Tumor necrosis factor alpha induces Warburg-like metabolism and is reversed by anti-inflammatory curcumin in breast epithelial cells.

The reprogramming of cellular metabolism in cancer cells is a well-documented effect. It has previously been shown that common oncogene expression can induce aerobic glycolysis in cancer cells. However, the direct effect of an inflammatory microenvironment on cancer cell metabolism is not known. Here, we illustrate that treatment of nonmalignant (MCF-10a) and malignant (MCF-7) breast epithelial cells with low-level (10 ng/ml) tumor necrosis factor alpha (TNF-α) significantly increased glycolytic reliance, lactate export and expression of the glucose transporter 1 (GLUT1). TNF-α decreased total mitochondrial content; however, oxygen consumption rate was not significantly altered, suggesting that overall mitochondrial function was increased. Upon glucose starvation, MCF7 cells treated with TNF-α demonstrated significantly lower viability than nontreated cells. Interestingly, these properties can be partially reversed by coincubation with the anti-inflammatory agent curcumin in a dose-dependent manner. This work demonstrates that aerobic glycolysis can be directly induced by an inflammatory microenvironment independent of additional genetic mutations and signals from adjacent cells. Furthermore, we have identified that a natural dietary compound can reverse this effect.

The clinical significance of inflammatory cytokines in primary cell culture in endometrial carcinoma.

Endometrial cancer is the most common malignancy of the female genital tract, and the incidence and mortality rates from this disease are increasing. Although endometrial carcinoma has been regarded as a tissue-specific disease mediated by female sex steroid pathways, considerable evidence implicates a role for an inflammatory response in the development and propagation of endometrial cancer. We hypothesized that if specific patterns of cytokine expression were found to be predictive of adverse outcome, then selective receptor targeting may be a therapeutic option. This study was therefore undertaken to determine the relationship between cytokine production in primary cell culture and clinical outcome in endometrial adenocarcinoma. Fresh endometrial tissues were fractionated into epithelial and stromal fractions and cultured. After 6-7 days, supernatants were collected and cells enumerated. Batched aliquots were assayed using ELISA kits specific for CSF-1, GMCSF, G-CSF, TNF-α, IL-6, IL-8, and VEGF. Data were compared using ANOVA, Fisher's exact, and log rank tests. Increased epithelial VEGF production was observed more often in tumors with Type 2 variants (p = 0.039) and when GPR30 receptor expression was high (p = 0.038). Although increased stromal VEGF production was detected more often in grade 3 endometrioid tumors (p = 0.050), when EGFR expression was high (p = 0.003), and/or when ER/PR expression was low (p = 0.048), VEGF production did not correlated with overall survival (OS). Increased epithelial CSF-1 and TNF-α production, respectively, were observed more often in tumors with deep myometrial invasion (p = 0.014) and advanced stage (p = 0.018). Increased CSF-1 (89.5% vs. 42.9%, p = 0.032), TNF-α (88.9% vs. 42.9%, p = 0.032, and IL-6 (92.3% vs. 61.5%, p = 0.052) also correlated with low OS. In Cox multivariate models, CSF-1 was an independent predictor of low survival when stratified by grade (p = 0.046) and histology (p = 0.050), and TNF-α, when stratified by histology (p = 0.037). In this study, high CSF-1, TNF-α, and IL-6 production rates identified patients at greatest risk for death, and may signify patients likely to benefit from receptor-specific therapy.

Abnormal glucose tolerance, white blood cell count, and telomere length in newly diagnosed, antidepressant-naïve patients with depression.

Chronic mood disorders have been associated with a shortened telomere, a marker of increased mortality rate and aging, and impaired cellular immunity. However, treatment may confound these relationships. We examined the relationship of glucose tolerance, white blood cell count and telomere length to depression in newly diagnosed, antidepressant-naïve patients. Subjects with major depression (n=15), and matched healthy control subjects (n=70) underwent a two-hour oral glucose tolerance test and evaluation of blood cell count and telomere content. The depression group had significantly higher two-hour glucose concentrations and a lower lymphocyte count than control subjects (respective means [SD] for two-hour glucose were 125.0mg/dL [67.9] vs 84.6 [25.6] (p<.001); for lymphocyte count 2.1×10(9)/L [0.6] vs 2.5×10(9)/L [0.7] p=.028). Telomere content was significantly shortened in the depression group (87.9 [7.6]) compared to control subjects (101.0 [14.3]; p<0.01). Abnormal glucose tolerance, lymphopenia and a shortened telomere are present early in the course of depression independently of the confounding effect of antidepressant treatment, supporting the concept of major depression as an accelerated aging disease.

