Telomeres and telomerase in head and neck squamous cell carcinoma: from pathogenesis to clinical implications.

Strongly associated with tobacco use, heavy alcohol consumption, and with high-risk human papillomavirus (HPV) infection, head and neck squamous cell carcinoma (HNSCC) is a frequently lethal, heterogeneous disease whose pathogenesis is a multistep and multifactorial process involving genetic and epigenetic events. The majority of HNSCC patients present with locoregional advanced stage disease and are treated with combined modality strategies that can markedly impair quality of life and elicit unpredictable results. A large fraction of those who undergo locoregional treatment and achieve a complete response later develop locoregional recurrences or second field tumors. Biomarkers that are thus able to stratify risk and enable clinicians to tailor treatment plans and to personalize post-therapeutic surveillance strategies are highly desirable. To date, only HPV status is considered a reliable independent predictor of treatment response and survival in patients with HNSCC arising from the oropharyngeal site. Recent studies suggest that telomere attrition, which may be an early event in human carcinogenesis, and telomerase activation, which is detected in up to 90 % of malignancies, could be potential markers of cancer risk and disease outcome. This review examines the current state of knowledge on and discusses the implications linked to telomere dysfunction and telomerase activation in the development and clinical outcome of HNSCC.

Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.

Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro.

Short-Term TERT Inhibition Impairs Cellular Proliferation via a Telomere Length-Independent Mechanism and Can Be Exploited as a Potential Anticancer Approach.

Telomerase reverse transcriptase (TERT), the catalytic component of telomerase, may also contribute to carcinogenesis via telomere-length independent mechanisms. Our previous in vitro and in vivo studies demonstrated that short-term telomerase inhibition by BIBR1532 impairs cell proliferation without affecting telomere length. Here, we show that the impaired cell cycle progression following short-term TERT inhibition by BIBR1532 in in vitro models of B-cell lymphoproliferative disorders, i.e., Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines (LCLs), and B-cell malignancies, i.e., Burkitt's lymphoma (BL) cell lines, is characterized by a significant reduction in NF-κB p65 nuclear levels leading to the downregulation of its target gene MYC. MYC downregulation was associated with increased expression and nuclear localization of P21, thus promoting its cell cycle inhibitory function. Consistently, treatment with BIBR1532 in wild-type zebrafish embryos significantly decreased Myc and increased p21 expression. The combination of BIBR1532 with antineoplastic drugs (cyclophosphamide or fludarabine) significantly reduced xenografted cells' proliferation rate compared to monotherapy in the zebrafish xenograft model. Overall, these findings indicate that short-term inhibition of TERT impairs cell growth through the downregulation of MYC via NF-κB signalling and supports the use of TERT inhibitors in combination with antineoplastic drugs as an efficient anticancer strategy.

TERT promoter mutations in head and neck squamous cell carcinoma: A systematic review and meta-analysis on prevalence and prognostic significance.

OBJECTIVES: To estimate the prevalence of two most common and mutually exclusive -124 C > T and -146 C > T TERT promoter mutations in HNSCC and analyse their prognostic role. MATERIALS AND METHODS: The databases Medline (via Ovid), Embase (via Ovid), Cochrane Library, Scopus, and Web of Science (Core Collection) were searched from inception to December 2022 to identify studies analysing TERT promoter mutations in HNSCC. Pooled prevalence of TERT promoter mutations and hazard ratio (sHR) of death/progression, with corresponding confidence intervals (CI), were estimated. RESULTS: The initial search returned 6416 articles, of which 17 studies, including 1830 patients, met the criteria for prevalence meta-analysis. Among them, 8 studies fitted the inclusion criterion to analyse the prognostic impact of TERT promoter mutations. Overall, 21% (95% CI: 12%-31%) of HNSCCs harboured TERT promoter mutation. TERT promoter mutations were more commonly found in oral cavity cancer (prevalence = 47%, 95% CI: 33%-61%), followed by laryngeal/hypopharyngeal cancer (prevalence = 12%, 95% CI: 4%-25%), while they were quite rare in oropharyngeal cancer (prevalence = 1%, 95% CI: 0%-4%). TERT promoter mutation -124 C > T was associated with a higher risk of death (sHR = 2.01, 95% CI: 1.25-3.23) and progression (sHR = 2.79, 95% CI: 1.77-4.40), while -146 C > T TERT promoter mutation did not show any significant correlation neither to overall nor progression-free survival. CONCLUSION: TERT promoter mutations were mainly topographically restricted to oral cavity cancer. -124 C > T was the most common TERT promoter mutation and was significantly associated to worse outcome in HNSCC.

