인체 유방암에서 Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1)과의 직접 결합을 통한 Nrf2의 안정화 연구

학위논문 (박사) -- 서울대학교 대학원 : 융합과학기술대학원 분자의학 및 바이오제약학과, 2020. 8. Surh Young-Joon.Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) specifically recognizes phosphorylated serine or threonine of a target protein and isomerizes the adjacent proline residue. Overexpression of Pin1 has been found in many types of malignancies, suggesting its oncogenic function. Recent studies have revealed constitutive activation of Nrf2, a transcription factor that regulates cellular redox homeostasis, in some transformed or cancerous cells, conferring an advantage for their growth and survival. Silencing of Pin1 by using siRNA or pharmacologic inhibition blocked the accumulation of Nrf2, thereby suppressing proliferation and clonogenicity of MDA-MB-231 human breast cancer cells and xenograft tumor growth in nude mice. Since Nrf2 harbours pSer/Thr-Pro motifs, I investigated whether Pin1 could directly interact with Nrf2 in the context of its implications in breast cancer development and progression. I found that Pin1 binds to Nrf2 which stabilizes this transcription factor by hampering proteasomal degradation. Notably, the interaction between Pin1 and Nrf2 was dependent on the phosphorylation of Nrf2 at Ser 215, 408 and 577. In another study, Keap1, the main inhibitor of Nrf2, was found to be phosphorylated at Ser 104 and Thr 277. These amino acids are preceded by proline and hence can be the putative binding sites for Pin1. I found the direct interaction between Keap1 and Pin1, and this was abolished upon substation of Ser 104 and Thr 277 with Alanine. The interaction of Nrf2 with Keap1 was markedly increased when Pin1 was downregulated. On the other hand, Keap1 knockout embryonic fibroblasts exhibited the enhanced interaction between Nrf2 and Pin1. Therefore, it is likely that Pin1 and Nrf2 may compete with each other for Keap1 binding. In conclusion, Pin1 plays a role in stabilization and constitutive activation of Nrf2 interfering the interaction between Nrf2 and Keap1.단백질의 인산화는 세포내 신호전달 단백질들의 활성에 영향을 미치며, 세포내 기능 조절을 위해 매우 중요한 역할을 담당한다. 인산화 부위 중 세린/트레오닌 다음에 특이적으로 프롤린 (pSer/Thr-Pro)이 위치할 경우 CDKs, MAPKs, and GSK-3β등과 같은 proline directed kinase들 (에 의해 인산화된다. 프롤린은 cis나 trans의 conformation을 모두 채택할 수 있어 단백질의 접힘이나 기능 활성을 조절한다. Pin1 (peptidyl-prolyl isomerase family of proteins)은 프롤린 잔기의 cis/trans의 이성화반응을 촉매하는 효소로서 인산화된 세린/트레오닌 다음에 프롤린이 오는 motif에 유일하게 결합하여 cis/trans 이성질화를 촉진한다. Pin1의 구조는 N-말단에 있는 WW 도메인과 C-말단에 있는 PPIase 도메인으로 이루어져 있으며, WW 도메인은 기질 단백질과 결합하고 PPIase 도메인은 인산화된 세린/트레오닌-프롤린 (pSer/Thr-Pro) 모티프의 프롤린 이성질화를 통한 구조 변화에 관여한다. Pin1에 의한 기질 단백질의 구조 변화는 단백질의 활성, 단백질 상호결합 및 세포내 분포와 안정성에 영향을 미친다. Pin1은 세포 주기 발달에서 중요한 역할을 하는 단백질들을 조절하는 것으로 알려져 세포내의 Pin1 단백질 발현 수준의 변화는 세포의 과도한 증식과 관련된 질병인 암의 발생 및 진행에 영향을 줄 수 있다 Nrf2는 산화적 스트레스에 의해 유발되는 염증, 암 발생, 심혈관계 질환, 및 당뇨등의 병리학적 과정에 관여하여 세포를 보호해 준다. 