Heparan sulfate proteoglycans and human breast cancer epithelial cell tumorigenicity

Heparan sulfate proteoglycans (HSPGs) are key components of the extracellular matrix that mediate cell proliferation, invasion, and cellular signaling. The biological functions of HSPGs are linked to their co-stimulatory effects on extracellular ligands (e.g., WNTs) and the resulting activation of transcription factors that control mammalian development but also associated with tumorigenesis. We examined the expression profile of HSPG core protein syndecans (SDC1–4) and glypicans (GPC1–6) along with the enzymes that initiate or modify their glycosaminoglycan chains in human breast cancer (HBC) epithelial cells. Gene expression in relation to cell proliferation was examined in the HBC cell lines MCF-7 and MDA-MB-231 following treatment with the HS agonist heparin. Heparin increased gene expression of chain initiation and modification enzymes including EXT1 and NDST1, as well as core proteins SDC2 and GPC6. With HS/Wnt interactions established, we next investigated WNT pathway components and observed that increased proliferation of the more invasive MDA-MB-231 cells is associated with activation of the Wnt signaling pathway. Specifically, there was substantial upregulation (>5-fold) of AXIN1, WNT4A, and MYC in MDA-MB-231 but not in MCF-7 cells. The changes in gene expression observed for HSPG core proteins and related enzymes along with the associated Wnt signaling components suggest coordinated interactions. The influence of HSPGs on cellular proliferation and invasive potential of breast cancer epithelial cells are cell and niche specific. Further studies on the interactions between HSPGs and WNT ligands may yield clinically relevant molecular targets, as well as new biomarkers for characterization of breast cancer progression.

Proteomic and other analyses to determine the functional consequences of deregulated kallikrein-related peptidase (KLK) expression in prostate and ovarian cancer

Rapidly developing proteomic tools are improving detection of deregulated kallikrein-related peptidase (KLK) expression, at the protein level, in prostate and ovarian cancer, as well as facilitating the determination of functional consequences downstream. Mass spectrometry (MS)-driven proteomics uniquely allows for the detection, identification and quantification of thousands of proteins in a complex protein pool, and this has served to identify certain KLKs as biomarkers for these diseases. In this review we describe applications of this technology in KLK biomarker discovery, and elucidate MS-based techniques which have been used for unbiased, global screening of KLK substrates within complex protein pools. Although MS-based KLK degradomic studies are limited to date, they helped to discover an array of novel KLK substrates. Substrates identified by MS-based degradomics are reported with improved confidence over those determined by incubating a purified or recombinant substrate and protease of interest, in vitro. We propose that these novel proteomic approaches represent the way forward for KLK research, in order to correlate proteolysis of biological substrates with tissue-related consequences, toward clinical targeting of KLK expression and function for cancer diagnosis, prognosis and therapies.

Novel use of faecal sterols to assess human faecal contamination in Antarctica : a likelihood assessment matrix for environmental monitoring

Wastewater containing human sewage is often discharged with little or no treatment into the Antarctic marine environment. Faecal sterols (primarily coprostanol) in sediments have been used for assessment of human sewage contamination in this environment, but in situ production and indigenous faunal inputs can confound such determinations. Using gas chromatography with mass spectral detection profiles of both C27 and C29 sterols, potential sources of faecal sterols were examined in nearshore marine sediments, encompassing sites proximal and distal to the wastewater outfall at Davis Station. Faeces from indigenous seals and penguins were also examined. Faeces from several indigenous species contained significant quantities of coprostanol but not 24-ethylcoprostanol, which is present in human faeces. In situ coprostanol and 24-ethylcoprostanol production was identified by co-production of their respective epi isomers at sites remote from the wastewat er source and in high total organic matter sediments. A C 29 sterols-based polyphasic likelihood assessment matrix for human sewage contamination is presented, which distinguishes human from local fauna faecal inputs and in situ production in the Antarctic environment. Sewage contamination was detected up to 1.5 km from Davis Station.

