Hanne Offenberg

TIMP-1 expression in human colorectal cancer is associated with TGF-B1, LOXL2, INHBA1, TNF-AIP6 and TIMP-2 transcript profiles

The balance of activity between the endogenous enzyme inhibitors known as tissue inhibitors of metalloproteinases and their targets, the matrix metalloproteinases, in the extracellular matrix is thought to play an important role in tumour cell invasion. Supporting this notion, we have shown that colorectal cancer patients have increased plasma levels of the tissue inhibitor of metalloproteinases‐1 (TIMP‐1), and that high plasma TIMP‐1 levels are associated with short colorectal cancer patient survival. However, although TIMP‐1 has been extensively studied in cancer, very little is known about how it is regulated. To further elucidate potential mechanisms of regulation of this protein, we did a number of experiments to look at associations between the transcript profile of TIMP‐1 with known matrix metalloproteinases (MMPs) as well as with expression profiles of other genes differentially regulated in human colorectal cancer (CRC) and the other TIMPs 2–4, which have also been associated with the progression of colorectal cancer. Genome‐wide expression profiling of 172 CRC and normal mucosa samples was used to identify transcript changes for the genes under investigation. We found that TIMP‐1 was up‐regulated in CRC samples compared with normal tissue, while TIMP‐2 was down‐regulated. Eight MMPs were up‐regulated in CRC compared with normal tissue. Correlating up‐regulated genes with the TIMP‐1 transcript, we identified 13 that were also up‐regulated in cancerous tissue. Among these were genes associated with the synthesis of extracellullar matrix, genes involved in the TGF‐beta signalling pathway, and genes that are likely transcribed by the tumour cells. These insights add to the complex picture emerging about the regulation of TIMPs in colorectal cancer.

Investigating the biomarker potential of glycoproteins using comparative glycoprofiling — application to tissue inhibitor of metalloproteinases-1

Cancer-induced alterations of protein glycosylations are well-known phenomena. Hence, the glycoprofile of certain glycoproteins can potentially be used as biomarkers for early diagnosis. However, there are a substantial number of candidates and the techniques for measuring their biomarker potential are limited, calling for new methods. Here, we have investigated the cancer marker potential of the glycoprofile of tissue inhibitor of metalloproteinase-1 (TIMP-1) using a method for comparative glycoprofiling. Glycoprofiles were obtained from plasma TIMP-1 of five healthy donors and five colorectal cancer (CRC) patients showing increased amounts of TIMP-1. Furthermore, the TIMP-1 glycoprofiles of media from two colon cancer cell lines (CCC) and a prostate cancer cell line were determined as disease references. TIMP-1 was purified from IgG-depleted samples using immuno affinity and gel electrophoresis and the glycoprofiling was performed using glycopeptide enrichment and mass spectrometry. The heterogeneous glycoprofiles of TIMP-1 were found to be highly conserved among the healthy donors, proving an ideal candidate marker and showed high reproducibility of the method. Numerous CCC-specific TIMP-1 glycans were observed illustrating cancer-induced changes. Unexpectedly, quantitation revealed that the glycoprofiles of healthy donors and CRC patients varied minimally. Considering the increased CRC TIMP-1 levels and the observed CCC-specific glycans, the lack of variation indicates that the increased amount of CRC TIMP-1 is not a direct product of the cancer cells. Hence, the TIMP-1 glycoprofile holds no biomarker potential for CRC when using plasma as the sample origin. This study clearly illustrates that the technique is capable of performing individualised site-specific glycan analysis and representing a new tool for biomarker investigation of low-abundant glycoproteins.

Identification of alternatively spliced TIMP-1 mRNA in cancer cell lines and colon cancer tissue

TIMP‐1 is a promising new candidate as a prognostic marker in colorectal and breast cancer. We now describe the discovery of two alternatively spliced variants of TIMP‐1 mRNA. The two variants lacking exon 2 (del‐2) and 5 (del‐5), respectively, were identified in human cancer cell lines by RT‐PCR. The del‐2 variant was, furthermore, detected in extracts from 12 colorectal cancer tissue samples. By western blotting additional bands of lower molecular mass than full‐length TIMP‐1 were identified in tumor tissue, but not in plasma samples obtained from cancer patients.

