Understandings of Immunomodulatory Pathways of Indoleamine 2,3‐dioxygenase (IDO) in Bladder Cancer Cells

Bladder cancer (BC) is the fourth most common cancer between men and the ninth in women, with a high recurrence rate and progression after conventional treatments. A growing body of evidence suggests that immunomodulatory molecules protect bladder tumors against immune surveillance by triggering signaling pathways that modulate locally the immune system. One important molecule is indoleamine 2,3‐dioxygenase (IDO), which promotes tryptophan breakdown, activating sensitive pathways such as GCN2 in immune cells. In addition, tryptophan breakdown leads to the production of kynurenine compounds, which activate aryl hydrocarbon receptor (AHR), promoting anergy and apoptosis on T cells and Natural killer cells. Although these IDO‐mediated pathways are well recognized in immune cells, they were not yet analyzed in bladder cancer cells. The use of IDO inhibitors arises as a promise anti‐tumor strategy, however, they have divergent effects depending on the activated pathway. Here, we analyzed the expression of IDO and the activation of its pathways in non‐invasive muscle and in invasive muscle bladder cancer cells, and the effect of two IDO inhibitors on these pathways. RT4 and T24 cells were maintained in RPMI medium and incubated for 48h with the following stimuli: IFN‐γ (1000U/mL), Methyl‐D‐tryptophan (MT) (500μM), Incb024360 analog (INCB) (1μM). Control cells were maintained in RPMI medium. After the stimuli, RNA was extracted and qRT‐PCR for IDO, CHOP (a marker of GCN2 activation), CYP1A1 (an AHR activation marker), and AHR was performed. T24 cells constitutively express higher levels of IDO when compared to RT4 cells (relative expression (RE) of 1.0±0.1 in RT4 vs. 73.8±17.0 in T24), accompanied by high expression of CHOP (RE of 1.0±1.2 in RT4 vs. 10.5±4.5 in T24) and CYP1A1 (RE 1.0±0.0 in RT4 vs. 28.2±1.5 in T24), with no differences in AHr expression. When stimulated with IFN‐γ, both RT4 and T24 cells showed increased IDO expression, CHOP and CYP1A1 (p<0.001). While INCB significantly reduced the IDO‐mediated pathways (CHOP and CYP1A1 expression), MT treatment increased CYP1A1 expression (p<0.05). IDO‐regulated pathways are differentiated in invasive and noninvasive bladder cancer cells. As such, adjuvant treatments to modulate IDO are indeed promising. The use of certain inhibitors, however, such as MT, while may inhibit tryptophan depletion, also lead to activation of AHR, promoting kynurenine‐like effects. The use of non AHR binding compounds, such as Incb024360 analogs are promising then proposed as an alternative and safer approach for regulating IDO‐mediated events without nonspecific bindings. Support or Funding Information Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .