급성전골수세포백혈병에서 분화와 PML-RARA 단백질 분해의 조절

Abstract

Acute promyelocytic leukemia (APL) is a malignant disease with abnormal accumulation of immature stage through reciprocal chromosomal translocation between Promylocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes. Although, all–trans retinoic acid (ATRA) and arsenic trioxide (ATO) have been shown to be remarkably effective as agents that cures APL, patients frequently develop drug resistance and relapse. Therefore, study for these drugs-induced molecular mechanisms and proposal of new therapeutic agent are necessary for completely remission of APL disease. Thus, it was to approach this goal from three aspects, First, anti-cancer effect of Korean red ginseng extract (KRGE) as therapeutic agent on APL cure, second, the role of Pin1 in PML-RARA degradation through ATRA-mediated PKA signaling activation, third, the role of PCGF2 in ATO-triggered PML-RARA sumoylation process. The overall goal of this thesis was to study on regulation of differentiation and PML-RARA protein degradation in APL. The purpose of this study was to identify the effect of KRGE on the APL cell line NB4 and investigate its molecular mechanisms. Upon KRGE treatment, induction of differentiation accompanies with cell cycle attenuation at the G1 phase in NB4 cells. Mechanistically, KRGE was shown to increase Cdk1b protein stability via downregulation of MYC-SKP2 dependent pathway. These finding suggest that KRGE may become an attractive candidates and assistant to cure APL. ATRA-induced PML-RARA protein degradation, protein kinase A (PKA) signaling and RARA phosphorylation cascade, and interaction of the peptidyl-prolyl-isomerase Pin1 with phosphorylated RARA have been emphasized by several studies. However, a sequential molecular mechanism from ATRA-induced PKA activation regard to PML-RARA phosphorylation, resulting in its degradation via binding of Pin1 that remains unknown. It has observed ATRA degrades PML-RARA protein via PKA signaling, such effect was reversed by Pin1 inhibitor. In addition, physical interaction between PML-RARA and Pin1 proteins enhanced in presence to ATRA, and Pin1 induces degradation of ATRA-induced PML-RARA phosphorylation, but not its mutants. Collectively, these results indicate that PKA signaling pathway, PML-RARA serine 581 and 873 phosphorylation, Pin1 activity, and PML-RARA-Pin1 interaction as a molecular mechanism for ATRA-induced PML-RARA protein degradation in APL. It has been previously reported that ATO degrades PML or PML-RARA protein through a Ubc9/SUMO-triggered Rnf4/Ubiquitin-mediated pathway and Polycomb group Pcgf2 gene interacts with Ubc9 to inhibit substrates sumoylation. It has been proposed that Pcgf2 may relate to ATO-mediated sumoylation of PML-RARA protein for its degradation. Thus, inhibition of PCGF2 results in enhanced PML-RARA protein degradation via increasing sumolyation and ubiquitination on PML region of PML-RARA, and subsequently accelerated degradation of PML-RARA protein. Reciprocal interaction of Pcgf2 with Ubc9 was disassociated by ATO exposure. In addition, UBC9 elevates