were cultivated under normal, folate deficient, and reconstituted conditions for different periods of time. The results were compared to cells continuously cultivated in standard folate medium. After 13 weeks in low folate, an increase in the phosphorylation of the histone H2AX was noted, indicative of an accumulation of DNA double strand breaks. DNA repair activity represented by the formation of those γ-H2AX clusters was maintained during the following 15 weeks of examination. However, the clustered arrangements of tags appeared to relax in a time-dependent manner. Parallel to the repair activity, the chromatin methylation activity increased as detected by H3K9me3 tags. The progress of DNA double strand repair was accompanied by a reduction of the detected nucleosome density around the γ-H2AX clusters, suggesting a shift from hetero- to euchromatin to allow access to the repair machinery. In conclusion, these data demonstrated a folate-dependent repair activity and chromatin re-organization on the SMLM nanoscale level. This offers new opportunities to further investigate folate-induced chromatin re-organization and the associated mechanisms.

Folate is an essential water-soluble vitamin in food and nutrition supplements. As a one-carbon source, it is involved in many central regulatory processes, such as DNA, RNA, and protein methylation as well as DNA synthesis and repair. Deficiency in folate is considered to be associated with an increased incidence of several malignancies, including cervical cancer that is etiologically linked to an infection with “high-risk” human papilloma viruses (HPV). However, it is still not known how a recommended increase in dietary folate after its deprivation affects the physiological status of cells. To study the impact of folate depletion and its subsequent reconstitution in single cells, we used quantitative chromatin conformation measurements obtained by super-resolution fluorescence microscopy, i.e., single molecule localization microscopy (SMLM). As a read-out, we examined the levels and the (re)positioning of γ-H2AX tags and histone H3K9me3 heterochromatin tags after immunostaining in three-dimensional (3D)-conserved cell nuclei. As model, we used HPV16 positive immortalized human keratinocytes that were cultivated under normal, folate deficient, and reconstituted conditions for different periods of time. The results were compared to cells continuously cultivated in standard folate medium. After 13 weeks in low folate, an increase in the phosphorylation of the histone H2AX was noted, indicative of an accumulation of DNA double strand breaks. DNA repair activity represented by the formation of those γ-H2AX clusters was maintained during the following 15 weeks of examination. However, the clustered arrangements of tags appeared to relax in a time-dependent manner. Parallel to the repair activity, the chromatin methylation activity increased as detected by H3K9me3 tags. The progress of DNA double strand repair was accompanied by a reduction of the detected nucleosome density around the γ-H2AX clusters, suggesting a shift from hetero- to euchromatin to allow access to the repair machinery. In conclusion, these data demonstrated a folate-dependent repair activity and chromatin re-organization on the SMLM nanoscale level. This offers new opportunities to further investigate folate-induced chromatin re-organization and the associated mechanisms.

A high-throughput matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS)-based method was here developed to genotype 16 high-risk human papillomavirus (HPV) types in cervical cytology specimens. This method was compared to a commercial kit, the Inno-LiPA HPV genotyping assay, which detects a broad spectrum of HPV types. HPV DNA was assessed by the two methods in a total of 325 cervical cytology specimens collected in PreservCyt® solution. The overall agreement was almost perfect (Cohen's k=0.86) in term of positive and negative cases. Indeed, HPV types 16, 35, 56 and 66 showed the highest agreement values (>0.80). The highest agreement values (K >0.80) were found for all 16 HPV types in single infections, but only for HPV 16, 35, 45 and 56 in multiple infections. In conclusion, the high-throughput MS-based method developed here is well-suited for broad spectrum HPV genotyping in large-scale epidemiological studies.

Transapical aortic valve implantation is being employed as a less invasive alternative to open heart surgery in high-risk patients with severe aortic stenosis. Here we report the case of an awake transapical aortic valve implantation in a patient with severe chronic obstructive pulmonary disease.

BACKGROUND: Glioblastoma Multiforme (GBM) is the most common malignant brain tumor in adults, but its aetiology remains unknown. Recent reports have demonstrated the presence of neurotropic viruses in glioma samples, leading to the hypothesis that viral infection could be involved in tumorigenesis. AIM OF THE STUDY: To assess the burden of HPV infection in a cohort of malignant gliomas. MATERIALS AND METHODS: Seventy-one fresh frozen tumour samples of 62 glioblastomas and 9 grade III astrocytomas have been retrospectively analyzed. The presence of HPV genome in tumour DNA was assessed by MY/GP nested PCR. PCR products were sequenced with Sanger technology in order to define the viral genotype involved in the infection. A PCR with genotype-specific primer sets was carried out as confirmation of the infection. RESULTS: Viral DNA was detected after PCR in 8 (11%) cases. According to our previous studies both low- and high risk genotypes were isolated. Sequencing analysis showed that HPV16 genome was present in 4 (50%) infected samples whereas the remaining 4 resulted positive to HPV6. All positive cases were diagnosed as primary glioblastomas, and no positivity was found in lower grade glioma samples. Median overall survival did not significantly differ between HPV-positive and HPV-negative patients. CONCLUSIONS: The presence of HPV in high grade glioma specimens deserves further investigation. The analysis of a large cohort of tumors, along with in vitro studies on glioblastoma cell lines, will allow to better clarify the role of HPV in gliomagenesis and assess its potential use as a prognostic factor.