High expression of sphingosine 1-phosphate receptors, S1P1 and S1P3, sphingosine kinase 1, and extracellular signal-regulated kinase-1/2 is associated with development of tamoxifen resistance in estrogen receptor-positive breast cancer patients.

Various studies in cell lines have previously demonstrated that sphingosine kinase 1 (SK1) and extracellular signal-regulated kinase 1/2 (ERK-1/2) interact in an estrogen receptor (ER)-dependent manner to influence both breast cancer cell growth and migration. A cohort of 304 ER-positive breast cancer patients was used to investigate the prognostic significance of sphingosine 1-phosphate (S1P) receptors 1, 2, and 3 (ie, S1P1, S1P2, and S1P3), SK1, and ERK-1/2 expression levels. Expression levels of both SK1 and ERK-1/2 were already available for the cohort, and S1P1, S1P2, and S1P3 levels were established by immunohistochemical analysis. High membrane S1P1 expression was associated with shorter time to recurrence (P=0.008). High cytoplasmic S1P1 and S1P3 expression levels were also associated with shorter disease-specific survival times (P=0.036 and P=0.019, respectively). Those patients with tumors that expressed high levels of both cytoplasmic SK1 and ERK-1/2 had significantly shorter recurrence times than those that expressed low levels of cytoplasmic SK1 and cytoplasmic ERK-1/2 (P=0.00008), with a difference in recurrence time of 10.5 years. Similarly, high cytoplasmic S1P1 and cytoplasmic ERK-1/2 expression levels (P=0.004) and high cytoplasmic S1P3 expression and cytoplasmic ERK-1/2 expression levels (P=0.004) were associated with shorter recurrence times. These results support a model in which the interaction between SK1, S1P1, and/or S1P3 and ERK-1/2 might drive breast cancer progression, and these findings, therefore, warrant further investigation.

Ras/Raf-1/MAPK pathway mediates response to tamoxifen but not chemotherapy in breast cancer patients.

PURPOSE: The expression and activation of the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway plays an important role in the development and progression of cancer, and may influence response to treatments such as tamoxifen and chemotherapy. In this study we investigated whether the expression and activation of the key components of this pathway influenced clinical outcome, to test the hypothesis that activation of the MAPK pathway drives resistance to tamoxifen and chemotherapy in women with breast cancer. EXPERIMENTAL DESIGN: Breast tumors from patients at the Glasgow Royal Infirmary and others treated within the BR9601 trial were analyzed for expression of the three Ras isoforms, total Raf-1, active and inactive forms of Raf-1 [pRaf(ser338) and pRaf(ser259), respectively], MAPK, and phospho-MAPK using an immunohistochemical approach. Analyses were done with respect to disease free-survival and overall survival. RESULTS: Expression and activation of the Ras pathway was associated with loss of benefit from treatment with tamoxifen but not chemotherapy. Overexpression of pRaf(ser338) was associated with shortened disease-free and overall survival time in univariate analyses. Multivariate analysis suggested pRaf(ser338) was independent of known prognostic markers in predicting outcome following tamoxifen treatment (P=0.03). CONCLUSION: This study suggests that activation of the Ras pathway predicts for poor outcome on tamoxifen but not chemotherapy, and identifies pRaf(ser338) as a potential marker of resistance to estrogen receptor-targeted therapy. In addition, it suggests that expression of pRaf(ser338) could identify patients for whom tamoxifen alone is insufficient adjuvant systemic therapy, but for whom the addition of chemotherapy may be of benefit.

An increase in N-Ras expression is associated with development of hormone refractory prostate cancer in a subset of patients.

Protein expression of H, K and N-Ras was assessed in hormone sensitive and hormone refractory prostate tumour pairs from 61 patients by immunohistochemistry. Expression of H-Ras and K- Ras was not associated with any known clinical parameters. In contrast an increase in N-Ras membrane expression in the transition from hormone sensitive to hormone refractory prostate cancer was associated with shorter time to relapse (p=0.01) and shorter disease specific survival (p=0.008). In addition, patients with an increase in N-Ras membrane expression had lower levels of PSA at relapse (p=0.02) and expression correlated with phosphorylated MAP kinase (p=0.010) and proliferation index (Ki67, p=0.02). These results suggest that in a subgroup patients N-Ras expression is associated with development of hormone refractory prostate cancer via activation of the MAP kinase cascade.

