No abstract

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

No abstract

The contribution of stromal cells to tumor progression is widely established while less is known about stromal changes that may influence the early phases of tumorigenesis such as incipient growth of (pre)neoplastic lesions or seeding and awakening of metastatic cells. We have previously characterized the pro-tumorigenic properties of lung fibroblasts identifying increased activity of the nuclear transporter Exportin-1 (XPO1) as a key driver in modulating metabolic properties of fibroblasts towards a tumor promoting phenotype through subcellular re-localization of p53 and p62/SQSTM1. Consistently, XPO1 inhibition prevented the ability of activated fibroblasts to stimulate migration of lung cancer cells and to increase the frequency of CD133+ cancer stem cells, thereby also reducing their in vivo pro-tumorigenic activity. To test the potential of therapeutic strategies based on modification of pro-tumorigenic microenvironments we exploited a mouse model of lung fibrosis based on single intranasal administration of an adenoviral vector expressing activated TGFβ. Fibrosis was efficiently induced in both nude and SCID mice and was associated with the creation of a pro-tumorigenic milieu as tested by tail vein injection of microenvironment-responsive lung cancer cell line LT73 one month after fibrosis induction. Mice treated with AdTGFβ showed increased number of tumor cells and/or CD133+ lung cancer stem cells two months after cancer cell seeding (p<0.05 for AdTGFβ vs. AdLacZ and medium alone). We also evaluated the modulation of immune cell subsets with multi parametric flow-cytometry detecting a consistent increase in neutrophils (Ly6-G+/Ly6-Clow) and a slight increase in monocyte derived macrophages (F4-80high/GR1-/CD11b+) and plasmacytoid dendritic cells (CD11c+/CD11b-) associated with upregulation of fibrosis-related miRNAs (miR-199a-5p) and induction of a pro-tumorigenic milieu. To test the efficacy of microenvironment modulating therapies we used the selective XPO1 inhibitor KPT-330. In vitro pharmacological inhibition of XPO1 was able to modulate expression of miRNAs in fibroblasts (including miR-199a-5p) and reduce fibroblast pro-tumorigenic secretory phenotype (reduction in IL-8, CXCL1, CCL2 and lactate production). For in vivo experiments animals were treated after induction of fibrosis but before injection of cancer cells to avoid confounding effects related to activity on cancer cells. XPO1 inhibition was able to convert the pro-tumorigenic potential of the fibrotic microenvironment and this was associated with a reduction of the levels of miR-199a-5p, decreased expression extracellular matrix-related genes (Sparc, Col1a1, Mmp2, Col6a1, Col6a3) and dampening of the inflammatory milieu. Overall these findings suggest potential therapeutic efficacy of XPO1 inhibition to revert the pro-tumorigenic activity of fibrotic lung microenvironments. Citation Format: Antonina Bruccoleri, Ornella Rondinone, Francesca Andriani, Federica Facchinetti, Monica Tortoreto, Gabriella Sozzi, Giulia Bertolini, Luca Roz. Fondazione IRCCS Ist. Inhibition of Exportin-1 function reverses the pro-tumorigenic potential of lung fibrotic microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1912.

Large cell carcinoma (LCC) is an aggressive lung cancer subtype with poor prognosis and no targeted therapies. We previously reported that tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we report that MMP1 is overexpressed specifically in LCC cell lines. Notably, silencing MMP1 expression in LCC cancer cell lines using shRNA revealed that MMP1 expression by LCC cells is necessary for induction of fibroblast senescence in coculture experiments with normal pulmonary fibroblasts, as revealed by the analysis of a panel of standard senescence markers, including β-galactosidase (SA-βgal) staining, permanent growth arrest and expression of senescence-associated secretory factors. Injecting control (shScr) or shMMP1 LCC cells into immunodeficient nude mice revealed that tumor growth, tumor take and cancer cell dissemination to the lung were reduced in shMMP1 H460 tumors compared to control tumors. We also observed fewer senescent fibroblasts in tumors from shMMP1 H460 cells using Sentragor staining, which allows identification of senescent cells in paraffin embedded tissues. Moreover, we found that recombinant active MMP1 in combination with TGF-β1 were sufficient to induce normal fibroblast senescence. In terms of the potential underlying mechanisms, treatment with the antioxidant n-acetyl cysteine (NAC) significantly attenuated the increase in SA-βgal+ fibroblasts elicited by co-stimulation with rMMP1 and TGF-β1, and its corresponding conditioned medium elicited a significantly lower growth and invasion in LCC cancer cells, revealing the oxidative stress implication in fibroblast senescence induction and associated pro-tumorigenic secretome. In summary, our results establish a new role for MMP1 in cancer and support that LCC cells elicit a tumor-supporting niche through the aberrant secretion of MMP1 and TGF-β1 to induce senescence in adjacent fibroblasts. Furthermore, we implicate oxidative stress in MMP1/TGF-β1-induced TAF senescence and support a novel therapeutic strategy in LCC based on targeting senescent TAFs. Citation Format: Marta Gabasa, Evette S. Radisky, Rafael Ikemori, Giulia Bertolini, Marselina Arshakyan, Alexandra Hockla, Paula Duch, Ornella Rondinone, Alejandro Llorente, Maria Maqueda, Alexandre Perera, Noemí Reguart, Luca Roz, Derek C. Radisky, Jordi Alcaraz. MMP1 and TGF-β1 cooperate to drive tumor progression in large cell carcinoma of the lung through fibroblast senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2515.