Interferon Regulatory Factor 9 Promotes Lung Cancer Progression via Regulation of Versican
Lung cancer is the leading cause of cancer-related death worldwide and accounts for more than 1.8 million deaths per year globally. It has been shown that the tumour microenvironment plays a crucial role in tumour progression and metastasis. Beside numerous cytokines, chemokines and other factors secreted by the tumour stroma, type I IFNs are ... strong immune modulators, showing anti-proliferative and pro-apoptotic properties. The transcription factor Interferon Regulatory Factor 9 (IRF9) is the key mediator in the canonical IFN pathway as it can bind directly to DNA at so called interferon-stimulated response elements (ISRE). Based on Kaplan-Meier estimators, high levels of IRF9 in lung cancer patients have been associated with a significantly lower survival. Using tissue microarrays, we could show that IRF9 is expressed in most of the lung cancer entities. In human lung cancer tissues, IRF9 is expressed in both the solid tumour part and the tumour microenvironment, where we identified strong expression of IRF9 in Tumour-Associated Macrophages. IFN treatment inhibited the proliferation of A549 and induced IRF9 expression, but it is also able to activate the expression of 2,000 other genes. To solely manipulate IRF9 expression, we used lentiviral particles to transduce the adenocarcinoma cell lines A549 and A427 to stably overexpress (A549/A427 LV IRF9) or to stably suppress IRF9 (A549/A427 shIRF9). Overexpression of IRF9 increased tumour cell proliferation and migration, whereas the knockdown of IRF9 accordingly led to a reduction. The transduction of murine lung cancer cell line CULA confirmed the oncogenic phenotype after overexpression; however, knockdown of murine Irf9 failed to regulate tumour cell proliferation and migration. RNA seq of transduced A549 identified 1544 regulated genes in IRF9-overexpressing cells, 662 regulated genes in IRF9-silenced cells and 117 genes that were commonly regulated, including the proteoglycan Versican (VCAN). Further analysis in human cell lines revealed a correlation between VCAN and IRF9 expression, and in silico analysis located potential ISRE in the promoter of VCAN. Chromatin-immunoprecipitation and luciferase studies proved that IRF9 binds at these sites and activates the expression of VCAN. According to the observations in transduced CULA cells, Irf9 overexpression induced Vcan upregulation, whereas the knockdown of Irf9 failed to regulate Vcan. Silencing of VCAN in transduced cells diminished IRF9-mediated proliferation and migration and was even stronger in IRF9-silenced cells. Additionally, we identified the tumour suppressor p21 to be affected by siVCAN transfection, indicating an IRF9-VCAN-p21 axis to regulate oncogenic behaviour in human lung adenocarcinoma. We applied transduced cells in a subcutaneous tumour model, where increased (A549 LV IRF9) and accordingly decreased (A549 shIRF9) tumour sizes were observed. In accordance with in vitro results, CULA LV Irf9 resulted in stronger tumour growth, but tumours from CULA shIrf9 did not differ from the control group. Naïve murine cancer cell line LLC1 was injected both intravenously and subcutaneously in Irf9 / mice to study the role of Irf9 in the tumour stroma. The loss of Irf9 did not lead to changes in artificial tumour progression, but led to a reduction of macrophages, monocytes and natural killer cells within the tumour composition, indicating its beneficial influence in the tumour microenvironment. Altogether, these results indicate that IRF9 influences tumour development and progression intrinsically and alters the cellular composition in the microenvironment. Tumour cell-specific inhibition of IRF9 provides a potential treatment for lung cancer, enabling higher specificity over a global inhibition of IFN pathway by bypassing collateral harm to beneficial stromal IFN and IRF9.