정상적인 상태에서 Nrf2는 저해단백질인 kelch-like ECH-associated protein 1 (Keap1)에 결합된 비활성 상태로 세포질내에 존재한다. 하지만 세포가 활성산소종 또는 친전자성 물질에 의해 자극되면 Nrf2의 인산화 또는 Keap1이 thiol modification을 통해 Keap1으로부터 Nrf2가 분리된다. 이후 Nrf2는 핵내로 이동하고, 핵내에 존재하는 small Maf 단백질과 복합체를 이룬후 항산화 효소의 프로모터 지역에 결합함으로써 phase II 항산화/해독화 효소의 발현을 조절한다. 그러나 스트레스에 반응하여 일시적으로 활성화되는 정상세포의 경우와 달리 악성 종양 세포에서 Nrf2의 과도한 지속적인 발현은 암세포의 성장을 촉진시키고 항암치료의 효과를 감소시킨다. Nrf2 단백질에도 다수의 Pin1 결합부위 (pSer/Thr-Pro motifs)가 존재하므로, Pin1과 Nrf2과의 상호작용과 이를 통한 유방암 증식 및 진행에 미치는 영향에 관하여 연구하였다. 특히, Nrf2 단백질의 세린 215, 408 and 577의 인산화 잔기가 Pin1 과의 결합 부위임을 확인하였고, Pin1과 Nrf2의 결합은 Nrf2의 프로테아좀에 의한 분해를 억제함으로써 Nrf2의 안정화에 관여함을 알 수 있었다. 한편, Nrf2의 주요 억제 단백질인 Keap1은 유방암 세포내에서 세린 104번 잔기와 트레오닌 277번 잔기에서의 인산화가 이루어짐을 ESI-LC-MS 분석을 통하여 확인하였고, 세린 104번 잔기와 트레오닌 277번 잔기의 인산화가 차단된 돌연변이 세포는 Pin1과의 직접적 결합이 Keap1 wild type 세포에 비해 현저히 감소함을 확인 할 수 있었다. 또한 Pin1 siRNA를 처리한 MDA-MB-231 세포에서는 control siRNA 그룹에 비해 Nrf2와 Keap1의 결합이 증가하였으며, Keap1 knockout 마우스 배아에서 분리한 섬유아세포(embryonic fibroblasts)에서는 Nrf2와 Pin1의 결합이 증가되었다. 이를 통하여 Pin1과 Nrf2는 Keap1과의 결합에 있어서 서로 경쟁적 역할을 함을 확인하였다. 그러므로 Pin1은 Nrf2와 Keap1사이의 결합을 방해하며 Nrf2 단백질의 활성화와 안정화에 관여할 것으로 사료된다.CHAPTER I General verview 1 1. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) in cancer 2 1.1. Pin1 has critical roles in breast cancer development and progression 2 1.2. Regulation of Pin1 gene expression in human breast cancer 3 1.3. Expression, post-translational modifications and subcellular localization of Pin1 4 1.4. The significance of Pin1 phosphorylation 8 1.5. Pin1 regulates signaling molecules associated with cancer and cancer stem cells. 8 1.6. Pin1 mediates drug resistance of breast cancer 9 1.7. Pin1 inhibitors 9 2. Nrf2 in cancer 12 2.1. Nrf2 promotes tissue invasion and metastasis 12 2.2. Role of Nrf2 in resistance to chemotherapy 13 2.3. Keap1 as an inhibitor of Nrf2 13 2.4. Keap1-independent regulation of Nrf2 14 2.5. Post-translational modifications of Nrf2 15 2.6. Protein stabilization of Nrf2. 17 REFERENCES 19 STATEMENT OF PURPOSE 36 CHAPTER II 37 Pin1 stabilizes Nrf2 in a Keap1 independent manner in breast cancer . 37 ABSTRACT 38 1. INTRODUCTION. 40 2. MATERIALS AND METHODS 43 3. RESULTS. 55 4. DISCUSSION. 99 REFERENCES. 104 CHAPTER III 111 H-Ras induces Nrf2-Pin1 interaction: Implications for breast cancer progression. 111 ABSTRACT. 112 1. INTRODUCTION. 113 2. MATERIALS AND METHODS 116 3. RESULTS. 124 4. DISCUSSION. 144 REFERENCES. 149 ABSTRACT IN KOREAN.. 160 CURRIULUM VITAE 165Docto