Amplified in breast cancer 1 in human epidermal growth factor receptor - positive tumors of tamoxifen-treated breast cancer patients.

PURPOSE: Amplified in breast cancer 1 (AIB1) is a member of the p160/steroid receptor coactivators family and is involved in estrogen-dependent gene transcription by reducing the antagonistic activity of tamoxifen-bound estrogen receptor-alpha (ER-alpha). The present study was carried out to test the hypothesis that AIB1 protein expression and/or gene amplification mediates tamoxifen resistance in breast cancer. EXPERIMENTAL DESIGN: Immunohistochemistry using AIB1 antibody and fluorescence in situ hybridization using probes specific for AIB1 and chromosome 20 was done on 402 ER-alpha-positive tamoxifen-treated breast cancers. RESULTS: AIB1 overexpression was not associated with relapse during treatment with tamoxifen. In contrast, high AIB1 expression in patients with human epidermal growth factor receptor (HER) 2- and HER3-overexpressing tumors or tumors expressing one or more of HER1, HER2, or HER3 (HER1-3 positive) was associated with an increased risk of relapse on tamoxifen [hazard ratio, 2.20; 95% confidence interval, 1.07-3.52 (P = 0.0416); hazard ratio, 2.42; 95% confidence interval, 1.32-4.43 (P = 0.0030), respectively]. AIB1 gene amplification was observed in 18 of 362 (5%) patients. High AIB1 gene copy number had no effect on overall or disease-free survival. CONCLUSIONS: Data presented here support a role for AIB1 expression on relapse during tamoxifen treatment in hormone-responsive HER-expressing clinical breast cancers and support clinical evidence, suggesting a cross-talk between ER-alpha and growth factor receptor pathways through changes in expression of specific coactivator proteins, such as AIB1. This study highlights the potential that tumor profiling, using multiple markers of treatment response, may improve patient selection for endocrine treatment, such as tamoxifen or aromatase inhibitors.

SIRT3 & SIRT7: Potential Novel Biomarkers for Determining Outcome in Pancreatic Cancer Patients.

PURPOSE: The sirtuin gene family has been linked with tumourigenesis, in both a tumour promoter and suppressor capacity. Information regarding the function of sirtuins in pancreatic cancer is sparse and equivocal. We undertook a novel study investigating SIRT1-7 protein expression in a cohort of pancreatic tumours. The aim of this study was to establish a protein expression profile for SIRT1-7 in pancreatic ductal adenocarcinomas (PDAC) and to determine if there were associations between SIRT1-7 expression, clinico-pathological parameters and patient outcome. MATERIAL AND METHODS: Immunohistochemical analysis of SIRT1-7 protein levels was undertaken in a tissue micro-array comprising 77 resected PDACs. Statistical analyses determined if SIRT1-7 protein expression was associated with clinical parameters or outcome. RESULTS: Two sirtuin family members demonstrated significant associations with clinico-pathological parameters and patient outcome. Low level SIRT3 expression in the tumour cytoplasm correlated with more aggressive tumours, and a shorter time to relapse and death, in the absence of chemotherapeutic intervention. Low levels of nuclear SIRT7 expression were also associated with an aggressive tumour phenotype and poorer outcome, as measured by disease-free and disease-specific survival time, 12 months post-diagnosis. CONCLUSIONS: Our data suggests that SIRT3 and SIRT7 possess tumour suppressor properties in the context of pancreatic cancer. SIRT3 may also represent a novel predictive biomarker to determine which patients may or may not respond to chemotherapy. This study opens up an interesting avenue of investigation to potentially identify predictive biomarkers and novel therapeutic targets for pancreatic cancer, a disease that has seen no significant improvement in survival over the past 40 years.

SIRT2: tumour suppressor or tumour promoter in operable breast cancer?

