Potential of Protein-based Anti-metastatic Therapy with Serpins and Inter α-Trypsin Inhibitors

Abstract

In this review we summarize the principles of anti-metastatic therapy with selected serpin family proteins, such as pigment epithelial-derived factor (PEDF) and maspin, as well as inter α-trypsin inhibitor (IαIs) light chains (bikunin) and heavy chains (ITIHs). Case-by-case, antimetastatic activity may be dependent or independent of the protease-inhibitory activity of the corresponding proteins. We discuss the incidence of target deregulation in different tumor entities, mechanisms of deregulation, context-dependent functional issues as well as in vitro and in vivo target validation studies with transfected tumor cells or recombinant protein as anti-metastatic agents. Finally, we comment on possible clinical evaluation of these proteins in adjuvant therapy.

Keywords: Adjuvant anti-metastatic therapy; bikunin; in vitro and in vivo target validation; inter α-trypsin inhibitors; maspin; pigment-epithelial derived factor; protein substitution therapy; review.

Figure 1. Anti-metastatic effects of pigment-epithelium derived factor (PEDF). PEDF can bind to several cell surface receptors and activate proapoptotic effectors such as CD95L, caspase 8, p53 and Bax. Activation of CD95L by PEDF has been shown in endothelial cells and tumor cells. In addition, PEDF inhibits VEGFR1 signaling in endothelial cells and anti-apoptotic protein Bcl2 in tumor cells. MMP2 and MMP9 inhibit PEDFand MT-MMP1 is inhibited by PEDF. Bax, Bcl2 associated X protein; CD95, cluster of differentiation 95; CD95L, CD95 ligand; ERK5, extracellularsignal regulated kinase 5; FLIP, FLICE-inhibitory protein; MEK5, mitogen-activated protein kinase 5; MMP2, 9, matrix metalloproteinases 2 and9; MT-MMP1, membrane-type matrix metalloproteinase 1; NFATc2, nuclear facor of activated T-cells c2; VEGF, vascular endothelial growth factor;VEGFR1, VEGF receptor 1.

Figure 2. Anti-metastatic effects of maspin. Maspin can be secreted, localized in the cytoplasm and the nucleus and can associate with the plasma membrane. In endothelial cells, maspin inhibits VEGFR and FGFR signaling. In tumor cells maspin inhibits a not yet identified receptor X which promotes adhesion, promotes internalisation of migration-promoting integrin β1, attenuates migration through inhibition of migration-promoting proteins Rac1 and cdc42 and can initiate an anti-invasive transcriptional program by chromatin modification (methylation) in the nucleus. Extracellularly, maspin can inhibit uPA. β1, Integrin β1; cdc42, cell division cycle 42; CH3, methyl; COL, collagen; FGF, fibroblast growth factor; FGFR1, FGF receptor 1; FN, fibronectin; Rac1, ras-related C3 botulinum substrate 1; uPA, urokinase-type plasminogen activator; VEGF, vascular endothelial growth factor; VEGFR1, VEGF receptor 1.

Figure 3. Composition of inter-α-trypsin inhibitors and serum-derived hyaluronan-associated proteins. ITα1 is composed of a CS backbone (Nacetylgalactosamine and glucuronic acid repeating disacharides), covalently linked to Bk and ITIH chains. SHAP is composed of HA (diglucuronic acid and N-acetyl-D-glucosamine repeating disacharides) covalently bound to ITIHs. CS, Chondroitin sulfate; HA, hyaluronic acid; ITαI, inter-α- trypsin inhibitor; SHAP, serum-derived hyaluronan-associated proteins.

Figure 4. Anti-metastatic effects of Bikunin (Bk). Bk inhibits PLMNR mediated conversion of PLMN to PLN as well as dimerization of CD44, MAPK activation and transcriptional activation of Bk. Bk, Bikunin; CD44, cluster of differentiation 44; HA, hyaluronic acid; MAPK, mitogen-activated protein kinase; PLMN, plasminogen; PLMNR, plasminogen receptor; PLN, plasmin; uPA, urokinase plasminogen activator.

Figure 5. Anti-metastatic functions of inter-trypsin inhibitor heavy chains (ITIHs). All ITIHs are secreted by tumor cells; in addition, ITIH2 can be located at membrane ruffles and at the periplasmic membrane and ITIH5 can be found in endocytic vesicles in the cytoplasm. ITIH5 increases integrin β1 expression, modulates activity of migration-related GTPases RhoA and Rac1 and epigenetically reprograms the transcriptional program of the tumor cell und supports transcription of tumor suppressor gene DAPK1. CH3, Methyl; DAPK1, death-associated kinase protein 1; ITIH, inter-trypsin inhibitor heavy chains; Rac1, ras-related C3 botulinum toxin substrate 1; Rho A, ras homologue A.

Figure 6. Expression of ITIH gene family members in tumor and matched normal samples by RNA sequencing of TCGA cohorts. Expression values (y-Axis) are provided as log2 normalized read counts. The red lines indicate a normalized read count of 100 separating very low from higher expression levels. Expression data are shown as box plots where the black line represent the data median, the black rectangle show the upper and lower 25% quartile (therefore, 50% of all data points are included in the black rectangle). All other data points, except for outliers lie within the upper and lower whiskers. Data are presented for the following tumor entities and matched normal tissues: bladder (408/19), breast (1100/112), cervical (306/3), colorectal (457/41), esophagus (185/11), head-and-neck: 522/44, lung adenocarcinoma (517/59), lung squamous cell carcinoma (501/51), rectum (167/10), stomach (415/359 and thyroid (509/59).