인간 유방암 세포주에서 Glut1에 의해 매개되는 암화 현상

Abstract

neu/Egr-1 야생형 (WT), neu/Egr-1 이형접합체 (het), neu/Egr-1 낙아웃 (KO). 본 연구자는 Neu/Egr-1 낙아웃 마우스와 neu/Egr-1 야생형, 이형접합체와의 종양 발생 시점, 마우스 당 발생한 종양 개수 등을 비교하였다. 또한 생체 외에서 Egr-1 의 기능을 조사하기 위해서 각 마우스의 유방암으로부터 neu/Egr-1 야생형 및 낙아웃 종양 세포주를 확립하였다. 이전 연구에 따르면, HER2/neu를 발현하지 않는 Egr-1 낙아웃 마우스의 유선에서 PTEN의 단백질이 감소하였고, Akt의 인산화가 증가를 관찰할 수 있었다. 따라서, neu/Egr-1 낙아웃 마우스는 neu/Egr-1 야생형 마우스와 비교하여 Egr-1/PTEN/Akt 신호 전달 경로를 조절함으로써 더 발암성을 가질 것이라고 예상했다. 그러나 예상과 달리, Egr-1 낙아웃은 in vivo에서 종양 발달을 지연시키고, in vitro에서 세포 성장 속도를 감소시켰다. 본 연구 결과는 Egr-1이 발암유전자로서 HER2/neu에 의한 유방암 발생에 필수적이고, 종양 발달에 필요하다는 것을 시사한다.|Breast cancer is the second leading cause of cancer-related death among women worldwide. Breast cancer is divided into four major subtypes: luminal A (estrogen receptor (ER) +, progesterone (PR) +, HER2 -), luminal B (ER+, PR+, HER2+), HER2-positive (ER-, PR-, HER2+), triple-negative (ER-, PR-, HER2-). The luminal A and B breast cancer subtypes consist of 60~70% of all breast cancers. The HER2-positive subtype occurs in about 20% of breast cancers. Because the triple-negative breast cancers (TNBCs) lack receptors for ER, PR or HER, treatment options for TNBCs are confined to cytotoxic chemotherapy. Therefore, it is important to find a target for the treatment of TNBCs. Glut1 is expressed at high levels in several types of cancer including breast cancers, especially TNBCs. The purpose of this study was to better understand the ‘Glut1-mediated tumorigenesis in human breast cancer’. To investigate alternative roles of Glut1 in breast cancer, I silenced Glut1 in triple-negative breast cancer cell lines using a short hairpin RNA (shRNA) system. Glut1 silencing was confirmed by Western blotting and qRT-PCR. Ablation of Glut1 resulted in decreased cell proliferation, glucose uptake, migration, and invasion through modulation of the EGFR/MAPK signaling pathway and integrin β1/Src/FAK signaling pathways. These results suggest that Glut1 not only plays a role as a glucose transporter, but also serves as a regulator of signaling cascades in the tumorigenesis of breast cancer. It suggests that Glut1 might be a therapeutic target for TNBCs. Therefore, I investigated the possibility of Glut1 as a therapeutic target in TNBC cell lines. I determined whether Glut1 silencing acted synergistically with chemotherapeutic agents to inhibit proliferation of TNBC cells. Contrary to my expectations, downregulation of Glut1 attenuated apoptosis and increased drug resistance via upregulation of p-Akt/p-GSK-3β (Ser9)/β-catenin/survivin. These results indicated that the potential of Glut1 as a therapeutic target should be carefully reevaluated. In Glut1 shRNA cells, Akt activity was significantly increased despite reduced EGFR activity. This was because PTEN levels have decreased through the EGFR/ERK/Egr-1 pathway. Egr-1 is a well-known transcription factor of PTEN and PTEN is a negative regulator of the PI3K/Akt pathway. Therefore, it can be speculated that Egr-1 would function as a tumor suppressor. Actually, Egr-1 is known as a tumor suppressor by directly regulating