PURPOSE: Sirtuins comprise a family of genes involved in cellular stress, survival and damage responses. They have been implicated in a range of diseases including cancer, with most information pertaining to their function in tumourigenesis being derived from in vitro studies, or model organisms. Their putative roles as tumour suppressors or tumour promoters remain to be validated in vivo. Little is known about their role in breast tumourigenesis. We sought to evaluate the seven sirtuin family members (SIRT1-7) in a human breast cancer cohort, in relation to clinico-pathological features and outcome of the disease. MATERIALS AND METHODS: Immunohistochemical analysis of SIRT1-7 protein levels was undertaken in 392 oestrogen receptor (ER+ve) and 153 ER-ve breast tumour samples. SIRT1-7 transcriptional levels were assessed in normal (n=25), non-malignant (n=73) and malignant (n=70) breast tissue using Relative Quantitative Real Time PCR. Statistical analyses determined if SIRT1-7 transcription or protein expression was associated with clinical parameters or outcome. RESULTS: In ER-ve tumours, high protein levels of nuclear SIRT2 were associated with reduced time to recurrence and disease-specific death. This association was only observed in Grade 3 tumours. In the ER+ve cohort, high SIRT2 nuclear levels were associated with shorter disease-free survival and time to recurrence whilst on Tamoxifen, in patients with Grade 3 tumours. Conversely, in Grade 2 tumours, high SIRT2 levels were associated with increased time to recurrence. CONCLUSIONS: Our data suggest that SIRT2 is the sirtuin predominantly involved in breast tumourigenesis and prognosis. It indicates that SIRT2 acts as a tumour suppressor or tumour promoter dependent upon breast tumour grade.

Pathfinder cells provide a novel therapeutic intervention for acute kidney injury.

Pathfinder cells (PCs) are a novel class of adult-derived cells that facilitate functional repair of host tissue. We used rat PCs to demonstrate that they enable the functional mitigation of ischemia reperfusion (I/R) injury in a mouse model of renal damage. Female C57BL/6 mice were subjected to 30 min of renal ischemia and treated with intravenous (i.v.) injection of saline (control) or male rat pancreas-derived PCs in blinded experimentation. Kidney function was assessed 14 days after treatment by measuring serum creatinine (SC) levels. Kidney tissue was assessed by immunohistochemistry (IHC) for markers of cellular damage, proliferation, and senescence (TUNEL, Ki67, p16(ink4a), p21). Fluorescence in situ hybridization (FISH) was performed to determine the presence of any rat (i.e., pathfinder) cells in the mouse tissue. PC-treated animals demonstrated superior renal function at day 14 post-I/R, in comparison to saline-treated controls, as measured by SC levels (0.13 mg/dL vs. 0.23 mg/dL, p<0.001). PC-treated kidney tissue expressed significantly lower levels of p16(ink4a) in comparison to the control group (p=0.009). FISH analysis demonstrated that the overwhelming majority of repaired kidney tissue was mouse in origin. Rat PCs were only detected at a frequency of 0.02%. These data confirm that PCs have the ability to mitigate functional damage to kidney tissue following I/R injury. Kidneys of PC-treated animals showed evidence of improved function and reduced expression of damage markers. The PCs appear to act in a paracrine fashion, stimulating the host tissue to recover functionally, rather than by differentiating into renal cells. This study demonstrates that pancreatic-derived PCs from the adult rat can enable functional repair of renal damage in mice. It validates the use of PCs to regenerate damaged tissues and also offers a novel therapeutic intervention for repair of solid organ damage in situ.

Interactions between MAP kinase and oestrogen receptor in human breast cancer.