Markers of field cancerization: proposed clinical applications in prostate biopsies.

Field cancerization denotes the occurrence of genetic, epigenetic, and biochemical aberrations in structurally intact cells in histologically normal tissues adjacent to cancerous lesions. This paper tabulates markers of prostate field cancerization known to date and discusses their potential clinical value in the analysis of prostate biopsies, including diagnosis, monitoring progression during active surveillance, and assessing efficacy of presurgical neoadjuvant and focal therapeutic interventions.

Coordinate regulation between expression levels of telomere-binding proteins and telomere length in breast carcinomas.

Telomere dysregulation occurs in both the in situ and invasive stages of many carcinomas, including breast. Knockout experiments have identified several telomere-associated proteins required for proper telomere function and maintenance, including telomere repeat-binding factor 1 and 2 (TRF1 and TRF2), protection of telomeres (POT1), and TRF1-interacting nuclear factor 2 (TIN2). Using telomere content assays and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we examined the relationship between telomere length and the mRNA levels of telomere-associated proteins in breast tumors. The levels of TRF2, TRF1, TIN2, and POT1 mRNA, but not telomerase reverse transcriptase (TERT) RNA, are inversely correlated with telomere content in breast tumors. Significant associations were identified between the mRNA levels of TRF1, TIN2, and POT1; however, there were no significant associations with the mRNA levels of TRF2 or TERT. These associations suggest that a complex transcriptional program coordinately regulates the expression of these mRNAs. We examined the promoter regions of the telomere-associated proteins to identify transcription factors consistent with the observed patterns of presumed coordinate expression. We demonstrated in human breast cancer cell lines that expressions of TRF1, TIN2, and POT1 are upregulated by dexamethasone, suggesting activation of the glucocorticoid receptor, whereas TERT, TRF2, TRF1, TIN2, and POT1 are upregulated by tumor necrosis factor-α (TNF-α), suggesting activation of the nuclear factor kappa B transcription factor. These findings link telomere content in breast tumors to the coordinate expression of several telomere-associated proteins previously shown to be negative regulators of telomere length in cell lines. The results further suggest a possible link between the expressions of the telomere-associated proteins and mediators of stress and inflammation.Telomere content assays and quantitative RT-PCR demonstrate that the levels of TRF2, TRF1, TIN2, and POT1 mRNA, but not telomerase reverse transcriptase (TERT) RNA, are inversely correlated with telomere content in breast tumors. Within human breast cancer cell lines, expressions of TRF1, TIN2, and POT1 are upregulated by dexamethasone, suggesting activation of the glucocorticoid receptor, whereas TERT, TRF2, TRF1, TIN2, and POT1 are upregulated by TNF-α, suggesting activation of the NFκB transcription factor. These findings link telomere content in breast tumors to the expression of several telomere-associated proteins previously shown to be negative regulators of telomere length in cell lines and suggest a link between the expressions of the telomere-associated proteins and mediators of stress and inflammation.

Early growth response 1 and fatty acid synthase expression is altered in tumor adjacent prostate tissue and indicates field cancerization.

BACKGROUND: Field cancerization denotes the occurrence of molecular alterations in histologically normal tissues adjacent to tumors. In prostate cancer, identification of field cancerization has several potential clinical applications. However, prostate field cancerization remains ill defined. Our previous work has shown up-regulated mRNA of the transcription factor early growth response 1 (EGR-1) and the lipogenic enzyme fatty acid synthase (FAS) in tissues adjacent to prostate cancer. METHODS: Immunofluorescence data were analyzed quantitatively by spectral imaging and linear unmixing to determine the protein expression levels of EGR-1 and FAS in human cancerous, histologically normal adjacent, and disease-free prostate tissues. RESULTS: EGR-1 expression was elevated in both structurally intact tumor adjacent (1.6× on average) and in tumor (3.0× on average) tissues compared to disease-free tissues. In addition, the ratio of cytoplasmic versus nuclear EGR-1 expression was elevated in both tumor adjacent and tumor tissues. Similarly, FAS expression was elevated in both tumor adjacent (2.7× on average) and in tumor (2.5× on average) compared to disease-free tissues. CONCLUSIONS: EGR-1 and FAS expression is similarly deregulated in tumor and structurally intact adjacent prostate tissues and defines field cancerization. In cases with high suspicion of prostate cancer but negative biopsy, identification of field cancerization could help clinicians target areas for repeat biopsy. Field cancerization at surgical margins on prostatectomy specimen should also be looked at as a predictor of cancer recurrence. EGR-1 and FAS could also serve as molecular targets for chemoprevention.