Telomere length and telomerase levels delineate subgroups of B-cell chronic lymphocytic leukemia with different biological characteristics and clinical outcomes.

BACKGROUND: B-cell chronic lymphocytic leukemia is a clinically heterogeneous disease; some patients rapidly progress and die within a few years of diagnosis, whereas others have a long life expectancy with minimal or no treatment. Telomere length and telomerase levels have been proposed as prognostic factors; however, very few cases have been characterized for both parameters and no study has analyzed the prognostic value of the telomere/telomerase profile. DESIGN AND METHODS: One hundred and seventy-three cases of chronic lymphocytic leukemia were characterized for telomere lengths and telomerase levels by real-time polymerase chain reaction. Data were correlated with established prognostic markers, IGVH mutational status and chromosomal aberrations, and clinical outcome. RESULTS: Telomere lengths were inversely correlated with telomerase levels (r(s) = -0.213; P = 0.012), and most of the cases of chronic lymphocytic leukemia with high levels (above median) of telomerase had short (below median) telomeres (P = 0.0001). Telomerase levels were higher and telomeres were shorter in unmutated IGVH cases than in mutated IGVH ones (P<0.0001). Chronic lymphocytic leukemias with 11q, 17p deletion or 12 trisomy had significantly higher levels of telomerase and shorter telomeres than those with no chromosomal aberration or the sole 13q deletion (P < 0.001). Telomere length/telomerase level profiles identified subgroups of patients with different clinical outcomes (P < 0.0001), even within the subsets of chronic lymphocytic leukemia defined by IGVH mutational status or chromosomal aberrations. Short telomere/high telomerase profile was independently associated with more rapid disease progression. CONCLUSIONS: Comprehensive analyses of telomeres, telomerase, chromosomal aberrations, and IGVH mutational status delineate groups of chronic lymphocytic leukemias with distinct biological characteristics and clinical outcomes. The telomere/telomerase profile may be particularly useful in refining the prognosis of chronic lymphocytic leukemia patients with mutated IGVH and no high-risk chromosomal aberrations.

Immune Activation, Exhaustion and Senescence Profiles as Possible Predictors of Cancer in Liver Transplanted Patients.

Liver transplanted (LT) patients for hepatocellular carcinoma (LT-HCC) or for other causes (LT-no-HCC) may develop post-transplantation malignancies. Although immune activation and senescence are frequently implicated in cancer development, no data is available on their possible role as biomarkers predictive of tumor onset in this setting. A total of 116 patients were investigated: the 45 LT-HCC patients were older than the 71 LT-non-HCC (p=0.011), but comparable for sex, HCV, HBV infection and immunosuppressive treatment. At baseline, the numbers of activated and senescent-like circulating cells were significantly higher in LT-HCC patients than in LT-no-HCC ones. After a median follow-up of 26.8 months, 6 post-transplant malignancies (PTM) occurred: 4 in LT-HCC (8.9%) and 2 in LT-no-HCC (2.8%) patients. Overall, subjects with high percentages of activated and exhausted T and B cells at baseline were at higher risk of PTM. Notably, within the LT-HCC group, a higher percentage of senescence-like T cells was also associated with cancer development. Moreover, patients with PTM had higher telomere erosion and higher levels of circulating PAMPs (16S rDNA) and DAMPs (mtDNA) when compared with matched patients without PTM. Overall, these findings suggest that immune activation and exhaustion may be useful to predict the risk of PTM occurrence, regardless of the cause of transplantation. In LT-HCC, T-cell senescence represents an additional risk factor for tumor onset.

Biological Predictors of De Novo Tumors in Solid Organ Transplanted Patients During Oncological Surveillance: Potential Role of Circulating TERT mRNA.