PURPOSE: The oestrogen receptor (ERα) may be activated in a ligand-dependent manner, via oestrogen, or in a ligand-independent manner, via signal transduction pathways. Mitogen Activated Protein Kinase (MAPK) is known to directly phosphorylate ERα at serine 118 in a ligand-independent manner. This study investigated the interaction between MAPK and ERα in breast cancer. MATERIALS & METHODS: Immunohistochemical experiments were undertaken to determine the expression of MAPK, pMAPK and pER(ser118) in breast tumours to determine their clinical relevance. Immunofluorescent experiments were performed, on MCF-7 breast cancer cells, to monitor the phosphorylation and localisation of MAPK and ERα in response to oestrogen, heregulin and a MAPK inhibitor. RESULTS: Oestrogen and Heregulin stimulated phosphorylation of ERα and its nuclear translocation, but heregulin induced this at levels much lower than those observed with oestrogen. Following stimulation with heregulin, but not oestrogen, treatment with MAPK inhibitor reduced the levels of nuclear pER(ser118). In cells treated with both oestrogen and heregulin, nuclear pER(ser118) was visible; but at levels comparable with heregulin treatment alone. CONCLUSION: This study confirms that ligand-mediated phosphorylation is associated with rapid nuclear localisation of ERα, due to oestrogen binding. ERα is phosphorylated at serine 118 in a ligand-independent manner. Preventing nuclear translocation of pMAPK reduced the levels of ligand-independent, but not ligand-dependent phosphorylation of ERα. Co-stimulation with both oestrogen and heregulin suggested that heregulin mediated signalling determines the subcellular localisation of ERα. Activation of ERα by direct phosphorylation may result in its rapid deactivation due to degradation or nuclear export.

Pre-transplant CDKN2A expression in kidney biopsies predicts renal function and is a future component of donor scoring criteria.

CDKN2A is a proven and validated biomarker of ageing which acts as an off switch for cell proliferation. We have demonstrated previously that CDKN2A is the most robust and the strongest pre-transplant predictor of post-transplant serum creatinine when compared to "Gold Standard" clinical factors, such as cold ischaemic time and donor chronological age. This report shows that CDKN2A is better than telomere length, the most celebrated biomarker of ageing, as a predictor of post-transplant renal function. It also shows that CDKN2A is as strong a determinant of post-transplant organ function when compared to extended criteria (ECD) kidneys. A multivariate analysis model was able to predict up to 27.1% of eGFR at one year post-transplant (p = 0.008). Significantly, CDKN2A was also able to strongly predict delayed graft function. A pre-transplant donor risk classification system based on CDKN2A and ECD criteria is shown to be feasible and commendable for implementation in the near future.

Is telomere length socially patterned? Evidence from the West of Scotland Twenty-07 Study.

Lower socioeconomic status (SES) is strongly associated with an increased risk of morbidity and premature mortality, but it is not known if the same is true for telomere length, a marker often used to assess biological ageing. The West of Scotland Twenty-07 Study was used to investigate this and consists of three cohorts aged approximately 35 (N = 775), 55 (N = 866) and 75 years (N = 544) at the time of telomere length measurement. Four sets of measurements of SES were investigated: those collected contemporaneously with telomere length assessment, educational markers, SES in childhood and SES over the preceding twenty years. We found mixed evidence for an association between SES and telomere length. In 35-year-olds, many of the education and childhood SES measures were associated with telomere length, i.e. those in poorer circumstances had shorter telomeres, as was intergenerational social mobility, but not accumulated disadvantage. A crude estimate showed that, at the same chronological age, social renters, for example, were nine years (biologically) older than home owners. No consistent associations were apparent in those aged 55 or 75. There is evidence of an association between SES and telomere length, but only in younger adults and most strongly using education and childhood SES measures. These results may reflect that childhood is a sensitive period for telomere attrition. The cohort differences are possibly the result of survival bias suppressing the SES-telomere association; cohort effects with regard different experiences of SES; or telomere possibly being a less effective marker of biological ageing at older ages.

Socio-economic status is associated with epigenetic differences in the pSoBid cohort.