sumoylation-mediated PML-RARA degradation, but its event was inhibited by expression of PCGF2. Moreover it has identified that Pcgf2 sumoylation practically participates in ATO-induced its loss. Taken together, PCGF2 negatively regulates ATO-induced PML-RARA degradation via Ubc9 inhibition. Along with these finding, suggestion for KRGE as medical assistant on APL cure, the role of Pin1 in ATRA-induced PML-RARA degradation, and PCGF2 as negatively regulator in ATO-triggered PML-RARA degradation are new and important discoveries in APL therapeutic model. |급성전골수세포백혈병 (acute promyelocytic leukemia, APL) 은 미분화 세포의 수와 PML-RARA 종양단백을 가진 악성 질환이다. 비록 급성전골수세포백혈병은 all-trans retinoic acid (ATRA) 와 arsenic trioxide (ATO) 같은 치료제의 높은 반응성으로 급성골수세포백혈병 (acute myelogenous leukemia, AML) 중에서 가장 예후가 좋다. 하지만 여전히 약물 내성과 재발 등의 극복해야 할 문제들이 남아있다. 그러므로, 새로운 치료 약제 개발과 분자 수준에서 질환 이해가 이 악성질환을 정복하는데 있어 도움이 될 것이다. 이러한 점에서 본 논문은 3가지 다른 접근을 통해 급성전골수세포백혈병의 이해를 기술하였다. 첫 번째, 천연 산물인 고려홍삼추출물의 치료제로서 가능성, 두 번째, ATRA, PKA, Pin1 매개되는 PML-RARA 단백 분해의 이해, 마지막으로, ATO 유도된 PML-RARA 단백 분해에서 polycomb group 인 PCGF2 유전자의 역할 규명이다. 우선 천연물인 KRGE (Korean red ginseng extracts)가 급성전골수세포백혈병 NB4 세포주에서 미치는 효과를 검정하였다. NB4 세포에 KRGE 처리는 세포 분화 유도를 수반하는 G1 단계에서 세포주기 억제를 보였다. 기전을 살펴보게 되면, KRGE 는 MYC-SKP2 의존적인 경로 억제를 통해 Cdkn1b 단백의 안정성을 높였다. KRGE 를 통한 Cdkn1b 단백 조절에 있어서 MYC-SKP2-Cdkn1b 의 상관관계를 증명하였다. 이 결과는 KRGE 가 APL 치료에 유익한 후보이자 도움이 된다고 제안하였다. ATRA 유도된 PML-RARA 종양단백 분해와 안정성에서 Pin1의 역할, PKA 활성화와 RARA 인산화 연속, 그리고 RARA 세린 77 인산화된 RARA와 Pin1의 결합들은 여러 연구를 통해 강조되었다. 그러나 ATRA 유도된 PKA 활성화가 PML-RARA 단백 인산화, 결과적으로 Pin1 단백 의 결합을 통해 분해가 되는지는 규명되지 않았다. ATRA가 PKA 신호를 통해 PML-RARA 종양단백을 억제와 이러한 결과가 Pin1 억제제로 인해 회복되는 것을 관찰하였다. 추가적으로 ATRA하에 야생형 PML-RARA과 Pin1 단백간의 물리적인 결합이 증가하였고, Pin1이 ATRA에 인산화된 PML-RARA의 분해를 유도하였다. 종합해보면, PKA 신호전달, PML-RARA 단백의 세린 581 과 873 인산화, Pin1 활성, 그리고 PML-RARA와 Pin1 의 연결은 급성전골수세포백혈병에서 ATRA 유도된 PML-RARA 종양단백 분해 기전으로 제안하였다. ATO는 PML 혹은 PML-RARA 단백을 Ubc9/SUMO 유발되어 Rnf4/Ubiquitin 매개된 경로를 통해 분해시키고, 그리고 polycomb group Pcgf2 단백은 Ubc9 단백과 결합하여 표적의 sumoylation 을 억제하였다가 보고되었다. 그러므로 ATO 에 매개되는 PML-RARA 단백의 스모화 (sumoylation)를 통한 분해과정에서 PCGF2 유전자의 역할을 규명하고자 하였다. 그래서 인위적인 PCGF2 억제는 PML-RARA 의 PML 부위에 스모화과 유비퀴틴화의 증가를 통해 PML-RARA 단백의 분해 가속화를 이끌었다. Pcgf2 와 Ubc9 단백의 상호결합은 ATO 에 의해서 분리되었다. 추가적으로 UBC9은 수모화 매개된 PML-RARA 단백 분해를 조절하고, 이러한 현상은 PCGF2 의 발현에 의해 억제하였다. 게다가 ATO에 의해서 PCGF2 수모화가 유도되어 손실된다고 증명하였다. 결론적으로 PCGF2는 ATO에 의해 유도되는 PML-RARA 종양단백 분해에 음성적인 조절자이고 UBC9의 활성 억제를 통해 PML-RARA 안정성을 조절됨을 기전으로 제시하였다. 이러한 결과들을 종합해보면, KRGE는 치료제로 제안, ATRA에 유도되는 PML-RARA 분해 과정을 정리, 그리고 PCGF2 유전자는 ATO에 의해 유발되는 PML-RARA 단백 분해에서 음성적인 역할로서 급성전골수세포백혈병의 치료 모델에 새롭고 중요한 발견이라 할 수 있다.; Acute promyelocytic leukemia (APL) is a malignant disease with abnormal accumulation of immature stage through reciprocal chromosomal translocation between Promylocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes. Although, all–trans retinoic acid (ATRA) and arsenic trioxide (ATO) have been shown to be remarkably effective as agents that cures APL, patients frequently develop drug resistance and relapse. Therefore, study for these drugs-induced molecular mechanisms and proposal of new