The positive feedback loop between Nrf2 and phosphogluconate dehydrogenase stimulates proliferation and clonogenicity of human hepatoma cells

© 2020, © 2020 Informa UK Limited, trading as Taylor & Francis Group.Recent studies report that nuclear factor-erythroid-2-related factor 2 (Nrf2) facilitates tumor progression through metabolic reprogramming in cancer cells. However, the molecular mechanism underlying the oncogenic functions of Nrf2 is not yet well understood. Some of the pentose phosphate pathway (PPP) enzymes are considered to play a role in the cancer progression. The present study was intended to explore the potential role of phosphogluconate dehydrogenase (PGD), one of the PPP enzymes, in the proliferation and migration of human hepatoma HepG2 cells. Genetic ablation of Nrf2 attenuated the expression of PGD at both transcriptional and translational levels. Notably, Nrf2 regulates the transcription of PGD through direct binding to the antioxidant response element in its promoter region. Nrf2 overexpression in HepG2 cells led to increased proliferation, survival, and migration, and these events were suppressed by silencing PGD. Interestingly, knockdown of the gene encoding this enzyme not only attenuated the proliferation and clonogenicity of HepG2 cells but also downregulated the expression of Nrf2. Thus, there seems to exist a positive feedback loop between Nrf2 and PGD which is exploited by hepatoma cells for their proliferation and survival. Treatment of HepG2 cells with ribulose-5-phosphate, a catalytic product of PGD, gave rise to a concentration-dependent upregulation of Nrf2. Collectively, the current study shows that Nrf2 promotes hepatoma cell growth and progression, partly through induction of PGD transcription.

Breast cancer cell debris diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor-associated macrophages

Chemotherapy is commonly used as a major therapeutic option for breast cancer treatment, but its efficacy is often diminished by disruption of patient's anti-tumor immunity. Chemotherapy-generated tumor cell debris could hijack accumulated tumor-associated macrophages (TAMs), provoking tumor recurrence. Therefore, reprogramming TAMs to acquire an immunocompetent phenotype is a promising strategy to potentiate therapeutic efficacy. In this study, we analyzed the proportion of immune cells in the breast cancer patients who received chemotherapy. To validate our findings in vivo, we used a syngeneic murine breast cancer (4T1) model. Chemotherapy generates an immunosuppressive tumor microenvironment in breast cancer. Here, we show that phagocytic engulfment of tumor cell debris by TAMs reduces chemotherapeutic efficacy in a 4T1 breast cancer model. Specifically, the engulfment of tumor cell debris by macrophages reduced M1-like polarization through heme oxygenase-1 (HO-1) upregulation. Conversely, genetic or pharmacologic inhibition of HO-1 in TAMs restored the M1-like polarization. Our results demonstrate that tumor cell debris-induced HO-1 expression in macrophages regulates their polarization. Inhibition of HO-1 overexpression in TAMs may provoke a robust anti-tumor immune response, thereby potentiating the efficacy of chemotherapy.

Docosahexaenoic acid induces expression of heme oxygenase-1 and NAD(P)H: Quinone oxidoreductase through activation of Nrf2 in human mammary epithelial cells

Docosahexaenoic acid (DHA), an omega-3 fatty acid abundant in fish oils, has diverse health beneficial effects, such as anti-oxidative, anti-inflammatory, neuroprotective, and chemopreventive activities. In this study, we found that DHA induced expression of two representative antioxidant/ cytoprotective enzymes, heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase (NQO1), in human mammary epithealial (MCF-10A) cells. DHA-induced upregulation of these enzymes was accompanied by enhanced translocation of the redox-sensitive transcription factor Nrf2 into the nucleus and its binding to antioxidant response element. Nrf2 gene silencing by siRNA abolished the DHA-induced expression of HO-1 and NQO1 proteins. When MCF-10A cells were transfected with mutant constructs in which the cysteine 151 or 288 residue of Keap1 was replaced by serine, DHA-induced expression of HO-1 and NQO1 was markedly reduced. Moreover, DHA activated protein kinase C (PKC)delta and induced Nrf2 phosphorylation. DHA-induced phosphorylation of Nrf2 was abrogated by the pharmacological PKC delta inhibitor rottlerin or siRNA knockdown of its gene expression. The antioxidants N-acetyl-L-cysteine and Trolox attenuated DHA-induced activation of PKC delta, phosphorylation of Nrf2, and and its target protein expression. In conclusion, DHA activates Nrf2, possibly through modification of critical Keap1 cysteine 288 residue and PKC delta-mediated phosphorylation of Nrf2, leading to upregulation of HO-1 and NQO1 expression