multiple tumor suppressors including TGFβ1, PTEN, p53 and fibronectin and is often inhibited in tumors. However, I also observed that Egr-1 contributed to the induction of EGFR gene, which serves as an oncogene. To determine whether Egr-1 functions as a tumor suppressor or an oncogene in breast tumors in vivo, I used mouse models of breast cancer induced by HER2/neu. HER2/neu is a well-known oncogenic inducer of human breast cancer. I generated 3 types of bigenic mice by crossing the MMTV-neu mice with Egr-1 knockout (-/-) mice; Breast cancer is the second leading cause of cancer-related death among women worldwide. Breast cancer is divided into four major subtypes: luminal A (estrogen receptor (ER) +, progesterone (PR) +, HER2 -), luminal B (ER+, PR+, HER2+), HER2-positive (ER-, PR-, HER2+), triple-negative (ER-, PR-, HER2-). The luminal A and B breast cancer subtypes consist of 60~70% of all breast cancers. The HER2-positive subtype occurs in about 20% of breast cancers. Because the triple-negative breast cancers (TNBCs) lack receptors for ER, PR or HER, treatment options for TNBCs are confined to cytotoxic chemotherapy. Therefore, it is important to find a target for the treatment of TNBCs. Glut1 is expressed at high levels in several types of cancer including breast cancers, especially TNBCs. The purpose of this study was to better understand the ‘Glut1-mediated tumorigenesis in human breast cancer’. To investigate alternative roles of Glut1 in breast cancer, I silenced Glut1 in triple-negative breast cancer cell lines using a short hairpin RNA (shRNA) system. Glut1 silencing was confirmed by Western blotting and qRT-PCR. Ablation of Glut1 resulted in decreased cell proliferation, glucose uptake, migration, and invasion through modulation of the EGFR/MAPK signaling pathway and integrin β1/Src/FAK signaling pathways. These results suggest that Glut1 not only plays a role as a glucose transporter, but also serves as a regulator of signaling cascades in the tumorigenesis of breast cancer. It suggests that Glut1 might be a therapeutic target for TNBCs. Therefore, I investigated the possibility of Glut1 as a therapeutic target in TNBC cell lines. I determined whether Glut1 silencing acted synergistically with chemotherapeutic agents to inhibit proliferation of TNBC cells. Contrary to my expectations, downregulation of Glut1 attenuated apoptosis and increased drug resistance via upregulation of p-Akt/p-GSK-3β (Ser9)/β-catenin/survivin. These results indicated that the potential of Glut1 as a therapeutic target should be carefully reevaluated. In Glut1 shRNA cells, Akt activity was significantly increased despite reduced EGFR activity. This was because PTEN levels have decreased through the EGFR/ERK/Egr-1 pathway. Egr-1 is a well-known transcription factor of PTEN and PTEN is a negative regulator of the PI3K/Akt pathway. Therefore, it can be speculated that Egr-1 would function as a tumor suppressor. Actually, Egr-1 is known as a tumor suppressor by directly regulating multiple tumor suppressors including TGFβ1, PTEN, p53 and fibronectin and is often