BACKGROUND: De novo tumors are a major cause of morbidity and mortality after long-term solid organ transplantation. Chronic immunosuppression strongly affects solid organ transplanted (SOT) patients' immune system by promoting immune evasion strategies and reactivations of viruses with oncogenic potential, ultimately leading to cancer onset. In this scenario, an oncological Surveillance Protocol integrated with biobanking of peripheral blood samples and evaluation of immunovirological and molecular parameters was activated for SOT patients at CRO-IRCCS Aviano, with the aim of identifying suitable biomarkers of cancer development. METHODS: An exploratory longitudinal study was designed based on two serial peripheral blood samples collected at least three months apart. Forty nine SOT patients were selected and stratified by tumor onset during follow-up. Spontaneous T-cell responses to EBV, CMV and tumor associated antigens, EBV-DNA and CMV-DNA loads, and circulating TERT mRNA levels were investigated. RESULTS: Significantly higher levels of circulating TERT mRNA were observed 3.5-23.5 months before and close to the diagnosis of cancer as compared to tumor-free patients. Plasmatic TERT mRNA levels >97.73 copies/mL at baseline were significantly associated with the risk of developing de novo tumors (HR=4.0, 95%C.I. = 1.4-11.5, p=0.01). In particular, the risk significantly increased by 4% with every ten-unit increment in TERT mRNA (HR=1.04, 95%C.I. = 1.01-1.07, p=0.01). CONCLUSIONS: Although obtained in an exploratory study, our data support the importance of identifying early biomarkers of tumor onset in SOT patients useful to modulate the pace of surveillance visits.

TERT Promoter Mutations and rs2853669 Polymorphism: Useful Markers for Clinical Outcome Stratification of Patients With Oral Cavity Squamous Cell Carcinoma.

OBJECTIVE: To date, no useful prognostic biomarker exists for patients with oral squamous cell carcinoma (OCSCC), a tumour with uncertain biological behaviour and subsequent unpredictable clinical course. We aim to investigate the prognostic significance of two recurrent somatic mutations (-124 C>T and -146 C>T) within the promoter of telomerase reverse transcriptase (TERT) gene and the impact of TERT single nucleotide polymorphism (SNP) rs2853669 in patients surgically treated for OCSCC. METHODS: The genetic frequencies of rs2853669, -124 C>T and -146 C>T as well as the telomere length were investigated in 144 tumours and 57 normal adjacent mucosal (AM) specimens from OCSCC patients. RESULTS: Forty-five tumours harboured TERT promoter mutations (31.3%), with -124 C>T and -146 C>T accounting for 64.4% and 35.6% of the alterations respectively. Patients with -124 C>T TERT promoter mutated tumours had the shortest telomeres in the AM (p=0.016) and showed higher risk of local recurrence (hazard ratio [HR]:2.75, p=0.0143), death (HR:2.71, p=0.0079) and disease progression (HR:2.71, p=0.0024) with the effect being potentiated by the co-occurrence of T/T genotype of rs2853669. CONCLUSION: -124 C>T TERT promoter mutation as well as the T/T genotype of the rs2853669 SNP are attractive independent prognostic biomarkers in patients surgically treated for OCSCC, with the coexistence of these genetic variants showing a synergistic impact on the aggressiveness of the disease.

Anti-Proliferative and Pro-Apoptotic Effects of Short-Term Inhibition of Telomerase In Vivo and in Human Malignant B Cells Xenografted in Zebrafish.

Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumor growth/progression through extra-telomeric functions. Our previous in vitro studies demonstrated that short-term TERT inhibition by BIBR1532 (BIBR), an inhibitor of TERT catalytic activity, negatively impacts cell proliferation and viability via telomeres' length-independent mechanism. Here we evaluate the anti-proliferative and pro-apoptotic effects of short-term telomerase inhibition in vivo in wild-type (wt) and tert mutant (terthu3430/hu3430; tert-/-) zebrafish embryos, and in malignant human B cells xenografted in casper zebrafish embryos. Short-term Tert inhibition by BIBR in wt embryos reduced cell proliferation, induced an accumulation of cells in S-phase and ultimately led to apoptosis associated with the activation of DNA damage response; all these effects were unrelated to telomere shortening/dysfunction. BIBR treatment showed no effects in tert-/- embryos. Xenografted untreated malignant B cells proliferated in zebrafish embryos, while BIBR pretreated cells constantly decreased and were significantly less than those in the controls from 24 to up to 72 h after xenotransplantation. Additionally, xenografted tumor cells, treated with BIBR prior- or post-transplantation, displayed a significant higher apoptotic rate compared to untreated control cells. In conclusion, our data demonstrate that short-term telomerase inhibition impairs proliferation and viability in vivo and in human malignant B cells xenografted in zebrafish, thus supporting therapeutic applications of TERT inhibitors in human malignancies.

Genetic Variants of the TERT Gene, Telomere Length, and Circulating TERT as Prognostic Markers in Rectal Cancer Patients.

Single-nucleotide polymorphisms (SNPs) in the TERT gene can affect telomere length and TERT expression and have been associated with risk and/or outcome for several tumors, but very few data are available about their impact on rectal cancer. Eight SNPs (rs2736108, rs2735940, rs2736098, rs2736100, rs35241335, rs11742908, rs2736122 and rs2853690), mapping in regulatory and coding regions of the TERT gene, were studied in 194 rectal cancer patients to evaluate their association with constitutive telomere length, circulating TERT mRNA levels, response to neoadjuvant chemoradiotherapy (CRT) and disease outcome. At diagnosis, the rs2736100CC genotype was associated with longer telomeres measured pre-CRT, while the rs2736100CC, rs2736108TT and rs2735940AA were associated with greater telomere erosion evaluated post-CRT. The rs2736108CC and rs2853690AA/GG genotypes, respectively associated with lower telomere erosion and lower levels of circulating TERT post-CRT, were also independently associated with a better response to therapy [OR 4.6(1.1-19.1) and 3.0(1.3-6.9)]. Overall, post-CRT, low levels (≤ median value) of circulating TERT and its stable/decreasing levels compared to those pre-CRT, were independently associated with a better response to therapy [OR 5.8(1.9-17.8) and 5.3(1.4-19.4), respectively]. Furthermore, post-CRT, patients with long telomeres (>median value) and low levels of circulating TERT had a significantly lower risk of disease progression [HR 0.4(0.1-0.9) and 0.3(0.1-0.8), respectively]. These findings suggest that TERT SNPs could be a useful tool for improving the selection of patients who could benefit from CRT and support the role of telomere length and circulating TERT mRNA levels as useful markers for monitoring the response to therapy and disease outcome in rectal cancer patients.

TERT Promoter Mutations Differently Correlate with the Clinical Outcome of MAPK Inhibitor-Treated Melanoma Patients.

Resistance is a major challenge in the management of mitogen-activated protein kinase inhibitor (MAPKi)-treated metastatic melanoma. Tumor genetic alterations can cause MAPK pathway reactivation, leading to lack of response and poor outcome. Characterization of the mutational profile in patients with melanoma might be crucial for patient-tailored treatment choices. Mutations in the promoter region of the telomerase reverse transcriptase gene (TERTprom) lead to increased TERT expression and telomerase activity and are frequent in BRAFV600 mutant melanoma. Reportedly, TERTprom, and BRAFV600 mutations cooperate in driving cancer progression and aggressiveness. We evaluated the effect of the TERTprom status on the clinical outcome in 97 MAPKi-treated melanoma patients. We observed that patients with the c.-146C > T mutation showed a significantly worse progression-free survival (PFS) compared to those carrying the c.-124C > T mutation and a two-fold increased risk of progression (median 5.4 vs. 9.5 months; hazard ratio (HR) 1.9; 95% confidence interval (CI) 1.2-3.2; p = 0.013). This trend was also observed for the overall survival (OS); melanoma patients with the c.-146C > T mutation showed a poorer prognosis compared to those with the c.-124C > T mutation (median 13.3 vs. 25.5 months; HR 1.9, 95% CI 1.1-3.3, p = 0.023). Our results disclose a different correlation of the two TERTprom mutations with MAPKi-treated melanoma patient outcome, highlighting a different impact of the pathway blockade.

