Telomerase inhibition by siRNA causes senescence and apoptosis in Barrett's adenocarcinoma cells: mechanism and therapeutic potential

BACKGROUND: In cancer cells, telomerase induction helps maintain telomere length and thereby bypasses senescence and provides enhanced replicative potential. Chemical inhibitors of telomerase have been shown to reactivate telomere shortening and cause replicative senescence and apoptotic cell death of tumor cells while having little or no effect on normal diploid cells. RESULTS: We designed siRNAs against two different regions of telomerase gene and evaluated their effect on telomere length, proliferative potential, and gene expression in Barrett's adenocarcinoma SEG-1 cells. The mixture of siRNAs in nanomolar concentrations caused a loss of telomerase activity that appeared as early as day 1 and was essentially complete at day 3. Inhibition of telomerase activity was associated with marked reduction in median telomere length and complete loss of detectable telomeres in more than 50% of the treated cells. Telomere loss caused senescence in 40% and apoptosis in 86% of the treated cells. These responses appeared to be associated with activation of DNA sensor HR23B and subsequent activation of p53 homolog p73 and p63 and E2F1. Changes in these gene regulators were probably the source of observed up-regulation of cell cycle inhibitors, p16 and GADD45. Elevated transcript levels of FasL, Fas and caspase 8 that activate death receptors and CARD 9 that interacts with Bcl10 and NFKB to enhance mitochondrial translocation and activation of caspase 9 were also observed. CONCLUSION: These studies show that telomerase siRNAs can cause effective suppression of telomerase and telomere shortening leading to both cell cycle arrest and apoptosis via mechanisms that include up-regulation of several genes involved in cell cycle arrest and apoptosis. Telomerase siRNAs may therefore be strong candidates for highly selective therapy for chemoprevention and treatment of Barrett's adenocarcinoma

SPAG5 as a prognostic biomarker and chemotherapy sensitivity predictor in breast cancer: a retrospective, integrated genomic, transcriptomic, and protein analysis

