Microphthalmia-associated Transcription Factor Regulates the Differentiation and Function of Myeloid-derived Suppressor Cells

다국어 초록 (Multilingual Abstract)

Myeloid-derived suppressor cell (MDSC) are immature immune suppressive cells observed in tumors, infections, and autoimmune diseases. They are identified as two major subtypes in mice, either granulocytic polymorphonuclear-MDSCs (PMN-MDSCs, Ly6G+Ly6ClowCD11b+), and monocytic-MDSCs (MO-MDSCs, Ly6G-Ly6ChighCD11b+) and both populations strongly suppress the immune response through diverse mechanisms. Microphthalmia-associated transcription factor (MITF), a basic/helix-loop-helix/leucine zipper (b-HLH-Zip) transcription factor, is mainly expressed in melanoma cells. MITF is well known to be a master regulator of melanogenesis, and it affects survival, metastasis, cell cycle arrest, and differentiation. Recently, MITF has also been reported to be expressed in nonpigment cells, including osteoclasts, mast cells, B-cells, and natural killer (NK) cells. However, the roles of MITF in immune cell differentiation and function remain unclear.

In this study, it was investigated to identify the role of MITF in the differentiation and activation of MDSCs during tumor development. It was revealed that MITF expression is significantly increased in MDSCs obtained from tumor tissue or splenocytes of tumor-bearing mice. In addition, MITF expression was induced by tumor cell-conditioned medium (TCCM) treatment during the generation of MDSCs from bone marrow (BM) cells in vitro. Treatment with IL-18 or IL-10 related with the tumor microenvironment (TME) markedly promoted the expression of MITF and MDSC activation markers. Moreover, statins, which have been known to enhance differentiation and activation of MDSCs, also induced MITF expression in BM-MDSCs. Besides, all-trans retinoic acid (ATRA), which has been known to reduce ROthe differentiation and function of tumor-associated MDSCs, suppressed the expression of MITF and activation markers of MDSCs. MITF overexpression in myeloid cells substantially promoted the immunosuppressive function of MDSCs, whereas shRNA-mediated knockdown of MITF in myeloid cells altered the immunoregulatory function of MDSCs. Furthermore, while berberine, which has been used in anticancer therapy, abrogated the immunosuppressive function of BM-MDSCs by inhibiting MITF expression, ML-329, an inhibitor of the MITF molecular pathway, suppressed the expression levels of MDSC activation markers. Similarly, nelfinavir that is recently known to suppress MITF expression altered the differentiation and activation of MDSCs through MITF inhibition. In contrast, an increase in MDSC number and activity was observed after treatment with 3-isobutyl-1-methylxanthine (IBMX), which is known to be a MITF inducer. Finally, it was observed that the tumor size is significantly reduced by a coinjection of 4T1 cells with MDSCs pretreated with MITF inhibitory small molecule, such as ML-329, compared to coinjection of 4T1 tumor cells with untreated MDSCs in tumor-bearing mice. In contrast, tumor progression is increased by a coinjection of 4T1 cells with MDSCs pretreated with MITF inducer, such as IBMX. Furthermore, immunohistochemical staining of human tumor tissues revealed that MITF+ MDSCs are more frequently observed in tumor tissues than in tumor-free draining lymph nodes (TDLNs) obtained from cancer patients. In summary, these results indicate that MITF regulates the differentiation and function of MDSCs and can be a novel therapeutic target for modulating MDSC activity in dysregulated tumor microenvironments.

국문 초록 (Abstract)