Critical dependence of blood-borne biomarker concentrations on the half-lives of their carrier proteins

Until recently, the low-abundance (LA) range of the serum proteome was an unexplored reservoir of diagnostic information. Today it is increasingly appreciated that a diagnostic goldmine of LA biomarkers resides in the blood stream in complexed association with more abundant higher molecular weight carrier proteins such as albumin and immunoglobulins. As we now look to the possibility of harvesting these LA biomarkers more efficiently through engineered nano-scale particles, mathematical approaches are needed in order to reveal the mechanisms by which blood carrier proteins act as molecular 'mops' for LA diagnostic cargo, and the functional relationships between bound LA biomarker concentrations and other variables of interest such as biomarker intravasation and clearance rates and protein half-lives in the bloodstream. Here we show, by simple mathematical modeling, how the relative abundance of large carrier proteins and their longer half-lives in the bloodstream work together to amplify the total blood concentration of these tiny biomarkers. The analysis further suggests that alterations in the production of biomarkers lead to gradual rather than immediate changes in biomarker levels in the blood circulation. The model analysis also points to the characteristics of artificial nano-particles that would render them more efficient harvesters of tumor biomarkers in the circulation, opening up possibilities for the early detection of curable disease, rather than simply better detection of advanced disease.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

PTRF/cavin-1 neutralizes non-caveolar caveolin-1 microdomains in prostate cancer

Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.

Pathological excretion patterns of urinary proteins in miners highly exposed to dinitrotoluene

A cohort of 161 underground miners who had been highly exposed to dinitrotoluene (DNT) in the copper-mining industry of the former German Democratic Republic was reinvestigated for signs of subclinical renal damage. The study included a screening of urinary proteins excreted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and quantitations of the specific urinary proteins α 1-microglobulin and glutathione-S-transferase α (GST α) as biomarkers for damage of the proximal tubule and glutathione-S-transferase π (GST π) for damage of the distal tubule. The exposures were categorized semiquantitatively (low, medium, high, and very high), according to the type and duration of professional contact with DNT. A straight dose-dependence of pathological protein excretion patterns with the semiquantitative ranking of DNT exposure was seen. Most of the previously reported cancer cases of the urinary tract, especially those in the higher exposed groups, were confined to pathological urinary protein excretion patterns. The damage from DNT was directed toward the tubular system. In many cases, the appearance of Tamm-Horsfall protein, a 105-kD protein marker, was noted. Data on the biomarkers α 1-microglobulin, GST α, and GST π consistently demonstrated a dose-dependent increase in tubular damage, which confirmed the results of screening by SDS-PAGE and clearly indicated a nephrotoxic effect of DNT under the given conditions of exposure. Within the cluster of cancer patients observed among the DNT-exposed workers, only in exceptional cases were normal biomarker excretions found.

Detoxification enzyme activities (CYP1A1 and GST) in the skin of humpback whales as a function of organochlorine burdens and migration status

The activities of glutathione-s-transferase (GST) and cytochrome P-450 1A1 (CYP1A1) enzymes were measured in freshly extracted epidermis of live-biopsied, migrating, southern hemisphere humpback whales (Megaptera novaeangliae). The two quantified enzyme activities did not correlate strongly with each other. Similarly, neither correlated strongly with any of the organochlorine compound groups previously measured in the superficial blubber of the sample biopsy core, likely reflecting the anticipated low levels of typical aryl-hydrocarbon receptor ligands. GST activity did not differ significantly between genders or between northward (early migration) or southward (late migration) migrating cohorts. Indeed, the inter-individual variability in GST measurements was relatively low. This observation raises the possibility that measured activities were basal activities and that GST function was inherently impacted by the fasting state of the sampled animals, as seen in other species. These results do not support the implementation of CYP1A1 or GST as effective biomarkers of organochlorine contaminant burdens in southern hemisphere populations of humpback whales as advocated for other cetacean species. Further investigation of GST activity in feeding versus fasting cohorts may, however, provide some insight into the fasting metabolism of these behaviourally adapted populations.

Meta-analysis of microarray data identifies GAS6 expression as an independent predictor of poor survival in ovarian cancer

Seeking new biomarkers for epithelial ovarian cancer, the fifth most common cause of death from all cancers in women and the leading cause of death from gynaecological malignancies, we performed a meta-analysis of three independent studies and compared the results in regard to clinicopathological parameters. This analysis revealed that GAS6 was highly expressed in ovarian cancer and therefore was selected as our candidate of choice. GAS6 encodes a secreted protein involved in physiological processes including cell proliferation, chemotaxis, and cell survival. We performed immunohistochemistry on various ovarian cancer tissues and found that GAS6 expression was elevated in tumour tissue samples compared to healthy control samples (P < 0.0001). In addition, GAS6 expression was also higher in tumours from patients with residual disease compared to those without. Our data propose GAS6 as an independent predictor of poor survival, suggesting GAS6, both on the mRNA and on the protein level, as a potential biomarker for ovarian cancer. In clinical practice, the staining of a tumour biopsy for GAS6 may be useful to assess cancer prognosis and/or to monitor disease progression.

