Proteomic Analysis of Thyroid Hormone-Induced Secretome in Cutaneous T Cell Lymphoma

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of T-cell non-Hodgkin lymphomas (T-NHL) derived from skin-homing mature T cells. Thyroid hormones (THs) are soluble factors found in the tumor microenvironment that can influence tumor progression. Previously, we characterized the transcriptional program triggered by THs in T-NHL (including CTCL) and found that TH membrane receptor, integrin αVβ3, induced cell proliferation, and angiogenic programs. Moreover, we recently showed that integrin αVβ3 activation by THs triggers JAK/STAT oncogenic pathways to promote T cell lymphoma dissemination. Particularly, the interaction of CTCL with the tumor microenvironment is crucial for their progression. Exploring how THs affect the CTCL microenvironment may reveal new therapeutic targets for these pathologies.

This study aims to determine and contrast the proteins secreted (i) under basal conditions and (ii) in the presence of THs with or without the integrin αVβ3 pharmacological inhibitor, cilengitide (cile). For this purpose, CTCL cells representing Mycosis Fungoides (MJ) and Sézary Syndrome (HuT78) subtypes were starved for 18 h and treated or not with physiological concentrations of THs (T4=100nM, T3=1nM) and 1.5 µM cile. After treatment, supernatants were collected for subsequent proteomic analysis using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Differential protein expression was analyzed using the limma package in RStudio (v 4.3.3), and functional enrichment analysis was performed using ShinyGO (v 0.75).

Under basal conditions, we identified 1249 unique peptides in HuT78 and MJ supernatants. We then contrasted secretomes and found that HuT78 and MJ supernatants are differentially enriched in 387 and 427 unique peptides, respectively (FDR<0.05). Both secretomes share enriched pathways related to metabolic reprogramming in cancer, upregulation of the IL-2 signaling, and vascularization signaling through VEGFA-VEGFR2. Differential expression analysis between both cell lines showed that HuT78 secretome is significantly enriched in proteins associated with HIF-1α signaling (e.g., PFK, TFRC, SLC2A1 and HK1), PDL-1/PD1 immune checkpoint (e.g., CSNK2A1, MAPK14 and ZAP70), and leukocyte transendothelial migration (e.g., ITGAL, VCL, EZR, VASP, VAV1 and MSN) (FDR<0.01), all of them strongly arguing for the involvement of the tumor microenvironment.

We performed differential protein expression analysis to identify TH-induced secretomes from the supernatants of CTCL-treated cells. We found significant up and downregulation of 21 and 114 proteins, respectively, in MJ upon TH treatment (FDR < 0.1, vs vehicle). We found that amongst the top regulated secreted proteins induced by THs (such as PSMA1, PSMA3, PSMA7, THY1, LCK, APP, and OTUB1) are associated with hematopoietic stem cell differentiation, response to IL-1, and T cell receptor signaling pathways (FDR<0.01).

Regarding HuT78 cells, we found that THs significantly increased 66 proteins and reduced the expression of 54 secreted proteins (FDR<0.1). We observed that HuT78 supernatants were enriched in proteins related to VEGFA-VEGFR2 signaling (FLNB, FAS, BCL2L1, and TXN), focal adhesion (FN1, FLNB, TLN1, THBS1 and FLNA) and TGF-beta signaling (FN1, UCHL5, THBS1 and CDK1) (FDR<0.05). Additionally, we evaluated the secretome from HuT78 cells that were cultured in the presence of THs and treated or not with cile for 24 hours. We found that integrin αVβ3 inhibition significantly up and down-regulated 427 and 255 proteins, respectively, in HuT78 supernatants (FDR<0.1). It is worth noting that many of the TH-induced secreted proteins, such as FN1 and THBS1, are associated with poor prognosis in various cancer types and the suppression of antitumor immunity. Interestingly, integrin αVβ3 inhibition with cile significantly decreases the expression of both proteins in the supernatants of HuT78 cells (FDR<0.1). Moreover, when we evaluated the transcriptional expression of these proteins in a CTCL patient database (GSE113113) we found it to be positively correlated with ITGB3 expression (p<0.05).

In summary, our study has shed light on the crucial role of THs as regulators of HuT78 and MJ secretome. These findings are of significant importance, as they provide a solid basis for further investigation into the role of these soluble factors and their receptors in the CTCL microenvironment.