골수유래억제세포 (MDSCs) 는 종양, 감염, 자가면역질환에서 관찰되는 미성숙 면역억제세포이다. 이들은 두 가지 유형 (PMN-MDSCs, MO-MDSCs)으로 나뉘며, 마우스에서 다양한 반응을 통해 강한 면역억제작용을 한다고 알려져 있다. Microphthalmia-associated transcription factor (MITF)는 주로 흑색종 세포에서 발현된다. MITF는 melanogenesis의 주요조절자로 잘 알려져 있으며, 생존, 전이, 세포 주기, 분화에 영향을 미친다. 최근 MITF는 파골세포, 비만세포, B세포, NK세포 등 멜라닌세포 이외에도 발현되며 중요한 기능을 한다고 발표된 바 있다. 그러나 MITF가 골수유래억제세포의 분화 및 기능에서의 역할은 아직까지 알려져 있지 않다.
본 연구에서는 종양 형성 시 골수유래억제세포의 분화와 활성에서 MITF의 역할에 대해 알아보았다. 종양 조직이나 비장세포에서 얻은 골수유래억제세포에서 MITF의 발현이 유의미하게 증가한 것을 관찰 하였다. 또한 in vitro에서 골수세포로부터 골수유래억제세포로 분화시키는 경우에 암 미세환경과 유사한 암 배지 처리에 의해 MITF의 발현이 증가함을 확인하였다. 암 미세환경과 관련된 IL-18과 IL-10은 MITF와 골수유래억제세포 활성마커의 발현을 촉진하였다. 또한, 골수유래억제세포의 분화와 활성을 높인다고 알려진 스타틴은 골수유래억제세포에서 MITF의 발현을 유도하였다. 이외에도 암조직내의 골수유래억제세포의 분화와 기능을 감소시키는 것으로 알려진 all-trans retinoic acid (ATRA)는 골수유래억제세포의 MITF와 활성마커의 발현을 억제하였다. 실제로 골수세포에서의 MITF 과발현은 골수유래억제세포에서의 면역억제기능을 유도하였다. 대조적으로, 골수세포에서 shRNA를 이용한 MITF 발현저해는 골수유래억제세포의 면역조절 기능을 변화시켰다. 또한, 항암 치료에 효능을 보이는 베르베린은 MITF의 발현을 저해함으로써 골수유래억제세포의 면역 억제기능을 저해하였고, MITF의 저해제인 ML-329는 골수유래억제세포의 활성마커의 발현 및 면역억제능을 저해하였다. 마찬가지로, MITF의 억제제로 스크리닝된 넬피나비르는 MITF 억제와 함께 골수유래억제세포의 분화와 활성을 억제하였다. 반대로 MITF의 유도제로 알려진 3-isobutyl-1-methylxanthin (IBMX)는 골수유래억제세포의 활성을 증가시켰다. 다음으로는 골수유래억제세포에 DMSO와 ML-329를 사전에 처리하고 마우스 유방암 세포주인 4T1과 함께 마우스에 주사하였다. DMSO를 처리한 경우는 4T1만 주사한 경우에 비해 종양 크기가 증가하였고 ML-329를 처리한 경우에는 종양 크기가 다시 4T1만 주사한 경우와 유사한 수준으로 감소하는 것을 관찰하였다. 이와는 대조적으로 IBMX를 전처리하여 4T1과 함께 마우스에 주사한 경우에는 종양 성장과 무게가 증가하였다. 마지막으로, MITF를 발현하고 있는 골수유래억제세포는 암 환자의 tumor draining lymph nodes (TDLNs)보다 종양 조직내에서 많이 관찰되었다. 이러한 결과는 MITF가 골수유래억제세포의 분화와 기능을 조절하며, 암 미세환경에서 골수유래억제세포에서의 MITF 발현조절은 암 치료에 대한 새로운 전략이 될 수 있음을 나타낸다.

목차 (Table of Contents)

• 1. Introduction = 1
• 1.1. Myeloid-derived suppressor cells (MDSCs) = 1
• 1.1.1. Differentiation of MDSCs = 1
• 1.1.2. Immunosuppressive function of MDSCs = 5
• 1.1.3. Targeting of MDSCs in the tumor microenvironment (TME) = 8
• 1.2. Microphthalmia-associated transcription factor (MITF) = 10
• 1.2.1. Overview of MITF = 10
• 1.2.2. Roles of MITF in immune cells = 14
• 2. Materials and methods = 15
• 2.1. Cell culture and reagents = 15
• 2.2. Mice and tumor models = 15
• 2.3. Tumor cell-conditioned medium (TCCM) = 16
• 2.4. RNA isolation and real-time PCR = 17
• 2.5. Western blot analysis = 19
• 2.6. Differentiation of murine bone marrow-derived MDSCs in vitro = 19
• 2.7. Isolation of MDSCs = 20
• 2.8. In vivo bioluminescence imaging = 20
• 2.9. Transfection assay = 21
• 2.10. Flow cytometry = 21
• 2.11. T-cell proliferation assay = 22
• 2.12. Measurement of ROS levels = 22
• 2.13. Patients and histological evaluation = 23
• 2.14. Survival analysis = 24
• 2.15. Statistical analysis = 24
• 3. Results = 25
• 3.1. MITF expression is increased in MDSCs during tumor development = 25
• 3.2. MITF expression is related to the immunosuppressive function of MDSCs in tumors = 31
• 3.3. MITF expression in two subtypes of MDSCs obtained from tumor-bearing mice = 33
• 3.4. MITF expression in BM-MDSCs is induced by undefined factors which are present in the TME = 36
• 3.5. MITF expression is related to MDSC activation under TME = 39
• 3.6. MITF expression is highly correlated with MDSC activation = 46
• 3.7. MITF overexpression enhances the immunosuppressive activity of BM-MDSCs = 51
• 3.8. MITF inhibition suppresses immune suppressive activity of BM-MDSCs = 55
• 3.9. Berberine reduces the immunosuppressive effect of BM-MDSCs by inducing MITF inhibition = 59
• 3.10. MITF inhibitor, ML-329, affects immune suppressive function of BM-MDSCs = 64
• 3.11. Nelfinavir affects the differentiation and activation of BM-MDSCs through MITF inhibition = 69
• 3.12. MITF inducer, IBMX, boosts immune suppressive function of BM-MDSCs = 72
• 3.13. The inhibition of MITF expression in MDSCs attenuates tumor-promoting activity of MDSCs in vivo = 77
• 3.14. Increased MITF expression in MDSCs enhances tumor growth = 82
• 3.15. High MITF expression is associated with poor survival in patients with breast cancer = 86
• 3.16. Enhanced MITF expression in MDSCs is linked to tumor development in a murine lung carcinoma model = 88
• 3.17. The number of MITF+MDSCs is elevated in tumor tissues from cancer patients = 94
• 4. Discussion = 97
• 5. References = 104