TERT promoter hotspot mutations and their relationship with TERT levels and telomere erosion in patients with head and neck squamous cell carcinoma.

PURPOSE: To evaluate the prevalence of two recurrent somatic mutations (-124 C>T and -146 C>T) within the promoter of the gene encoding telomerase reverse transcriptase (TERT) as well as their relationship with TERT level, telomeres length, and outcome in patients with head and neck squamous cell carcinomas (HNSCCs). METHODS: We evaluate the prevalence of TERT promoter mutations, TERT levels, and telomere length in paired cancer tissue and adjacent mucosa (AM) in a series of HNSCCs. RESULTS: Cancer tissue and AM specimens from 105 patients were analyzed. Telomere length and TERT mRNA levels were estimated using real-time polymerase chain reaction. TERT promoter mutations were assessed using Sanger sequencing. Out of 105 cases, 101 were considered suitable for the analysis. TERT promoter harbored mutations in 12 tumors (11.9%), with -124 C>T and -146 C>T accounting for 83.3% and 16.7% of the alterations, respectively. No mutations were detected in AM samples. The prevalence of TERT promoter mutations was significantly higher in oral cavity SCCs (10 out of 27 tumors; 37%), and telomere length in AM was shorter in patients with tumors carrying TERT promoter mutations than in patients with unmutated TERT promoter cancers (p = 0.023). TERT levels in tumor did not significantly differ according to the mutational status of TERT promoter. No significant association was found between TERT promoter status and overall survival. CONCLUSION: TERT promoter mutations are most likely a late event in tumor development, occurring in a context of critically short telomeres, mostly in patients with oral cavity SCC. TERT levels, but not TERT promoter mutational status impact clinical outcome.

Immune senescence and immune activation in elderly colorectal cancer patients.

In our previous study, we found that low thymic output and short telomere length were associated with a higher risk of tumor in elderly cancer patients. Here, we aimed to examine in depth the impact of immunological and biological senescence and immune activation on disease outcome in elderly patients with colorectal cancer (CRC).Peripheral blood samples from 81 CRC patients were studied for immune activation, immune senescence and recent thymic emigrant(RTE) CD4 and CD8 cells by flow cytometry. T-cell receptor rearrangement excision circle (TREC) levels and telomere lengths were measured by real-time PCR. Plasma levels of microbial translocation markers, LPS and sCD14, were quantified by ELISA. While TREC levels and telomere length were not prognostic of disease outcome, high percentages of immune senescent and immune activated CD8 cells were associated with a higher risk of a negative event (relapse, progression, or death) in all studied patients and disease relapse in I-III staged patients. Levels of sCD14 and LPS were higher in patients who will experience a negative event than in patients who will not. In conclusion, in elderly CRC patients higher immunological senescence and immune activation negatively impact the disease outcome; how these characteristics influence the antineoplastic treatments remains to be investigated.

Predictive and prognostic significance of telomerase levels/telomere length in tissues and peripheral blood in head and neck squamous cell carcinoma.

A growing body of evidence indicates that the expression of TERT, the catalytic subunit of telomerase, is a biological marker of progression in several cancers. We investigated the predictive and prognostic role of TERT levels and telomere length in tissues and peripheral blood in patients with head and neck squamous cell carcinoma (HNSCC). High TERT levels in cancer tissues were independently associated with worse response to therapy (odds ratio [OR]:6.26), regional failure (hazard ratio [HR]:5.75), progression (HR:2.12), and death (HR:3.53). Longer telomeres in the mucosa surrounding the tumor (SM) were independently associated with a lower risk of mucosal failure (HR:0.39). While telomere length in peripheral blood mononuclear cells (PBMC) significantly decreased with age, no correlation was found between age and telomere length in SM. No associations were found between TERT levels in plasma and telomere length in PBMC and the prognostic variables. High levels of TERT transcripts in cancer cells represent a reliable prognostic marker for identifying HNSCC patients with risk of progression. The altered relationship of telomere length to age in SM compared with PBMC suggests that in a subset of cases the phenotypically normal SM constitutes an acquired telomere-shortened epithelial field prone to genetic instability.

Genetic, Epigenetic, and Immunologic Profiling of MMR-Deficient Relapsed Glioblastoma.

