In spite of considerable evidence on the regulation of immunity by thyroid hormones, the impact of the thyroid status in tumor immunity is poorly understood. Here, we evaluated the antitumor immune ...responses evoked in mice with different thyroid status (euthyroid, hyperthyroid, and hypothyroid) that developed solid tumors or metastases after inoculation of syngeneic T lymphoma cells. Hyperthyroid mice showed increased tumor growth along with increased expression of cell cycle regulators compared to hypothyroid and control tumor-bearing mice. However, hypothyroid mice showed a higher frequency of metastases than the other groups. Hyperthyroid mice bearing tumors displayed a lower number of tumor-infiltrating T lymphocytes, lower percentage of functional IFN-γ-producing CD8
+
T cells, and higher percentage of CD19
+
B cells than euthyroid tumor-bearing mice. However, no differences were found in the distribution of lymphocyte subpopulations in tumor-draining lymph nodes (TDLNs) or spleens among different experimental groups. Interestingly, hypothyroid TDLN showed an increased percentage of regulatory T (Treg) cells, while hyperthyroid mice displayed increased number and activity of splenic NK cells, which frequency declined in spleens from hypothyroid mice. Moreover, a decreased number of splenic myeloid-derived suppressor cells (MDSCs) were found in tumor-bearing hyperthyroid mice as compared to hypothyroid or euthyroid mice. Additionally, hyperthyroid mice showed increased cytotoxic activity, which declined in hypothyroid mice. Thus, low levels of intratumoral cytotoxic activity would favor tumor local growth in hyperthyroid mice, while regional and systemic antitumor response may contribute to tumor dissemination in hypothyroid animals. Our results highlight the importance of monitoring the thyroid status in patients with T cell lymphomas.
Key messages
T cell lymphoma phenotype is paradoxically influenced by thyroid status.
Hyperthyroidism favors tumor growth and hypothyroidism rises tumor dissemination.
Thyroid status affects the distribution of immune cell types in the tumor milieu.
Thyroid status also modifies the nature of local and systemic immune responses.
Cholesterol contributes to neuronal membrane integrity, supports membrane protein clustering and function, and facilitates proper signal transduction. Extensive evidence has shown that cholesterol ...imbalances in the central nervous system occur in aging and in the development of neurodegenerative diseases. In this work, we characterize cholesterol homeostasis in the inner ear of young and aged mice as a new unexplored possibility for the prevention and treatment of hearing loss. Our results show that cholesterol levels in the inner ear are reduced during aging, an effect that is associated with an increased expression of the cholesterol 24-hydroxylase (CYP46A1), the main enzyme responsible for cholesterol turnover in the brain. In addition, we show that pharmacological activation of CYP46A1 with the antiretroviral drug efavirenz reduces the cholesterol content in outer hair cells (OHCs), leading to a decrease in prestin immunolabeling and resulting in an increase in the distortion product otoacoustic emissions (DPOAEs) thresholds. Moreover, dietary supplementation with phytosterols, plant sterols with structure and function similar to cholesterol, was able to rescue the effect of efavirenz administration on the auditory function. Altogether, our findings point towards the importance of cholesterol homeostasis in the inner ear as an innovative therapeutic strategy in preventing and/or delaying hearing loss.