therapeutic agent are necessary for completely remission of APL disease. Thus, it was to approach this goal from three aspects, First, anti-cancer effect of Korean red ginseng extract (KRGE) as therapeutic agent on APL cure, second, the role of Pin1 in PML-RARA degradation through ATRA-mediated PKA signaling activation, third, the role of PCGF2 in ATO-triggered PML-RARA sumoylation process. The overall goal of this thesis was to study on regulation of differentiation and PML-RARA protein degradation in APL. The purpose of this study was to identify the effect of KRGE on the APL cell line NB4 and investigate its molecular mechanisms. Upon KRGE treatment, induction of differentiation accompanies with cell cycle attenuation at the G1 phase in NB4 cells. Mechanistically, KRGE was shown to increase Cdk1b protein stability via downregulation of MYC-SKP2 dependent pathway. These finding suggest that KRGE may become an attractive candidates and assistant to cure APL. ATRA-induced PML-RARA protein degradation, protein kinase A (PKA) signaling and RARA phosphorylation cascade, and interaction of the peptidyl-prolyl-isomerase Pin1 with phosphorylated RARA have been emphasized by several studies. However, a sequential molecular mechanism from ATRA-induced PKA activation regard to PML-RARA phosphorylation, resulting in its degradation via binding of Pin1 that remains unknown. It has observed ATRA degrades PML-RARA protein via PKA signaling, such effect was reversed by Pin1 inhibitor. In addition, physical interaction between PML-RARA and Pin1 proteins enhanced in presence to ATRA, and Pin1 induces degradation of ATRA-induced PML-RARA phosphorylation, but not its mutants. Collectively, these results indicate that PKA signaling pathway, PML-RARA serine 581 and 873 phosphorylation, Pin1 activity, and PML-RARA-Pin1 interaction as a molecular mechanism for ATRA-induced PML-RARA protein degradation in APL. It has been previously reported that ATO degrades PML or PML-RARA protein through a Ubc9/SUMO-triggered Rnf4/Ubiquitin-mediated pathway and Polycomb group Pcgf2 gene interacts with Ubc9 to inhibit substrates sumoylation. It has been proposed that Pcgf2 may relate to ATO-mediated sumoylation of PML-RARA protein for its degradation. Thus, inhibition of PCGF2 results in enhanced PML-RARA protein degradation via increasing sumolyation and ubiquitination on PML region of PML-RARA, and subsequently accelerated degradation of PML-RARA protein. Reciprocal interaction of Pcgf2 with Ubc9 was disassociated by ATO exposure. In addition, UBC9 elevates sumoylation-mediated PML-RARA degradation, but its event was inhibited by expression of PCGF2. Moreover it has identified that Pcgf2 sumoylation practically participates in ATO-induced its loss. Taken together, PCGF2 negatively regulates ATO-induced PML-RARA degradation via Ubc9 inhibition. Along with these finding, suggestion for KRGE as medical assistant on APL cure, the role of Pin1 in ATRA-induced PML-RARA degradation, and PCGF2 as negatively regulator in ATO-triggered PML-RARA degradation are new and important discoveries in APL therapeutic model.