BioScience2005

Expression cloning of novel regulators of 92 kDa type IV collagenase expression

R.R. Nair, D.D. Boyd

Abstract

Overexpression of the 92 kDa type IV collagenase (MMP-9) contributes to cancer progression. However, to date, there are few known regulators of expression of this metalloproteinase. We employed an expression library comprising 500000 cDNA clones to screen for novel regulators of MMP-9 expression. HT1080 cells were transiently co-transfected with an MMP-9 promoter-luciferase reporter and pools of the cDNA expression library. Positive-scoring pools were subdivided in secondary and tertiary screens, after which the regulatory cDNAs were identified by DNA sequencing. This brief review illustrates the utility of expression cloning in identifying specific regulators of MMP-9 expression.

  • cancer
  • expression cloning strategy
  • HT1080 cells
  • 92 kDa type IV collagenase
  • matrix metalloproteinase-9 (MMP-9)
  • tumour progression

Introduction

The matrix metalloproteinase (MMP) family, consisting of >24 human MMPs, is formed of zinc-binding endopeptidases capable of degrading ECM (extracellular matrix) components including collagen, fibrinogen and proteoglycans [1]. One member of this family, the 92 kDa type IV collagenase [MMP-9 (matrix metalloproteinase-9)], has long been recognized for its ability to degrade the ECM of basement membranes during tumour invasion. More recently, in addition to the extracellular matrix proteins, a large number of bioactive non-matrix proteins including pro-growth factors, cytokines and cell adhesion molecules have been identified as MMP-9 substrates. Indeed, MMP-9 contributes to a variety of pathological conditions including cancer, infectious diseases, wound healing, inflammation and vascular diseases [2] via its role in angiogenesis, invasion, metastasis and immunity [3].

Regulation of MMP-9 activity

The MMP-9 gene is encoded on chromosome 20 and its expression is under the control of a 2.2 kb upstream regulatory sequence harbouring binding sites for AP-1 (activator protein 1), NF-κB (nuclear factor κB), Sp1 (specificity protein 1) and PEA3/Ets (where PEA3 stands for polyoma enhancer activator 3) [4]. Transcription of the gene generates a 2.5 kb transcript that is translated into the latent 92 kDa protein product, which is subsequently activated by the removal of 73 amino acids at the N-terminus by several enzymes including stromelysin, MMP-2 and cathepsin G [5]. The activity of the resultant (active) enzyme, in turn, is subjected to titration by a variety of endogenous inhibitors in the TIMPs (tissue inhibitor of metalloproteinases 1–3) [6].

Strategies to interfere with MMP-9

Considering the wealth of data implicating MMP-9 in tumour progression, there is a real need to identify the biological cues ‘driving’ expression of this gene in cancer. At the same time, it must be recognized that since some members of the MMPs are protective against cancer, targeting the entire family in an indiscriminate fashion will almost certainly fail therapeutically. We have taken the view that scanning the expressed human genome for novel regulators of MMP-9 expression could allow for the selective targeting of this key metalloproteinase, ultimately benefiting patients at high risk for tumour dissemination.

Expression cloning strategy

To parse the expressed human gene for novel regulators of MMP-9 expression, HT1080 cells are co-transfected with cDNA pools and a luciferase reporter regulated by 2.2 kb of MMP-9 upstream sequence and subsequently assayed for luciferase activity. This promoter sequence includes all the regulatory elements necessary for appropriate MMP-9 expression [4]. In the primary screen, a cDNA pool modulating MMP-9 promoter activity is identified and this cDNA pool subdivided and again subjected to a secondary screen. This process is repeated a third time yielding a single clone regulating MMP-9 expression which is then identified by DNA sequencing.

Using this method, we have identified several putative regulators (Table 1) some of which have been validated by demonstrating modulation of the endogenous gene in response to cDNA overexpression and siRNA approaches. Interestingly, both SM22 and SIRT1, while repressing MMP-9 expression, have little effect on a separate metalloproteinase (MMP-2) arguing that these regulators show some selectivity for the former collagenase.

View this table:
Table 1 Different putative regulators of MMP-9 identified using expression cloning strategy

Conclusions

There is compelling in vitro and in vivo evidence for a role of MMP-9 in tumour progression. However, enzyme inhibitors have shown little success in the clinic probably because other protective metalloproteinases are just as efficiently targeted with these broad-spectrum inhibitors. Thus, there is a need to identify specific biological cues in cancer that ‘drive’ MMP-9 expression (but not that of the ‘protective’ collagenases). Indeed, with improved diagnostics and public awareness, we can expect increasing numbers of patients with earlier disease, a cohort of which are at high risk for tumour progression. Such patients could very well benefit from therapies specifically targeting MMP-9 expression while sparing the expression of ‘protective’ collagenases.

Footnotes

  • Research Colloquia: Research Colloquia at BioScience2005, held at SECC Glasgow, U.K., 17–21 July 2005. Edited by F. Antoni (Edinburgh, U.K.), C. Cooper (Essex, U.K.) and M. Schweizer (Heriot-Watt, U.K.). The first five papers featured in this Section were presented as a part of the Free Radicals and Cyclic Nucleotide Signalling Pathways: Nitric Oxide, Reactive Oxygen Species, cAMP and cGMP Pfizer-Sponsored Research Colloquium.

Abbreviations: ECM, extracellular matrix; MMP, matrix metalloproteinase

References

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