Thyroid cancer (TC) is the most common endocrine malignancy and its incidence is increasing. Despite well-differentiated forms, including papillary thyroid carcinomas (PTCs), follicular thyroid carcinomas (FTCs), have an excellent prognosis, with more than 90% cured by therapy, a small percentage (approx. 5%) of tumours becomes resistant to radioiodine therapy (radioactive iodine resistant-RAIR), the main treatment for these patients. Poorly differentiated thyroid carcinomas (PDTC), that invariably evolve in RAIR tumours, and anaplastic thyroid carcinomas (ATC), which are by definition RAIR, show limited options of treatment.
The scientific community believes that a small population of cells (cancer stem-like cells, CSCs) within the tumour is responsible for resistance to chemo-radiotherapy, recurrence and metastasis. Various studies supported this concept, thus strengthening the importance to characterize CSC populations in each cancer type. ATC, besides being the most aggressive among all thyroid cancers, and one of the most aggressive among all human cancer types, display stem-like features. ATCs features high levels of stemness markers and a stem-like behaviour. Recently, a CSC population has been identified in human ATCs samples, based upon the ability of this subpopulation to express high aldehyde dehydrogenase activity, to form thyrospheres in low adherence conditions and to form tumours in serial transplantation experiments. ATCs CSCs are more abundant and more aggressive than the CSC population found in PTCs 30. In TC, stemness features correlate with the acquisition of Epithelial-to-Mesenchymal Transition (EMT) 78.
We have previously shown that the pro-inflammatory cytokine IL-8 induces stemness features in TC, through the induction of EMT 43. We also found that IL-8 induces stemness properties in TC through the up-regulation of the SLUG EMT transcriptional factor 43. Consistently, we found that IL-8 stimulation of TC cells induces the expression of stemness-related genes, including OCT4, SOX2, and NANOG. SOX2 and OCT4 stand at the top of a hierarchy governing the regulation of self-renewal, pluripotency and de- differentiation 79. The roles of OCT4 and SOX2 have been described in various tumours 80.
Here, we generated OCT4 and SOX2-overexpressing PTC and ATC cell line models to characterize the effects of the two transcriptional factors in TC, and to obtain TC cells with enhanced stemness features, with which to study the features of TC stem-like cells. OCT4/ SOX2 TC cells show an increase in the proliferation rate and also in the death rate. These apparently contrasting results suggest that some of the genes activated in response to OCT4 and SOX2 may be responsible for an antiproliferative response such as apoptosis, senescence or other forms of cell-cycle arrest 74. This phenomenon has been
defined as “reprogramming-induced senescence” 74. Our data also support the finding that reprogramming factors can trigger apoptosis. It has been demonstrated for example that BAX is upregulated in response to the expression of OCT4, SOX2 and KLF4, and that the co-expression of the BAX antagonist BCL2 results in enhanced reprogramming efficiency 75. It is possible that also in TC cells, the expression of the reprogramming factors OCT4 and SOX2 induces cell death by increasing the amount of pro- apoptotic proteins. We are currently analysing this possibility.
In both ATC (8505c) and PTC (TPC-1) cells, enforced expression of OCT4/SOX2 caused an increase in cell motility, as assessed by migration assays. Moreover, OCT4/SOX2 induced an enhancement of stemness features, including self-renewal and tumorigenic potential, as assessed by self-renewal and limiting dilution assays and tumorigenicity experiments in immunodeficient mice.
There is increasing evidence that CSCs are responsible for the aggressive behaviour of tumours. This is not only due to CSC resistance to anti-cancer therapy and increased metastatic activity, but also to the ability of CSCs to evade the immune system. CSCs possess features similar to normal stem cells in their ability of inducing immune modulation. Unfortunately, possession of these features by CSCs contributes to their escape from the immune system recognition.