PURPOSE: In-depth characterization of recurrent glioblastoma (rGBM) might contribute to a better understanding of the mechanisms behind tumor progression and enable rGBM treatment with targeted drugs.Experimental Design: In this study, GBM samples were collected at diagnosis and recurrence from adult patients treated with Stupp protocol. Expression of mismatch repair (MMR) proteins was evaluated by IHC, followed by whole exome sequencing (WES) of tumor samples showing loss of MSH6 reactivity. Established genetic, epigenetic, and immunologic markers were assessed by standard methods and correlated with loss of MMR proteins and patient survival. RESULTS: Expression of MMR proteins was partially or completely lost in 25.9% rGBM samples. Specifically, 12 samples showed partial or total MSH6 expression reduction. Conversely, 96.4% of GBM samples at diagnosis expressed MMR markers. WES disclosed lack of variants in MMR genes in primary samples, whereas two MSH6-negative rGBM samples shared a c.3438+1G>A* splicing MSH6 variant with a potential loss of function effect. MSH6-negative rGBM specimens had high tumor mutational burden (TMB), but no microsatellite instability. In contrast, GBM samples with partial loss of MMR proteins disclosed low TMB. MMR-deficient rGBM showed significant telomere shortening and MGMT methylation and are characterized by highly heterogeneous MHC class I expression. CONCLUSIONS: Multilevel profiling of MMR-deficient rGBM uncovered hypermutated genotype uncoupled from enriched expression of immune-related markers. Assessment of MHC class I expression and TMB should be included in protocols aiming to identify rGBM patients potentially eligible for treatment with drugs targeting immune-checkpoint inhibitors.

Extra-telomeric functions of telomerase in the pathogenesis of Epstein-Barr virus-driven B-cell malignancies and potential therapeutic implications.

The Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus causally linked to a broad spectrum of both lymphoid and epithelial malignancies. In order to maintain its persistence in host cells and promote tumorigenesis, EBV must restrict its lytic cycle, which would ultimately lead to cell death, selectively express latent viral proteins, and establish an unlimited proliferative potential. The latter step depends on the maintenance of telomere length provided by telomerase. The viral oncoprotein LMP-1 activates TERT, the catalytic component of telomerase. In addition to its canonical role in stabilizing telomeres, TERT may promote EBV-driven tumorigenesis through extra-telomeric functions. TERT contributes toward preserving EBV latency; in fact, through the NOTCH2/BATF pathway, TERT negatively affects the expression of BZLF1, the master regulator of the EBV lytic cycle. In contrast, TERT inhibition triggers a complete EBV lytic cycle, leading to the death of EBV-infected cells. Interestingly, short-term TERT inhibition causes cell cycle arrest and apoptosis, partly by inducing telomere-independent activation of the ATM/ATR/TP53 pathway. Importantly, TERT inhibition also sensitizes EBV-positive tumor cells to antiviral therapy and enhances the pro-apoptotic effects of chemotherapeutic agents. We provide here an overview on how the extra-telomeric functions of TERT contribute to EBV-driven tumorigenesis. We also discuss the potential therapeutic approach of TERT inhibition in EBV-driven malignancies.

Different apoprotein(a) isoform proportions in serum and carotid plaque.

INTRODUCTION: Cardio- and/or cerebro-vascular risk are associated with high lipoprotein (a) [Lp(a)] levels and low-molecular-weight (LMW) apo(a) isoforms. Aims of this study were to evaluate the deposition of apo(a) isoforms and apoprotein B (apo B) in atherosclerotic plaque from patients (males and females) who had carotid endarterectomy for severe stenosis, and to identify differences between patients classified by gender and divided according to the stability or instability of their plaques. MATERIALS AND METHODS: We determined lipids, apo B and Lp(a) in serum and plaque extracts from 55 males and 25 females. Apo(a) was phenotyped and isoforms were classified by number of kringle IV (KIV) repeats. RESULTS: Lp(a) levels were higher in female serum and plaque extracts than in male samples, while apo B levels were lower. More Lp(a) than apo B deposition was observed in plaque after normalization for serum levels. Thirty-one different apo(a) isoforms were detected in our patients, with a double band phenotype in 94% of cases. In both sexes, the low/high (L/H) molecular weight apo(a) isoform expression ratio was significantly higher in plaque than in serum. Females with unstable plaques had higher Lp(a) levels in both serum and tissue extracts, and fewer KIV repeats of the principal apo(a) isoform in the serum than the other female group or males. CONCLUSIONS: In both sexes, the same apo(a) isoforms are found in serum and atherosclerotic plaque, but in different proportions: in plaque, LMW apo(a) is almost always more strongly accumulated than HMW apo(a), irrespective of any combination of apo(a) isoforms in double band phenotypes or Lp(a) serum levels. Moreover, serum and tissue Lp(a) levels were higher in females than in males, and particularly in the group with unstable plaques.

