Choreographing metastasis to the tune of LTBP

Abstract

Latent Transforming Growth Factor beta (TGFβ) Binding Proteins (LTBPs) are chaperones and determinants of TGFβ isoform-specific secretion. They belong to the LTBP/Fibrillin family and form integral components of the fibronectin and microfibrillar extracellular matrix (ECM). LTBPs serve as master regulators of TGFβ bioavailability, functioning to incorporate and spatially pattern latent TGFβ at regular intervals within the ECM, and actively participate in integrin-mediated stretch activation of TGFβ in vivo. In so doing they create a highly patterned sensory system where local changes in ECM tension can be detected and transduced into focal signals. The physiological role of LTBPs in the mammary gland remains largely unstudied, however both loss and gain of LTBP expression is found in breast cancers and breast cancer cell lines. Importantly, elevated LTBP1 levels appear in two gene signatures predictive of enhanced metastatic behavior. LTBP may promote metastasis by providing the bridge between structural and signaling components of the epithelial to mesenchymal transition (EMT).

Figure 1

Assembly and function of latent-TGFβ. a The tripartite large latent complex (LLC) is formed early in the rough endoplasmic reticulum (RER), via disulphide bonding first between two TGFβ pro-protein monomers to form pro-TGFβ, and then between pro-TGFβ and the 8-cys domain of LTBP. In the Golgi, furin mediates the cleavage of pro-TGFβ into dimeric TGFβ and its propeptide, latency associated protein (LAP). These two homodimers associate non-covalently to form the small latent complex (SLC). LTBP acts as a chaperone, ensuring proper folding and disulphide bonding between LAP and TGFβ. LAP is also glycosylated in the Golgi at mannose-6-phosphate groups, which may be important in extracellular signaling. The mature LLC is then secreted. b The LLC positions itself within the extracellular matrix (ECM) first by LTBP binding through its N-terminus (N) to fibronectin and later through its C-terminus (C) binding to N-terminus of fibrillin in microfibrils. This maintains extracellular TGFβ in its latent state. c Latent TGFβ is activated via integrins in the stretch mechanism. Upon mechanical signal from the actin cytoskeleton, integrins bind to RGD sites on LAP. As LTBP maintains its hold on the ECM during this process, the two opposing forces allow a conformational change in LAP that releases TGFβ from the complex, activating it and allowing it to bind its receptor, TGF-βR

Figure 2

Members of the LTBP/Fibrillin family. Four LTBPs and three fibrillins are known. LTBPs have internal promoter initiation sites (black arrows) and undergo alternative splicing giving rise to splice variants (pink dotted lines). LTBP and fibrillin contain 8-Cys repeats (red globules) and the EGF-like repeats. EGF-like repeats can either bind to calcium (grey rods), giving the protein its rigidity, or can remain unbound (green rods). Hybrid domains (brown globules) have combined features of 8-Cys and EGF-like repeats. The small latent TGFβ-LAP complex (SLC) binds to a specific 8-Cys domain found only in LTBP 1, 3 and 4, adjacent to the fibrillin binding C-terminal region. LTBPs also have a central hinge region for additional flexibility. The hinge and the hybrid domains form a major site for ECM attachment and play a role in the stretch response when interacting with integrins. Fibrillins contain a unique C-terminal conserved region (blue rods and purple octagons). RGD domains (yellow arrowheads) present in LAP and in fibrillins are functional and bind to integrins. Although present in LTBP their integrin binding function has not been established

Figure 3

Expression pattern of LTBP-1 during mammary development, using LTBP1L-LacZ mouse model. a LTBP1L-LacZ (blue) is expressed in the mammary mesenchyme surrounding the embryonic (E) E12.5 (left) mammary bud, and persists at this site under the developing nipple E17.5. LTBP1L-LacZ becomes additionally expressed within the luminal epithelium of the rudimentary sprout as the lumen first forms. b During puberty, LTBP1L-LacZ is expressed in quiescent ductal luminal cells but not in the terminal end buds. c During pregnancy, LTBP1L-LacZ expression is high in the luminal cells of ducts and side branches, but is absent from secretory structures such as alveoli. d LTBP1L-LacZ is not expressed during lactation, but is seen in a subset of cells during involution

Figure 4

Model for potential LTBP interactions with the ECM in mammary gland. Integrins are multifunctional proteins, which can specify cellular responses in multiple ways. a Cells use integrin α5β1 (brown) to bind directly to fibronectin (FN) in the ECM. Mammary cells express αVβ5 (yellow) and αVβ3 (orange), which in other cells have been shown to bind to the RGD domains (yellow arrowheads) of LAP. b αVβ5 (yellow) and αVβ3 (orange) interactions can also form between the cell surface and fibrillins. The integrin-LLC association with fibronectin and fibrillins provide extra footholds between the cell and the matrix. Different matrix molecules assemble distinct complements and arrangements of integrins. In the case of fibronectin it is hypothesized that stretch mediated integrin activation of TGFβ will stimulate the EMT transcriptional program to reinforce migratory behavior