We have shown in vitro that thyroid hormones (THs) regulate the balance between proliferation and apoptosis of T lymphoma cells. The effects of THs on tumor development have been studied, but the ...results are still controversial. Herein, we show the modulatory action of thyroid status on the in vivo growth of T lymphoma cells. For this purpose, euthyroid, hypothyroid, and hyperthyroid mice received inoculations of EL4 cells to allow the development of solid tumors. Tumors in the hyperthyroid animals exhibited a higher growth rate, as evidenced by the early appearance of palpable solid tumors and the increased tumor volume. These results are consistent with the rate of cell division determined by staining tumor cells with carboxyfluorescein succinimidyl ester. Additionally, hyperthyroid mice exhibited reduced survival. Hypothyroid mice did not differ significantly from the euthyroid controls with respect to these parameters. Additionally, only tumors from hyperthyroid animals had increased expression levels of proliferating cell nuclear antigen and active caspase 3. Differential expression of cell cycle regulatory proteins was also observed. The levels of cyclins D1 and D3 were augmented in the tumors of the hyperthyroid animals, whereas the cell cycle inhibitors p16/INK4A (CDKN2A) and p27/Kip1 (CDKN1B) and the tumor suppressor p53 (TRP53) were increased in hypothyroid mice. Intratumoral and peritumoral vasculogenesis was increased only in hyperthyroid mice. Therefore, we propose that the thyroid status modulates the in vivo growth of EL4 T lymphoma through the regulation of cyclin, cyclin-dependent kinase inhibitor, and tumor suppressor gene expression, as well as the stimulation of angiogenesis.
Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in ...short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCζ, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCζ nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.
Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in ...short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCzeta, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCzeta nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.PUBLICATION ABSTRACT
Bexarotene (Bex) is an oral RXR agonist that is effective for the treatment of early and advanced-stage CTCL. However, Bex is associated with the unavoidable side effect of hypothyroidism in about ...95% of pts, which is prophylactically managed with the administration of thyroid hormone (TH). Paradoxically, we found that physiological levels of TH increase the proliferation of CTCL by activating both the nuclear TRA receptor and the membrane integrin αVβ3 in these cells. Here, we determined the influence of TH replacement therapy on the anti-lymphoma activity of Bex, an unknown topic with clinical implications.
Results: In standard culture conditions, Bex decreases the proliferation and viability of CTCL cell lines HuT78 and MJ. We conducted RNA-sequencing in HuT78 cells treated with Bex (vs. vehicle) to understand the mechanism/s underpinning these effects. We found that Bex regulates pathways related to “cell proliferation and differentiation” (e.g. REL, SCD, CCND1) and “immune system” (e.g. TBX21, IFNG, MX1) , which we independently validated. This suggests that Bex treatment could also impact anti-neoplastic immunity by increasing INF-gamma release from CTCL cells. We then conducted the same experiment culturing HuT78 and MJ in TH-depleted culture conditions and observed a higher effect of Bex in decreasing proliferation and viability; supporting the notion that Bex should not be administered with TH replacement. However, hypothyroidism is associated with marked immunosuppression that could favour CTCL progression and potentially affect the immune modulator effect of Bex that we described above.
We thus determined the impact of TH replacement on the anti-lymphoma effect of Bex using a murine CTCL model. We implanted murine EL4 TCL cells in the hypodermis of immunocompetent C57BL/6 mice. Once tumors developed, mice were randomized into treatment with vehicle (Veh), Bex with no TH replacement (Bex) and Bex with TH replacement (BexT4+). TH replacement was done with oral administration of T4. We measured T4 in these mice plasma by RIA to assure that mice with T4 replacement reach physiological levels and those with no T4 develop hypothyroidism. Compared to Veh mice, the administration of Bex significantly decreased lymphoma growth in both conditions although slightly better in mice without T4 replacement (p<0.001 and p<0.01) for Bex and BexT4+, respectively). However, mice with Bex alone that developed hypothyroidism showed a significant decrease of CD8+CD44+ T-cells (p<0.05 vs. BexT4+) and increase of myeloid-derived suppressor cells in the tumor microenvironment and draining lymph nodes. Since infiltration of CD8+ cells decreases with CTCL progression, our data indicates that Bex treatment should be administered with T4 replacement to avoid a negative immune microenvironment.
