Breast Tumor Cell Invasion and Pro-Invasive Activity of Cancer-Associated Fibroblasts Co-Targeted by Novel Urokinase-Derived Decapeptides

Abstract

Among peritumoral cells, cancer-associated fibroblasts (CAFs) are major facilitators of tumor progression. This study describes the effects of two urokinase-derived, novel decapeptides, denoted as Pep 1 and its cyclic derivative Pep 2. In a mouse model of tumor dissemination, using HT1080 fibrosarcoma cells, Pep 2 reduced the number and size of lung metastases. Specific binding of fluoresceinated Pep 2 to HT1080 and telomerase immortalised fibroblasts (TIF) cell surfaces was enhanced by αv overexpression or abolished by excess vitronectin, anti-αv antibodies or silencing of ITGAV αv gene, identifying αv-integrin as the Pep 2 molecular target. In 3D-organotypic assays, peptide-exposed TIFs and primary CAFs from breast carcinoma patients both exhibited a markedly reduced pro-invasive ability of either HT1080 fibrosarcoma or MDA-MB-231 mammary carcinoma cells, respectively. Furthermore, TIFs, either exposed to Pep 2, or silenced for αv integrin, were impaired in their ability to chemoattract cancer cells and to contract collagen matrices, exhibiting reduced α-smooth muscle actin (α-SMA) levels. Finally, peptide exposure of αv-expressing primary CAFs led to the downregulation of α-SMA protein and to a dramatic reduction of their pro-invasive capability. In conclusion, the ability of the novel decapeptides to interfere with tumor cell invasion directly and through the down-modulation of CAF phenotype suggests their use as lead compounds for co-targeting anti-cancer strategies.

Modulation of Cellular Function by the Urokinase Receptor Signalling: A Mechanistic View

“…A thourough conformational analysis of the CP-derived, anti-migratory peptides suggested the design of a novel cyclic peptide denoted uPAcyclin, corresponding to the N-terminal region of CPp, with the S138E substitution in a stabilized, putative bioactive conformation. The uPAcyclin has anti-migratory and anti-invasive properties in culture and prevents lung metastases in nude mice through its interaction with the αv-integrin subunit ( Belli et al, 2020 ). Furthermore, the novel peptide induces a partial reversion of the Cancer-Associated Fibroblasts (CAF) phenotype and markedly reduces the pro-invasive ability of peritumoral CAFs from breast cancer patients in combination with MDA-MB-231 mammary adenocarcinoma cells in organotypic assays ( De Vincenzo et al, 2019 ; Belli et al, 2020 ).…”

“…The uPAcyclin has anti-migratory and anti-invasive properties in culture and prevents lung metastases in nude mice through its interaction with the αv-integrin subunit ( Belli et al, 2020 ). Furthermore, the novel peptide induces a partial reversion of the Cancer-Associated Fibroblasts (CAF) phenotype and markedly reduces the pro-invasive ability of peritumoral CAFs from breast cancer patients in combination with MDA-MB-231 mammary adenocarcinoma cells in organotypic assays ( De Vincenzo et al, 2019 ; Belli et al, 2020 ). Independent evidence showed the multiple activities of an 8-mer linear peptide corresponding to residues 136–143 of human uPA and denoted Å6.…”

αV-Integrin-Dependent Inhibition of Glioblastoma Cell Migration, Invasion and Vasculogenic Mimicry by the uPAcyclin Decapeptide

“…The U87-MG and U251-MG cell lines were purchased from ECACC (European Collection of Authenticated Cell Cultures, Salisbury, UK) and ATCC (American Type Culture Collection, Manassas, VA, USA), respectively, whereas the C6, HEK-293, and U138-MG cells were obtained from ICLC (Interlab Cell Line Collection, National Institute for Cancer Research, Genoa, Italy). The HEK-293/αV stable transfectants have been described elsewhere [25].…”

“…Thus, in the present work, we investigated the effects of the anti-migratory uPAcyclin decapeptide, binding to the αV integrin subunit [25]. This cyclic decapeptide is derived from a non-catalytic region of the human urokinase (uPA) plasminogen activator.…”

Therapeutic Strategies Targeting Urokinase and Its Receptor in Cancer

“…Pep 1 and Pep 2 bind specifically to the αv integrin subunit, and TIFs ability to chemoattract cancer cells and contract collagen matrices is reduced by αv integrin silencing. Interestingly, TIFs or primary CAFs exposure to these peptides reduced α-smooth muscle actin levels downregulating their matrix contracting ability [125]. Due to the clear-cut effects of Pep 1 and Pep 2 on tumor cell invasion, the possibility of their use as therapeutic lead compounds should be investigated in-depth.…”