Anchorage-independent triple-negative breast cancer (TNBC) cells exhibit elevated levels of the tryptophan (TRP) catabolizing enzyme tryptophan 2,3-dioxygenase 2 (TDO2) compared to the same cells grown in two-dimensional culture. Tracing of 13C11-TRP demonstrated that anchorage-independent culture and/or inflammatory cytokines that activate nuclear factor kappa-light-chain-enhancer of activated B (NFκB) increase TRP catabolism and production of downstream catabolites such as kynurenine (KYN), which activate the aryl hydrocarbon receptor (AhR). TDO2 expression is heterogeneous within TNBC cell lines. To determine the function of TDO2, both pharmacologic inhibition and genetic manipulation were conducted. TDO2 knockdown revealed a compensatory increase in indoleamine 2,3-dioxygenase 1 (IDO1), a non-homologous TRP catabolizing enzyme, indicating that dual inhibition of these two enzymes is necessary to reliably block TRP catabolism. Thus, we tested a newly developed TDO2/IDO1 dual inhibitor, AT-0174, and found that it effectively inhibits TNBC TRP catabolism. Furthermore, AT-0174 treatment or AhR inhibitor significantly decreased TNBC anchorage-independent survival, invasive capacity, and expression of mesenchymal genes and protein, while exogenous KYN increased invasion through AhR-mediated ZEB1 expression. Thus, dual inhibition of TDO2/IDO1 may prove efficacious against TNBC progression.

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2024
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Supplementary data

Supplementary Table S1

shRNA used in this study.

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Supplementary Table S2

qPCR primers used in this study.

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Supplementary Table S3

Differential gene expressions of scRNA-seq for MDA-MB-453 treated with IL1β+TNFα vs. Vehicle

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Supplementary Table S4

Significant GSEA of Bulk RNA-seq for BT549 (under suspension condition) treated with AT-0174 vs. Vehicle

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Supplementary Table S5

Differential gene expression list of Bulk RNA-seq for BT549 (under suspension condition) treated with AT-0174 vs. Vehicle

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Supplementary Table S6

Significant GSEA full list of Bulk RNA-seq for BT549 shTDO2-82 vs. shSCR control

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Supplementary Table S7

Differential gene expression list of Bulk RNA-seq for BT549 shTDO2-82 vs. shSCR Control

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Supplementary Table S8

Significant GSEA full list of Bulk RNA-seq for BT549 shTDO2-98 (IDO1 comp) vs. shTDO2-82

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Supplementary Table S9

Differential gene expression list of Bulk RNA-seq for BT549 shTDO2-98 (IDO1 comp) vs. shTDO2-82

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Supplementary Table S10

Gene co-expression with TDO2 list of single-cell RNA-seq for MDA-MB-453 treated with Vehicle

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Supplementary Figure S1

Gene enrichment set analysis (GSEA) for MDA-MB-453 forced suspension culture vs. attached culture.

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Supplementary Figure S2

Increased tryptophan catabolism and accumulation of kynurenine and other downstream catabolites.

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Supplementary Figure S3

Tryptophan catabolism to kynurenine was enhanced in cells surviving in the anchorage independent condition over time.

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Supplementary Figure S4

Compound AT-0174 decreased secreted KYN in TDO2 overexpression (OE) cells.

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Supplementary Figure S5

AT-0174 significantly decreased extracellular tryptophan catabolites produced by MDA-MBA-453 cells.

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Supplementary Figure S6

Pharmacologic inhibition or genetic knockdown of TDO2 reduces TNBC anchorage independent growth.

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Supplementary Figure S7

AT-0174 reduced invasion in TDO2 OE cells.

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Supplementary Figure S8

Treatment of AT-0174, epacadostat or Stemreginin reduces invasion in SUM159PT cells.

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Supplementary Figure S9

The AhR ChIP seq dataset shows high enrichments at the promoter region of ZEB1 transcript.

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Supplementary Figure S10

AhR, ZEB1 and TDO2 expression in clinical breast cancer database.

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Supplementary Figure S11

Genetic knockdown or pharmacological inhibition of TDO2 or AhR decreased ZEB1 mRNA expression but increased while IDO1 compensated; KYN treatment increased ZEB1 expression.

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