Background: Although the use of proliferation markers/profiles has been recommended when choosing the appropriate systemic-treatment for breast cancer (BC), the best molecular-marker/test that should be used needs to be identified. Methods: To identify factors that drive proliferation and its associated features in BC an artificial neural network (ANN) based integrative data-mining methodology was applied to three cohorts [(Nottingham-discovery (ND), Uppsala and METABRIC (Molecular Taxonomy of Breast Cancer International Consortium)]. The most prominent genes in the resulting interactome-map were then identified. Given that SPAG5 was associated with many features of proliferation, featured prominently in the interactome-map and has a fundamental role in mitotic-progression,, we hypothesized that it could be a better indicator of proliferation activity. (BC). Subsequently to test if it could provide a more accurate guide for the delivery of systemic therapies in BC, we investigated the clinico-pathological utility of SPAG5: gene copy number aberrations (CNAs); mRNA and protein expression, in over 10,000 BCs. Integrated analysis of SPAG5-gene CNAs, transcript and protein expression was conducted in the ND cohort (n=171) and validated in the METABRIC cohort (n=1980). In addition, the associations of SPAG5 CNAs, transcript and/or protein with BC specific survival (BCSS), disease free survival (DFS) and/or distant relapse free survival (DRFS) were analysed in multiple cohorts including Uppsala (n=249), METABRIC, three-untreated lymph node (LN) negative cohorts (n=684), a combined multicentre clinical data set (n=5439), Nottingham historical early-stage-primary BC (Nottingham-HES-BC; n=1650), Nottingham oestrogen receptor (ER) negative BC (n=697), Nottingham anthracycline-Neoadjuvant-chemotherapy (Nottingham-AC-Neo-ACT; n=200), and MD Anderson Cancer Centre Taxane/anthracycline (MDACC-T/AC-Neo-ACT; n=508) cohorts. The association of SPAG5 transcript and protein expression with pathological response rate (pCR) were also tested in [MDACC-T/AC-Neo-ACT (n=508) and the phase II trial NCT00455533; n=253)] and [Nottingham-AC-Neo-ACT (n=200)] cohorts; respectively. Findings: SPAG5 gene gain/amplification at the Ch17q11·2 locus was found in 10.4% of BC (206/1980 (; METABRIC) and was reported in 19·4% of PAM50-HER2 (46/237) and 17·8% of PAM50-LumB (87/488). SPAG5-CNA gain/amplification and high SPAG5-transcript and SPAG5-protein were associated with increased risk of death from BC [Uppsala; (HR (CI 95%): 1·50 (1·18-1·92); p=0·00010, METABRIC; (HR (CI 95%): 1·68 (1·40-2·01) p<0·0001), and Nottingham-HSE-BC; (HR (CI 95%): 1·68 (1·32-2·12), p<0·0001); respectively]. Multivariable Cox regression models, including other validated-prognostic factors, (Uppsala: age, size, LN-stage, genomic grade index (GGI), ER, TP53 mutation and MKi67; METABRIC: age, size, LN-stage, histologic-grade, ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), hormone-therapy, chemotherapy, interaction term of SPAG5 and both chemotherapyy and hormonotherapy; Desmedt-untreated LN- cohort: ER, Nottingham prognostic index (NPI), 76-gene prognostic signature (Veridex) and Adjuvant-Online (AOL); Nottingham-HES-BC: menopausal status, size, LN- stage, histologic-grade, ER, PR, HER2, ki67, hormone-therapy, chemotherapy, interaction term of SPAG5 and both chemotherapy[y and hormonotherapy), showed that high SPAG5-transcript and high SPAG5-protein were associated with shorter BCSS [Uppsala: (HR (CI 95%): 1·62 (1·03-2·53) p=0·036); METABRIC: (HR (CI 95%): 1·27 (1·02-1·58) p=0·034); Desmedt-untreated LN- cohort: (HR (CI 95%): 2·34 (1·24-4·42) p=0·0090), and Nottingham-HES-BC (HR (CI 95%): 1·73 (1·23-2·46) p=0·0020); respectively]. In ER-negative-BC with high SPAG5-protein, administration of anthracycline-adjuvant-chemotherapy had reduced the risk of death by 60% compared to chemotherapy-naive (HR (95% CI): 0·37 (0·20-0·60); p=0·0010). A multivariable Cox regression analysis, which included other validated prognostic factors for chemotherapy (e.g., menopausal status, size, lymph node stage, histologic grade, ER, PR, HER2, Bcl2, chemotherapy, interaction term of SPAG5 and both chemotherapy[y), revealed that SPAG5-transcript+ was independently associated with decreased risk of DRFS after receiving Taxane/anthracycline-Neo-ACT [MDACC-T/AC-Neo-ACT: (HR (CI 95%): 0·68 (0·48-0·97); p=0·0070)]. In multivariable logistic regression analysis, both SPAG5-transcript+ and SPAG5-protein+ and were independent predictors for higher pCR after combination-cytotoxic chemotherapy [MDACC-T/AC-Neo-ACT: (OR (95% CI) 1·71 (1·07-2·74); p=0·024), and Nottingham-AC-Neo-AC: (OR (95% CI): 8·75 (2·42-31); p=0·0010); respectively]. Interpretation: SPAG5 is a novel amplified gene on Ch17q11.2 in PAM50-LumB and PAM-HER2 BC, and its transcript and protein products are independent prognostic and predictive biomarkers, with potential clinical utility as a biomarker for combination cytotoxic chemotherapy sensitivity, especially in ER- BC

Vertebral Bone Marrow Fat Is independently Associated to VAT but Not to SAT: KORA FF4—Whole-Body MR Imaging in a Population-Based Cohort

The objective of the current study was to assess the relationship of bone marrow adipose tissue (BMAT) content to abdominal fat depots, including visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), as well as cardiovascular risk factors (CVRF) beyond physical activity in a population-based cohort study undergoing whole-body magnetic resonance (MR) imaging. Subjects of the Cooperative Health Research in the Augsburg Region (KORA) FF4 study without known cardiovascular disease underwent fat fraction quantification in vertebrae (BMATL1/L2) via a 2-point T1-weighted volumetric interpolated breath-hold examination (VIBE) Dixon sequence. The same MR sequence was applied to quantify VAT and SAT volume. Subjects&rsquo; characteristics, including physical activity, were determined through standardized exams and self-assessment questionnaires. Univariate and multivariate linear regression were applied. In the cohort of 378 subjects (56 &plusmn; 9.1years; 42.1% female), BMATL1/L2 was 54.3 &plusmn; 10.1%, VAT was 4.54 &plusmn; 2.71 L, and SAT was 8.10 &plusmn; 3.68 L. VAT differed significantly across BMATL1/L2 tertiles (3.60 &plusmn; 2.76 vs. 4.92 &plusmn; 2.66 vs. 5.11 &plusmn; 2.48; p &lt; 0.001), there was no significant differences for SAT (p = 0.39). In the fully adjusted model, VAT remained positively associated with BMATL1/L2 (&beta; = 0.53, p = 0.03). Furthermore, BMATL1/L2 was associated with age (&beta; = 5.40 per 10-years, p &lt; 0.001), hemoglobin A1c (HbA1c; &beta; = 1.55 per 1%, p = 0.04), lipids (&beta; = 0.20 per 10 mg/dL triglycerides; &beta; = 0.40 per 10 mg/dL low-density lipoprotein (LDL); &beta; =&minus;3.21 lipid-lowering medication; all p &lt; 0.05), and less physical activity (&beta; = 3.7 &ldquo;no or nearly no exercise&rdquo; as compared to &ldquo;&ge;2 h per week, regularly&rdquo;, p = 0.003); gender was not significantly different (p = 0.57). In the population-based cohort, VAT but not SAT were associated with higher BMATL1/L2 independently of physical activity and other cardiovascular risk factors. Further, BMATL1/L2 increased with older age, less physical activity, higher HbA1c, and increased lipids but decreased with lipid-lowering medication

Changes in Bone Marrow Fat upon Dietary-Induced Weight Loss

Background: Bone marrow fat is implicated in metabolism, bone health and haematological diseases. Thus, this study aims to analyse the impact of moderate weight loss on bone marrow fat content (BMFC) in obese, healthy individuals. Methods: Data of the HELENA-Trial (Healthy nutrition and energy restriction as cancer prevention strategies: a randomized controlled intervention trial), a randomized controlled trial (RCT) among 137 non-smoking, overweight or obese participants, were analysed to quantify the Magnetic Resonance Imaging (MRI)-derived BMFC at baseline, after a 12-week dietary intervention phase, and after a 50-week follow-up. The study cohort was classified into quartiles based on changes in body weight between baseline and week 12. Changes in BMFC in respect of weight loss were analysed by linear mixed models. Spearman&rsquo;s coefficients were used to assess correlations between anthropometric parameters, blood biochemical markers, blood cells and BMFC. Results: Relative changes in BMFC from baseline to week 12 were 0.0 &plusmn; 0.2%, &minus;3.2 &plusmn; 0.1%, &minus;6.1 &plusmn; 0.2% and &minus;11.5 &plusmn; 0.6% for Q1 to Q4. Across all four quartiles and for the two-group comparison, Q1 versus Q4, there was a significant difference (p &lt; 0.05) for changes in BMFC. BMFC was not associated with blood cell counts and showed only weaker correlations (&lt;0.3) with metabolic biomarkers. Conclusion: Weight loss is associated with a decrease of BMFC. However, BMFC showed no stronger associations with inflammatory and metabolic biomarkers