Salivary tissue inhibitor of metalloproteinases‐1 localization and glycosylation profile analysis

Holten‐Andersen L, Thaysen‐Andersen M, Jensen SB, Buchwald C, Højrup P, Offenberg H, Nielsen HJ, Brünner N, Nauntofte B, Reibel J. Salivary tissue inhibitor of metalloproteinases‐1 localization and glycosylation profile analysis. APMIS 2011; 119: 741–9.

Identifying sources and estimating glandular output of salivary TIMP-1

Objective. Tissue inhibitor of metalloproteinases 1 (TIMP‐1) has been identified as a potential biomarker in diseases such as cancer, cardiovascular diseases and diabetes. Since TIMP‐1 resides in most tissues and bodily fluids, we evaluated the potential of using saliva to obtain reproducible TIMP‐1 measurements in a non‐invasive manner. Material and methods. Samples of unstimulated and stimulated whole saliva and saliva collected from individual glands were analysed for TIMP‐1 content. A TIMP‐1 ELISA was validated for use in saliva testing and the most optimal sampling and handling procedures for reproducible measurements identified. Western blotting and MALDI‐TOF mass spectrometry were used for confirmatory analyses. Results. The TIMP‐1 ELISA was found suitable for saliva measurements. All saliva secretions contained TIMP‐1, but in different concentrations ranging from 2.81 ng/mL in submandibular/sublingual saliva to 173.88 ng/mL in parotid saliva. TIMP‐1 concentrations were influenced to a varying degree by fluctuations in flow. We found the lowest output in submandibular/sublingual saliva stimulated with 0.5 % citric acid (3.56 ng/min) and highest output in chewing‐stimulated whole saliva (267.01 ng/min). Conclusion. This study shows that saliva contains authentic TIMP‐1, the concentration of which was found to depend on gland type and salivary flow. Stimulated whole saliva is suggested as a reliable and easily accessible source for TIMP‐1 determinations in bodily fluids.

Biomarkers for therapeutic efficacy.

Abstract Depending on the tumour type, a larger or smaller number of cancer patients receive chemotherapy with systemic toxicity as the only effect. In that situation, an alternative, not necessarily medical, treatment would have been a better choice – and toxicity (and financial resources) could have been spared by withholding ineffective drugs. One of the reasons for this apparent paradigm is that the tumour cells of each cancer pa- tient may show different sensitivity/resistance towards different chemotherapeutic drugs, i.e. breast cancer or colorectal cancer is not only breast or colorectal cancer. With our increasing biological insight and understanding, it has become apparent that each patients tumour tissue is unique and as a consequence, each patients tumour cell sensitivity/resistance to- wards chemotherapeutic drugs may be different. As of today there is no method in routine clinical use to predict the sensitivity/resistance to chemotherapy in its broad sense in the individual patient. This chapter will describe several different DNA, RNA, protein and cell based assay methodologies and marker molecules that have been brought forward as potential predictive assays/markers to be used to select the most effective drugs for the individual cancer patient.

A TIMP-1 splice variant transcript : Possible role in regulation of TIMP-1 expression

A splice variant of tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA lacking exon 2 (TIMP-1-v2) has been identified in human cancer cells and in colorectal and breast cancer tumors. The purpose of this study was (1) to study the level of full length TIMP-1 and TIMP-1-v2 transcripts in colorectal tumors; (2) to investigate if TIMP-1-v2 is translated to protein. Full length TIMP-1 and TIMP-1-v2 mRNA levels were compared between colorectal tumors and normal mucosa by Q-PCR. Both full length TIMP-1 and TIMP-1-v2 transcripts were upregulated in tumor tissue. However, the level of TIMP-1-v2 relative to full length TIMP-1 was higher in normal compared to tumor tissue. Translation of TIMP-1-v2 to protein was analyzed in CHO cells. In this system, no TIMP-1-v2 protein was produced. Thus, the variant transcript seems to be an untranslated mRNA. These findings suggest that alternative splicing of TIMP-1 pre-mRNA to TIMP-1-v2 mRNA might be involved in regulating TIMP-1 expression.