BACKGROUND: Epigenetic programming and epigenetic mechanisms driven by environmental factors are thought to play an important role in human health and ageing. Global DNA methylation has been postulated as an epigenetic marker for epidemiological studies as it is reflective of changes in gene expression linked to disease. How epigenetic mechanisms are affected by psychological, sociological and biological determinants of health still remains unclear. The aim of this study was to investigate the relationship between socio-economic and lifestyle factors and epigenetic status, as measured by global DNA methylation content, in the pSoBid cohort, which is characterized by an extreme socio-economic and health gradient. METHODS: DNA was extracted from peripheral blood leukocytes using the Maxwell® 16 System and Maxwell® 16 Blood DNA Purification kit (Promega, UK). Global DNA methylation was assessed using Methylamp™ Global DNA Methylation Quantification Ultra kit (Epigentek, USA). Associations between global DNA methylation and socio-economic and lifestyle factors were investigated in linear regression models. RESULTS: Global DNA hypomethylation was observed in the most socio-economically deprived subjects. Job status demonstrated a similar relationship, with manual workers having 24% lower DNA methylation content than non-manual. Additionally, associations were found between global DNA methylation content and biomarkers of cardiovascular disease (CVD) and inflammation, including fibrinogen and interleukin-6 (IL-6), after adjustment for socio-economic factors. CONCLUSIONS: This study has indicated an association between epigenetic status and socio-economic status (SES). This relationship has direct implications for population health and is reflected in further associations between global DNA methylation content and emerging biomarkers of CVD.

Telomere attrition and decreased fetuin-A levels indicate accelerated biological aging and are implicated in the pathogenesis of colorectal cancer.

PURPOSE: Increasing chronological age is a risk factor for many types of cancer including colorectal. An understanding of the biology of aging and factors which regulate it may provide insight into cancer pathogenesis. The role of telomere biology in both the cancer and aging process could prove useful in this regard. EXPERIMENTAL DESIGN: Using quantitative PCR, we determined telomere length in the peripheral blood leukocytes of 64 colorectal cancer (CRC) patients and 1,348 controls. We also measured telomere length in 32 colorectal tumor samples and matched normal tissue. We aimed to assess whether telomere lengths were reflected in circulating mediators of inflammation and redox control factors, including fetuin-A, a circulating modulator of calcium homeostasis. RESULTS: CRC patients had shorter telomeres [adjusted mean ratio of relative telomere repeat copy number to single-copy gene number (RelT/S) = 0.61] compared with chronologically older controls (mean age = 75, adjusted mean RelT/S = 0.70; ANCOVA, P = 0.004). Telomere length in tumor tissue [median = 0.43, interquartile range (IQR) = 0.40] was significantly shorter than adjacent normal tissue (median = 0.65, IQR = 0.28; P = 0.004). Patients with low fetuin-A levels were shown to have significantly shorter telomeres (P = 0.041). Patients with rectal tumors had significantly higher levels of fetuin-A than those with colonic tumors (P = 0.045). CONCLUSIONS: We have observed that patients with CRC display clear evidence of telomere attrition compared with controls. This is congruent with accelerated biological aging in the pathogenesis of CRC. An imbalance in redox control mechanisms and calcium homeostasis may be a contributing factor to telomere dynamics in our patients. Furthermore, fetuin-A levels can be used to distinguish between colon and rectal cancers.

Pancreatic-derived pathfinder cells enable regeneration of critically damaged adult pancreatic tissue and completely reverse streptozotocin-induced diabetes.

We demonstrate that intravenous delivery of human, or rat, pancreas-derived pathfinder (PDP) cells can totally regenerate critically damaged adult tissue and restore normal function across a species barrier. We have used a mouse model of streptozotocin (STZ)-induced diabetes to demonstrate this. Normoglycemia was restored and maintained for up to 89 days following the induction of diabetes and subsequent intravenous delivery of PDP cells. Normal pancreatic histology also appeared to be restored, and treated diabetic animals gained body weight. Regenerated tissue was primarily of host origin, with few rat or human cells detectable by fluorescent in situ hybridization (FISH). Crucially, the insulin produced by these animals was overwhelmingly murine in origin and was both types I and II, indicative of a process of developmental recapitulation. These results demonstrate the feasibility of using intravenous administration of adult cells to regenerate damaged tissue. Critically, they enhance our understanding of the mechanisms relating to such repair and suggest a means for novel therapeutic intervention in loss of tissue and organ function with age.

Accelerated telomere attrition is associated with relative household income, diet and inflammation in the pSoBid cohort.