Breast field cancerization: isolation and comparison of telomerase-expressing cells in tumor and tumor adjacent, histologically normal breast tissue.

Telomerase stabilizes chromosomes by maintaining telomere length, immortalizes mammalian cells, and is expressed in more than 90% of human tumors. However, the expression of human telomerase reverse transcriptase (hTERT) is not restricted to tumor cells. We have previously shown that a subpopulation of human mammary epithelial cells (HMEC) in tumor-adjacent, histologically normal (TAHN) breast tissues expresses hTERT mRNA at levels comparable with levels in breast tumors. In the current study, we first validated a reporter for measuring levels of hTERT promoter activity in early-passage HMECs and then used this reporter to compare hTERT promoter activity in HMECs derived from tumor and paired TAHN tissues 1, 3, and 5 cm from the tumor (TAHN-1, TAHN-3, and TAHN-5, respectively). Cell sorting, quantitative real-time PCR, and microarray analyses showed that the 10% of HMECs with the highest hTERT promoter activity in both tumor and TAHN-1 tissues contain more than 95% of hTERT mRNA and overexpress many genes involved in cell cycle and mitosis. The percentage of HMECs within this subpopulation showing high hTERT promoter activity was significantly reduced or absent in TAHN-3 and TAHN-5 tissues. We conclude that the field of "normal tissue" proximal to the breast tumors contains a population of HMECs similar in hTERT expression levels and in gene expression to the HMECs within the tumor mass and that this population is significantly reduced in tissues more distal to the tumor.

Markers of fibrosis and epithelial to mesenchymal transition demonstrate field cancerization in histologically normal tissue adjacent to breast tumors.

Previous studies have shown that a field of genetically altered but histologically normal tissue extends 1 cm or more from the margins of human breast tumors. The extent, composition and biological significance of this field are only partially understood, but the molecular alterations in affected cells could provide mechanisms for limitless replicative capacity, genomic instability and a microenvironment that supports tumor initiation and progression. We demonstrate by microarray, qRT-PCR and immunohistochemistry a signature of differential gene expression that discriminates between patient-matched, tumor-adjacent histologically normal breast tissues located 1 cm and 5 cm from the margins of breast adenocarcinomas (TAHN-1 and TAHN-5, respectively). The signature includes genes involved in extracellular matrix remodeling, wound healing, fibrosis and epithelial to mesenchymal transition (EMT). Myofibroblasts, which are mediators of wound healing and fibrosis, and intra-lobular fibroblasts expressing MMP2, SPARC, TGF-ß3, which are inducers of EMT, were both prevalent in TAHN-1 tissues, sparse in TAHN-5 tissues, and absent in normal tissues from reduction mammoplasty. Accordingly, EMT markers S100A4 and vimentin were elevated in both luminal and myoepithelial cells, and EMT markers α-smooth muscle actin and SNAIL were elevated in luminal epithelial cells of TAHN-1 tissues. These results identify cellular processes that are differentially activated between TAHN-1 and TAHN-5 breast tissues, implicate myofibroblasts as likely mediators of these processes, provide evidence that EMT is occurring in histologically normal tissues within the affected field and identify candidate biomarkers to investigate whether or how field cancerization contributes to the development of primary or recurrent breast tumors.

Organ-wide telomeric status in diseased and disease-free prostatic tissues.