Considering the cellular effects of TH are exerted through TRA and integrin αVβ3 that can be independently modulated pharmacologically, we investigated whether integrin αVβ3 inhibition would be sufficient to blunt the TH-induced decrease on the antineoplastic efficacy of Bex. The lack of expression of integrin αVβ3 in normal T-cells offers a rationale for a selective effect on CTCL cells while sparing immune cells. In HuT78 and MJ cell lines, either αVβ3 silencing by siRNAs or pharmacologic inhibition with the clinical drug cilengitide avoided the decrease in the anti-CTCL effect of Bex upon TH supplementation. Moreover, gene expression analysis (RNA-seq and PCR) demonstrated that αVβ3 silencing induced higher (vs. Bex) up-regulation of “immune ” genes like TBX21 and INFG, or down-regulation of “proliferation” genes like REL and CCND1 .
We thus tested if this mechanism can be therapeutically capitalized to improve Bex treatment in our CTCL murine model. We implanted 24 C57BL/6 mice with murine EL4 TCL cells and randomized them to vehicle (Veh), Bex with TH replacement in combination with cilengitide 40mg/kg (BexT4+Cil) or without cilengitide (BexT4+). At end of treatment, BexT4+Cil mice showed significantly smaller tumors compared with Veh and BexT4+ mice (p<0.001 and p<0.05, respectively). Importantly, the anti-tumoral immune response of CD8+CD44+ T-cells in the CTCL microenvironment and draining lymph nodes remained similar in BexT4+Cil vs. BexT4+ mice. Our data indicates that inhibition of the integrin αVβ3 is an effective strategy to improve Bex-based treatments in CTCL.
Cerchietti:Celgene: Research Funding; Lymphoma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment.
Abstract
Despite the development of novel therapies, breast cancer (BC) tumors are treated with conventional therapy eventually. Due to tumor heterogeneity, it is necessary to define new biomarkers ...to identify groups with different molecular characteristic and on this basis improve the treatment decision. The impact of thyroid status in BC growth has previously been studied. In particular, alterations of thyroid function during chemotherapy for BC treatment have been found and it has been demonstrated that T3 induces chemosensitization of BC cells. However, the relationship between thyroid hormones (THs) membrane receptor, integrin αvβ3, and BC response to chemotherapy remains unclear. The aim of this study is to evaluate the relationship between THs and integrin αvβ3 expression as prognostic value in BC patients. The mRNA expression of ITGB3 in the TCGA-PanCancer Atlas BC dataset (1084 patients) was associated with worse overall survival. Moreover, ITGB3 mRNA expression was significantly correlated with genes encoding multidrug resistance transporter proteins MDR1 and BCRP, (Pearson, R score >0.3 and p<0,05). The diagnostic role of ITGB3 in BC was assessed by receptor operating characteristic (ROC) curve analysis. ITGB3 mRNA levels were higher in non-responder patients treated with anthracycline (AUC=0,673, p=1.8e-10) and taxanes (AUC=0.675, p=1.9e-07). To confirm the association between the integrinαvβ3-mediated effects and multidrug resistance protein transporters, we conducted in vitro assays ofMDA-MB-231 cells treated with physiological concentrations of THs and evaluated their action on MDR1 protein levels and Rhodamine efflux as an indicator of the transporter activity. We found that THs induce MDR1 protein expression and transporter activation and this effect is blocked by the presence of the integrin selective inhibitor cilengitide. Furthermore, in MDA-MB-231 doxorubicin-resistant cells THs induce MDR1 and BCRP greater protein expression than in MDA-MB-231 parental cells and this effect is abrogated by cilengitide. Therefore, we found that integrin β3 is correlated with worse overall survival and response to anthracyclines and taxanes pointing out its role as acancer biomarker with potential clinical utility. In patients with high integrin β3 mRNA expression, we found a significant correlation with proteins involved in drug transport such as MDR1 and BCRP. We also demonstrated that THs through integrin αvβ3 modulate the expression and activity of drug transporters. These results pointout the role of THs, and their membrane receptor, in BC response to treatments and introduce a new biomarker with potential clinical significance.
Presentation: No date and time listed
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