Interaction between Peptidyl-prolyl Cis-trans Isomerase NIMA-interacting 1 and GTP-H-Ras: Implications for Aggressiveness of Human Mammary Epithelial Cells and Drug Resistance

Aberrant activation of Ras has been implicated in aggressiveness of breast cancer. Among Ras isoforms (H-, K-, and N-), H-Ras has been known to be primarily responsible for invasion and metastasis of breast cancer cells. Phosphorylation of serine (Ser) or threonine (Thr) is a key regulatory mechanism responsible for controlling activities and functions of various proteins involved in intracellular signal transduction. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1, Pin1 changes the conformation of a subset of proteins phosphorylated on Ser/Thr that precedes proline (Pro). In this study we have found that Pint is highly overexpressed in human breast tumor tissues and H-Ras transformed human mammary epithelial (H-Ras MCF10A) and MDA-MB-231 breast cancer cells. Notably, Pin1 directly bound to the activated form of H-Ras harbouring a Ser/Thr-Pro motif. Pharmacologic inhibition of Pin1 reduced clonogenicity of MDA-MB-231 human breast cancer cells. Paclitaxel accelerates apoptosis in Pin1 silenced H-Ras MCF10A cells. MDR genes (MDR1 and MRP4) were significantly downregulated in MDA-MB-231 cells stably silenced for Pin1. We speculate that Pint interacts with GTP-H-Ras, thereby upregulating the expression of drug resistance genes, which confers survival advantage and aggressiveness of breast cancer cells under chemotherapy.

Stabilization of C/EBP beta through direct interaction with STAT3 in H-Ras transformed human mammary epithelial cells

Signal transducer and activator of transcription 3 (STAT3) plays important roles in cancer-associated inflammation by controlling expression of proinflammatory cytokines and chemokines. Recent studies suggest that C/EBP13 (CCAAT-enhancer binding protein beta) and STAT3 synergistically stimulate cancer cell proliferation and epithelial-mesenchymal transition. C/EBP13 is a leucine-zipper transcription factor that regulates expression of a variety of inflammatory cytokines or chemokines, such as IL-8, G-CSF (granulocyte colony stimulating factor), and GM-CSF (granulocyte macrophage colony stimulating factor) which induce neutrophil infiltration and differentiation. However, molecular mechanisms by which STAT3 and C/EBP13 cooperatively interact had not been fully elucidated. In this study, we found that the level of C/EBP13 protein, but not that of its mRNA transcript, was decreased in the absence of STAT3 in H Ras transformed human mammary epithelial (H -Ras MCF10A) cells. In addition, silencing STAT3 dramatically induced ubiquitination of C/EBP13 for proteasomal degradation. Furthermore, direct inter action between STAT3 and C/EBP13 was confirmed by immunoprecipitation and proximity ligation assays. Taken together, these results suggest that STAT3 stabilizes C/EBP13, thereby promoting cancer-associated inflammation. (c) 2021 Elsevier Inc. All rights reserved.

Tumor Promoting Effects of Sulforaphane on Diethylnitrosamine-Induced Murine Hepatocarcinogenesis

Nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor involved in protection against initiation of carcinogenesis in normal cells. Notably, recent studies have demonstrated that aberrant activation of NRF2 accelerates the proliferation and progression of cancer cells. The differential effects of NRF2 on multi-stage carcinogenesis have raised a concern about the validity of NRF2 activators for chemoprevention. This prompted us to assess the effects of sulforaphane (SFN), a prototypic NRF2 activating chemopreventive phytochemical, on experimentally induced carcinogenesis. In the present study, SFN was daily injected intraperitoneally (25 mg/kg) for 3 months to male C57BL/6 mice at 6 months after single intraperitoneal administration of a hepatocarcinogen, diethylnitrosamine (DEN). The liver to body weight ratio, tumor growth, and the number and the size of hepatomas measured at 9 months after DEN administration were significantly higher in SFN-treated mice than those in vehicle-treated mice. Moreover, the expression of NRF2, its target protein NAD(P)H:quinone oxidoreductase 1, and the cell proliferation marker, proliferating cell nuclear antigen was further elevated in DEN plus SFN-treated mice. These results suggest that once hepatocarcinogenesis is initiated, SFN may stimulate tumor progression