BACKGROUND: Telomere attrition occurs early in the development of prostatic adenocarcinoma. However, little is known about either telomere status in benign prostatic hyperplasia (BPH), or the spatial and organ-wide distribution of potential telomere aberrations throughout all areas of prostatic glands affected by cancer or BPH. METHODS: Slot blot titration assay was used to determine telomere DNA content (TC), a proxy for telomere length, in macrodissected tissue consisting of 54 normal samples from 5 disease-free prostates, 128 BPH samples from 4 non-cancerous prostates, and 45 tumor, 73 BPH, and 4 prostatic intraepithelial neoplasia (PIN) samples from 5 cancerous prostates. RESULTS: Compared to TC in normal prostate samples (n = 54; TC mean = 0.98), tumor samples displayed telomere attrition (n = 45; TC mean = 0.67). TC in PIN samples was similar to tumors. TC in BPH samples from cancerous prostates was similar to TC in tumors and also displayed telomere shortening (n = 73; TC mean = 0.76), whereas BPH samples from non-cancerous prostates displayed longer telomeres (n = 128; TC mean = 1.06). In prostates affected by adenocarcinoma, areas of potential telomere attrition occurred in histologically normal tissues through the entire gland. However, three-dimensional zoning revealed a pattern of increasing TC as a function of distance from the primary (index) tumor. CONCLUSIONS: Spatial distributions of TC in prostate specimens indicate a complex "field effect" with varying contributions from both cancer and BPH. The observation that telomere length variations occur in fields of histologically normal tissues surrounding the tumor is of clinical importance, as it may have implications for the diagnosis and focal therapy of prostate cancer.

Telomere DNA content in prostate biopsies predicts early rise in prostate-specific antigen after radical prostatectomy for prostate cancer.

OBJECTIVE: To determine whether measurement of telomere DNA content (TC) in prostate biopsy tissue predicts prostate-specific antigen (PSA) recurrence in men after undergoing radical prostatectomy for prostate cancer. METHODS: Slot blot titration assay was used to quantitate TC in archived diagnostic prostate needle biopsy specimens for subjects (n = 103) diagnosed with prostate cancer and who subsequently underwent radical prostatectomy between 1993 and 1997. TC was compared to the clinical outcome measure; PSA recurrence, defined as an increase in PSA > or = 0.2 ng/mL on 2 or more consecutive measurements post-prostatectomy, was observed retrospectively, for a mean follow-up period of 114 months (range, 1-165). RESULTS: In the cohort, 46 subjects had a PSA recurrence. In a univariate Cox proportional hazards model, low TC (< 0.3 of standard) demonstrated a significant risk for PSA recurrence (HR = 1.94; 95% CI: 1.02-3.69, P = .04). In a subset analysis of men with biopsy Gleason sum < or = 6 (n = 63; 25 recurrences), a univariate Cox proportional hazards model demonstrated that low TC had a greater risk of PSA recurrence (HR = 4.53; 95% CI: 2.00-10.2, P < .01). In a multivariate Cox proportional hazards model, low TC was also significantly associated with PSA recurrence in this subset after controlling for preoperative PSA levels (HR = 6.62; 95% CI: 2.69-16.3, P < .01). CONCLUSIONS: Low TC measured in prostate biopsy tissue predicts early likelihood of post-prostatectomy PSA recurrence in a retrospective analysis, and in men with biopsy Gleason sum < or = 6 disease it is also independent of preoperative PSA level.

Mammary field cancerization: molecular evidence and clinical importance.

The term "field cancerization" originally denoted the presence of histologically abnormal tissue/cells surrounding primary tumors of the head and neck. Similar concepts with different and continuously changing definitions have been used for other types of tumors including breast adenocarcinoma, where field cancerization presently denotes the occurrence of molecular alterations in histologically normal tissues surrounding areas of overt cancer. Human mammary tissue morphology lends itself to the proposed concepts of field cancerization, which may include the gradual accumulation of genetic and other aberrations in stationary epithelial cells with intact morphology, or the spread of histologically normal yet genetically aberrant epithelial cells within mammary tissue. In this report, we review published molecular genetic, epigenetic, and gene expressional data in support of field cancerization in human mammary tissues. We then discuss the clinical implications of mammary field cancerization, including its source for potential biomarkers with diagnostic/prognostic potential, and its relationship to surgical margins and disease recurrence. We conclude with a future outlook on further research on mammary field cancerization addressing experimental methods, as well as the development of possible models and integrated approaches to gain a better understanding of the underlying mechanisms with the ultimate goal of developing clinical applications.

