The epithelial-mesenchymal transition (EMT) is a key process implicated in cancer metastasis and therapy resistance. Recent studies have emphasized that cells can undergo partial EMT to attain a ...hybrid epithelial/mesenchymal (E/M) phenotype - a cornerstone of tumour aggressiveness and poor prognosis. These cells can have enhanced tumour-initiation potential as compared to purely epithelial or mesenchymal ones and can integrate the properties of cell-cell adhesion and motility that facilitates collective cell migration leading to clusters of circulating tumour cells (CTCs) - the prevalent mode of metastasis. Thus, identifying the molecular players that can enable cells to maintain a hybrid E/M phenotype is crucial to curb the metastatic load. Using an integrated computational-experimental approach, we show that the transcription factor NRF2 can prevent a complete EMT and instead stabilize a hybrid E/M phenotype. Knockdown of NRF2 in hybrid E/M non-small cell lung cancer cells H1975 and bladder cancer cells RT4 destabilized a hybrid E/M phenotype and compromised the ability to collectively migrate to close a wound in vitro. Notably, while NRF2 knockout simultaneously downregulated E-cadherin and ZEB-1, overexpression of NRF2 enriched for a hybrid E/M phenotype by simultaneously upregulating both E-cadherin and ZEB-1 in individual RT4 cells. Further, we predict that NRF2 is maximally expressed in hybrid E/M phenotype(s) and demonstrate that this biphasic dynamic arises from the interconnections among NRF2 and the EMT regulatory circuit. Finally, clinical records from multiple datasets suggest a correlation between a hybrid E/M phenotype, high levels of NRF2 and its targets and poor survival, further strengthening the emerging notion that hybrid E/M phenotype(s) may occupy the 'metastatic sweet spot'.
Activation of the NRF2 pathway through gain-of-function mutations or loss-of-function of its suppressor KEAP1 is a frequent finding in lung cancer. NRF2 activation has been reported to alter the ...tumor microenvironment. Here, we demonstrated that NRF2 alters tryptophan metabolism through the kynurenine pathway that is associated with a tumor-promoting, immune suppressed microenvironment. Specifically, proteomic profiles of 47 lung adenocarcinoma (LUAD) cell lines (11
mutant and 36
wild-type) revealed the tryptophan-kynurenine enzyme kynureninase (KYNU) as a top overexpressed protein associated with activated NRF2. The siRNA-mediated knockdown of
, the gene encoding for NRF2, or activation of the NRF2 pathway through siRNA-mediated knockdown of
or via chemical induction with the NRF2-activator CDDO-Me confirmed that NRF2 is a regulator of KYNU expression in LUAD. Metabolomic analyses confirmed KYNU to be enzymatically functional. Analysis of multiple independent gene expression datasets of LUAD, as well as a LUAD tumor microarray demonstrated that elevated KYNU was associated with immunosuppression, including potent induction of T-regulatory cells, increased levels of PD1 and PD-L1, and resulted in poorer survival. Our findings indicate a novel mechanism of NRF2 tumoral immunosuppression through upregulation of KYNU.
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in a broad range of inflammatory and oncologic diseases. MIF is unique among cytokines in terms of its ...release profile and inflammatory role, notably as an endogenous counter-regulator of the anti-inflammatory effects of glucocorticoids. In addition, it exhibits a catalytic tautomerase activity amenable to the design of high affinity small molecule inhibitors. Although several classes of these compounds have been identified, biologic characterization of these molecules remains a topic of active investigation. In this study, we used in vitro LPS-driven assays to characterize representative molecules from several classes of MIF inhibitors. We determined that MIF inhibitors exhibit distinct profiles of anti-inflammatory activity, especially with regard to TNFα. We further investigated a molecule with relatively low anti-inflammatory activity, compound T-614 (also known as the anti-rheumatic drug iguratimod), and found that, in addition to exhibiting selective MIF inhibition in vitro and in vivo, iguratimod also has additive effects with glucocorticoids. Furthermore, we found that iguratimod synergizes with glucocorticoids in attenuating experimental autoimmune encephalitis, a model of multiple sclerosis. Our work identifies iguratimod as a valuable new candidate for drug repurposing to MIF-relevant diseases, including multiple sclerosis.