A biomarker panel to discriminate between systemic inflammatory response syndrome and sepsis and sepsis severity

Introduction: In this study, we report on initial efforts to discover putative biomarkers for differential diagnosis of a systemic inflammatory response syndrome (SIRS) versus sepsis; and different stages of sepsis. In addition, we also investigated whether there are proteins that can discriminate between patients who survived sepsis from those who did not. Materials and Methods: Our study group consisted of 16 patients, of which 6 died and 10 survived. We daily measured 28 plasma proteins, for the whole stay of the patients in the ICU. Results: We observed that metalloproteinases and sE-selectin play a role in the distinction between SIRS and sepsis, and that IL-1, IP-10, sTNF-R2 and sFas appear to be indicative for the progression from sepsis to septic shock. A combined measurement of MMP-3, -10, IL-1, IP-10, sIL-2R, sFas, sTNF-R1, sRAGE, GM-CSF, IL-1 and Eotaxin allows for a good separation of patients that survived from those that died (mortality prediction with a sensitivity of 79% and specificity of 86%). Correlation analysis suggests a novel interaction between IL-1a and IP-10. Conclusion: The marker panel is ready to be verified in a validation study with or without therapeutic intervention.

Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. (C) 2012 Elsevier B.V. All rights reserved.

Saliva as an emerging biofluid for clinical diagnosis and applications of MEMS/NEMS in salivary diagnostics

Saliva as a biological fluid is gaining wider acceptance for diagnosing diseases. The growing interest in saliva as a biological fluid is due to its noninvasiveness, ease of use, cost-effectiveness, and multiple sample collection possibilities as well as minimal risk to health care professionals of contracting infectious organisms such as HIV and Hep B. However, the clinical translation of saliva is hampered by our lack of understanding of the biomolecular transportation from blood into saliva, the diurnal variations of biomolecules present in saliva, and relatively low levels of analytes (100th to a 1000th fold less than in blood). We provide information on the current status of salivary research, salivary diagnostics empowered by nanotechnology, and future prospects in this emerging field of saliva diagnostics.

DNA methylation at the novel CpG sites in the promoter of MED15/PCQAP gene as a biomarker for head and neck cancers

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P, 0.05 and Mann-Whitney test P, 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P, 0.01) and 0.63 (P, 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC. © the authors.

A novel saliva-based microRNA biomarker panel to detect head and neck cancers

Background MicroRNAs (miRNAs) are known to play an important role in cancer development by post-transcriptionally affecting the expression of critical genes. The aims of this study were two-fold: (i) to develop a robust method to isolate miRNAs from small volumes of saliva and (ii) to develop a panel of saliva-based diagnostic biomarkers for the detection of head and neck squamous cell carcinoma (HNSCC). Methods Five differentially expressed miRNAs were selected from miScript™ miRNA microarray data generated using saliva from five HNSCC patients and five healthy controls. Their differential expression was subsequently confirmed by RT-qPCR using saliva samples from healthy controls (n = 56) and HNSCC patients (n = 56). These samples were divided into two different cohorts, i.e., a first confirmatory cohort (n = 21) and a second independent validation cohort (n = 35), to narrow down the miRNA diagnostic panel to three miRNAs: miR-9, miR-134 and miR-191. This diagnostic panel was independently validated using HNSCC miRNA expression data from The Cancer Genome Atlas (TCGA), encompassing 334 tumours and 39 adjacent normal tissues. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic capacity of the panel. Results On average 60 ng/μL miRNA was isolated from 200 μL of saliva. Overall a good correlation was observed between the microarray data and the RT-qPCR data. We found that miR-9 (P <0.0001), miR-134 (P <0.0001) and miR-191 (P <0.001) were differentially expressed between saliva from HNSCC patients and healthy controls, and that these miRNAs provided a good discriminative capacity with area under the curve (AUC) values of 0.85 (P <0.0001), 0.74 (P < 0.001) and 0.98 (P < 0.0001), respectively. In addition, we found that the salivary miRNA data showed a good correlation with the TCGA miRNA data, thereby providing an independent validation. Conclusions We show that we have developed a reliable method to isolate miRNAs from small volumes of saliva, and that the saliva-derived miRNAs miR-9, miR-134 and miR-191 may serve as novel biomarkers to reliably detect HNSCC. © 2014 International Society for Cellular Oncology.