Telomere length and pulse pressure in newly diagnosed, antipsychotic-naive patients with nonaffective psychosis.

INTRODUCTION: Recent studies suggest that in addition to factors such as treatment side effects, suicide, and poor health habits, people with schizophrenia may have an increased risk of diabetes prior to antipsychotic treatment. Diabetes is associated with an increased pulse pressure (PP) and a shortened telomere. We tested the hypothesis that prior to antipsychotic treatment, schizophrenia and related disorders are associated with a shortened telomere, as well as an increased PP. METHODS: Telomere content (which is highly correlated with telomere length) and PP were measured in newly diagnosed, antipsychotic-naive patients with schizophrenia and related disorders on first clinical contact and in matched control subjects. Both groups were also administered an oral glucose tolerance test. RESULTS: Compared with control subjects, the patients with psychosis had decreased telomere content and an increased PP. As previously reported, they also had increased glucose concentrations at 2 hours. These differences could not be attributed to differences in age, ethnicity, smoking, gender, body mass index, neighborhood of residence, socioeconomic status, aerobic conditioning, or an increased cortisol concentration in the psychotic subjects. DISCUSSION: These results suggest that prior to antipsychotic use, nonaffective psychosis is associated with reduced telomere content and increased PP, indices that have been linked to an increased risk of diabetes and hypertension.

Genomic instability demonstrates similarity between DCIS and invasive carcinomas.

PURPOSE: To assess telomere DNA content (TC) and the number of sites of allelic imbalance (AI) as a function of breast cancer progression. EXPERIMENTAL DESIGN: TC and AI were determined in 54 histologically normal tissues, 10 atypical ductal hyperplasias (ADH), 122 in situ ductal carcinomas (DCIS) and 535 invasive carcinomas (Stage I-IIIA). RESULTS: TC was altered in ADH lesions (20%), DCIS specimens (53%) and invasive carcinomas (51%). The mean number of sites of AI was 0.26 in histologically normal group tissue, increased to 1.00 in ADH, 2.94 in DCIS, and 3.07 in invasive carcinomas. All groups were statistically different from the histologically normal group (P < 0.001 for each); however, there was no difference between DCIS and the invasive groups. CONCLUSIONS: Genomic instability increases in ADH and plateaus in DCIS without further increase in the invasive carcinomas, supporting the notion that invasive carcinomas evolve from or in parallel with DCIS.

Telomere DNA content predicts breast cancer-free survival interval.

BACKGROUND: Telomeres are nucleoprotein complexes that protect chromosome ends from degradation and recombination. Critically shortened telomeres generate genomic instability. It has been postulated that the extent of telomere DNA loss is related to the degree of genomic instability within a tumor and therefore may presage clinical outcome. The objective of this investigation was to evaluate the hypothesis that telomere DNA content (TC) in breast tumor tissues predicts breast cancer-free survival interval. MATERIALS AND METHODS: Slot blot titration assay was used to quantitate TC in 530 archival breast tumor tissues in a population-based cohort. The relationships between TC, 12 risk factors for breast cancer adverse events (i.e., death due to breast cancer, breast cancer recurrence, or development of a new primary breast tumor), and breast cancer-free survival interval were evaluated by Fisher's exact test, log-rank analysis, and univariate and multivariate Cox proportional hazards models. RESULTS: TC was independent of each of the 12 risk factors. Ethnicity, tumor-node-metastasis stage, estrogen receptor, progesterone receptor, and p53 status, chemotherapy sequence, adjuvant therapy, and TC each conferred significant relative hazards. The best overall multivariate Cox proportional hazards model included TC, p53 status, tumor-node-metastasis stage, and estrogen receptor status as independent predictors of breast cancer-free survival interval (P < 0.00005). Low TC (< or =200% of standard), relative to the high-TC group (>200% of standard), conferred an adjusted relative hazard of 2.88 (95% confidence interval, 1.16-7.15; P = 0.022) for breast cancer-related adverse events. CONCLUSIONS: TC in breast cancer tissue is an independent predictor in this group of breast cancer-free survival interval.

Assessment of the frequency of allelic imbalance in human tissue using a multiplex polymerase chain reaction system.