Here, we demonstrated that stemness is closely linked with immunosuppressive phenotype in TC cells. In fact, TC cells with increasing aggressive features also display increased immunsoppressive functions. The immunosuppressive phenotype of TC cells is characterized by increased expression of PD-L1, PD-L2, IDO, observed in PTCs versus normal thyroid cells, and in ATCs versus PTCs. Moreover, OCT4/SOX2 overexpressing TC cells display increased immunosuppressive features in comparison to parental cells. Not only were immunosuppressive molecules increased in OCT4/SOX2 TC cells, but also immunosuppressive activity on lymphocytes was enhanced. In fact, lymphocyte vitality was significantly reduced when co-cultured with OCT4/SOX2 TC cells when compared to control cells. This effect was not observed when lymphocytes were treated with conditioned media form TC cultures, but only when lymphocytes were co-cultured with TC cells. These observations indicate that OCT4 and SOX2 can increase the immunomodulatory functions of TC cells, possibly by the engagement of cell-cell contact mechanisms (i.e. PD-L1 and/or PD-L2). In support of these findings, we analysed the behaviour of immunosuppressive molecules in a mouse model of TC, in which the TRK-T1 oncogene is placed under the transcriptional control of the thyroid-specific TG gene promoter, thus causing the occurrence of thyroid hyperplasia and thyroid tumours. By comparing the immunosuppressive features of normal and pathological
the thyroids derived from the TRK-T1 mice with respect the control thyroids. Importantly, the increase in PD-L1 correlated with a decrease of CD8+ T lymphocytes infiltrating TG TRK-T1 thyroids. Therefore, TC display immunosuppressive features, and these features correlate with TC aggressiveness; moreover stemness features could enhance immunosuppressive ability of TC.
A limited number of reports indicate that the immunomodulatory properties of CSCs derived from several tissues could be linked specifically to SOX2 and/or OCT4 expression. Zhong et al. found that both SOX2 and PD-L1 were expressed at a markedly higher level in hepatocellular carcinoma (HCC) tissues in comparison to adjacent non-tumour tissues 81. Moreover, the expression levels of both genes were correlated with each other. Knockdown of SOX2 reduced the cell proliferation ability and induced apoptosis of HCC cells 81. Noteworthy, the depletion of SOX2 also reduced the expression of PD-L1. Further analysis showed that there is a consensus SOX2 binding site in the promoter region of PD-L1. It has been also demonstrated that SOX2 directly bound to the PD-L1 promoter through the consensus SOX2 motif 81. Further evidence by luciferase reporter assays revealed that SOX2 promoted the transcription activity of PD-L1 promoter region through the SOX2 motif. Collectively, these data provide a novel insight into the function and the interplay of SOX2 and PD-L1 in HCC. Moreover, there are also evidence that PD-L1 can regulate stemness. In fact, specific knockdown of PD-L1 using shRNA revealed its critical role in the expression of OCT4 and NANOG in breast CSCs 82. Conversely, these factors could be induced upon PD-L1 ectopic expression in cells that are normally PD-L1 negative. Most importantly, down-regulation of PD-L1 compromised the self-renewal capability of breast CSCs in vitro and in vivo as shown by tumoursphere formation assay and extreme limiting dilution assay, respectively. This study demonstrates a novel role for PD-L1 in identifying and sustaining CSCs of breast 82.
There is growing interest toward therapies that target the CSC population within tumours. Moreover, immunotherapies directed against the PD-1/PD- L1/PD-L2 circuit raised great expectations in the cure of various cancer types given the successful results obtained in the first clinical trials. Our data indicate that targeting stemness pathways in TC might not only inhibit its malignant features, but also limit its immunosuppressive capacity.
LIST OF PUBLICATIONS
Liotti F, Collina F, Pone E, La Sala L, Franco R, Prevete N, Melillo RM. “Interleukin-8, but not the Related Chemokine CXCL1, Sustains an Autocrine
Circuit Necessary for the Properties and Functions of Thyroid Cancer Stem Cells”
Stem Cells. 2016 Aug 31. [Epub ahead of print]. Online ISSN: 1549-4918
ACKNOWLEDGMENTS
I carried out my study in the Molecular Medicine and Medical Biotechnology Department of Federico II University of Naples.
Foremost, I would like to express my sincere gratitude to my tutor Prof. Rosa Marina Melillo for the continuous support of my PhD study and research, for her patience, and immense knowledge. Her guidance helped me in all the time of research and writing of this thesis. I want to thank Dott. Liotti Federica and Dott. Prevete Nella for their active participation and support in my PhD project for helping me in the experiments and stimulated discussion. I also want to thank all people that I meet during my PhD experience.
I also want to thank Francesco for his love.
Last but not the least my gratitude goes to my family for their encouragement and faith in me.
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