Abstract
Myocarditis is an inflammatory disease of the myocardium and a major cause of sudden death in young adults. It is characterized by the presence of immune infiltrates in the myocardium and ...often progresses to dilated cardiomyopathy (DCM). Despite the inflammatory nature of this autoimmune disease, immunosuppressive treatments with glucocorticoids (GCs) such as dexamethasone (Dex) have not been very effective in preventing myocarditis and progression to DCM. In addition, some patients develop resistance to GCs. We hypothesized that macrophage migration inhibitory factor (MIF) may play a role in resistance to GCs, as it is the only known pro-inflammatory cytokine to be induced by GCs. Importantly, MIF counter-regulates GC-mediated immunosuppression. Using the experimental autoimmune myocarditis (EAM) model, we observed that MIF-/- mice treated with Dex were highly resistant to disease and progression to DCM. In addition, we observe lower expression of CCL3 mRNA in MIF-/- mice treated with Dex compared with wild-type mice during the onset of EAM, which indicates that MIF promotes recruitment of inflammatory cells to the myocardium. Our results suggest that therapeutic inhibition of MIF may increase the efficacy of GC treatment. This study will allow us to better understand the mechanism by which MIF affects treatment of myocarditis by inhibiting the therapeutic effects of GCs.
Macrophage Migration Inhibitory Factor (MIF) has previously been shown to be a promising therapeutic target of inhibition for treatment of chronic inflammatory autoimmune diseases such as Multiple ...Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE), a mouse model for MS. MIF is unique in that it is a proinflammatory cytokine upregulated in response to Glucocorticoids (GCs), the predominant treatment for the acute episodes of MS. We investigated the efficacy of recently developed small molecule MIF inhibitors both independently and in conjunction with glucocorticoid treatment in attenuating the clinical disease severity of EAE. EAE was induced in wild type mice followed by treatment with the Dexamethasone (Dex) and a small molecule MIF inhibitor following clinical disease onset. Antigen-specific splenocytes were harvested and cultured for cytokine ELISPOT assay. Small molecule MIF inhibitors were found to be effective at attenuating clinical EAE disease both in conjunction with glucocorticoid treatment and independently of glucocorticoid treatment. Our data demonstrates the effectiveness of small molecule MIF inhibitors at reducing clinical pathology in EAE and we hope this data will serve as a precursor to clinical application of small molecule MIF inhibitors in MS.
Abstract
Background
Approximately 20% of lung adenocarcinoma (LUAD) is negative for the lineage-specific oncogene Thyroid transcription factor 1 (TTF-1) and exhibits worse clinical outcome with a low ...frequency of actionable genomic alterations. To identify molecular features associated with TTF-1–negative LUAD, we compared the transcriptomic and proteomic profiles of LUAD cell lines. SRGN , a chondroitin sulfate proteoglycan Serglycin, was identified as a markedly overexpressed gene in TTF-1–negative LUAD. We therefore investigated the roles and regulation of SRGN in TTF-1–negative LUAD.
Methods
Proteomic and metabolomic analyses of 41 LUAD cell lines were done using mass spectrometry. The function of SRGN was investigated in 3 TTF-1–negative and 4 TTF-1–positive LUAD cell lines and in a syngeneic mouse model (n = 5 to 8 mice per group). Expression of SRGN was evaluated in 94 and 105 surgically resected LUAD tumor specimens using immunohistochemistry. All statistical tests were 2-sided.
Results
SRGN was markedly overexpressed at mRNA and protein levels in TTF-1–negative LUAD cell lines (P < .001 for both mRNA and protein levels). Expression of SRGN in LUAD tumor tissue was associated with poor outcome (hazard ratio = 4.22, 95% confidence interval = 1.12 to 15.86, likelihood ratio test, P = .03), and with higher expression of Programmed cell death 1 ligand 1 (PD-L1) in tumor cells and higher infiltration of Programmed cell death protein 1–positive lymphocytes. SRGN regulated expression of PD-L1 as well as proinflammatory cytokines, including Interleukin-6, Interleukin-8, and C-X-C motif chemokine 1 in LUAD cell lines; increased migratory and invasive properties of LUAD cells and fibroblasts; and enhanced angiogenesis. SRGN was induced by DNA demethylation resulting from Nicotinamide N-methyltransferase–mediated impairment of methionine metabolism.
Conclusions
Our findings suggest that SRGN plays a pivotal role in tumor–stromal interaction and reprogramming into an aggressive and immunosuppressive tumor microenvironment in TTF-1–negative LUAD.
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