BACKGROUND: It has previously been hypothesized that lower socio-economic status can accelerate biological ageing, and predispose to early onset of disease. This study investigated the association of socio-economic and lifestyle factors, as well as traditional and novel risk factors, with biological-ageing, as measured by telomere length, in a Glasgow based cohort that included individuals with extreme socio-economic differences. METHODS: A total of 382 blood samples from the pSoBid study were available for telomere analysis. For each participant, data was available for socio-economic status factors, biochemical parameters and dietary intake. Statistical analyses were undertaken to investigate the association between telomere lengths and these aforementioned parameters. RESULTS: The rate of age-related telomere attrition was significantly associated with low relative income, housing tenure and poor diet. Notably, telomere length was positively associated with LDL and total cholesterol levels, but inversely correlated to circulating IL-6. CONCLUSIONS: These data suggest lower socio-economic status and poor diet are relevant to accelerated biological ageing. They also suggest potential associations between elevated circulating IL-6, a measure known to predict cardiovascular disease and diabetes with biological ageing. These observations require further study to tease out potential mechanistic links.

Cellular senescence in pretransplant renal biopsies predicts postoperative organ function.

Older and marginal donors have been used to meet the shortfall in available organs for renal transplantation. Post-transplant renal function and outcome from these donors are often poorer than chronologically younger donors. Some organs, however, function adequately for many years. We have hypothesized that such organs are biologically younger than poorer performing counterparts. We have tested this hypothesis in a cohort of preimplantation human renal allograft biopsies ( n = 75) that have been assayed by real-time polymerase chain reaction for the expression of known markers of cellular damage and biological aging, including CDKN2A, CDKN1A, SIRT2 and POT1. These have been investigated for any associations with traditional factors affecting transplant outcome (donor age, cold ischaemic time) and organ function posttransplant (serum creatinine levels). Linear regression analyses indicated a strong association for serum creatinine with pre-transplant CDKN2A levels ( p = 0.001) and donor age ( p = 0.004) at 6 months post-transplant. Both these markers correlated significantly with urinary protein to creatinine ratios ( p = 0.002 and p = 0.005 respectively), an informative marker for subsequent graft dysfunction. POT1 expression also showed a significant association with this parameter ( p = 0.05). Multiple linear regression analyses for CDKN2A and donor age accounted for 24.6% ( p = 0.001) of observed variability in serum creatinine levels at 6 months and 23.7% ( p = 0.001) at 1 year posttransplant. Thus, these data indicate that allograft biological age is an important novel prognostic determinant for renal transplant outcome.

AKT activation predicts outcome in breast cancer patients treated with tamoxifen.

Oestrogen receptor (ERalpha) expression is a strong predictor of response to endocrine therapy. The PI3K/AKT/mTOR signal transduction pathway has been implicated in endocrine resistance in vitro. The present study was carried out to test the hypothesis that AKT activation mediates tamoxifen resistance in clinical breast cancer. Immunohistochemistry (IHC) using AKT1-3, pan-AKT, pAKT (Thr-308), pAKT (Ser-473), pER (Ser-167), and pHER2 antibodies was performed on 402 ERalpha-positive breast carcinomas from patients treated with tamoxifen. High pAKT (Ser-473) activity (p = 0.0406) and low AKT2 expression (p = 0.0115) alone, or in combination [high pAKT (Ser-473)/low AKT2; 'high-risk' patient group] (p = 0.0014), predicted decreased overall survival in tamoxifen-treated patients with ERalpha-positive breast cancers. There was no significant association between tumour levels of AKT expression or activity and disease-free survival (DFS); however, the 'high-risk' patient group was significantly more likely to relapse (p = 0.0491). During tamoxifen treatment, neither AKT2 nor pAKT predicted DFS. Finally, activation of AKT, via phosphorylation, was linked to activation of both HER2 and ERalpha in this patient cohort. The data presented here show that the PI3K/AKT/mTOR pathway is associated with relapse and death in ERalpha-positive breast cancer patients treated with tamoxifen, supporting in vitro evidence that AKT mediates tamoxifen resistance. Patients with a 'high-risk' expression profile were at increased risk of death (hazard ratio 3.22, p = 0.002) relative to 'low-risk' patients, highlighting the potential that tumour profiling, with multiple IHC markers predictive of therapeutic response, may improve patient selection for endocrine therapies, eg tamoxifen or aromatase inhibitor-based treatments.