Heregulin-β1 Activates NF-E2-related Factor 2 and Induces Manganese Superoxide Dismutase Expression in Human Breast Cancer Cells via Protein Kinase B and Extracellular Signal-regulated Protein Kinase Signaling Pathways

Heregulin-beta 1, a ligand of ErbB-2 and ErbB-3/4 receptors, has been reported to potentiate oncogenicity and metastatic potential of breast cancer cells. In the present work, treatment of human mammary cancer (MCF-7) cells with heregulin-beta 1 resulted in enhanced cell migration and expression of manganese superoxide dismutase (MnSOD) and its mRNA transcript. Silencing of MnSOD abrogated clonogenicity and migrative ability of MCF-7 cells. Heregulin-beta 1 treatment also increased nuclear translocation, antioxidant response element binding and transcriptional activity of NF-E2-related factor 2 (Nrf2). A dominant-negative mutant of Nrf2 abrogated heregulin-beta 1-induced MnSOD expression. Treatment with heregulin-beta 1 caused activation of protein kinase B (Akt) and extracellular signal-regulated protein kinase (ERK). The pharmacological inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase 1/2, which are upstream of Akt and ERK, respectively, attenuated heregulin-beta 1-induced MnSOD expression and nuclear localization of Nrf2. In conclusion, heregulin-1 induces upregulation of MnSOD and activation of Nrf2 via the Akt and ERK signaling in MCF-7 cells, which may confer metastatic potential and invasiveness of these cells.

Interaction of Nrf2 with dimeric STAT3 induces IL-23 expression: Implications for breast cancer progression

Persistent activation of STAT3 and Nrf2 is considered to stimulate the aggressive behavior of basal-like breast cancer (BLBC). However, the precise mechanism underlying sustained overactivation of these transcription factors and their roles in breast cancer progression remain elusive. Analysis of the TCGA multi-omics data showed that high levels of STAT3 and Nrf2 mRNA were correlated with elevated expression of P-STAT3(Y705) and Nrf2 target proteins in breast cancer patients. Our present study demonstrates a unique interaction between Nrf2 and STAT3 in the maintenance and progression of BLBC. RNA sequencing analysis identified the gene encoding IL-23A upregulated by concurrent binding of STAT3 and Nrf2 to its promoter. IL-23A depletion also showed the similar phenotypic changes to those caused by double knockdown of both transcription factors. In conclusion, the STAT3-Nrf2 interaction accelerates BLBC growth and progression by augmenting IL-23A expression, which underscores the importance of subtype-specific molecular pathways in human breast cancer.Y

Alternative regulation of HIF-1α stability through Phosphorylation on Ser451

The hypoxia-inducible factor (HIF-1 alpha) functions as a master regulator of oxygen homeostasis. Oxygen dependent hydroxylation of HIF-1 alpha is tightly regulated by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). The prolyl hydroxylation facilitates the recruitment of the von HippelLindau (VHL) protein, leading to ubiquitination and degradation of HIF-1 alpha by the proteasomes. Besides prolyl hydroxylation, phosphorylation of HIF-1 alpha is another central post-translational modification, which regulates its stability under hypoxic conditions as well as normoxic conditions. By use of LC/MS/MSbased analysis, we were able to identify a specific serine residue (Ser451) of HIF-1 alpha phosphorylated under hypoxic conditions. Using plasmids expressing wild type (WT), non-phosphorylatable mutant HIF1 alpha (S451A), and phosphomimetic mutant HIF-1 alpha (S451E), we demonstrated that the phosphorylation at Ser451 is important in maintaining the HIF-1 alpha protein stability. Notably, phosphorylation at S451 interrupts the interaction of HIF-1 alpha with PHD and pVHL. A phosphomimetic construct of HIF-1 alpha at Ser451 (S451E) is significantly more stable than WT HIF-1 alpha under normoxic conditions. Cells transfected with unphosphorylatable HIF-1 alpha exhibited significantly lower HIF-1 transcriptional activity than WT cells and markedly reduced tumor cell migration. Further, tumors derived from the phosphomimetic mutant cells grew faster, whereas the tumors derived from non-phosphorylatable mutant cells grew slower than the control tumors, suggesting that the phosphorylation of HIF-1 alpha at the Ser451 site is critical to promote tumor growth in vivo. Taken together, our data suggest an alternative mechanism responsible for the regulation of HIF-1 alpha stability. (C) 2021 Elsevier Inc. All rights reserved.