inhibited in tumors. However, I also observed that Egr-1 contributed to the induction of EGFR gene, which serves as an oncogene. To determine whether Egr-1 functions as a tumor suppressor or an oncogene in breast tumors in vivo, I used mouse models of breast cancer induced by HER2/neu. HER2/neu is a well-known oncogenic inducer of human breast cancer. I generated 3 types of bigenic mice by crossing the MMTV-neu mice with Egr-1 knockout (-/-) mice; 유방암은 전세계 여성들의 암 관련 사망 원인 중 두 번째로 큰 원인이다. 유방암은 luminal A, luminal B, HER2 양성, 삼중음성의 네 가지 주요 아형으로 분류된다. Luminal A 및 luminal B 유방암 아형은 전체 유방암의 60~70%를 차지한다. HER2 양성 아형은 유방암의 약 20%에서 발생한다. 삼중음성유방암은 에스트로젠 수용체, 프로제스테론 수용체, HER2 수용체가 없기 때문에 이 아형의 치료는 세포 독성 화학요법에 국한된다. 따라서, 삼중음성유방암의 치료 타겟을 찾는 것은 중요하다. Glut1은 유방암, 특히 삼중음성유방암을 표함한 여러 유형의 암에서 높은 수준으로 발현된다. 본 연구의 목적은 ‘인간 유방암 세포주에서 Glut1에 의해 매개되는 암화 현상’을 알아보고자 함이다. 유방암에서 Glut1의 포도당수송체 이외의 역할을 연구하기 위해, shRNA 시스템을 이용하여 삼중음성유방암 세포주에서 Glut1을 낙다운시켰다. Glut1 발현 억제는 웨스턴블랏과 qRT-PCR로 확인하였다. Glut1의 발현 억제는 EGFR/MAPK 신호전달 경로와 integrin β1/Src/FAK 신호전달 경로의 조절을 통한 세포 증식, 포도당 흡수, 이동 및 침윤을 감소시켰다. 본 연구 결과는 Glut1이 포도당수송체 역할을 할 뿐만 아니라 유방의 종양 발생에서의 신호전달 경로의 조절 인자 역할을 한다는 것을 의미한다. 이는 Glut1이 삼중음성유방암의 치료 타겟이 될 수 있음을 시사한다. 따라서, 본 연구자는 삼중음성유방암 세포주에서 Glut1이 치료 타겟으로서의 가능성이 있는지에 관한 연구를 진행하였다. Glut1의 발현 억제가 삼중음성유방암 세포의 증식을 억제하기 위해 화학치료요법과 상승작용하는지 확인하였다. 예상과 다르게, Glut1의 발현 억제는 세포예정사를 감소시키고, p-Akt/p-GSK-3β (Ser9)/β-catenin/survivin 의 상향 조절을 통해 약물에 대한 내성을 증가시켰다. 위 결과는 Glut1의 치료 표적으로서의 가능성이 신중하게 재평가되어야 한다는 것을 암시한다. Glut1 발현 억제 세포주에서, EGFR활성의 감소에도 불구하고 Akt 활성이 증가한 것을 확인하였는데, 이것은 EGFR/ERK/Egr-1 신호전달 경로를 통해서 PTEN의 발현이 감소했기 때문이다. Egr-1은 PTEN의 잘 알려진 전사인자이며, PTEN은 PI3K/Akt의 음성 조절 인자이다. 따라서, Egr-1은 종양 억제 인자로서 기능할 것이라고 추측할 수 있었다. 실제로, Egr-1은 TGFβ1, PTEN, p53, fibronectin 을 포함한 여러 종양 억제 인자를 직접 조절함으로써 종양 억제 기능을 한다고 알려져 있으며, 종종 종양에서 발현이 억제된 것을 확인할 수 있다. 그러나, 본 연구자는 Egr-1이 발암유전자인 EGFR유전자의 발현을 유도한다는 것도 관찰하였다. Egr-1이 유방 종양에서 종양 억제 인자로 작용하는지, 발암유전자로 작용하는지 확인하기 위해 HER2/neu에 의해 유도된 유방암 마우스 모델을 사용했다. HER2/neu는 인간 유방암의 잘 알려진 종양 유도인자이다. 본 연구자는 MMTV-neu 마우스와 Egr-1 낙아웃 마우스를 교배시켜 세 가지 유형의 이원성 마우스를 만들었다; neu/Egr-1 wild type (+/+) (neu/Egr-1 WT), neu/Egr-1 heterozygote (+/-) (neu/Egr-1 het) and neu/Egr-1 knockout (neu/Egr-1 KO). Neu/Egr-1 KO mice were compared with neu/Egr-1 WT or het mice with regard to onset of tumor appearance and the number of tumors per mouse. In addition, to examine the role of Egr-1 in vitro, I established neu/Egr-1 WT and KO tumor cell lines derived from the breast tumors developed in each mice. Ju et al. previously observed that PTEN protein level was downregulated and phosphorylated Akt was upregulated in the mammary glands of Egr-1 deficient mice which do not express HER2/neu. Hence, I expected that neu/Egr-1 KO mice would have more tumorigenic properties by modulating Egr-1/PTEN/Akt signaling axis compared with neu/Egr-1 WT mice. Contrary to my expectations, Egr-1 deletion delayed the tumor development in vivo and decreased the rate of cell growth in vitro. These results suggest that Egr-1 plays a role in HER2/neu driven mammary tumorigenesis as an oncogene and is required for tumor development.