Genomic instability can generate chromosome breakage and fusion randomly throughout the genome, frequently resulting in allelic imbalance, a deviation from the normal 1:1 ratio of maternal and paternal alleles. Allelic imbalance reflects the karyotypic complexity of the cancer genome. Therefore, it is reasonable to speculate that tissues with more sites of allelic imbalance have a greater likelihood of having disruption of any of the numerous critical genes that cause a cancerous phenotype and thus may have diagnostic or prognostic significance. For this reason, it is desirable to develop a robust method to assess the frequency of allelic imbalance in any tissue. To address this need, we designed an economical and high-throughput method, based on the Applied Biosystems AmpFlSTR Identifiler multiplex polymerase chain reaction system, to evaluate allelic imbalance at 16 unlinked, microsatellite loci located throughout the genome. This method provides a quantitative comparison of the extent of allelic imbalance between samples that can be applied to a variety of frozen and archival tissues. The method does not require matched normal tissue, requires little DNA (the equivalent of approximately 150 cells) and uses commercially available reagents, instrumentation, and analysis software. Greater than 99% of tissue specimens with >or=2 unbalanced loci were cancerous.

A novel loss-of-function mutation in TP53 in an endometrial cancer cell line and uterine papillary serous carcinoma model.

The etiology of carcinoma of the uterine endometrium (ECa) is poorly understood. However, loss of apoptosis is one of the major factors that allow cancer cells to survive and progress. Hec50co, a poorly differentiated human ECa cell line, is widely used in the investigation of ECa. Previously, Hec50co xenograft tumor model in nude mice developed an advanced phenotype, similar to that of uterine papillary serous carcinoma (UPSC). Importantly, loss-of-function mutation in tumor suppressor TP53 was found in 20-30% of all ECa and >90% of UPSC. Thus, understanding the status of TP53 in Hec50co is essential for using Heco50co as a model for UPSC. To obtain an accurate genotype-phenotype status of TP53 in Hec50co, we performed mutation and functional analysis of TP53 gene of Hec50co by RT-PCR, genomic-PCR, and cloning and expression of mutant and wildtype TP53 alleles. We found a novel 42-bp deletion mutation in the exon6-intron6 splice junction of TP53 (TP53.del42bp) leading to a 113-bp exon6-deleted/skipped transcript was identified in Hec50co. In addition, the other TP53 allele in Hec50co is inactivated through a large deletion. Adenovirus (AD) harboring wildtype full-length TP53 cDNA induces caspase-dependent apoptosis; while the AD-TP53.del42bp allele does not. In addition, messenger RNA of TP53.del42bp allele is stable whereas the protein product of TP53.del42bp allele is made but not stable. Taken together, we demonstrate that Hec50co is a TP53-null cell line possessing one TP53.del42bp allele and the other lost allele and therefore provides an excellent model to dissect the molecular and cellular bases of UPSC and other p53-null cancers.

Diagnostic significance of allelic imbalance in cancer.

Allelic imbalance (AI), a deviation from the normal 1:1 ratio of maternal and paternal alleles, occurs in virtually all solid and blood-borne malignancies. The frequency and spectrum of AI in a tumor cell reflects the karyotypic complexity of the cancer genome. Hence, many investigations have assessed the extent of AI to analyze differences between normal and tumor tissues in a variety of different organs. In this review, the authors describe established and emerging technologies used to assess the extent of AI in human tissues, and their application in the diagnosis of cancer. The four major methods to be reviewed represent powerful and widely used tools for the identification of allelic imbalances accompanying cancer initiation and progression. These are fluorescent in situ hybridization, comparative genomic hybridization, single nucleotide polymorphism arrays and the use of microsatellite markers. For each method, the authors provide a brief description of the approach and elaborate on specific studies that highlight its utility in the diagnosis of human cancers.

Telomeres: prognostic markers for solid tumors.

