Modulating ER And PPAR Pathways In Tumor Cells: The Effect On Macrophage Activation State And Pro- Tumor Function.

Abstract

Owing to its aggressive resistance to therapies, lung cancer is the leading cause of cancer deaths with 5-year survival rate of 17.5%. Non-small cell lung cancer (NSCLC) is a common subtype of lung cancer. Almost 80% of lung cancer cases are NSCLC cases. There has been evidence that the estrogen receptor (ER) signaling pathway and peroxisome proliferative activation receptor (PPAR) signaling pathway have an important role to play in cancer and inflammation. Previous reports have shown that activation of PPAR provides an anti-proliferative effect on epithelial cells. However, there have been reports that PPAR activating agents like pioglitazone shift the macrophage paradigm towards the M2 like/ pro-tumorigenic phase, an effect that is undesirable. Estrogen can enhance the proliferation of lung cancer cells through activation of the human epidermal growth factor receptor (EGFR) pathway, by inducing EGFR ligand release. It also makes the tumor microenvironment more tumorigenic by enhancing activation of M2 macrophages. To curb the action of pioglitazone on M2 macrophages, an estrogen receptor inhibitor like Fulvestrant can be used to avoid ER signaling, shifting the macrophages more to the M1 like/ pro-inflammatory phase. It is still not clear how ER and PPAR pathways are modulated in macrophages and lung tumor cells. We investigated the levels of M1 and M2 cytokines produced by macrophages and used conditioned medium from tumor cells to identify the ligands that were modulated by treatment with pioglitazone and fulvestrant. AREG, IL-10, VEGF and IL-1b were detected as the M2 prominent ligands modulating the crosstalk between tumor cells and macrophages. These ligands were expressed in reduced amounts in presence of the combination of fulvestrant and pioglitazone. We also showed that combination treatment leads to downregulation of COX-2/PGE2 in tumor cells and macrophages. The combination also targets proliferative pathways. Estradiol levels were curbed in presence of the combination. There was also a significant downregulation of COX-2 in the presence of neutralizing antibodies to AREG and IL-1β. This suggests that AREG and IL-1β, the prominent ligands that are modulated by the drug combination, are likely responsible for the COX-2 downregulation. Pharmacologically, the combination shifts the pro-tumorigenic macrophages towards the pro-inflammatory macrophages phenotype, which could make the tumor microenvironment less supportive of tumor progression. Analysis of cell proliferation via culturing of macrophages media with tumor cells showed a significant decrease in tumor cell proliferation when macrophages were pre-treated with pioglitazone and fulvestrant. Interestingly, we also observed that VEGF could be one of the cytokines involved in maintenance of M2-like/ pro-tumorigenic microenvironment. Dual therapy also strongly affected the cell migration of tumor cells in presence of macrophage conditioned media. From the evidence gathered above, we believe that the drugs could worked synergistically to affect cell proliferation, cell migration and shift the M1/M2 balance in the NSCLC tumor microenvironment. Therefore, we suggest a new combination therapy that targets both tumor cells and macrophages in NSCLC. It would be worthwhile to further investigate its effect in xenograft models of lung cancer.