Thyroid Hormones Maintain The CTCL Malignant Phenotype Through Membrane- and Nuclear-Initiated Transcriptional Programs

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 crucial regulators of cellular differentiation, growth and metabolism. We recently found that THs stimulate the proliferation and metabolism of T cells and that this pathway is co-opted by T-NHL cells to maintain their malignant phenotype. Accordingly, a decrease in TH levels affects the proliferation of T-NHL cells. We also found that THs modulate these functions by activating both canonical nuclear (TR) and membrane receptors (mTR, that for most cells is represented by integrin αvβ3). We now hypothesized that the characterization of the relative contribution of these receptors to the malignant phenotype of T-NHLs will allow for a more precise therapeutic intervention. The study presented here was conducted to determine (i) whether THs are pro-survival factors in CTCL and (ii) the relative contribution of TR and mTR to the effect of THs in CTCLs.

To test this hypothesis, we first analyzed the expression of TR and mTR in CTLC cells (HuT 78, MJ and HuT102) vs. normal T cells (from peripheral blood) by qRT-PCR and immunoblotting. We found that CTCL expressed 2 to 10 fold more THRA, ITGAV and ITGB3 mRNA and protein than their normal counterpart (p<0.05, T test). Moreover, in a tissue microarray of 52 CTCLs, the integrin avβ3 was overexpressed in 92% of the cases. To differentiate between nuclear vs. membrane-initiated effects, CTCL cells were treated with physiological concentrations of free and cell impermeable agarose-bound T3/T4 (TH-ag). After 24h of treatment, cell proliferation increased 20-38% in HuT-78 cells and 18-32% in MJ cells with both forms of THs (p<0.05 for both comparisons vs. control). Long-term exposure (96h) to free TH and TH-ag accelerated HuT-78 doubling time by 17-25% and 16-22%, respectively (p<0.05 for both comparisons). By time course analysis of mRNA abundance (1, 2, 3, 6, 12 and 24 h post TH), we found that TH-induced phenotypic changes were associated with 1.5 to 3 fold up-regulation of proliferation markers such as PCNA, CCND1, CCND2, CCND3, CCNE2 and CCNB1 in addition to a concomitant decrease of the tumor suppressor genes TP53 and CDKN1A.

To determine the specific transcriptional programs regulated by mTR and TR, T-NHLs were treated with free T3/T4 (for total TR activity) and agarose-coupled T3/T4 (for mTR activity) for 24h and analyzed by mRNA-sequencing. Differentially regulated genes were further analyzed using Ingenuity Pathway Analysis. We found the TNFR and interleukin-2 pathways (e.g. TNFRSF19, TNFRSF9, IL2RA) among the top programs regulated by free THs through canonical nuclear TR activation whereas mTR activation upregulated genes involved in angiogenesis induction (e.g. PDGFR, VEGFA, VEFGB), lymphocyte proliferation/differentiation and DNA replication (e.g. IL4, DOK2). We independently validated these genes in HuT-78 and MJ cell lines by qRT-PCR. Taken together, these data suggest that TR and mTR regulate distinct transcriptional programs that are complimentary towards CTCL proliferation.

Considering that (i) mTR are over-expressed in CTCL vs. normal T cells (in contrast to canonical nuclear TR that are broadly expressed) and (ii) mTR regulate pro-survival pathways in CTLC, we wished to determine whether mTR inhibition will be sufficient to decrease CTCL proliferation. We therefore tested the effect of mTR abrogation by using siRNA to ITGAV and ITGB3 as well as the effect of pharmacological mTR inhibitors (i.e: RGD peptide and Tetrac) in CTCL proliferation. We found that mTR inhibition with siRNA or compounds affected mTR-induced transcriptional programs. In fact, the TH-ag mediated upregulation of VEGFB, VEGFA, DOK2 and IL4 mRNA levels were totally abrogated in CTCL cells transfected with the siRNA to ITGAV and ITGB3. Moreover, almost 100% of the TH-ag-induced and 80% of the free-TH-induced CTCL proliferative effect was abrogated by ITGAV- and/or ITGB3-targeting siRNA.

Taken together these data suggest that THs are important inducers of CTCL proliferation by transcriptionally regulating major survival pathways, and inhibition of mTR could constitute a selective chemotherapy-free treatment for CTCLs.