Solid tumors continue to affect millions of people worldwide. Increasingly sophisticated diagnostic tools contribute to the high incidence rates for some tumor types, and treatment options continue to expand. However, the progression of solid tumors represents a challenge for the appropriate treatment of individual patients because of the relative inaccuracy of current prognostic markers, including the widely used Tumor-Nodes-Metastasis (TNM) staging system, to predict the course of disease. As a result, both over- and undertreatment are clinical realities in the management of patients diagnosed with solid tumors. Therefore, population-based screening programs that increase the overall cancer incidence rates are controversial, as they may do little to improve the patient's quality of life. Consequently, there is a strong need to develop novel and independent markers of prognosis. In this context, we review the use of telomeres as prognostic markers for solid tumors, including cancers from lung, breast, prostate, colon, brain and head and neck. Telomeric sequences, the repetitive DNA at the end of human chromosomes, are mediators of genomic stability and can undergo length alterations during tumor initiation and progression. In a number of studies reviewed here, these alterations, measured as telomere attrition and elongation, have been shown either to be associated with clinical markers of disease progression or to be independent markers of cancer prognosis. We conclude from these studies that careful assessment of telomere length or its proxies, such as telomere DNA content, will be part of novel risk assessment and prognostic modalities for patients with solid tumors.

Telomere content correlates with stage and prognosis in breast cancer.

PURPOSE: To evaluate the hypothesis that telomere DNA content (TC) in breast tumor tissue correlates with TNM staging and prognosis. EXPERIMENTAL DESIGN: Slot blot assay was used to quantitate TC in 70 disease-free normal tissues from multiple organ sites, and two independent sets of breast tumors containing a total of 140 samples. Non-parametric Rank-Sums tests, logistic regression and Cox proportional hazards models were used to evaluate the relationships between TC and tumor size, nodal involvement, TNM stage, 5-year survival and disease-free interval. RESULTS: TC in 95% of normal tissues was 75-143% of that in the placental DNA standard, whereas only 50% of tumors had TC values in this range. TC was associated with tumor size (p=0.02), nodal involvement (p<0.0001), TNM stage (p=0.004), 5-year overall survival (p=0.0001) and 5-year disease-free survival (p=0.0004). A multivariable Cox model was developed using age at diagnosis, TNM stage and TC as independent predictors of breast cancer-free survival. Relative to the high TC group (>123% of standard), low TC (<101% of standard) conferred an adjusted relative hazard of 4.43 (95% CI 1.4-13.6, p=0.009). Receiver operating characteristic curves using thresholds defined by the TC distribution in normal tissues predicted 5-year breast cancer-free survival with 50% sensitivity and 95% specificity, and predicted death due to breast cancer with 75% sensitivity and 70% specificity. CONCLUSIONS: TC in breast cancer tissue is an independent predictor of clinical outcome and survival interval, and may discriminate by stage.

Protease nexin-1 expression is altered in human breast cancer.

BACKGROUND: Urokinase-type Plasminogen Activator (uPA), a serine protease, plays a pivotal role in human breast cancer metastasis by mediating the degradation of extracellular matrix proteins and promoting cell motility. In more advanced breast cancers, uPA activity is significantly up regulated and serves as a prognostic indicator of poor patient outcome. Classically, regulation of uPA activity, especially in breast cancers, is thought to be mediated by Type 1 Plasminogen Activator Inhibitor (PAI-1). However, we have recently found that a lesser known natural inhibitor of uPA, Protease Nexin 1 (PN-1), is expressed in normal human mammary tissue. Based on this observation, we investigated if PN-1 is also expressed in human breast cancers where it may contribute to the regulation of uPA and participate in the development of a metastatic phenotype. RESULTS: Using quantitative real-time PCR analysis, we measured PN-1 mRNA expression in tissues obtained from 26 human breast tumor biopsies and compared these values with those obtained from 10 normal breast tissue samples. Since both PAI-1 and uPA expression levels are known to be elevated in metastatic breast cancer, we also measured their levels in our 26 tumor samples for direct comparison with PN-1 expression. We found that PN-1 expression was elevated over that found in normal mammary tissue; an increase of 1.5- to 3.5-fold in 21 of 26 human breast tumors examined. As anticipated, both PAI-1 and uPA mRNA levels were significantly higher in the majority of breast tumors; 19 of 26 tumors for PAI-1 and 22 of 26 tumors for uPA. A quantile box plot of these data demonstrates that the elevated PN-1 expression in breast tumor tissues directly correlates with the increased expression levels found for PAI-1 and uPA. CONCLUSION: The fact that PN-1 expression is elevated in human breast cancer, and that its increased expression is directly correlated with increases measured for PAI-1 and uPA, suggests that PN-1 may contribute to the regulation of uPA-mediate tumor cell motility and metastatic spread.