Pancreatic cancer-derived S-100A8 N-terminal peptide: a diabetes cause?

BACKGROUND: Our aim was to identify the pancreatic cancer diabetogenic peptide. METHODS: Pancreatic tumor samples from patients with (n=15) or without (n=7) diabetes were compared with 6 non-neoplastic pancreas samples using SDS-PAGE. RESULTS: A band measuring approximately 1500 Da was detected in tumors from diabetics, but not in neoplastic samples from non-diabetics or samples from non-neoplastic subjects. Sequence analysis revealed a 14 amino acid peptide (1589.88 Da), corresponding to the N-terminal of the S100A8. At 50 nmol/L and 2 mmol/L, this peptide significantly reduced glucose consumption and lactate production by cultured C(2)C(12) myoblasts. The 14 amino acid peptide caused a lack of myotubular differentiation, the presence of polynucleated cells and caspase-3 activation. CONCLUSIONS: The 14 amino acid peptide from S100A8 impairs the catabolism of glucose by myoblasts in vitro and may cause hyperglycemia in vivo. Its identification in biological fluids might be helpful in diagnosing pancreatic cancer in patients with recent onset diabetes mellitus.

Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells.

Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumorigenesis through extra-telomeric functions. The possible therapeutic effects of BIBR1532 (BIBR), a powerful TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein-Barr virus (EBV)-driven B-cell malignancies. Our aim was to characterize the biological effects of TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt's lymphoma (BL) cell lines. We found that BIBR selectively inhibits telomerase activity in TERT-positive 4134/Late and 4134/TERT+ LCLs and EBV-negative BL41 and EBV-positive BL41/B95.8 BL cell lines. TERT inhibition led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately to increased apoptosis, compared with mock-treated control cells. All these effects occurred within 72 h and were not observed in BIBR-treated TERT-negative 4134/TERT- and U2OS cells. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone. In conclusion, TERT inhibition impairs cell cycle progression and enhances the pro-apoptotic effects of chemotherapeutic agents in TERT-positive cells. These results support new therapeutic applications of TERT inhibitors in EBV-driven B-cell malignancies.

Pancreatic cancer-associated diabetes mellitus: an open field for proteomic applications.

BACKGROUND: Diabetes mellitus is associated with pancreatic cancer in more than 80% of the cases. Clinical, epidemiological, and experimental data indicate that pancreatic cancer causes diabetes mellitus by releasing soluble mediators which interfere with both beta-cell function and liver and muscle glucose metabolism. METHODS: We analysed, by matrix-assisted laser desorption ionization time of flight (MALDI-TOF), a series of pancreatic cancer cell lines conditioned media, pancreatic cancer patients' peripheral and portal sera, comparing them with controls and chronic pancreatitis patients' sera. RESULTS: MALDI-TOF analysis of pancreatic cancer cells conditioned media and patients' sera indicated a low molecular weight peptide to be the putative pancreatic cancer-associated diabetogenic factor. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of tumor samples from diabetic and non-diabetic patients revealed the presence of a 1500 Da peptide only in diabetic patients. The amino acid sequence of this peptide corresponded to the N-terminal of an S-100 calcium binding protein, which was therefore suggested to be the pancreatic cancer-associated diabetogenic factor. CONCLUSIONS: We identified a tumor-derived peptide of 14 amino acids sharing a 100% homology with an S-100 calcium binding protein, which is probably the pancreatic cancer-associated diabetogenic factor.